SVMP Book Chapter
SVMP Book Chapter
SVMP Book Chapter
Chakraborti · Naranjan S. Dhalla
Editors
Proteases in
Physiology
and
Pathology
Proteases in Physiology and Pathology
Sajal Chakraborti • Naranjan S. Dhalla
Editors
Proteases in Physiology
and Pathology
Editors
Sajal Chakraborti Naranjan S. Dhalla
Department of Biochemistry and Biophysics St. Boniface Hospital Research Centre,
University of Kalyani University of Manitoba, Faculty of
Kalyani, West Bengal, India Health Sciences, College of Medicine
Institute of Cardiovascular Sciences
Manitoba, Winnipeg, Canada
The history of research on proteases is relatively old, which has been initiated in
the late eighteenth century, although in recent times it has gained a tremendous
momentum because of their widespread applications, especially in biotechnology
and medicine. There are many ways in which proteases elicit both the beneficial and
detrimental effects on the functioning of living beings, and this has prompted
researchers to study their roles in health and disease.
Recent research revealed that about 2% of all gene products are proteases, indi-
cating that it is one of the important functional groups of proteins. Notably, it seems
difficult to know how a protease can be distinguished from another related one. On
one side, scientists are engaged in understanding the basic mechanisms of the
potentiality of different types of proteases in a variety of disease progression and
evaluation of relevant therapeutics; on the other side, researchers are trying to
answer two fundamental questions: How does knowledge of one protease help in
the understanding of related proteases? How can a novel protease’s role be truly
ascertained?
Research on renin inhibitors as potential anti-hypertensive drugs started in the
early 1970s. Some early peptide-like inhibitors showed significant inhibitory activ-
ity towards renin but lacked adequate bioavailability. After decades of research, the
first bioavailable renin inhibitor aliskiren was approved and marketed in 2007; how-
ever it was discontinued in 2011 due to its side effects. Notably, the success of
inhibitors of angiotensin-converting enzyme (ACE) and β-blockers for the treat-
ment of hypertension have supported the concept that protease inhibitors can prove
useful as successful drugs. Peptidomimetic inhibitors, which bind at the active site
of matrix metalloproteases (MMPs), have been tested, and most MMP inhibitors in
ix
x Preface
xi
xii Contents
xv
Part I
Regulation of Proteases in Health and Disease
Physiological and Pathological
Functions of Mitochondrial Proteases 1
Clea Bárcena, Pablo Mayoral, Pedro M. Quirós,
and Carlos López-Otín
Abstract
Mitoproteases display an essential role in the preservation of mitochondrial
homeostasis under regular and stress conditions. These enzymes perform tightly
regulated proteolytic reactions by which they participate in mitochondrial pro-
tein trafficking, processing and activation of proteins, protein quality control,
regulation of mitochondrial biogenesis, control of mitochondrial dynamics,
mitophagy, and apoptosis. In this chapter, we have revised the physiological
functions of the intrinsic mitochondrial proteases, analyzing their roles in the
different compartments of this organelle and their connection to human pathol-
ogy, primarily cancer, neurodegenerative disorders, and multisystemic diseases.
Keywords
Mitochondria • Mitoproteases • Mitochondrial dynamics • Cancer • Aging •
Neurodegenerative disorders
1.1 Introduction
functions, fundamental for the generation of most of the cell supply of adenosine
triphosphate (ATP), the source of chemical energy within the cells.
The complex structure and the relevance of the mitochondrial function within the
organisms justify the need to set an organization of the molecules that collaborate in
the maintenance of the assembly and function of this energy machine, among which
proteases are gaining an increasing attention. Proteases have been considered for
many years just as performers of the catabolic reactions in the organisms; however,
we are currently beholding a surprising and unexpected increment in the studies of
these enzymes, positioning them as multifunctional molecules carrying essential
functions in health and disease [1–3]. The study of proteases contained in the mito-
chondria, named mitoproteases, has also experienced an exciting expansion. Until
very recently, they were seen only as members of the quality control system of
mitochondria, in charge of the degradation of misfolded and damaged proteins or
being responsible for the processing of proteins imported from cytosol into mito-
chondria [4]. Nevertheless, in recent years, we have witnessed an impressive prog-
ress in the knowledge of their functions, becoming increasingly evident that they are
not merely actors of the catabolic functions of mitochondria. Instead, mitoproteases
perform precise and tightly regulated proteolytic roles that determine time-specific
functions of regulatory proteins.
Due to this increased knowledge and interest in the study of mitochondrial pro-
teases, we have recently proposed the concept of mitodegradome to define the com-
plete set of proteases and homologues that function in mitochondria from cells and
tissues of an organism [5]. The mitodegradome would therefore modulate in an
efficient and irreversible way the function of mitochondria in order to adapt these
organelles to the diverse stress situations that they face, especially given the fact that
there is an absence of de novo synthesis of mitochondria. This characteristic is of
special relevance in post-mitotic tissues, endowing mitochondria and the mitode-
gradome an important role in health and aging. Consistent with this, malfunction or
deregulation of mitochondrial proteases has been associated with numerous pathol-
ogies such as metabolic impairments, cancer, inflammatory diseases, and neurode-
generative disorders [6–8], as well as with the control of aging and longevity [9].
Mitoproteases have the common feature of their location in the mitochondria,
which can be exclusive or shared with the cytosol. Beyond this characteristic, they
form an assorted group attending to their proteolytic diversity. To set some clarity in
this complex grouping, we have recently organized the mitoproteases according to
their function, localization, and proteolytic nature in three groups: intrinsic or resi-
dent mitoproteases, which exert their function exclusively in this organelle regard-
less of the compartment in which they act; the pseudo-mitoproteases, which have a
protease structure but are catalytically impaired; and transient mitoproteases, which
are translocated into mitochondria only in some particular circumstances [5].
Among these three groups of defined mitoproteases, the intrinsic or resident
mitoproteases are the most relevant, and it will be the main focus of this chapter. All
the enzymes that belong to this group exert their function essentially in mitochon-
dria, being considered as the bona fide mitochondrial proteases. In this group,
we can find 20 enzymes, divided into 1 cysteine, 7 serine, and 12 metalloproteases.
1 Physiological and Pathological Functions of Mitochondrial Proteases 5
The only member in the subgroup of cysteine proteases is the deubiquitinase USP30,
which participates in the quality control system in the outer membrane [10]. By
contrast, the metalloproteases are represented by members of seven different
families, which exert a variety of functions in mitochondria. This subgroup includes
the processing peptidases PMPCB, MIPEP, XPNPEP3, METAP1D, ATP23, and
OMA1; the ATP-dependent proteases AFG3L2, SPG7, and YME1L1; the oligopep-
tidases NLN and PITRM1; and the relatively unknown enzyme OSGEPL1. Finally,
the serine proteases are represented by beta-lactamase (LACTB); the processing
peptidases IMMPL1, IMMPL2, and PARL; the ATP-dependent proteases LONP1
and CLPP; and the quality control protease HTRA2. Interestingly, many of these
proteolytic enzymes are widely distributed from bacteria to mammals. In fact, there
are bacterial orthologues of nearly all human mitochondrial proteases, including
different oligopeptidases and aminopeptidases belonging to the M3 and M24 metal-
loprotease families; the highly conserved families of ATP-dependent proteases,
such as Lon, ClpP, and FtsH proteases (orthologous of LONP1, CLPP, and the AAA
proteases AFG3L2, SPG7, and YME1L1, respectively); the ATP-independent
stress-response metalloprotease HtpX (orthologous of OMA1); and the serine pro-
teases DegP and DegS (orthologous of HTRA2) (Table 1.1).
Functionally, the mitoproteases include the classical proteases involved in the
import of proteins to mitochondria and in the protein quality control system.
However, it is now recognized that mitoproteases exert a variety of functions within
mitochondria, including mitochondrial protein trafficking, processing and activa-
tion of proteins, protein quality control, regulation of mitochondrial biogenesis,
control of mitochondrial dynamics, mitophagy, and apoptosis [5].
The present chapter discusses the different proteolytic functions of mitoprote-
ases in the mitochondrial compartments, focusing on recent advances of the study
of this group of enzymes. Additionally, we present an overview of the role of these
enzymes in human pathology.
Fig. 1.1 Mitochondrial proteases in the inner membrane. Intrinsic mitochondrial proteases
that exert their role in the inner membrane. The proteolytic activities of mitoproteases in this com-
partment are vastly diverse, participating in protein import, mitochondrial dynamics, and regula-
tion of apoptosis and mitophagy
Among the quality control proteases, we can also find ATP23 (XRCC6BP1) [24,
25]. Although the function of this metalloprotease in mammals has not been con-
firmed yet, its yeast orthologue participates in the quality control of mitochondria
through the degradation of lipid transfer proteins that are highly conserved in the
course of evolution. The high functional conservation that is usually found among
mitoproteases from yeast to mammals suggests that ATP23 might play a similar role
in mammalian mitochondria [25].
NLN is supposed to be an oligopeptidase that functions in the intermembrane
space. It is also known as neurolysin or mitochondrial oligopeptidase M and can be
found both in the cytosol and inside of mitochondria [26].
Finally, among the mitoproteases from the intermembrane space, we can find
LACTB, whose function has not been completely defined yet. However, as it is
known to form internal filaments, it has been proposed to possess a structural func-
tion through the compartmentalization and structuration of the intramitochondrial
membrane [27].
The mitoproteases located in the inner membrane are diverse and participate in
several if not all mitochondrial processes that occur in this compartment (Fig. 1.1).
Among them, we can find the ATP-dependent proteases mAAA (SPG7 y AFG3L2)
1 Physiological and Pathological Functions of Mitochondrial Proteases 9
and iAAA (YME1L1), which participate in the quality control of the inner
membrane, in the mitochondrial biogenesis, and in the regulation of mitochondrial
dynamics. iAAA, and its only subunit YME1L1 protease, has its active site oriented
to the intermembrane space, whereas mAAA, composed in humans of AFG3L2 and
SPG7 (or paraplegin), has its active site oriented to the matrix [28, 29]. mAAA and
iAAA proteases exert their quality control role through different mechanisms. First
of all, these proteases degrade damaged or non-assembled subunits of the electron
transport chain, so their absence provokes the malfunction of oxidative phosphory-
lation (OXPHOS) caused by the accumulation of defective complexes [29–31].
Besides, mAAA subunit AFG3L2 is critical to the maintenance of inner membrane
integrity under aberrant protein accumulation caused by the loss of temporal and
spatial coordination in the assembly of the oxidative phosphorylation complexes
[32]. AAA proteases also have a role in mitochondrial biogenesis. Some years ago,
it was observed that yeast cells without mAAA had a respiratory deficiency as a
consequence of damaged mitochondrial translation as well as a defective processing
of the ribosomal subunit MrpL32 [33]. It was later defined that this protease con-
trols the assembly of mitochondrial ribosomes and the synthesis of mitochondrially
encoded respiratory chain subunits [32]. mAAA protease, and in particular its sub-
unit AFG3L2, also has a role in calcium homeostasis as it induces mitochondrial
fragmentation through the processing of OPA1, thereby decreasing calcium uptake
[34]. Also, in the case of iAAA (YME1L1), it degrades the translocase subunit
TIM17A as a consequence of the stress response, decreasing protein import into
mitochondria [35]. Additionally, YME1L1 protease also modulates cardiolipin lev-
els and the resistance to apoptosis by degrading PRELID1 (known as Ups1 in yeast),
a protein that prevents apoptosis by complexing to TRIAP1, a p53-regulated pro-
tein, and mediating this way the intramitochondrial transport of phosphatidic acid,
necessary for cardiolipin synthesis and consequently apoptosis resistance [36].
Mitoproteases in the inner membrane also have a role in mitochondrial dynamics
(Fig. 1.1). OMA1, an ATP-independent protease, is a stress-response protease that
functions together with the AAA proteases. It is activated under different stress
conditions, such as oxidative and heat stress, and membrane depolarization [37].
As a result, it carries the proteolytic cleavage of all long forms of OPA1, inhibiting
this way the fusion process [38–40]. OMA1 negatively regulates itself by auto-
processing in order to control and limit the stress response [37]. The processing of
the inner membrane fusion protein OPA1 by OMA1 and/or YME1L1, as well as the
regulation of the abundance of mitofusins and DRP1 at the outer membrane, serves
to regulate mitochondrial dynamics. In particular, the processing of OPA1 by
YME1L1 seems to provide a connection between mitochondrial dynamics and
OXPHOS function dependent on the metabolic state [41]. Actually, it has been
recently reported that depolarization of the mitochondrial membrane leads to OMA1
activation, and depending on the energy status of the cell (ATP levels), YME1L1
will degrade OMA1 (high levels) or OMA1 will degrade YME1L1 (low levels),
allowing cells to adapt mitochondrial dynamics to distinct cellular insults [42]. It has
also been described that a third and unknown cysteine protease is able to cleave OPA1
in the C-terminal region, apparently an event that occurs in the liver after a meal.
10 C. Bárcena et al.
The exact nature of this additional processing of OPA1 is still unknown; however, it
seems to be dependent on mitofusin 2 and independent of OMA1 [43]. In conso-
nance to this, cells from Oma1-deficient mice are unable to process and inactivate
OPA1. For this reason, these cells are protected against mitochondrial fragmenta-
tion, and consequently they show an increase in highly connected mitochondria
[39]. On the other hand, Yme1l1-deficient mice cells have constantly activated
OMA1, so they show fragmented mitochondria [44]. As a consequence of this regu-
lation of mitochondrial dynamics by OMA1 and AAA proteases, Oma1-null mice
have an unbalanced OPA1 processing, being unable to adapt mitochondrial dynam-
ics to stress conditions in the cells. As a result, these mutant mice are obese and have
a defective thermogenesis, proving the connection between the control of metabolic
homeostasis and the regulation of mitochondrial dynamics [39, 45]. Very recently,
it has also been described that loss of Oma1 in yeast cells leads to elevated ROS
levels and activation of stress survival responses in a TORC1-mediated way, linking
mitochondrial quality control and TOR signaling in the response to stress stimuli [46].
Mitoproteases of the inner membrane also participate in the regulation of apop-
tosis by different mechanisms (Fig. 1.1). For example, mitochondrial fusion serves
to maintain the integrity of cristae, avoiding this way the release of pro-apoptotic
proteins and protecting against apoptosis. Probably for this reason, the absence of
YME1L1 leads the processing of OPA1 by OMA1, causing a loss of the cristae
integrity and mitochondrial fragmentation and eventually an increased susceptibil-
ity to apoptosis [44]. OMA1 also has a role in the regulation of mitophagy, as altera-
tion of its activity stabilizes OPA1 and prevents mitochondrial fragmentation, a
process that is required in order to elicit mitophagy [47]. On the contrary, it can be
activated by the pro-apoptotic proteins BAX and BAK [48] and by other stress
stimuli [37]. As a result of the degradation of OPA1, cytochrome c is released pro-
moting apoptosis. Consequently, deficiency in this metalloprotease protects against
apoptotic stimuli [39, 49].
PARL is a rhomboid intramembrane protease located in the inner membrane and
with known roles in mitophagy and autophagy. In the first case, its function is
related to the degradation of the mitochondrial kinase PINK1, which triggers the
binding of parkin protein to depolarized mitochondria in order to induce mitophagy.
Other mitoproteases participate in the processing of PINK1. Thus, under normal
conditions, PINK1 enters the mitochondria, and once there, it is processed by MPP
[50]. In addition, it is cleaved and consequently destabilized by PARL, leading to its
release from mitochondria in order to be degraded by the ubiquitin-proteasome sys-
tem in the cytosol [51]. Afterwards, PINK1 is recycled in a process still poorly
understood involving other mitoproteases such as mAAA, CLPP, and LONP1 [50].
PARL also regulates mitophagy by participating in the degradation of PGAM5 [52],
a phosphatase that activates mitophagy and necrosis under stress conditions. Its
degradation by PARL would therefore prevent mitophagy [53, 54]. Finally, PARL
also has a role in the regulation of apoptosis in a HTRA2-independent manner,
through the remodeling of cristae and the control of cytochrome c release during
apoptosis [55, 56]. Accordingly, mice deficient in PARL show an increased apopto-
sis that leads to a premature death [55].
1 Physiological and Pathological Functions of Mitochondrial Proteases 11
Fig. 1.2 Matrix mitochondrial proteases. Intrinsic mitochondrial proteases situated mainly in
the matrix. Their roles in this compartment are predominantly the processing of imported peptides,
maintenance of mtDNA, and degradation of misfolded or damaged proteins
The last mitoproteases from the inner membrane are IMMP1L and IMMP2L,
processing peptidases that eliminate hydrophobic signals from proteins that have
been imported into the intermembrane space after being processed by MPP [57].
These two mitoproteases also promote the assembly of yeast translocase inner
membrane (TIM) complexes through the cleavage of specific carboxy-terminal
subunits [58].
Among the ATP-dependent proteases that exert their function in the mitochondrial
matrix, we can mention LONP1 and CLPP, which participate in quality control,
mitochondrial biogenesis, and mitochondrial stress response (Fig. 1.2). LONP1 is a
serine protease highly conserved through evolution. It participates in the quality
control of the matrix by degrading oxidized, misfolded or mutated proteins, ensur-
ing cell viability. Therefore, it can respond to different stress stimuli that can be
potentially harmful [59, 60], as well as to normal conditions that require a recondi-
tioning of the protein homeostasis in mitochondria. LONP1 has been shown to
degrade several proteins in mammals, including succinate dehydrogenase subunit 5
(SDH5), aconitase, glutaminase C, cytochrome c oxidase isoform COX4–1, ste-
roidogenic acute regulatory protein (StAR), mitochondrial transcription factor A
(TFAM), cystathionine-β-synthase (CBS), heme oxygenase-1, and 5-aminolevulinic
12 C. Bárcena et al.
acid synthase (ALAS1) [61–68]. Probably due to this wide spectrum of action, mice
knockout for LONP1 exhibit an early embryonic lethality. However, mice heterozy-
gous for the deletion of Lonp1 show alterations in mitochondrial respiration and in
the OXPHOS system, probably causing an inability for metabolic reconversion in
malignant cells and thereby showing a decreased tumoral susceptibility. This study
has clearly demonstrated the indispensable role of LONP1 in life and disease [69].
This mitoprotease also has a role in mitochondrial biogenesis, as it functions as a
DNA-binding protein that upon stress conditions is released from mtDNA and
degrades the transcription factor TFAM. This way, LONP1 controls mtDNA main-
tenance as well as mitochondrial gene expression [68, 70, 71].
The other ATP-dependent protease with proteolytic function in the mitochon-
drial matrix is the serine protease CLPP (Fig. 1.2). It is known that CLPP forms the
complex CLPXP together with the chaperone CLPX; however, its function is still
not completely clear. It has been related to the degradation of misfolded proteins
and, consequently, to the mitochondrial unfolded protein response (UPRmt) in
Caenorhabditis elegans [72]. However, it has been recently reported that its deletion
in DARS2-deficient mice, a mouse model of UPRmt, alleviates their mitochondrial
cardiomyopathy, suggesting that CLPP does not participate in mammalian UPRmt
[73]. It has also been described that CLPP absence in mice provokes hearing loss,
infertility, and growth retardation, probably through the accumulation of CLPX
subunits and mtDNA [74].
Apart from these two ATP-dependent proteases, mitochondrial matrix also needs
processing peptidases engaged in the import of proteins to mitochondria and oligo-
peptidases that deal with the peptides that result from the degradation of damaged
proteins by LONP1, CLPP, and mAAA proteases (Fig. 1.2). Among them we find
PITRM1, an oligopeptidase from the pitrilysin family. This metalloprotease also
shows a presequence processing role that is critical for correct mitochondrial func-
tion [75–77]. PITRM1 degrades the mitochondrial amyloid β-protein in human
cells [78] and, in yeast, it has also been observed that amyloid β-protein can in turn
inhibit the yeast orthologue of PITRM1, impairing this way the processing activity
of MPP. This would trigger the accumulation of preproteins in mitochondria, link-
ing accumulation of amyloid β-protein to mitochondrial dysfunction [77].
As explained before, most of mitochondrial proteins are encoded in the nucleus
DNA. In order to be translocated into the mitochondria after being synthesized in
the cytosol, these proteins carry mitochondrial import signals that, once inside of
the organelle, need to be removed. This action is performed by the so-called mito-
chondrial processing peptidases, a group of enzymes that includes PMPCB, MIPEP,
METAP1D, and XPNPEP3 [79]. PMPCB (also known as β-MPP) is responsible for
the processing of the majority of mitochondrial proteins, thus being the most impor-
tant mitochondrial protein peptidase [80]. It forms a heterodimer with its non-
protease homologue PMPCA (α-MPP), resulting in the MPP complex [81]. PMPCA,
although without protease activity, facilitates the proteolytic processing of PMPCB
by recognizing and binding to the mitochondrial targeting presequences from the
imported proteins [82]. As already stated, PMPCB is the major mitochondrial pro-
cessing peptidase. However, there are other processing peptidases with more defined
1 Physiological and Pathological Functions of Mitochondrial Proteases 13
targets and essential roles in the maturation and stabilization of the imported pro-
teins. Among them, we can mention the mitochondrial intermediate peptidase or
MIPEP (Oct1 in yeast) that cleaves octapeptides from cytosolic proteins imported
into the mitochondria, whereas the aminopeptidase XPNPEP3 (also known as
Icp55) cleaves one single amino acid from the amino terminus of MPP-processed
substrates [83, 84]. Finally, the mitochondrial Met aminopeptidase METAP1D (also
known as MAP1D) cleaves the Met residue from the amino terminus of some proteins
encoded in the mitochondrial DNA, generating functionally active proteins [85].
Another proteolytic enzyme that functions in the mitochondrial matrix is
OSGEPL1, whose role is still unknown although some studies suggest that it could
participate in the mtDNA maintenance, as demonstrated for its orthologous proteins
Kae1(OSGEP)/YgjD in multiple organisms [86].
conditions that are found in tumors in a HIF1α-mediated way [64]. More specifi-
cally, it has been observed that LONP1 induces the necessary metabolic switch in
tumor cells and protects them from entering senescence [69]. Indeed, it was recently
described that increase in LONP1 levels in tumor cells facilitates survival to apop-
tosis through the increase stability of the HSP60-mtHSP70 complex [103].
of PINK1 and PGAM5. Finally, the deubiquitylase USP30 has also been implicated
in Parkinson’s disease by opposing to parkin-mediated autophagy, thereby promoting
this neurodegenerative disorder [13].
Apart from their implication in Parkinson’s disease, PARL and HTRA2 have
also been linked to striatal neuronal injury cerebral ischemia [110]. Additionally,
intragenic deletions in IMMP2L are known to cause Tourette syndrome, while
SNPs and CNVs in this gene have been associated with several neurodevelopmental
disorders as well as with autism [111–113]. Meanwhile, the already exposed role of
PITRM1 in the degradation of amyloid β-protein accumulation involves an obvious
implication in the pathology of Alzheimer’s disease [78].
The ATP-dependent protease mAAA has also been implicated in neurological
disorders by mutations in its two different subunits. Mutations in both AFG3L2 and
SPG7 cause spastic paraplegia and ataxia disorders [114–116], whereas mutations
in SPG7 have also been related to chronic ophthalmoplegia owing to the associated
defects in mtDNA maintenance [117, 118]. Indeed, deletion of Afg3l2 subunit in
mice causes hyperphosphorylation of Tau protein and defective mitochondrial
anterograde transport, leading to a neurological alteration that resembles axonal
degeneration disorders observed in humans. Deletion in mice of the other mAAA
subunit, Spg7, also causes axonopathy and abnormal mitochondria, developing a
neurodegenerative disorder [119, 120]. More recent studies with mice deficient for
the mAAA subunits have demonstrated their relevance for neurological function
and their implication in cerebellar degeneration [121]. Apart from its relevance in
correct axonal maintenance, AFG3L2 has also been implicated in dementia and
neurodegeneration through its essential role in mitochondrial protein synthesis and
Purkinje cells’ survival [122–125]. Interestingly, antioxidants such as
N-acetylcysteine or vitamin E have been observed to restore mitochondrial function
in neurons with a depletion in AFG3L2, opening new horizons for therapeutic
approaches in neurodegenerative disorders [122].
Finally, the essential role of mitochondria in every tissue of the body implies that its
incorrect function is related to several other metabolic and multisystemic diseases.
For example, type 2 diabetes has been related to mutations or SNPs in SPG7 and
PARL. Moreover, mutations in SPG7 have also been linked to coronary artery dis-
ease [126, 127]. Furthermore, it was recently reported that cardiac-specific ablation
of Yme1l1 in mice induced dilated cardiomyopathy and heart failure through an
increase in mitochondrial fragmentation due to constant activation of OMA1 and
consequent OPA1 proteolysis. Interestingly, deletion of Oma1 rescued these altera-
tions [128]. Additionally, the essential function of OMA1 in the adaptation of mito-
chondrial dynamics to different stress stimuli is consistent with the observation that
mice deficient in this mitoprotease show an obesity phenotype when fed a high-fat
diet. The observed obesity is also accompanied with an impaired thermogenesis, an
increment in hepatic steatosis and a marked alteration of glucose metabolism [39, 45].
16 C. Bárcena et al.
1.4 Conclusions
The study of mitoproteases has experienced an enormous expansion in the last years
owing to the discovery that their functions are much more complex and refined than
the merely catabolic reactions involved in the quality control of mitochondria. In
fact, mitoproteases exert essential roles in order to regulate the energetic responses
to different internal and external stresses, as well as to control the trafficking
between cytosol and mitochondria, the correct mitonuclear communication, and the
tight coordination of apoptotic signals. The complexity that this area of knowledge
was acquiring made it necessary to establish an organization of the proteases located
in mitochondria. For this reason, we have recently classified the mitoproteases by
their spatial action, distinguishing between intrinsic or resident mitoproteases,
pseudo-mitoproteases, and transient or roaming mitoproteases [5]. In the first
group, we have included those mitoproteases that exert their function mainly in
mitochondria, even though some of them can be sporadically found in cytosol. The
second group encloses molecules with a protease structure but with an impaired
catalytic activity. Finally, the group of transient or roaming mitoproteases includes
those mitoproteases that although their main function takes place in a different part
of the cell, they translocate into mitochondria in some situations such as apoptosis
or autophagy. In this chapter, we have revised the physiological function of intrinsic
mitoproteases according to the compartment of mitochondria where they exert their
main function (Table 1.1). This way, we have compiled the proteolytic activities that
can be found in the outer membrane, in the intermembrane space, in the inner mem-
brane, and in the matrix. In all compartments, we can detect mitoproteases with
functions related to mitochondrial dynamics, trafficking and processing of other
peptides, quality control, apoptosis or mitophagy.
Finally, we have conducted a revision of the implication of the different mitopro-
teases in pathology (Table 1.1). In this context, we have revised the implication of
mitoproteases in cancer, where they can act as protumoral (METAP1D, CLPP, and
LONP1) and as tumor suppressors (OMA1 and IMMP2L) or may exert a dual role
(HTRA2). Also, we have studied the relation of mitoproteases in neurodegenerative
disorders, including Parkinson’s disease (HTRA2, PARL, and USP30), spastic
paraplegia and ataxia (AFG3L2 and SPG7), dementia (AFG3L2), or Tourette
1 Physiological and Pathological Functions of Mitochondrial Proteases 17
Acknowledgments The work in our laboratory was supported by grants from Ministerio de
Economía y Competitividad and Instituto de Salud Carlos III (RTICC). We also thank the generous
support by J.I. Cabrera. The Instituto Universitario de Oncología is supported by Fundación
Bancaria Caja de Ahorros de Asturias.
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The Role of Matrix Metalloproteinase-2
and Metalloproteinase-9 in Embryonic Neural 2
Crest Cells and Their Derivatives
Rotem Kalev-Altman, Efrat Monsonego-Ornan,
and Dalit Sela-Donenfeld
Abstract
Neural crest cells (NCCs) are transient cell populations that are initially residing
at the dorsal-most part of the neural tube of the developing vertebrate embryo. At
well-defined time points, NCCs detach from the neural tube as they undergo
epithelial-to-mesenchymal transition (EMT) and migrate in distinct pathways to
their final destinations. There, this unique cell population differentiates into a
great variety of cell types including bone and cartilage tissues of the head and
face, connective tissue of the heart, skin melanocytes, adipocytes, enteric neu-
rons, and most of the peripheral sensory neurons, glia, and Schwann cells. Matrix
metalloproteinases (MMPs) are a large family of matrix-degrading enzymes,
which are divided into several subfamilies according to their structure and sub-
strate specificity. The gelatinases subfamily, which includes MMP-2 and MMP-9
solely, is the most investigated group. Both MMP-2 and MMP-9 were previously
reported to be expressed in embryonic NCCs and to have a role in their EMT and
R. Kalev-Altman
Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture,
Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel
The Institute of Biochemistry and Nutrition, The Robert H. Smith Faculty of Agriculture,
Food and Environment, The Hebrew University of Jerusalem,
P.O.B 12, Rehovot 76100, Israel
E. Monsonego-Ornan (*)
The Institute of Biochemistry and Nutrition, The Robert H. Smith Faculty of Agriculture,
Food and Environment, The Hebrew University of Jerusalem,
P.O.B 12, Rehovot 76100, Israel
e-mail: efrat.mo@mail.huji.ac.il
D. Sela-Donenfeld (*)
Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture,
Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel
e-mail: dalit.seladon@mail.huji.ac.il
migration processes. In this review we present the most recent data regarding the
role of MMP-2 and MMP-9 in embryonic NCCs and in their various derivatives
in later embryonic stages and in adults.
Keywords
Neural crest cells • Matrix metalloproteinases • MMP-2 • MMP-9 • Gelatinases
• Osteoblasts • Chondrocytes • Melanocytes • Adipocytes • Enteric nervous sys-
tem • Glial cells • Schwann cells • Embryo • Epithelial-to-mesenchymal
transition
2.1 Introduction
Neural crest cells (NCCs) are a transient cell population that resides at the dorsal-
most part of the neural tube (NT) of the developing vertebrate embryo. The NCCs
detach from the neuroepithelium in a gradual synchronized manner from head to
tail and migrate in distinct well-characterized pathways throughout the embryonic
axis to their final destinations [1–3]. There, this unique cell population differentiates
into a great variety of cell types including most of the peripheral sensory neurons,
glia and Schwann cells, smooth muscle cells, connective tissue of the heart, skin
melanocytes, and the secreting cells of several endocrine tissues as the thymus,
thyroid, and adrenal glands [2, 4, 5]. Moreover, cranial NCCs contribute also to the
majority of bone and cartilage tissues of the head and face such as the frontal, nasal,
premaxillary, maxillary, mandibular, and temporal bones; Meckel’s cartilage; jaw,
ear, and hyoid cartilages; and tooth dentin [4, 6–10].
Due to their fundamental contribution to this vast number of cell types and
organs, their appropriate formation and migration are crucial for normal embryonic
development. Defects in these processes will lead to various birth disorders and
pathologies such as different heart defects, albinism, malformation of craniofacial
structures including cleft palate/lip, mal-innervation of the digestive tract (which
leads to Hirschsprung’s disease), and several other congenital syndromes like
DiGeorge, Treacher Collins, and Waardenburg [5, 9]. Furthermore, some of the
most aggressive tumors derive from NCC origin (i.e., glioblastoma, melanoma, cra-
nial chondrosarcoma); as such, the process of NCC migration together with their
ability to differentiate into many derivatives is in common with metastatic cancer
cells [11, 12].
In order to engage in migration, NCCs undergo epithelial-to-mesenchymal tran-
sition (EMT) after which they acquire motility. EMT is a major process involved in
many physiological and pathological conditions in embryos and adults and requires
fundamental changes in the cells and their surrounding environment, such as break-
age of cell-cell interactions, rearrangement of cell cytoskeleton, and remodeling of
the extracellular matrix (ECM) [13–15]. Therefore, EMT is mandatory for NCCs in
order to transform from being immotile neuroectodermal cells with typical cell-cell
and cell-matrix connections to mesenchymal-separated cells with the ability to
2 The Role of Matrix Metalloproteinase-2 and Metalloproteinase-9 in Embryonic… 29
migrate throughout various stereotypic pathways. The NCC EMT process involves
degradation of the basal membrane around the dorsal NT, rearrangement of their
cytoskeleton (i.e., via the modulation of RhoA/Rho-kinase, calponin, alpha-catenin),
loss of adhesion molecules, and remodeling of the ECM [2, 16–22]. Major signaling
factors such as BMP and Wnt, as well as several transcriptional regulators like Snail
and Sox9, were also found to act as regulators of NCC EMT [23–30]. However, the
putative executers of these processes to trigger the cell separation and migration are
still much less investigated.
Specifically, the gelatinase catalytic site has a fibronectin domain which improves
the degradation of denatured collagen (gelatin). Finally, the hemopexin domain,
found in the c-terminal of MMPs, is attached through the hinge region to the cata-
lytic domain and is folded to a structure of four-bladed β propeller which is involved
in substrate recognition and TIMP binding. MT-MMPs (MMP-14, MMP-15, MMP-
16, and MMP-24) have an additional domain that anchors them to the cell membrane
as a transmembrane domain or glycosylphosphatidylinositol (GPI) [32, 33, 38].
Members of the MMP family are implicated in many physiological processes
which involve proteolytic cleavage or remodeling of the ECM during embryogene-
sis, angiogenesis, and tissue repair, via their involvement in cell proliferation, dif-
ferentiation, apoptosis, and migration [39, 40]. They also control key reproductive
events such as ovulation, embryo implantation, breast involution, and more [32, 41].
On the other hand, imbalance between the expression/activity of MMPs and TIMPs
is linked to different pathological conditions such as in tumor invasion and metasta-
sis, arthritis, nephritis, fibrosis, endometriosis, and diseases such as multiple sclero-
sis and Alzheimer’s [41–44].
Fig. 2.1 The expression of MMP-2 and MMP-9 in migrating chick NCCs. (A, A′)
Immunofluorescence labeling of MMP-2 protein in the cranial level of a 12-somite-stage whole-
mount chick embryo, using MMP-2 antibody (green); MMP-2 is expressed in actively migrating
NCCs as they detach from the NT (arrowheads) and during their migration (arrows). Panel A′ is
an enlargement of the boxed areas in panel A. (B) An ex-vivo explant of NCCs obtained from the
hindbrain of 6–8-somite-stage embryo showing immunofluorescence labeling of MMP-9 protein,
using MMP-9 antibody (green); MMP-9 is expressed in actively migrating NCCs as they detach
from the explanted NT (arrowheads) and also during their migration as separated mesenchymal
cells (arrows). Panel B′ is an enlargement of the boxed areas in panel B. Blue staining (DAPI)
represents cell nuclei
and during, their migration in the cranial and trunk axis levels (Fig. 2.1B, B′). The
role of MMP-9 in executing NCC EMT and further migration was examined using
loss and gain-of-function methods; addition of a specific MMP-9 pharmacological
inhibitor or an antisense morpholino oligomer against MMP-9 mRNA inhibited the
EMT and migration processes of NCCs, in explants and in-vivo. In contrast, addi-
tion of exogenous MMP-9 to NCC progenitors was sufficient to promote their
migration ahead of time, as well as to rescue the effect of the MMP-9 inhibition on
NCC migration. By analyzing possible substrates for MMP-9, our findings strongly
suggested that MMP-9 executes NCC migration by degrading N-cadherin and lam-
inin, two adhesion proteins, and that their degradation and/or downregulation in
cell-cell and cell-basal lamina interactions, respectively, has been demonstrated to
be necessary for NCC EMT [21, 48–50]. Together, these studies have shown that
both gelatinases MMP-2 and MMP-9 play a role in executing NCC migration in the
chick embryo.
Albeit the demonstrated role of MMP-2 and MMP-9 in early stages of NCC
development in the chick embryo, their expression pattern or function in NCCs of
other species, such as mouse, frog (Xenopus laevis), or teleost fish (Zebra fish), was
not yet reported. Notably, in the zebra fish embryo, MMP-2 was found to be
32 R. Kalev-Altman et al.
expressed from as early as one-cell stage through at least the 72 first hours of devel-
opment, and its inhibition via injection of MMP-2 antisense morpholino oligomer
greatly impaired zebra fish development [51]. In agreement with these data, Keow
and colleagues have used a probe to tag the active form of MMP-2 and demon-
strated the distribution of active MMP-2 in the developing zebra fish embryo [52].
The localization patterns of active MMP-2 that was presented in this study seemed
reminiscent of the migration streams of NCCs. Therefore, similarly to the chick
embryo, a role for MMP-2 in executing zebra fish NCC migration is possible
although not directly tested. Furthermore, MMP-9 was also found to be expressed
in the zebra fish embryo, throughout the first 5 days postfertilization [53]. In this
study, the researchers showed that in 10-somite-stage old embryo, MMP-9 expres-
sion was localized to the anterior midline region of the embryonic mesoderm,
whereas later on, MMP-9 was expressed around the head and eyes. These patterns
of MMP-9 may implicate for its expression and role in the different stages of zebra
fish NCC development.
Both MMP-2 and MMP-9 have been identified and characterized in different NCC
derivatives including craniofacial cartilage and bone, cardiac cushion cells which
contribute to the heart valves and septum, melanocytes, adipocytes, enteric neurons,
and different neural supporting cells such as glia and Schwann cells. We present
here the main findings according to gelatinases’ tissue localization and function.
NCCs from the cranial axial level generate the majority of bone and cartilage cra-
niofacial tissues including tooth dentin. The cranial NCC population migrates from
different regions of the developing brain: the forebrain, midbrain, and hindbrain
domains. NCCs that arise from the forebrain and rostral midbrain colonize the fron-
tonasal and periocular regions, while caudal midbrain-derived NCCs populate the
maxillary component of the first pharyngeal arch. Collectively, these NCCs give rise
to the upper jaw, palatal mesenchyme, and extrinsic ocular muscles. The hindbrain
is transiently partitioned into seven segments called rhombomeres. NCCs from
these rhombomeres migrate in discrete segregated streams into the first through
sixth pharyngeal arches, where they form the jaw, middle ear, hyoid, and thyroid
cartilages [6, 8, 54, 55]. Different markers and signaling transducers were reported
to be involved in the specification of cranial NCCs. This includes Sox10, Notch,
Neuregulin1, BMP2, Neurogenin2, Hox genes, and more [56, 57]. For example,
Hoxa2 was found to be essential for proper patterning of structures derived from the
cranial NC since targeted inactivation of Hoxa2 resulted in lethality at birth and
malformations of craniofacial elements derived from cranial NC [58–60].
2 The Role of Matrix Metalloproteinase-2 and Metalloproteinase-9 in Embryonic… 33
Fig. 2.2 The expression of MMP-2 and MMP-9 in mouse chondrocyte, rat chondrosarcoma,
and human melanoma cell lines. Immunofluorescence labeling of MMP-2 and MMP-9 proteins
using MMP-2 (A, C, E) or MMP-9 (B, D, F) antibodies, in mouse chondrocyte cell line ATDC5
(red) (A, B), rat chondrosarcoma cell line (green) (C, D), and human melanoma cell line A375SM
(green) (E, F). Blue staining (DAPI) represents cell nuclei
of bone density. Yet, mild craniofacial defects have also been reported, such as
shorter upper and lower jaws, nose, and overall skull length, which may imply on
NCC developmental defects, although this has not been specifically examined [77,
78]. In support of this finding, a genetic missense mutation in the human MMP-2
gene has been described as causing inherited osteolytic/arthritic syndrome, which
was suggested to be caused by the impairment of MMP-2 activity in bone and ECM
remodeling [79]. Noticeably, patients with this syndrome suffer from several facial
defects, which may also be due to the loss of MMP-2 in developing cranial NCCs.
All of this data suggests a role of MMP-2 in the development of cranial NCCs.
However, whether the MMP-2-KO has any effect on the early stages of NCC migra-
tion has not been examined yet.
In addition, Vu and coworkers found that MMP-9-KO mice presented shorter
long bones, as the tibia and femurs, compared with those of wild-type littermates.
These bones had a lengthened zone of hypertrophic cartilage in their growth plates
due to inhibition of chondrocyte apoptosis which is normally regulated by MMP-9.
Yet, similar to the MMP-2-KO mice, no clear cranial bone defect was reported [76].
One possible explanation for the normal NCC phenotypes in the mutants is the
presence of the other gelatinase in NCC but not in the hypertrophic chondrocytes,
2 The Role of Matrix Metalloproteinase-2 and Metalloproteinase-9 in Embryonic… 35
which may compensate for the ablated gene and enables cell migration and ECM
remodeling during NCC development. Therefore, future studies will be needed to
investigate whether double KO for MMP-2 and MMP-9 genes affects cranial NCC
migration and leads to major craniofacial defects in the mice. Surprisingly, such
double KO line was not yet reported in the literature, which may indicate for its
lethality.
The possible involvement of MMP-2 and MMP-9 in craniofacial development
was also demonstrated in zebrafish embryos [80]. In this study, the researchers
examined the mechanism by which antenatal usage of glucocorticoids (GC) induces
teratogenic effects, as was previously described as causing fetal growth restriction
and cleft palate [81]. Hillegass and colleagues have shown that GC treatment
increases MMP-2, MMP-9, and MMP-13 mRNA levels and causes abnormal cra-
niofacial development [80]. Although these studies have not directly examined the
relation to NCC development, these data strongly suggest that GC-induced increase
in MMPs affects cranial NCC ontogeny and therefore results in abnormal craniofa-
cial development.
2.4.2 Heart
During cardiac development, NCCs that arise from the mid-otic up to the caudal end
of the third somite level, termed “cardiac NCCs”, migrate beneath the ectoderm into
pharyngeal arches III, IV, and VI. Eventually, a subset of these cells invades the
outflow tract of the developing heart and contributes to the formation of the heart
valves and septum [5, 82–85]. The importance of NCCs to heart development was
demonstrated in a series of studies in different embryonic models in which pre-
migratory cardiac NCCs were ablated, resulting in a subsequent loss of NCC deriva-
tives in the developing cardiovascular system leading to defects of the cardiac
outflow tract, valve formation, and aortic arch arteries [86–90].
Yet, data on a possible link between cardiac NCC and MMP-2/MMP-9 activity
is so far limited to avian embryos; MMP-2 mRNA was found to be expressed in
cardiac NCCs of quail and chick embryos, and its activity levels were increased
with the initiation of cushion NCC migration [91–93]. Moreover, Cai and Brauer
have demonstrated how injection of a general MMP inhibitor (KB8301) to chick
embryos at stages before cardiac NCCs begin to migrate caused major anomalies in
cardiac morphogenesis, which was correlated with a decrease in MMP activity [94].
These data demonstrate that MMP-2 is expressed and acts during chick cardiac
morphogenesis and suggests that perturbation of endogenous MMP activity may
lead to NCC-related congenital defects. Further studies will be required in other
embryonic models to analyze how conserved is the activity of MMP-2 in cardiac
NCC, whether MMP-9 is also implicated in cardiac NCC development, and what is
the mechanism of action of MMP-2 in heart morphogenesis.
36 R. Kalev-Altman et al.
2.4.3 Melanocytes
The NC is the major, if not the sole source of all pigment cells in the developing
vertebrate, except those of the retina. The melanocyte progenitors reside in the ven-
tral most part of the dorsal NT and are the last ones to emigrate in a dorsolateral
migratory pathway [95, 96]. All pre-migratory NCCs in the dorsal NT express
FoxD3, Sox10, and Pax3. The NCC-derived melanoblasts continue to express
Sox10 and Pax3 during their delamination and migration, while they downregulate
FoxD3 and upregulate Mitf when they exit the NT [2, 97, 98].
Many signaling cues and migration regulators have been reported to affect mela-
nocyte specification and migration [95, 97, 99]. Yet, data regarding MMP-2/
MMP-9 in embryonic melanocytes is limited. The expression of MMP-2 was so far
reported in melanophores of frog embryos (Xenopus laevis), where it was also
shown to promote melanophore migration [100]. This study used a chemical
genomic screen and a functional approach to discover modulators of melanophore
migration and identified one molecule, termed NSC 84093 that selectively inhibited
melanophore migration via inhibition of MMP-2.
Much more knowledge exists regarding gelatinases in adult melanocytes. For
instance, Simonetti et al. [101] analyzed the expression patterns of MMP-2 and
MMP-9 in benign and invasive lesions of human melanomas. The results showed
that invasive melanoma cells express MMP-2 and MMP-9 and that MMP-2 is pres-
ent in both the melanoma cells and in the tumor-surrounding stromal or host cells
[101]. In addition, MMP-9 was found to be variably expressed in the radial growth
phase of primary melanoma cells, indicating that MMP-9 is correlated with early
invasive stages of melanoma [102]. On the other hand, a different study argued that
MMP-9 expression is absent during early stages of melanoma but present in
advanced stages [103].
These data are in agreement with other studies which demonstrated MMP-2 and
MMP-9 in melanoma cell lines or in-vivo [38, 104–106]. For example, both gelati-
nases were investigated for their involvement in canine cutaneous melanocytic
tumors and were found to be expressed in both benign and malignant lesions.
However, MMP-2 expression was significantly higher in benign melanocytic tumors
than in malignant counterpart. In contrast, MMP-9 expression was elevated in
malignant melanocytic tumors compared with benign tumors [38]. These results
suggested that malignancy is associated with an increase of MMP-9 and a decrease
of MMP-2 expression and that a switch in gelatinases’ expression profile might
occur during tumor progression, involving not only emergence of MMP expression
but also its downregulation. Furthermore, our lab also found that both MMP-2 and
MMP-9 are expressed in human melanoma cell line A375SM (Fig. 2.2E, F, respec-
tively). The large amount of data regarding MMP-2 and MMP-9 in adult melano-
cytes as compared to embryos may result from a restricted activity of gelatinases
only to pathological conditions. However, as melanoma invasion resembles in many
instances to embryonic NCC migration [12], it is highly possible that the limited
knowledge on expression/activity of gelatinases in embryonic melanocytes is due to
insufficient research in this field.
2 The Role of Matrix Metalloproteinase-2 and Metalloproteinase-9 in Embryonic… 37
2.4.4 Adipocytes
In contrast to other NCC derivatives, the adipocytes lineage of NCCs has not been
carefully explored in the past. Billon et al. [107] have demonstrated that in the quail,
both cephalic and trunk NCCs can differentiate into adipocytes ex-vivo, in an explant
system [107]. These adipocytes had been shown to express the typical adipocyte
differentiation markers such as CEBPα, PPARγ, and FABP4. Moreover, the
researchers used Sox10-Cre transgenic mice to map NCC derivatives in order to
genetically demonstrate that mice NCCs contribute to adipocytes also in normal
development in-vivo [107].
Studies in adult tissues have shown that adipocytes express both gelatinases
[108–111]. Human adipocytes and pre-adipocytes release MMP-2 and MMP-9, and
this secretion is modulated during adipocyte differentiation. Moreover, inhibition of
these MMP activities resulted in a blockage of adipocyte differentiation [108].
Secretion of both gelatinases was also increased during adipocyte differentiation of
murine 3T3F442A pre-adipocyte cell line [108]. Differentiation of adipocytes leads
to cell hyperplasia which is one of the changes in adipose tissue that leads to obe-
sity, along with hypertrophy of the cells and increased angiogenesis [112, 113]. As
different MMPs in general, and MMP-2 and MMP-9 in particular, have a central
role in angiogenesis [76, 114–116], it was speculated that both gelatinases have a
dual role in the development of adipose tissue and obesity by increasing the differ-
entiation rate of pre-adipocytes to adipocytes on the one hand and by increasing
angiogenesis on the other [108].
However, the role of MMP-2 in adipogenesis in-vivo remains elusive with con-
troversial data. For instance, nude mice that were injected with MMP-2 knockdown
pre-adipocytes showed small decrease in adipose tissue markers, but the tissue itself
did not differ in weight or size from control [110]. In contrast, other studies showed
that mice lacking MMP-2 or its activator MMP-14 presented impaired adipose tis-
sue development [117–119].
Moreover, the expression of TIMP-1 was also found in pre-adipocytes and adi-
pocytes and was strongly inhibited when pre-adipocytes entered differentiation
[108, 120]. Furthermore, TIMP-1 expression in 3T3F442A pre-adipocyte cell line
was also associated with enhanced gelatinase expression and activity, which may
suggest its untraditional role as a gelatinases activator [120].
The enteric nervous system which innervates the gastrointestinal tract consists of
different neurons and glial cells that are distributed in two intramuscular plexuses
along the gut and control the smooth muscle contractile activity [121, 122]. Back in
1954, Yntema and Hammond were the first to reveal that the neurons which inner-
vate the gut are originated from vagal NCCs [123]. They demonstrated that upon
ablation of the vagal region of the NC in avian embryos, enteric ganglia failed to
form along the gut. Other studies also showed that vagal NCCs provide the majority
38 R. Kalev-Altman et al.
of enteric precursors, whereas sacral NCCs arising caudal to the 28th somite con-
tribute a smaller number of cells to the hindgut region only [124–126]. Vagal and
sacral NCCs express different transcription regulators such as Sox10, endothelin
receptor B (EndRb), Phox2b, receptor tyrosine kinas (RET), and low-affinity nerve
growth receptor p75 [125, 127–129]. Different studies show that inappropriate NCC
development and/or migration leads to mal-innervation of the gut and may also
result in Hirschsprung disease, a congenital disease characterized by the absence of
enteric ganglia (aganglionosis) along variable lengths of distal colon [2, 121, 122].
This congenital aganglionosis, which occurs in 1 in 5000 live births, is limited to the
rectosigmoid colon in 80% of cases and most commonly presents with the failure of
a newborn to pass meconium within 48 h of life [130, 131].
MMP-2/MMP-9 activity was found to be required for enteric NCC migration
and network formation [125, 132]. At first, a broad-spectrum hydroxamate-based
MMP inhibitor, named GM6001, was used in order to examine the possible role of
MMPs in the migration of enteric mouse and rat NCCs and in the formation of the
neural network within the developing gut [132]. Next, the researchers examined
specifically the involvement of either MMP-3, MMP-8, or MMP-2/MMP-9 in these
processes, using specific pharmacological inhibitors, and revealed that MMP-3 or
MMP-8 inhibition had no effect on enteric NCC migration, whereas MMP-2/
MMP-9 inhibition significantly decreased the distance that enteric NCCs migrated
in the developing gut and the complexity of the neural network that the cells formed
[125].Whether impaired activities of MMP-2/MMP-9 in enteric NCCs may be asso-
ciated directly or indirectly with gut aganglionosis awaits further research.
The NC is the source of all glial cells of the peripheral nervous system, including
the peripheral nerves and the sensory, sympathetic, parasympathetic, and enteric
ganglia [133]. NCCs that migrate from the NT in a ventral direction give rise to
neurons in dorsal root sensory ganglia and to peripheral glial cells [134]. The
peripheral glial cells include three types of cells, the satellite cells of the sensory and
autonomic ganglia, the Schwann cells lining the peripheral nerves, and the enteric
ganglia [135, 136].
MMPs in general, and MMP-2 and MMP-9 in particular, were reported previ-
ously in Schwann cells [137–139]. For example, Asundi et al. [137] have shown that
syndecan-3 shedding in Schwann cells, a process that can have important conse-
quences on cell adhesion, morphology, and migration, is being mediated by the
proteases of the MMP family. The researchers also reported that analysis of MMP
expression by gene microarray techniques has shown that Schwann cells express
MMP-14 mRNA at very high levels. However, they mentioned that other mRNAs,
including MMP-2 and MMP-9, were absent from Schwann cells [137]. Contradicting
data was demonstrated in a different study, in which both MMP-2 and MMP-9 were
found to increase the neurite-promoting potential of Schwann cell basal lamina and
were also found to be upregulated in a degenerated nerve [139]. Furthermore,
2 The Role of Matrix Metalloproteinase-2 and Metalloproteinase-9 in Embryonic… 39
Table 2.1 The expression and role of MMP-2 and MMP-9 in NCC derivatives. Summary of
the data presented in this review regarding the expression and role of MMP-2 and MMP-9 in vari-
ous NCC derivatives; cranial cartilage and bone, connective tissue of the heart, melanocytes, adi-
pocytes, enteric neurons, and glial cells. Other NCC derivatives as the secreting cells of different
endocrine glands as the thymus, thyroid, and adrenal are not discussed in this review
NCC derivative Tissue/cell type MMP-2 MMP-9 References
Cranial cartilage Palatal folds of E13.5–15.5 mouse + – [65]
and bone embryo
Meckel’s cartilage and mandibles of + + [66, 73]
E13–15 mouse embryos
Odontoblasts during odontogenesis – + [75]
in rats
Zebra fish craniofacial structures + + [80]
Heart Quail and chick cushion cells + – [91–94]
Melanocytes Frog melanophores + – [100]
Benign and invasive human + + [101–106]
melanoma lesions
Canine cutaneous melanocytic + + [38]
tumors
Adipocytes Pre-adipocytes during their + + [108, 118,
differentiation into adipocytes 119]
(human and murine)
Enteric neurons Enteric nervous system formation in + + [125, 132]
mouse embryos
Glial cells Schwann cells of rats, mice, and + + [137–140]
humans
MMP-2 immunoreactivity was also found in Schwann cells in human brain tissues
and was suggested to play a role in antiproliferative activity which may balance
between proteolytic and protease inhibitor activity; such a balance is crucial for
both normal neuronal development and for neuronal response to injury [138].
Indeed, studies using damaged peripheral nerves were conducted to clarify the
exact role of MMPs in Schwann cells. For example, Kobayashi et al. examined the
role of MMPs in the development of mechanical allodynia through myelin-binding
protein (MBP) degradation after rat L-5 spinal nerve crush (L-5 SNC) injury [140].
After conducting the L-5-SNC injury, the researchers found increase in MMP-9 but
not in MMP-2 or MMP-7-mRNA levels, two other known regulators of MBP deg-
radation. While they found no gelatinolytic activity of MMP-9 in uninjured nerves,
the L-5-SNC injury caused major increased gelatinolytic activity of MMP-9. On the
other hand, moderate gelatinolytic activity of MMP-2 was observed before the
injury, and no change was noticed afterward [140].
Finally, MMP-3, which is a known activator of MMP-9 [141], was found to be
expressed in microglial cells and ischemic neurons [142]. Another novel finding in
this study was the presence of MMP-2 in normal astrocytes. These results suggested
that MMP-2 is important in the repair process and could contribute to angiogenesis
and glial scar formation (Table 2.1).
40 R. Kalev-Altman et al.
As was described before, MMP-2 and MMP-9 are secreted as latent zymogens and
require an in-situ cleavage of the pro-domain for activation. This cleavage is often
made by another already activated MMP or by several serine proteases. Two known
activators of MMP-2 are MT1-MMP and MT3-MMP, which are also known as
MMP-14 and MMP-16, respectively [143–146]. In a study conducted on mouse
fibroblasts, the activation of MMP-2 by both MMP-14 and MMP-16 was found to
form a ternary complex with the inhibitor TIMP-2, which is bound to the catalytic
domain of the MT-MMP. Then, the pro-MMP-2 binds through its hemopexin
domain to the C-terminus end of the same TIMP, and finally, a second-free MMP-14
molecule adjacent to the complex cleaves the pro-domain of the MMP-2 and acti-
vates it [146]. A different study demonstrated how MMP-2 expression is mediated
by MMP-16. The researchers showed how MMP-16 suppression resulted in decrease
in MMP-2-mRNA levels, whereas in parallel, TIMP-2 levels increased. In concomi-
tant, an increase in MMP-2-mRNA levels and decrease in TIMP-2 levels were
shown upon MMP-16-overexpression treatment, suggesting that MMP-16 posi-
tively regulates MMP-2 function [145, 147].
Despite the fact that the similarity between MMP-2 and MMP-9 regarding their
activity and structure may suggest resemblance in their activation, no data has been
demonstrated yet linking MMP-14 or MMP-16 directly to MMP-9 activation pro-
cess. However, other mechanisms of MMP-9 activation are known. For example,
MMP-9 is known to be activated indirectly via the MMP-14/MMP-2 axis; after
MMP-14 activates pro-MMP-2 together with TIMP-2, the activated MMP-2 acti-
vates in turn the pro-MMP-9 [148, 149]. Another known mechanism in which pro-
MMP-9 is being activated is via the MMP-14/MMP-13 cascade in which MMP-14
activates pro-MMP-13, and then activated MMP-13 cleaves directly pro-MMP-9 to
its active form [150, 151]. Activation of pro-MMP-9 by other MMPs besides
MMP-2 and MMP-13 has also been reported in several organs and cell types. This
includes MMP-1, MMP-3, MMP-7, MMP-10, and MMP-26 [148–150, 152–154].
In consonant with the data gathered on MMP-2 and MMP-9 expression and roles
in NCCs, both of their activators, MMP-14 and MMP-16, were also found to be
expressed in migrating NCCs and in some of their derivatives. Data gathered from
frog embryos show expression of MMP-14 at the timing of NCC migration as well
as at later stages, and it was further demonstrated that MMP-2 expression is subse-
quent to the expression of MMP-14 in NCCs of these embryos [143, 155]. Moreover,
MMP-14 expression was also found in migrating frog melanocytes [100].
Furthermore, MMP-16 was reported to be expressed in the frog embryo during
development and to activate MMP-2 [145]. However, this expression has never been
demonstrated to correlate with migrating NCCs or with NCC markers. In addition,
very recently our lab found that MMP-16 is expressed and has a role in executing
NCC migration in the avian embryo [156]. In this study, we demonstrated that
MMP-16 is expressed in migrating cranial chick NCCs, and by using loss- and
2 The Role of Matrix Metalloproteinase-2 and Metalloproteinase-9 in Embryonic… 41
2.6 Conclusion
The gelatinases, MMP-2 and MMP-9, are expressed and/or play important roles
during NCC EMT and early migration in various embryonic species. Alternation in
the expression and activity of these proteases in older embryos or adults in different
NCC derivatives (i.e., cranial structures, melanocytes, heart septum and valves, adi-
pocytes, enteric neurons, glial cells, etc.) leads to impaired development or malfor-
mations of various tissues. Yet, further investigations are needed in order to
determine whether the role of these gelatinases in early stages of NCC development
is linked to the later functioning of the different NCC derivatives or whether the
early and late activities of MMP-2 and MMP-9 are two independent processes.
Moreover, it is important to uncover the conservation in their roles in different spe-
cies and the way they are regulated by their activators and inhibitors in the unique
NCC population and descendants. Finally, other derivatives of the NCCs such as
smooth muscle cells, the thymus and adrenal glands, and more were out of the
scoop of this review, and the plausible expression and role of MMP-2 and MMP-9 in
these cell types and tissues should be further examined.
42 R. Kalev-Altman et al.
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The Matrix Metalloproteinase and Tissue
Inhibitors of Metalloproteinase Balance 3
in Physiological and Pathological
Remodeling of Skeletal Muscles
Hala S. Alameddine
Abstract
Skeletal muscle is a highly plastic tissue that undergoes physiological or patho-
logical remodeling in response to various stimuli such as exercise, immobiliza-
tion, injury, disease, or aging. This remodeling process implies subtle or more
profound changes to skeletal muscle structure and composition that involves
extracellular matrix (ECM) degradation by matrix metalloproteinases. The bal-
ance between matrix metalloproteinases (MMPs) and their physiological inhibi-
tors, the tissue inhibitors of metalloproteinases (TIMPs), regulates tissue
homeostasis. Upregulation of MMPs and/or TIMPs correlates with vascular
growth and enlargement in endurance-exercised individuals or with inflamma-
tion and regeneration of muscle fibers in injured or diseased muscles. They, fur-
ther, contribute to the development of fibrosis by regulating cytokine/chemokine
production and release/activation of growth factors. Those induce phenotypic
transformation and favor the production of ECM components. It is, therefore,
important to define the exact pattern of MMP/TIMP expression and regulation in
normal and diseased muscles in order to identify potential targets for therapeutic
approaches or biomarkers for specific disease entities and therapeutic
follow-up.
Keywords
MMPs • TIMPs • Skeletal muscle remodeling • Muscle injury • Muscle
diseases
3.1 Introduction
Skeletal muscle is the most abundant tissue in the human body and is prone to modi-
fications in response to variation of functional demands, traumatic injury, aging, or
disease. Development, hypertrophy, hyperplasia, atrophy, degeneration/regenera-
tion, or fibrosis are all remodeling events that modify tissue composition, architec-
ture, vascularization, or innervation. Variations of fiber size, gene expression,
contractile parameters, or capillary density result from signaling events that influ-
ence cell-cell and/or cell-matrix interactions and modify the balance between
MMPs and TIMPs.
Considered for a long time as a stable tissue, skeletal muscle has a remarkable
capacity to adapt to different types of stimuli throughout life. Since their initial
formation until the end of life in aging individuals, the development and adaptation
of skeletal muscles are governed by an interactive cross talk between these muscles
and their environment. The amplitude of cellular, molecular, or structural modifica-
tions induced by remodeling stimuli varies from subtle changes of gene expression
and cell and tissue architecture to more profound modifications leading, for exam-
ple, to total reconstitution of adult skeletal muscle tissue after severe trauma. The
course of remodeling includes modifications to the ECM that surrounds muscle
fibers, to the morphology of skeletal muscle fibers (formation, maturation, atrophy,
or hypertrophy), or to muscle architecture (necrosis, regeneration, angiogenesis,
fibrosis) and function (shift from slow to fast and vice versa, decrease or improve-
ment of contractile parameters).
The ECM, formed by a complex and dynamic network of macromolecules,
engulfs normal adult skeletal muscle fibers and serves as a support tissue. It is not a
static structure. It contributes to structural integrity of muscle fibers and plays a role
in signal transduction. It also has an essential role in key aspects of cell biology
through the production, degradation, or remodeling of ECM components. Physical
and biochemical properties of the ECM vary with the functional status of skeletal
muscles. Modifications of ECM composition or physical properties such as rigidity,
porosity, topography, and insolubility may have functional repercussions on
anchorage-dependent biological functions such as cell division, cell migration, and
tissue polarity. Defects in ECM components (laminin and type VI collagen), or
other proteins participating to the molecular link between ECM and nucleus (lam-
inin A-C or Emerin), via cytoskeletal elements (intermediate filaments, desmin), are
responsible of several types of muscular diseases [1–7]. The MMP/TIMP balance
regulates cellular microenvironment that affects myoblast proliferation, migration,
and differentiation and potentially modifies cell fate. Cells exposed to cryptic frag-
ments resulting from ECM hydrolysis, to growth factors liberated from the ECM or
to cytokines or chemokines, respond to these stimuli by modifying their functional
status and regulating MMP/TIMP expression [8, 9].
Enzymes of both serine and matrix metalloproteinase family degrade ECM com-
ponents. Collectively, the different MMP family members degrade all ECM compo-
nents despite a certain substrate specificity of each member [10]. Tissue homeostasis
is characterized by the balance between basal levels of MMP expression/activity
3 The Matrix Metalloproteinase and Tissue Inhibitors of Metalloproteinase Balance… 51
and their physiological inhibitors, the TIMPs. Transient increase of MMPs associ-
ates with various physiological or pathological remodeling situations such as ovula-
tion, uterine resorption after pregnancy, bone development, angiogenesis, mammary
involution, wound healing and exercise, immobilization, and injury or disease of
skeletal muscles [11–24]. In normal situations, bursts of MMP expression under-
score the tight regulation of MMP/TIMP expression and the essential role played by
MMP/TIMP balance in tissue homeostasis. Strong evidence also points to the role
played by MMPs in the pathogenic process in inflammatory diseases and other
pathological conditions with focal or generalized tissue destruction. The degrada-
tion of certain ECM components (laminin, fibronectin) [25, 26], while others are
preserved (laminin(s) that serves as a guidance cue for re-innervating axons) [27,
28] during the degeneration/regeneration process, indicates that selective regulation
of MMP expression/activation probably occurs for the accomplishment of a specific
task.
As far as we know from mice models of MMP deficiency, these enzymes are
dispensable for embryogenesis probably because of functional redundancy, enzy-
matic compensation, or adaptive development. However, studies in animal models
point to their essential role after birth as regulators of microenvironmental changes
in remodeling tissues including development or disease [29]. MMPs are involved in
cell migration by creating space for cells to migrate. They modulate cell functions
by producing specific substrate-cleavage fragments with independent biological
activity. They regulate tissue architecture through effects on the ECM and intercel-
lular junctions and can activate, deactivate, or modify the activity of signaling mol-
ecules. MMP and related ADAM (a disintegrin and metalloproteases) and ADAM-TS
(ADAMs with thrombospondin repeats) families are involved in shedding growth
factors, which are synthesized as cell-membrane-bound precursor forms, or cell-
surface-adhesion molecules such as syndecan-1. The wide range of MMP targets
that range from peptide growth factors going through tyrosine kinase receptors, cell
adhesion molecules, cytokines and chemokines, as well as other MMPs and unre-
lated proteases has considerably extended their role. MMPs have evolved from
simple hydrolytic enzymes to regulators of signal transduction, of innate and adap-
tive immunity, and modifiers of cellular/molecular phenotype.
Fig. 3.1 Expression of MMP-2, MMP-14, and TIMP-2 in primary cultures of human myogenic
cells, immunofluorescence (panel A), zymography (Panel B), and immunoblotting (Panel C)
Panel A: immunolocalization of MMP-14 and TIMP-2 in human myogenic cells (myoblasts) and
(myotubes). MMP-14 labeling is patchy and spreads along the surface of the cells, whereas
TIMP-2 is detected in certain cell nuclei. Panel B: MMP-2 is secreted into conditioned medium of
myoblasts and myotubes. Panel C: MMP-14 is detected in both myoblasts and myotubes. MW
molecular weight, Mb myoblasts, Mt myotubes
Myogenic cells of various animal species ranging from mice to men express MMP-
1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-14, and MMP-16 [21, 40–
47]. Human and murine myoblasts and myotubes constitutively express MMP-14,
TIMP-2, and pro-MMP-2 (Fig. 3.1, unpublished results) but not MMP-9. However,
myoblasts of different embryological origins may have different MMP expression
3 The Matrix Metalloproteinase and Tissue Inhibitors of Metalloproteinase Balance… 53
patterns [34]. Myoblasts isolated from masseter have a constitutive MMP-9 expres-
sion, whereas myoblasts isolated from limb muscles do not [40, 44]. In the latter,
MMP-9 is induced by TNF-α [48], phorbol ester [21, 43], and factors released from
damaged muscles [21, 49]. Similarly, tumorigenic human rhabdomyosarcoma and
C2C12 mouse myogenic cells express different MMP levels and variety that corre-
late with different invasive or fusion capacities [34, 50, 51]. Myoblasts also respond
differently to cytokines and growth factors. Serum fibronectin, platelet-derived
growth factor-BB (PDGF-BB), transforming growth factor-β (TGF-β), or insulin
growth factor-1 (IGF-1) do not affect MMP-9 expression in adult mouse myoblasts,
whereas tumor necrosis factor-α (TNF-α) and basic fibroblast growth factor (β-FGF)
induce MMP-9 expression. However, none of these growth factors affects MMP-1
or MMP-2 expression [52].
In normal adult skeletal muscles, the exact panel of MMP/TIMP proteins/activ-
ity/mRNA expressed is still not clearly defined. However, one can assume that basal
levels of MMP/TIMP protein/mRNA would be detected in the various cell types
composing the muscle. Gelatinases A and B—both proteins and activity—are the
most documented together with MMP-1 and MMP-14, but data about other MMPs
remain elusive and often restricted to mRNA expression. In normal adult muscles,
in situ zymography, which does not allow the distinction between MMP-2 and
MMP-9, reveals a weak gelatinase activity in the sarcoplasm, endomysium, and
perimysium [19, 33, 53]. Intracellularly, gelatinase activity localizes to mononucle-
ated cells, presumably fibroblasts, endothelial or satellite cells, and patchy spots
within the sarcoplasm [33]. It probably corresponds to MMP-2 which is the only
gelatinase detected by gel zymography in normal muscle extracts [21]. Recent
reports confirm intracellular localization of MMP-2 in type I and type II fibers
where it is more prominent [54].
MMP-2-deficient mice. They present as normal, fertile, but have slower growth rate
[61]. Closer examination reveals marked age-related bone density loss, bone abnor-
malities, craniofacial defects, sclerotic cranial sutures, and articular cartilage
destruction [62] resembling human Multicentric Osteolysis with Arthropathy
(MOA) [63]. This explains slower growth rate that may have functional repercus-
sion on skeletal muscle architecture. Indeed parallel alignment and skeletal muscle
fiber organization depend on mechanical tension induced by bone growth [64].
MMP-2 and MMP-14 double mutant mice [65] die immediately after birth with
respiratory failure, abnormal blood vessels, accumulation of ECM components, and
small centrally nucleated muscle fibers that remind of myofiber immaturity in cen-
tronuclear myopathies. Central nucleation is a characteristic feature of incomplete
maturation of myofibers during development. It can be due to gene defects or to the
absence of appropriate vascularization. Besides the indirect effect these proteins can
have on muscle fiber maturation through vascular defects, in vitro data indicate they
have a complementary not an overlapping role on myotube formation [65].
The third set of proteins that influences myotube formation is TIMP-3/MMP-9.
TIMP-3 regulates myogenesis via miR-206-TIMP3-TACE-TNF-α-p38 signaling
pathway. It acts by regulating autocrine release of TNF-α, known to induce MMP-9
upregulation [48]. The perturbation of TIMP-3 downregulation, which occurs tran-
siently during myoblast fusion or muscle regeneration, blocks TNF-α release, p38
MAPK activation, myogenic gene expression, and myotube formation. On the con-
trary, supplementation at physiological concentration of TNF-α rescues myoblast
differentiation regulated by miR-206 that promotes myogenesis and mediates
TIMP-3 downregulation [66]. The active role of these proteins in myogenic cell
fusion is corroborated in regenerating soleus in which overexpression of TIMP-3
impairs the release of TNF-α, downregulates myogenic gene expression, and delays
the formation of new fibers.
Finally, the addition of TIMP-1, TIMP-2, or TIMP-3 or overexpression of RECK,
the membrane-localized MMP inhibitor, inhibits myotube formation [47]. Myogenic
regulatory factors (MRFs), which determine myogenic cell fate, regulate RECK
expression: MyoD suppresses promoter activity while MRF4 activates it [67].
Physical and chemical signals allow the body to increase or decrease skeletal mus-
cle size throughout adulthood. In adult mammals, an increase in muscle mass occurs
primarily as an increase in muscle fiber size (hypertrophy) rather than muscle fiber
number (hyperplasia) [70]. The most vigorous muscle hypertrophy is accompanied
by ECM remodeling [73], in response to increase of anabolic hormonal signaling or
strength training. Hemodynamic and mechanical stimuli activate satellite cells,
increase angiogenesis, favor the proliferation of capillary-associated cells, and reg-
ulate MMP activity in the absence of any significant inflammation. Among the
enzymes modified by physical activity are MMP-2 and MMP-14 [70, 74, 75].
MMP-2 is increased with high-intensity exercise and is particularly prominent in
skeletal muscles with high percentage of fast muscle fibers [16]. MMP-9 is induced
after a single bout of exercise [72, 76]. High-intensity exercise is required to pro-
mote the expression of MMP-2 in skeletal muscles, and the influence of exercise on
MMP-2 expression is dominant in muscles containing a high percentage of fast
fibers [16]. Visualization of gelatinase activity by high-resolution in situ zymogra-
phy localized MMP-2 activity in myonuclei in endurance-trained rat soleus mus-
cles, while activated satellite cells are identified as the source of MMP-9 upregulation
that occurs within few hours of exercise [53]. Endurance training induces various
biochemical modifications, hypertrophy, and transition of muscle fiber types accom-
panied by an increase of capillary density [77]. In this whole system setting, auto-
crine/paracrine networks may explain the induction of angio-myogenesis. In
response to non-injurious exercise, moderately and highly trained athletes have sys-
temic pro-inflammatory molecules produced in plasma [78], but there is no clear
evidence indicating that exercise induces an inflammatory response in muscle tissue
[79]. Induction of mild inflammatory response after a single bout of exercise is rapid
and resolves within 24 h. With chronic loading, both muscle and tendon show adap-
tive changes with increased collagen production and MMP/TIMP changes indica-
tive of matrix turnover [79]. Pro-inflammatory molecules and MMPs can mediate
mobilization of stem cells [80] and angiogenesis [81, 82]. Peroxisome-proliferator-
activated receptor (PPAR)-δ activation induces direct transcriptional activation of
MMP-9 which, at turn, degrade insulin-like growth factor-binding protein (IGFBP)-
3. This results in IGF-1 receptor (IGF-1R) activation in surrounding target cells,
augmented angiogenesis, and improved regeneration [83]. Various other stimuli
induce MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-12, and MT1-MMP in
vascular tissues [18].
In skeletal muscle, expression at basal levels of MMP-2, TIMP-1, IGF-1, and
ciliary neurotrophic factor, conveys an advantage for aged persons to adapt to resis-
tance training [84]. Specific MMP-1 and MMP-9 polymorphisms correlate with
increased body mass and muscle strength in elderly exercised women. Increased
3 The Matrix Metalloproteinase and Tissue Inhibitors of Metalloproteinase Balance… 57
cell body mass and maximal isometric strength correlate with G insertion in MMP-1
(G+/−1607), while homozygote for 21 or less CA repeats in MMP-9 (cytosine-
adenine microsatellite) gains more isometric strength than carriers of longer micro-
satellites [85]. On the contrary, muscle fiber atrophy in response to unloading,
immobilization, denervation, or aging involves MMP-2 activity [86, 87] although
both MMP-2 and MMP-9 gelatinases are upregulated [88–92]. In such cases, MMP-
2-deficient mice show preserved laminin and type IV collagen and less muscle atro-
phy than MMP-9-deficient mice [93]. Upregulation of MMP-2 activity is reported
in denervated muscles after stretching associated or not to electrical stimulation
[86], but this response seems to be restricted to atrophic muscles because single or
repetitive stretching sessions fail to regulate MMP-2 levels in normal muscles [94].
fully active soluble 50 kDa form, which retain its ability to process MMP-2, follows
TIMP-2 reduction [12] suggesting the implication of MMP-2/MMP-14/TIMP-2 in
either myoblast fusion or angiogenesis. Additionally, MMP-3 and TIMP-1 are
upregulated 24 h after injury, and then TIMP-1 decreases 2 days later, whereas
MMP-3 increase is followed by a decrease of active MMP-3 [101]. Other MMPs
such as MMP-10 and MMP-13, the murine homologue of human MMP-1, are
important for efficient muscle regeneration [102, 103]. MMP-13 expression is pro-
nounced after myoblast fusion and myotube formation in vitro [103], and its levels
correlate with the extent of tissue damage [104]. MMP-10 is also critical for skeletal
muscle maintenance and regeneration [102]. MMP-10-deficient muscles display
impaired recruitment of endothelial cells, reduced levels of ECM proteins, dimin-
ished collagen deposition, and decreased fiber size, which collectively contribute to
delayed muscle regeneration. MMP-10 acts via a CXCR4/SDF1 signaling axis that
proved essential for efficient skeletal muscle regeneration [105].
Damaging exercise induced by downhill running in human volunteers releases
MMP-2, MMP-9, TIMP-1, and MMP-2/TIMP-2 complex into circulation [106].
The acute increase of TIMP-1, MMP-2, and TIMP-2 does not correlate with muscle
damage, but the elevated levels of circulating MMP-9 correlate with the increase of
the number of circulating leukocytes induced by exercise.
Finally, MMPs are largely involved in tendon degeneration, rupture, and healing
[107, 108]. Diagnostic and therapeutic opportunities in orthopedics are provided by
MMPs and TIMPs [109] with clear correlation between increase in MMPs and poor
healing in rotator cuff muscles [110]. The increase of MMP-1 and MMP-13 in rota-
tor cuff synovial fluid correlates with decreased healing in the tendon-bone repair
site. On the contrary, blockade of MMPs improves tendon-to-bone healing by
increasing fibrocartilage and collagen organization and improving the scar strength
[111, 112].
inflammation [24, 114]. This makes them good targets for the control of inflamma-
tory conditions [115].
All inflammatory myopathies have in common the upregulation of MMP-9 in
muscle tissue. High MMP-2 and MMP-9 levels are reported in muscle homogenates
of polymyositis, dermatomyositis, and inclusion body myositis (IBM) patients
[116–118], but distinct MMP expression patterns are revealed by immunolabeling.
MMP-9 antibodies label atrophic myofibers [117, 119] although Choi et al. [118]
report that labeling is restricted to CD8+ cytotoxic T cells. MMP-2 labeling has
similar distribution but weaker intensity, whereas MMP-7 antibodies strongly label
myofibers invaded by inflammatory cells only in polymyositis [119]. Further,
MMP-1 overexpression is localized in mesenchymal cells resembling fibroblasts
and around the sarcolemma. MMP-1 and MMP-9 protein overexpression is con-
firmed by transcript upregulation in polymyositis, in dermatomyositis, and to a
lesser extent in IBM [117]. Finally, muscle biopsies of sporadic inclusion body
myositis (s-IBM) patients show a co-localization of MMP-2 and beta-APP labeling
in rimmed vacuoles, indicating a potential involvement of MMP-2 in the formation
of amyloid deposits [118]. TIMP levels remain unchanged and MMP levels in the
serum are not modified [117].
Muscular dystrophies (MD) are a group of myopathies that weaken the musculo-
skeletal system and hamper locomotion. Progressive skeletal muscle weakness,
defects in muscle proteins, and loss of muscle cells and tissue usually characterize
skeletal muscles of affected patients [120]. Muscular dystrophies do not have an
available treatment or cure, but physiotherapy, aerobic exercise, and low-intensity
catabolic steroids help to maintain muscle tone [121].
Duchenne muscular dystrophy (DMD) is the emblematic form of these diseases.
It is the most frequent and most severe X-linked recessive muscle disorder as it
affects 1 in 3500 newborn boys and leads to death in the second or third decade of
life [122, 123]. DMD and its milder form Becker muscular dystrophies (BMD) are
due to mutations in the dystrophin gene leading, respectively, to the absence or the
expression of a partially functional protein. Dystrophin is a constitutive element of
the “dystrophin-glycoprotein complex” that anchors the cytoskeleton to the ECM
through the outer membrane of muscle cells. Thereby, modifications that affect
these proteins result in defective scaffolding that leads to the disruption of the cell
membrane during muscle contraction and eventual weakening and wasting of myo-
fibers. Interestingly, a number of severe forms of hereditary neuromuscular diseases
such as Duchenne or Fukuyama muscular dystrophy have secondary abnormalities
of dystroglycan [124–127] identified as an MMP target. In patients with sarcogly-
canopathy, MMPs process β-dystroglycan and liberate a 30 kDa proteolytic frag-
ment [128]. The disruption of the link between extracellular matrix and cell
membrane hence leads to deleterious evolution to which MMPs/TIMPs participate
in DMD or other dystrophies raising important interrogations about their regulation
60 H.S. Alameddine
Fig. 3.2 TIMP-1 immunolocalization in normal (Control) and DMD muscles showing increased
labeling of endomysial space with TIMP-1 antibody in DMD muscles by comparison to control
muscles. Note the labeling of intramuscular vessels and nerves
and role in these diseases and their potential use as biomarkers or targets in these
pathologies. Their role in pathological and physiological processes involving the
dystrophin-glycoprotein complex has been reviewed recently [129].
In DMD patients, muscle fibers are lost and replaced by fibro-fatty tissue as a
consequence of recurrent cycles of degeneration-regeneration cycles and exhaus-
tion of regenerative capacity. Continuous necrosis-regeneration with its inflamma-
tory component, cytokine production, generation of reactive oxygen species, release
of growth factors, and production and activation of MMPs/TIMPs perpetuates con-
stant remodeling of skeletal muscles. The restoration of dystrophin to the sarco-
lemma can be achieved by cell or gene therapy and result in the interruption of these
vicious cycles [130–133].
Dystrophic muscles exhibit MMP-2 and MMP-9 in both x-linked muscular dys-
trophy (mdx) and canine x-linked muscular dystrophy (CXMD) [19, 21], whereas
only MMP-2 is reported in normal muscles. MMP-9 is also upregulated in the serum
of adult mdx mice [38] and is overexpressed throughout life span (Alameddine,
unpublished results), and gelatinase activity is localized in necrotic fibers, inflam-
matory cells, and endomysium [33]. Differences in MMP expression patterns have
been associated with different time course and amplitude of inflammation and
regeneration in mdx muscles and experimental injuries [11]. Other MMP, ADAMTS,
and TIMP genes are differently regulated in mdx muscles including MMP-3, MMP-
8, MMP-9, MMP-10, MMP-12, MMP-14, MMP-15, Adamts2, and TIMP-1 mRNA
or activity that is increased. On the contrary, MMP-11, Adamts1, Adamts5, Adamts8,
TIMP-2, and TIMP-3 mRNA are downregulated [37]. CXMD muscles also evi-
denced upregulation of MMP regulatory molecules, MT1-MMP, TIMP-1, TIMP-2,
and RECK [19].
In DMD muscles, TIMP-1, TIMP-2, and MMP-2 transcripts are upregulated, and
MMP-2 activity is increased [134]. TIMP-1 levels are also elevated in the serum,
plasma, and muscles of muscular dystrophy patients like in patients with fibrotic
diseases (Fig.3.2 unpublished results). It correlates with TGF-β1 levels in DMD and
congenital muscular dystrophy (CMD) but not with Becker muscular dystrophy
patients [135]. TGF-β1 is released from decorin that is degraded by MMP-2.
3 The Matrix Metalloproteinase and Tissue Inhibitors of Metalloproteinase Balance… 61
TGF-β1 modulates the ability of cells to synthesize various ECM components and
may modify the protein pattern produced by DMD fibroblasts upon their transfor-
mation to myofibroblasts. It increases MMP-7 that contributes to fibrosis
[136–138].
of various MMP family members in the serum and CSF of ALS patients failed to
establish a correlation between MMPs and TIMP-1 expression and the age of
patients despite the elevation of MT1-MMP, MMP-2, MMP-9, and TIMP-1 in the
serum of the majority of cases particularly in mild ALS patients. There is, however,
a positive correlation between increased levels and disease duration [153]. The dif-
ference of MMP-2 and MMP-9 levels is particularly significant between mild and
severe subgroups establishing MMP-9 as a candidate therapeutic target for ALS
[150].
Monitoring MMP expression in transgenic mice models of ALS indicates they
can be used as biomarkers of disease evolution and follow-up of treatments [154–
157]. High gelatinase levels are the common factor linking pathology, response to
oxidative stress and cytokine release in symptomatic mice [155]. MMP-9 levels are
modulated throughout the course of disease progression in mice. MMP-9 elevation
in the sera corresponds to an early stage of disease progression and declines at end
stage [156], observation that reminds MMP-2 and MMP-9 increase in mild rather
than in severe cases in human patients [153]. At last, in a mouse model of neur-
aminidase 1 deficiency which results in a neurodegenerative disease in human indi-
viduals, muscle biopsies show expansion of epimysial and perimysial spaces,
extensive sarcolemmal invaginations, and infiltration of myofibers by fibroblast-like
cells and ECM. MMP-2, MMP-9, and cathepsin increase in mice muscles associ-
ates with a “unique” pattern of muscle damage, progressive cytosolic fragmenta-
tion, and overt myofiber atrophy that may explain neuromuscular manifestations
reported in patients with type II form of sialidosis [158].
Normal wound healing involves transitory and highly organized response consisting
of interrelated dynamic phases with overlapping time courses that lead to tissue
replacement [159]. All successive stages, (1) hemostasis, (2) inflammation, (3)
migration and proliferation, and (4) remodeling, necessitate high levels of extracel-
lular proteolytic activity [160]. Interference with any of the inflammatory, prolifera-
tive, or regenerative phase jeopardizes the success of the repair process possibly
leading to fibrosis. Building up of fibrosis results from deregulation between syn-
thesis and hydrolysis of ECM components [161] normally accomplished by MMPs
and enzymes of the plasminogen activation system [162–164]. Dysregulated MMP/
TIMP balance [33, 162] and excessive or low-grade inflammation contribute to the
development of fibrosis and exacerbation of pathological features or organ dysfunc-
tion [114, 165] in dystrophic or severely injured muscles.
3 The Matrix Metalloproteinase and Tissue Inhibitors of Metalloproteinase Balance… 63
The initiation and progression of tissue remodeling involve a prior step of degrada-
tion and reorganization of the ECM scaffold to which inflammation is an indispens-
able contributor. However, if the inflammatory process is continuous, it may result
in deleterious evolution of the disease with organ dysfunction and exacerbation of
pathological features.
In a number of experimental injury models or different muscle pathologies,
MMP overexpression correlates with the intensity of the inflammatory reaction or
the persistence of low-grade inflammatory reaction [11, 21, 36, 38, 166]. Considering
that white blood cells produce MMPs to facilitate their migration [167] and regulate
their function [168, 169], one may predict that MMP levels increase concomitantly
to inflammation. Indeed, MMP-9 is produced by inflammatory cells [170] and
stored in granules [171–173] ready to be released during cell transmigration through
vessel/capillary walls. Inflammatory cells also regulate MMP-9 production in a
manner that resembles sequential variations of MMP-9 at the early stages of muscle
regeneration [174]. Of the muscle diseases cited here-above, MMP elevation associ-
ates with inflammation in muscular dystrophies and inflammatory myopathies, but
not in neurogenic myopathies in which it associates with tissue remodeling [116,
151–153].
During skeletal muscle degeneration/regeneration cycles in animal models or
dystrophic patients, the presence of inflammatory cells [175–178] correlates with
high MMP-9 in blood vessels, mononuclear cells, regenerating fibers [11, 19, 21,
38, 179], and the serum of dystrophic mice and DMD patients [166, 180]. MMP-1
signal and TIMP-1 and MMP-2 mRNAs localize in areas of degeneration/regenera-
tion and high density of macrophages, whereas TIMP-2 transcripts distribute more
homogeneously in mesenchymal fibroblasts [134, 179]. Similar to what is observed
in dystrophic muscles, several MMPs including MMP-1, MMP-2, MMP-7, and
MMP-9 are increased in correlation with the density of inflammatory cells in these
myopathies [117, 119, 181].
Recent evidence indicates a potential benefit of controlling muscle inflammation
or inhibiting MMP-9 activity by various natural or chemical compounds to improve
skeletal muscle regeneration in dystrophic animal models [36–38, 182]. However,
the potential use of MMP inhibition should be considered with great precaution as
it may prove deleterious at the long term. The beneficial effect observed upon
MMP-9 inhibition in young dystrophic mice [39] is completely reversed by its long-
term inhibition which results in the development of fibroadipogenic tissue in old
MMP-9-deficient mdx mice. Further, experimental data clearly indicate certain
MMPs are necessary for the regenerative process [102, 105] and for improving
myogenic cell engraftment in dystrophic skeletal muscles [34, 35].
64 H.S. Alameddine
The robust regenerative ability of skeletal muscle can be impaired in case of severe
injuries or in dystrophic muscles in which massive muscle loss yields a reparative,
stabilizing fibrotic response that proceeds more rapidly than the growth of new mus-
cle fibers. Recurrent wound healing response in dystrophic muscles generates, in
cascade, molecular modifications that influence tissue reconstitution. In the first
stage, a dysregulation between hydrolysis and synthesis of ECM components occurs
in favor of hydrolysis and is followed by a resolution phase during which inhibitors
are upregulated. Therefore, continuous cycles of necrosis-regeneration in dystro-
phic muscles may be associated to increase of both MMPs and TIMPs, as observed
in DMD muscles [180].
Inflammatory cells contribute to the development of fibrosis by producing cyto-
kines/chemokines [178, 183–185] that upregulate MMPs which, at turn, regulate
the cytokines, their ligands, and receptors [186, 187]. MMPs also activate growth
factors such as transforming growth factor beta (TGF-β) and connective tissue
growth factor (CTGF) [188–190]. Each of these growth factors is reported to induce
phenotypic transformation of myoblasts into myofibroblasts [191, 192], whereas
CTGF also induces MMP upregulation [193]. Additionally, activated MMPs and a
disintegrin and metalloproteinase (ADAM) signal through their receptors and
downstream mitogen-activated kinases to activate the transcription of immediate-
early genes that mediate fibrosis [194]. ADAM-17 (tumor necrosis factor-α convert-
ing enzyme, TACE, or MT4-MMP) and ADAM-12 are influential actors in the
pathogenesis of inflammatory and fibrous connective tissue diseases. TACE overex-
pression and activation activate Epidermal Growth Factor Receptor (EGFR) and
stimulate type I collagen expression [195]. It induces transcriptional regulation of
MMP-2 and ADAM-12 that activates TGF-β signaling independently of its protease
activity [188]. TNF-α release by TACE stimulates collagen synthesis in fibroblasts
[196], whereas TNF-α inhibition significantly reduces necrosis [197, 198], the lev-
els of TGF-β1, and type I collagen mRNA in mdx mice [199].
Serious indications, although not proven experimentally, indicate that TIMPs are
involved in muscle fibrosis. Substantial proliferation of fibrotic tissue correlates
with the elevation of TIMP-1 in the serum and endomysium of muscles of DMD
patients and dystrophic mouse models [180] and (Fig. 3.2). In cells derived from
heart muscles, all four TIMPs are reported to induce fibroblast cell proliferation, but
only TIMP-2 upregulates collagen production [200]. This needs to be confirmed in
skeletal muscle tissue.
A subtle balance between MMPs and TIMPs operates at the intracellular and extra-
cellular levels to regulate homeostasis of skeletal muscle tissue. The role of certain
MMPs has been clarified in skeletal muscle tissue remodeling. MMP-2 and its acti-
vator MMP-14 are linked to angiogenesis and vessel growth [201], and MMP-2
3 The Matrix Metalloproteinase and Tissue Inhibitors of Metalloproteinase Balance… 65
conduct clinical trials using MMP inhibition for the treatment of muscular dystro-
phies. This is particularly important to highlight that the inhibition of all the other
MMPs investigated until now proved to have detrimental effect on skeletal muscle
regeneration/maturation [102, 201].
Acknowledgments The author wishes to thank K.Mamchaoui and V. Mouly for generously pro-
viding primary human myogenic cells and the AFM-Telethon (Association Française contre les
Myopathies) for financial support.
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Role of BMP1/Tolloid like Proteases
in Bone Morphogenesis and Tissue 4
Remodeling
Abstract
BMP-1/ tolloid like proteases collectively referred as BTPs are members of the
astacin family of metalloproteases belonging to the metzincin clan. Four mem-
bers of BTP are identified in humans; BMP-1, mTLD, mTLL1 and mTLL2.
The BTPs are involved in several pathophysiological diseases including bone
morphogenesis, fibrosis, tissue remodeling and tumor progression. BTPs are
important regulators to activate several growth factors and helps to release
anti-angiogenic fragments from parent proteins. Three dimensional structure of
BTPs reveal the presence of a highly unusual disulphide bridge present within
the cysteine-rich loop region in the active site. The activity of BTPs is controlled
mainly by activators. The only endogenous inhibitor of mammalian BTPs is
α2-macroglobulin. Several small molecular inhibitors of BTPs have been
reported. Further studies will help to explore the full spectrum of activities of
BTPs which will help in recognition of BTPs as new targets for future
therapies.
Keywords
BMP1/Tolloid like Proteases (BTPs) • Morphogenesis • Tissue remodeling •
Cancer activators • Inhibitors
4.1 Introduction
Fig. 4.1 Domain structure of BTPs. The BTPs consists of a signal peptide, pro-peptide, an prote-
ase/ catalytic domain followed by several CUB (for complement C1r/C1s, uEGF, BMP-1) and
EGF (epidermal growth factor) domains
Fig. 4.2 Multiple Sequence alignment of protease domain of BTPs and astacin. Identical (con-
served), strong and weak residues represented as green, cyan and orange colour respectively.The
abbreviation for species are the same as used in Table 4.1
Fig. 4.3 X-ray structure of BMP-1 solved at 1.27 Å resolution. Disulphide bridges and important
residues in substrate recognition involved at active site are represented as stick. Catalytic zinc is
represented as CPK model
BMP-1 (Fig. 4.3) indicates it has a deep active site cleft within which is the three
conserved His residues binding to the catalytic zinc. But unlike the prototypical
protease astacin and other members of the family that a conserved tyrosine in
BMP-1 is not involved in binding zinc at the active site [6]. BTPs differ from the
other astacin like proteases in having a highly unusual additional disulphide bond in
the active site region. This occurs in the cysteine-rich loop region and is believed to
control the access of substrates through change in conformation [6]. Like other
astacin like proteases, the catalytic domain of BMP-1 consist of a 100 residue
80 S. Chakraborty et al.
N-terminal domain and a C-terminal domain separated by the active site cleft. The
N-terminal domain contains a five stranded β- sheet as also two long helices. The
C-terminal domain contains few regular secondary structures. The two disulphide
bonds present in the other astacin members is conserved in BMP-1 (Cys64-Cys84 and
Cys42-Cys198) with another additional disulphide bridge between Cys62 and Cys65 [7].
BMP-1/tolloid like proteinases (BTPs) has been detected in species from
Drosophila to human. Procollagen C-proteinase (also called bone morphogenetic
protein-1) of the astacin family cleaves procollagen and non-collagen substrates
into the insoluble fibrillary collagen. It also processes prolysyl oxidase to its active
form which is responsible for catalyzing the cross-linking of collagen fibrils thereby
contributing to the structural stability of collagen [7]. BTPs are important regulators
to activate several growth factors such as BMP-2/−4, TGF-β/1, GDF-8/−11 and
IGFs and helps to release anti-angiogenic fragments from parent proteins [8].
This chapter summarises the recent advances on the structure, function and diverse
role of BTPs of the astacin family and discusses their role as potential targets in
therapeutic use. Table 4.1 describes the different members of the BTP’s reported so
far in human.
The sequence similarity among the metalloprotease domain of several BTPs and
astacin is indicative of the fact that the metalloprotease domain structure of BTPs is
similar to that of astacin. Among the proteases of known three dimensional struc-
tures, digestive enzyme astacin (PDB ID: 1AST, 1QJJ and 1QJI share 35% sequence
identity with TLL-1/BMP-1) is most closely related to BTPs. All BTPs are charac-
terised by the presence of a highly unusual disulphide bridge between two adjacent
cysteine residues present in the cysteine-rich loop region of the active site.
The protease domain of BTPs containing about 200 amino acid residues consist of
two sub-domains separated by a deep active site cleft with the catalytic zinc at its
bottom.
Bone morphogenetic protein-1 (BMP-1) was initially found in bone extracts. It can
induce bone formation at ectopic sites. BMP1 is a zinc-dependent metalloprotein-
ase. The BMP-1 molecule contains an astacin-like catalytic domain, three CUB
domains and an EGF (epidermal growth factor) domain which is located between
the second and third CUB domains (Fig. 4.1). The CUB 1 domain is required for the
secretion of the molecule. Glu483 on the β4-β5 loop of CUB2 is essential for the
4 Role of BMP1/Tolloid like Proteases in Bone Morphogenesis and Tissue Remodeling 81
Comparing the structure of catalytic domain of astacin from crayfish Astacus asta-
cus it is found to be closely related to BMP-1/TLL-1 (Fig. 4.4).Three histidines, a
tyrosine and a water molecule coordinated with Zn2+ion at the active site is unique
in astacins and serralysins enzyme family. Surprisingly these structural homologs
have different substrate specificity. Astacins specificities are dominated by small
aliphatic side chain in P1′ and a proline in P2′, whereas BMP-1/TLL-1 prefers
strictly an Asp in P1′ and Asp/Glu in P2′ site [12]. Furthermore, BMP-1 crystal and
molecular structure reveals a unique conformation of a small cysteine-rich loop
within the active site cleft, which suggests that the flap movement is required to
control substrate recognition. Therefore in comparison to astacin, BMP-1/TLL-1
protease’s substrate specificity governed not only by S1’ pocket but also registered
by a unique cysteine-rich loop. Although they have conserved catalytic residues but
the most structural differences between BMP-1 and astacin are around the substrate-
binding pocket. A confirmed basic residue in S1′ fits well with the specificity of
BMP-1 to counter opposite charge acidic amino acid, aspartate in the P1′ position.
Fig. 4.4 Superimposition of TLL-1(pink) on BMP-1 (cyan). Important residues involved in sub-
strate recognition are represented as stick. Catalytic zinc is represented as CPK model
82 S. Chakraborty et al.
BMP-1 S1’ pocket is larger than astacin due to open conformational loop
near Arg176.
In summary the cysteine-rich loop and the disulfide bonded network of BMP-1
clamps its substrate into a favourable position for catalysis [6].The unusual hydro-
phobic character and the flexibility of the cysteine-rich loop have not been observed
for astacin or other related matrix metalloproteases. This loop controls substrate
selectivity, therefore may guide in designing potent inhibitors of BMP-1/TLL-1
proteases against potential human metalloproteases anti-targets.
BTPs have involved as interesting therapeutic agents for treatment of healing bone
fractures, periodontal bone defects, enhancing bone response around alloplastic
materials implanted in bone and also in prevention of osteoporosis. In dentistry,
bone morphogenetic proteins have been tested in periodontal, implant and restor-
ative endodontic procedures [13].
The expression of a large number of proteases including BMP-1 and mTLD, prote-
ase inhibitors and growth factors can positively or negatively be modulated by TGF-
β1 [17, 18]. The upregulation of some essential ECM matrix proteins like collagen
4 Role of BMP1/Tolloid like Proteases in Bone Morphogenesis and Tissue Remodeling 83
and fibronectin can be induced by TGF-β1 [8]. The activation of TGF-β1 can take
place both in a proteolytic or non-proteolytic manner. BMP1 plays an important role
in the proteolytic pathway [19]. It is also known that BTPs contribute to the mainte-
nance of high levels of TGF-β1 by stimulating the degradation of soluble betaglycan
and CD109, two antagonists of TGF-β [16]. Several members of the TGF-β or IGF
superfamilies can be triggered by BTPs that targets different pathophysiological
processes such as morphogenesis, muscle growth, tumor progression and dorso-
ventral patterning [20–22]. BTPs can also help to degrade several proteins that
shows angiogenic properties. BTPs can convert prolactin and growth hormones into
effective anti-angiogenic molecules [23] and remove the pro-metastatic potential of
angiopoietin-like protein2 [24].
The involvement of BTPs in a wide range of diseases in human have been reported.
Since BMP-1 also known as Procollagen C-Proteinase have been shown to be
responsible for a number of diseases in human, BMP-1 can be used as new target for
drug therapy.
4.5.1 Fibrosis
Bone disorders have been attributed to the role of BTPs. Missense mutations in the
BTPs cleavage sites of procollagenα1 and α2 chains have been describes in osteo-
genesis imperfect (OI) [30]. This leads to delay in procollagen processing which
results in increase in bone mineral density together with susceptibility of bone fracture.
Recent studies have identified homozygous missense mutations in BMP-1/mTLD in
84 S. Chakraborty et al.
4.5.3 Cancer
BMP-1 includes a group of signature extracellular matrix proteins linked with tumor
metastasis has been confirmed by proteomic study [34]. BMP-1 and PCPE-1are
desmoplastic markers associated with colorectal cancer [35]. microRNA and miR-
194 downregulation of BMP-1 expression reduce metastasis of lung by reducing the
level of TGF-β activation and MMP-2/−9 expression [36]. Angiogenesis is an
important component of metastasis. PCPE-1 is one of the five proteins required for
formation of endothelial cell lumen [37]. Results indicate that BMP-1 and PCPE-1
are noted components of tumor microenvironment. Future studies will unfold the
inhibition of BMP-1 activity and its significant impact on metastasis and cancer.
4.6.1 Activators
4.6.2 Inhibitors
The involvement of the BMP-1 /tolloid like proteases (BTPs) in several chronic
fibrotic conditions like pulmonary, renal and liver fibrosis, scleroderma and muscle
wasting indicates that these enzymes can be good therapeutic targets for the design-
ing of potent, specific inhibitors at physiological condition. BTP activities are con-
trolled by substrate specific activators rather than inhibitors. BTPs are therapeutic
targets to prevent deposition of excess collagen in fibrosis. Cardiac fibrosis has been
related to BTPs and associated proteins. BTPs are associated with bone genetic
disorders. BMP-1 and PCPE-1 (Procollagen C-proteinase enhancers) are thus
important components of the tumor microenvironment. Future studies will help to
unveil if the inhibition of BMP-1and PCPE-1 can have an important role in tumor
progression and metastasis.The designing of specific BTP inhibitors for the treat-
ment of metalloprotease-associated diseases is an important goal for future disease
management.
Acknowledgement The authors sincerely thank Dr. Indrani Sarkar of Narula Institute of
Technology for her useful suggestions in preparation of the chapter.
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Abstract
N-Myristoyltransferase (NMT) catalyzes the amide-linked addition of the myris-
toyl (C14:0) moiety to the amino-terminal glycine of substrate molecules. The
upregulation of NMT in cancerous states was originally reported by our group,
and subsequently this protein has drawn significant interest as a therapeutic tar-
get. However, the regulation of this enzyme in various physiological states is still
in a state of infancy. NMT possesses PEST sequences and is a target for m-
calpain-mediated degradation. The two isoforms, NMT1 and NMT2, interact
differentially with the proteases caspase-3 and m-calpain. Recently, we have
shown that the interaction of these isoforms (i.e., NMT1 and NMT2) with cas-
pases regulates co- and posttranslational protein myristoylation. In this chapter,
we have discussed the findings of the regulation of NMT functions by various
proteases and the effects of proteolytic processing of NMT on its biochemical
behavior.
S. Kumar
Department of Pathology and Laboratory Medicine, College of Medicine,
University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada
Department of Chemistry and Biochemistry, University of the Sciences,
Philadelphia, PA 19104, USA
U. Das • J.R. Dimmock
College of Pharmacy and Nutrition, University of Saskatchewan,
Saskatoon, SK S7N 5C9, Canada
R.K. Sharma (*)
Department of Pathology and Laboratory Medicine, College of Medicine,
University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada
e-mail: rajendra.sharma@usask.ca
Keywords
Caspase • Calpain • N-Myristoyltransferase • Lipidation • Protein
myristoylation
5.1 Introduction
The process of protein lipidation acts as a critical regulator for proper targeting of
numerous cellular proteins to their destined sub-cellular localizations i.e., to various
membranes, membrane domains, and organelles [1, 2]. The lipid moiety attached in
the prevalent forms of lipidic modification in eukaryotes includes (1) a 14-carbon
myristoyl chain, (2) a 16-carbon palmitoyl chain, (3) isoprenoids (15-carbon farne-
syl or 20-carbon geranylgeranyl), and (4) glycosylphosphatidylinositol (GPI)
anchors [3]. The addition of the C14:0 (myristoyl) group to proteins, referred to as
“myristoylation,” is unique among these processes. A stable amide bond between
the C14:0 group and the exposed amino-terminal glycine renders the half-life of the
myristoyl moiety on the protein equivalent to the half-life of the polypeptide chain
backbone [4, 5]. The catalytic subunit of cAMP-dependent protein kinase was the
very first myristoylated protein discovered, blocked at the N-terminus by
n-tetradecanoic acid, and a similar modification was soon reported in the β-subunit
of calcineurin [6, 7]. Furthermore, isoform-specific protein myristoylation has been
shown for many proteins to aid in their tissue-specific cellular roles [8, 9].
The process of protein myristoylation is catalyzed by the enzyme
N-myristoyltransferase (NMT; EC 2.3.1.97) and involves the covalent transfer of a
myristoyl group (C14:0) to the amino-terminal glycine moiety of substrate protein
molecules [4, 5, 10–12]. The enzyme is found ubiquitously distributed in lower and
higher eukaryotes [13]. The availability of an N-terminal exposed glycine moiety is
an absolute requirement and the modification usually occurs on a general consensus
motif of GXXXS/T (where X is any amino acid) [4, 14]. However, the availability
of Ser/Thr at position 5, downstream of N-terminal acceptor glycine, is not a man-
datory requirement. In such cases, Asp/Gln at the penultimate position to the accep-
tor glycine acts as a facilitator of myristoylation [9, 15, 16]. Protein myristoylation
takes place in either of the following two ways: (i) in a co-translational manner,
when the protein chain being synthesized on the ribosome (after the synthesis of up
to 100 amino acids) is subjected to the removal of the initiator methionine, by the
enzyme methionine aminopeptidase, thus exposing a penultimate N-terminal gly-
cine, and (ii) in a post-translational fashion, when an internal glycine within a poly-
peptide is exposed following a proteolytic cleavage event, mostly in the apoptotic
states [12, 17, 18].
The process of protein myristoylation follows the ordered reaction mechanism,
the apoenzyme first binds to the ligand myristoyl-CoA (MYR) allowing to form a
binary complex of NMT-MYR which subsequently binds to protein/peptide sub-
strates. The catalytic transfer of myristoyl from the donor MYR to acceptor glycine
occurs through direct nucleophilic addition-elimination reaction. After the
5 Role of Proteases in the Regulation of N-Myristoyltransferase 91
Fig. 5.1 N-Myristoyltransferase structure showing bound myristoyl-CoA in the N-terminal half
of the enzyme
NMT, in the mammalian species, exists in two major isoforms, NMT1 and NMT2
[12]. The two human isoforms (hNMT1 and hNMT2) reflect divergence only in the
amino-terminal domains and share an overall ~77% identity [21]. These two iso-
zymes, hNMT1 and hNMT2, are encoded by different genes, have been cloned and
show differential specificities. The knockdown of hNMT1 is detrimental to tumor
growth in vivo and is thus considered a potential novel cancer target [22–26]. The
knockdown of hNMT1 also results in defective myelopoiesis in mice, thus indicat-
ing its essentiality in cellular development [27]. Among the two isoforms of human
NMTs, hNMT1, but not hNMT2, exists in multiple isoforms. hNMT2 appears as a
92 S. Kumar et al.
single 65 kDa protein, whereas four distinct isoforms (from 49 to 68 kDa) of
hNMT1 are observed in cells [21]. The first report on the multiple isoforms of NMT
came from studies on bovine brain NMT, and further, isozymes with varying size
and tissue distribution have been reported from a variety of sources [21, 28–31].
Five isoforms of NMT exist in the murine leukemia cell line L1210, whereas two
isoforms were reported in bovine brain cortex [29, 32]. Later it was reported that the
bovine brain contains a heterogeneous mixture of NMT subunits [30]. To date it is
not fully understood how the in vivo regulations are conferred on to the NMT
isoforms.
N-Myristoylation lies upstream of multiple pro-proliferative and oncogenic path-
ways, and thus NMT is a validated target for therapeutic interventions in cancer [12,
23–26]. The selective knockdown using small interfering RNAs (siRNAs) against
NMT isozymes in human cells shows that targeting NMT isozyme induces apopto-
sis, with depletion of hNMT2 having a 2.5-fold greater response than hNMT1, and
that the loss of hNMT2 shifts the expression of the Bcl-family proteins toward
apoptosis [24]. The silencing of hNMT1 inhibits cell replication associated with a
loss of activation of c-Src and its target FAK. Also the signaling through the c-Raf/
MEK/ERK/Elk pathway is reduced [24]. However, the two isoforms have been sug-
gested only to have partially overlapping functions, and hNMT1 is more critical for
tumor cell proliferation [24]. It is recently reported that in HeLa cells, upon NMT
inhibition, the cell death occurs via apoptosis which is following, or concurrent
with, accumulation in the G1 phase [33]. In response to NMT inhibition, the cell
cycle regulation-associated proteins are downregulated, whereas the proteins
involved in the endoplasmic reticulum stress and unfolded protein response are
upregulated in the HeLa cells as well as breast (MCF-7, MDA-MB-231) and colon
(HCT116) cancer cell lines [33].
This chapter discusses how the interaction of NMTs with various proteases regu-
lates the biochemical behavior of N-myristoyltransferase and why cells need to
allow for the proteolytic processing in demand to diversified physiological
requirements.
The sequence of both human NMT isoforms contains a higher percentage of Pro
(P), Glu (E), Ser (S), and Thr (T) amino acid residues forming a PEST sequence
which may act as an intramolecular signal for rapid proteolytic degradation [34].
Calpains, Ca2+-dependent neutral proteases, recognize PEST signature and are
responsible for the degradation of PEST-containing proteins [35]. An earlier study
from our laboratory suggested that bovine cardiac NMT1 activity is completely
abolished by m-calpain in vitro [36]. Degradation of NMT by m-calpain is shown to
be inhibited by the calpain inhibitor, calpastatin [36]. Furthermore, we have reported
earlier that in colorectal adenocarcinomas, the activity and expression of m-calpain
is significantly higher [37]. The two isoforms, NMT1 and NMT2, in human colorec-
tal adenocarcinoma tissues and human colon cancer cell lines (HCCLs) show
5 Role of Proteases in the Regulation of N-Myristoyltransferase 93
1 2 3 4 5 6
A WB: Ab-m-Calpain
B WB: Ab-Caspase 3
Fig. 5.2 Interaction between NMTs (NMT1 and NMT2) and proteases (m-calpain and caspase-3)
by immunoprecipitation analysis in human colon cancer. Lanes 1 and 4, human normal colorectal
sample; lanes 2 and 5, human adenocarcinoma samples; and lanes 3 and 6, HT29 colon cancer cell
line (For details see Selvakumar et al. [38])
differential interactions with m-calpain and caspase-3 [38]. It was observed that
NMT1 interacts with m-calpain in normal, adenocarcinoma, and HT29 colon cancer
cell line (Fig. 5.2A, lanes 1–3), whereas NMT2 does not (Fig. 5.2A, lanes 4–6).
These findings suggest that the two NMT isoforms may regulate cellular signaling
differently and have been discussed elsewhere in details [39]. It is also observed that
overexpression of calpastatin downregulates calpain activation but increases
caspase-3-like activity and also accelerates the appearance of apoptotic nuclear
morphology [40]. Besides being degraded by calpain, calpastatin may also be a
target for degradation by caspases during apoptosis [41]. It is possible that calpain
might be indirectly activated by caspase-3 via calpastatin degradation.
Immunoprecipitation analysis shows that both in normal and cancerous samples,
NMT2 interacts with caspase-3 (Fig. 5.2B, lanes 4–6), whereas NMT1 does not
(Fig. 5.2B, lanes 1–3). These findings reveal that NMTs may be involved in the
calpain/caspase-mediated pathway during the development of cancer [38, 39]. The
differential interaction of the two isoforms of the NMT with m-calpain and caspase-
3 is remarkable. m-Calpain showed to interact with NMT1 only, whereas NMT2
with caspase-3, indicating that a differential regulation may exist for NMT1 and
NMT2 by m-calpain and caspase-3 [38] (Fig. 5.3).
A recent study has shown that both forms of human NMT (i.e., NMT1 and
NMT2) are cleaved upon execution of apoptosis [42]. The timing of the cleavage of
NMTs is in parallel with that of the apoptotic marker PARP-1, suggesting it is due
to the action of caspases. The cleavages of NMT1 and NMT2 result in the decrease
of molecular mass by ∼11 and ∼10 kDa, respectively. The general and caspase-8-
specific inhibitors completely block the cleavage of NMT1, whereas caspase-3-
specific inhibitor shows only partial protection against fragmentation. However,
NMT2 cleavage is abrogated by all of these caspase inhibitors [42]. The findings
suggest that NMT1 is a substrate for both caspase-3 and caspase-8 and NMT2 is
likely a substrate of caspase-3 but not caspase-8. Identification of the amino-termi-
nal residues of the cleaved fragments revealed that caspase-8-mediated NMT1
cleavage (∼48 kDa) occurs mainly after Asp72 with minor cleavage at Asp38 [42].
However, it is suggested that the Asp38-NMT1 fragment generated by purified cas-
pase-8 might be nonphysiological. The caspase-3-mediated NMT1 cleavage results
in a single ∼48 kDa fragment corresponding to a cleavage at Asp72. N-terminal
94 S. Kumar et al.
Fig. 5.3 Schematic illustration of interaction between NMTs (NMT1 and NMT2) with m-calpain
and caspase-3. NMT exists in two major isoforms, NMT1 and NMT2, which interact differentially
with calpain and caspase 3
sequencing of the caspase-3 cleavage products of NMT2 revealed that the process-
ing of NMT2 was at both Asp25 and Asp67 [42]. The primary sequence information
reveals that the caspase cleavage sites for both NMTs are located in the amino-ter-
minus portion of the molecule. The caspase cleavage, either by caspase-3 or cas-
pase-8, does not involve the carboxy-terminal catalytic domain of NMTs, and the
caspase-cleaved enzymes retain their catalytic activity [42]. Of particular interest in
the N-terminus of the enzymes, there is a presence of a poly-lysine block in the
cleaved portion of the enzyme. This poly-lysine block is the part of the ribosomal
targeting signal in consistence with a co-translational protein myristoylation model.
The co-translational myristoylation requires the proximity of NMT within the pro-
tein synthetic complex (ribosomal complex) to timely capture its substrate when
prompted by an appropriate N-myristoylation consensus signal available on a pro-
spective nascent polypeptide [31]. The cleavage of the NMT1 leads to a cytosolic
relocalization (>55%) from the predominantly membrane-bound form (64%),
whereas NMT2 relocalizes to membranes (>80%) from the predominantly cytosolic
(62%) when cleaved [31].
We have shown that the amino-terminal region of the catalytic module of NMT1
shows variability both in length and nature of the amino acids among the ortholo-
gous NMTs (Fig. 5.4) [43]. The minimal sequence length upstream of a 310αA’
conserved region is found in Trypanosoma brucei and Trypanosoma cruzi. The T.
brucei and T. cruzi NMTs lack the sequence length parallel to the region corre-
sponding to the first 28 amino acids of the catalytic domain of human NMT1
(Fig. 5.4). Using biochemical analyses on the purified recombinant proteins, we
have shown that the deletion of the 28 residues at N-terminus enhances the
5 Role of Proteases in the Regulation of N-Myristoyltransferase 95
Fig. 5.4 Sequence comparisons of the N-terminal region orthologous NMTs from divergent spe-
cies reflecting differences in the sequence lengths. The first conserved residues across all sequences
are shown in red boxes (For details see Kumar and Sharma [43])
5.4 Summary
A
hNMT1s
∆28-hNMT1s
4000
2000
1000
0
0 2000 4000 6000 8000 10000
MYR [nM]
B hNMT1s
∆28-hNMT1s
4000
Reaction Velocity (RFU)
3000
2000
1000
0
0 10 20 30 40 50
Peptide [µM]
Fig. 5.5 Enzymatic activity of the catalytic domain (hNMT1s) and N-terminal truncation in cata-
lytic domain (Δ28-hNMT1s) was measured under standard assay conditions as described (43). (a)
Michaelis-Menten analysis of the myristoyl-CoA (MYR) and (b) Michaelis-Menten analysis of
peptide substrate by the proteins used in study (For details see Kumar and Sharma [43])
usually occurs after a proteolytic cleavage event, which allows for the unmasking of
a glycine residue at internal positions, mostly in the apoptotic states. The proteolytic
cleavage of NMTs itself alters its sub-cellular localization either through the removal
of ribosome-targeting domain containing the poly-lysine region in NMT1 or that of
a negatively charged domain upstream of the poly-lysine domain in NMT2. The ribo-
some binding and/or membrane binding is mediated by poly-lysine domain and the
caspase-mediated cleavage and of NMT1, and therefore the removal of the polybasic
domain of hNMT1 translocates the caspase-cleaved NMT1 to cytosol from its pri-
marily membrane-bound state. In cells undergoing apoptosis, a disproportionate
number of substrates become quickly available following proteolytic cleavage and
require being N-myristoylated to perform their physiological functions. This
5 Role of Proteases in the Regulation of N-Myristoyltransferase 97
increases the demand for the myristoylation process, and to meet physiological
requirements for myristoylation of the substrates, cells could undergo an increased
synthesis of the NMT enzyme. However, the rate of polypeptide synthesis may not
match the requirements within the rapid time scales of apoptosis. The removal of the
ribosome-binding poly-lysine domain might represent the controlling features for
the rapid relocalization of NMTs for the prompt post-translational myristoylation of
substrate proteins available after the caspase cleavage events during apoptosis.
Furthermore, the proteolytic processing as observed earlier may serve to meet the
increased demands of myristoylation by removal of the N-terminal regulatory
regions, and therefore enhancing the turnover rates of peptide substrates.
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Role of Tissue Factor-FVIIa Blood
Coagulation Initiation Complex 6
in Cancer
Abstract
Since a century ago, an intricate relationship exists between cancer progression
and thromboembolism. In various case studies, thromboembolic complications
have been found to maintain an intricate relationship with the progression of
various tumours like breast, lung, colon and glioblastoma. Moreover, coagula-
tion factors have also been reported to be involved for metastatic augmentation
complications in cancer patients with elevated levels of complication in cancer-
associated thrombosis. Production and protease activity of various coagulation
factors like thrombin and tissue factor (TF)-FVIIa complex affect tumour pro-
gression and propagation actively. TF exerts both coagulant as well as PAR2-
dependent cancerous activity by eliciting various cell survival signalling
pathways, like P42/44MAPK and PI3K/AKT. However, the molecular elucida-
tion of the role of these coagulation factors in cancer-associated thrombosis and
metastatic progression has not been understood till date.
Keywords
Blood coagulation • Cancer • Factor VIIa • Microvesicles • Macrophages •
Signalling • Tissue factor
6.1 Introduction
The phenomenon of blood coagulation has been studied enormously in the last
few decades. Although intrinsic factors involved in blood coagulation are not fully
understood, its relation to various other pathophysiological and systemic disorders
has been uncovered through several research studies. Blood coagulation is a com-
plex series of enzymatic cascade reactions processed either by one or both the
pathways: intrinsic and extrinsic. The concept of the coagulation cascade was first
proposed in the year of 1964, which deals with the conversion of inactive protease
precursors (zymogen) into their activated form. The extrinsic coagulation process
is initiated by tissue factor (TF) on the cell surface [1]. In veins and arteries,
coagulation factor VII (FVII) circulates as zymogen (inactive), and an endothelial
layer forms a lining between the bloodstream and the extravascular layer where
TF, the receptor for FVII, resides. Damage of the endothelial lining exposes TF to
the blood, where it binds with its ligand FVII and form TF-FVIIa proteolytic
active binary complex. This activated complex then activates FX to FXa, which
binds with its cofactor FVa, and converts prothrombin into thrombin. Further,
thrombin activates platelets and proteolytically activates soluble fibrinogen to
insoluble fibrin, which eventually in conjugation with activated platelets forms
mesh-like structure, known as blood clot [2]. In the intrinsic pathways, FVIIIa
with its ligand FIXa forms tenase FVIIIa-FIXa complex in the presence of Ca2+ on
membrane surface, which plays a crucial role to generate FXa and finally leads to
activation of downstream cascade in a similar fashion that of extrinsic pathways.
TF-FVIIa binary complex formation is the key step to initiate blood coagulation,
where FVIIa acts as serine protease; on the other hand, TF acts as cofactor for
FVIIa. Free FVIIa has very less proteolytic activity, but TF allosterically modifies
FVIIa conformation in such a manner that FVIIa proteolytic activity gets enhanced
several folds than that of free form. Structurally, FVII consists of four domains:
GLA domain (contains gamma-carboxyglutamate-rich residues), two epidermal
growth factor domains (EGF1 and EGF2) and serine protease (SP) domain as
shown in Fig. 6.1. Although allosteric modifications imparted by TF on FVIIa are
still under investigation, so far it is known that upon activation of FVIIa from
FVII, a proteolytic cleavage occurs in the single peptide bond between Arg152 and
Ile153(16) that results to the formation of two peptide chains (heavy chain and light
chain) linked covalently by a single disulphide bond. The newly generated
N-terminal Ile153(16) of FVIIa (heavy chain) makes a salt bridge with Asp343(194)
by spontaneous insertion of its N-terminal tail into the cavity (the chymotrypsin
numbering is denoted in superscript with parentheses). It has been reported that
TF-binding promotes N-terminal insertion, which accounts for allosteric regula-
tion of FVIIa [3]. Like FVII, human FX and FIX also belong to same chymotryp-
sin homology family, having the catalytic triad of residues, namely, His(57), Asp(102)
and Ser(195), in the protease domain (heavy chain). FX circulates in blood as zymo-
gen form; upon proteolytic cleavage between Arg194(15) and Ile195(16) by TF-FVIIa
complex, it gets activated to form FXa, an active serine protease with 52-residue
activation peptide release. FIX is also a single- chain protein, in which
6 Role of Tissue Factor-FVIIa Blood Coagulation Initiation Complex in Cancer 103
Fig. 6.1 Structure of TF-FVIIa with membrane lipid bilayer. In the left panel, cartoon diagram of
FVIIa, showing four domains GLA (green), EGF1 (pink), EGF2 (blue) and serine protease (SP)
(cyan) domain. Full length TF is shown in blue colour ribbon representation. The catalytic triad
residues (His57, Ser195 and Asp102) are located in SP domain in TF-FVIIa complex, as shown in
stick representation in the right panel. Bound calcium ions with Gla residues of GLA domain are
shown in red colour ball representation
Till now the impact and contribution of coagulation proteases TF-FVIIa and its
association with various cellular signalling pathways have been well accepted to get
detail picture of this complex as a signal transducer [19–21]. During endothelial
disruption, TF acts as a cofactor for FVIIa and forms TF-FVIIa protease-activated
complex, which initiates various signalling pathways by cleaving and activating
proteinase-activated receptor (PAR) family proteins [22, 23], which are seven trans-
membrane domain cellular receptors that get activated by self-ligation of the proteo-
lytically cleaved end of extracellular amino terminus. TF-FVIIa activates directly
by cleaving after arginine residue of sequence NH2-SSKGRSLIGKV-COOH of
PAR2 as shown in Fig. 6.2. PAR2 is generally present in human epithelial cells of
neuronal, intestinal, airway and vascular tissues. Its expression level gets elevated in
injured tissues or after inflammatory mediator treatment [24–26]. Here exposure of
the tethered ligand that folds back to the second extracellular loop results to activa-
tion of PAR2. Unlike PAR2, PAR1 gets indirectly activated by TF-FVIIa proteolytic
complex through thrombin and FXa, cleaving after arginine residue in the sequence
NH2-LDPRSFLLRN-COOH, as shown in Fig. 6.2. PAR1 is the prototypical recep-
tor of thrombin, but is also activated by several other proteases, such as activated
protein C, MMP-1 (matrix metalloproteinase) and plasmin. Activation of specific
PAR subtypes is cancer dependent, for example, gene expression and their regula-
tion evoked by TF-FVIIa through PAR2 in MDA-MB-231 cells occur in glioblas-
toma cell lines through thrombin-mediated activation of PAR1 [27]. TF-FVIIa,
FXa, trypsin and tryptase activate PAR2, whereas thrombin and plasmin activate
PAR4 [28]. In mouse, PAR3 has been found to serve as a cofactor for PAR4 [29],
but in human it has been reported that PAR3 may also get directly activated by
thrombin [30]. After activation, PARs interact to heterotrimeric G-proteins, which
6 Role of Tissue Factor-FVIIa Blood Coagulation Initiation Complex in Cancer 105
Fig. 6.2 Protein structures of proteinase-activated receptors (PAR1 and PAR2). Amino acid
sequences and the protease cleavage site for receptor activation are shown in figure. Scissors sym-
bol indicate the cleavage sites after arginine residue
leads to initiate further signalling events [28]. PARs expression level is usually
found higher in various cancer types. Several evidences have revealed the intricate
correlation between aggressive behaviour of cancer cells and PAR expression [31–
34]. Ternary coagulation complex (TF-FVIIa-FXa) also cleaves and activates PAR2.
TFPI (tissue factor pathway inhibitor) suppresses PAR2 activation by targeting ter-
nary complex (involving FXa); however, the PAR2 signalling induced by TF-FVIIa
binary complex is not affected by TFPI, because binary complex is independent of
FXa.
Substantial evidences exist that TF plays pivotal role in the development of onco-
genic processes and specifically tumour metastasis [35]. Most human epithelial can-
cers have high levels of TF, and the experimental studies clearly demonstrate that
TF-driven tumour angiogenesis enhances tumour growth and promotes metastasis.
It is important to note that TF could enhance cell migration through activation of
PARs but may have the potential to initiate the migration in protease-independent
manners like binding with integrins, decreasing the interactions with extracellular
matrix by activating MMPs. Investigators reported that TF-dependent migratory
ability of SW620 colorectal cancer cells [36], glioma cells [37] and MDA-MB231
breast cancer cells [35] appears to be dependent on both the proteolytic activity of
FVIIa and the activation of PAR2. Experimentally it is evident that protein kinase C
106 A. Roy et al.
phosphorylates the serine residues of the cytoplasmic tail of TF [38]; however, the
role of the cytoplasmic domain of TF in stimulating signalling and TF-FVIIa asso-
ciation in gene expression is not completely understood yet [39]. It is also reported
that both intra- and extracellular domains of TF are essential for the prometastatic
function of TF [40, 41]. More precisely both TF and FVIIa contribute to tumour
progression through various signalling pathways and play a vital role in regulating
the activity of various oncogenes.
Fig. 6.3 Different protease-mediated factors (TF-FVIIa, FXa and thrombin) can activate various
cellular signalling through the activation of PAR family. Some of the pathways involving PAR2
activation are PI3K/AKT, P42/44 and P38 MAPK which leads to the enhanced transcriptional
activation of various genes (VEGF, UPAR, IL-8, etc.). In addition to this, cytoskeletal alteration
through Rac-1 also contributes to cancer progression. Another receptor PAR1 (activated by throm-
bin) also contributes to tumour progression by increasing various antiapoptotic proteins
and tumour PAR1 significantly decreases the tumour cell metastatic potential.
Analogous observations were made in mouse models that support the PAR1 involve-
ment in melanoma and breast cancer metastases [62]. Surprisingly, it has been men-
tioned that MMP-1 may act as a relevant PAR1 activator in the microenvironment
of cancer cells [63]. Although PAR1 signalling induces a similar series of proteins
in breast cancer cells, the activation of TF-FVIIa/PAR2 axis appears to elicit a more
efficient production of these angiogenesis and immune regulators [27]. In mice har-
bouring a mammary tumour virus promoter-driven polyoma middle T antigen
(PyMT) cassette, PAR2 deficiency resulted in delay of angiogenic switch and a
concomitant reduction in tumour growth [46].
Lan et al. have reported that PAR2-induced cancer cell migration requires miR-
125b involvement by targeting Gab2 and that NSun2- dependent RNA methylation
contributes to the downregulation of miR-125b by PAR2 signalling. This study pro-
poses the involvement of a novel epigenetic mechanism in which the altering
expression of miRNA-125b modulates the cancer cell migratory ability [64]. All
these studies suggest the importance of this binary complex as a key regulator dur-
ing cancer progression. While TF-dependent tumour growth is critically dependent
on PAR2 activation, TF-dependent metastasis is dependent on the formation of
thrombin [65]. TF-mediated PAR2 signalling is reported to be dependent to some
extent on β1-integrins, as PAR2 signalling could be diminished using a β1-integrin-
inhibiting antibody [65].
Yokota et al. reported in a hyperthrombotic mouse model (thrombomodulin defi-
cient) that a thrombin-mediated TF-dependent metastasis is associated with the
hyperactivity of platelets and the formation of platelet leukocyte aggregates [66].
Activated PARs in blood cells, cancer and vessel wall cells by thrombin lead to the
transcriptional activation of many proangiogenic genes such as VEGF and its recep-
tor (VEGFR), MMP-2, basic fibroblast growth factor (bFGF), MAP, TF, angiopoi-
etin-
2 (Ang-2) and PI3 kinases [67–72]. In vitro studies reported that VEGF
secretion from platelets and cancer cells occurs within few minutes of activation
[69]. Furthermore, PAR activation by thrombin induces production of reactive oxy-
gen species (ROS) via elevated expression of hypoxia-induced factor-1 (HIF-1)
[73]. HIF-1 activates the transcription and expression of VEGF gene in response to
fatty acid metabolism (arachidonic acid) [74]. These lines of experimental key find-
ings altogether suggest that the presence of TF and blood coagulation enzymes in
the microenvironment of tumour-associated cells plays a pivotal role in the neoplas-
tic progression, mainly through the stimulation of PAR1 and PAR2 receptors.
Fig. 6.4 Cancer cell-derived microvesicles generation. Cancer cells secreting eMVs containing
proteins, RNAs and miRNAs fuse with the recipient cells to alter its phenotype
Over the past few years, emerging evidences indicate that these MVs act as an
important contributor to various disease progression like atherosclerosis, liver dis-
eases, cardiovascular anomalies, acute and chronic kidney injuries and cancer [87–
90]. Microvesicles are capable of inducing antiapoptotic properties to the recipient
cells [91, 92]. PAR2 activation in hypoxic cancer cells results in microvesicle gen-
eration [93]. Previous reports have already demonstrated the direct involvement of
PAR2 in cancer cell proliferation and migration, although the detail mechanism still
remains unelucidated [94]. PAR activation leads to the generation of extracellular
microvesicles with both procoagulant and pro-cancerous activity.
Experimental and clinical experiments performed in the last 3–4 decades have
established the vivid role of the blood clotting system in supporting tumour progres-
sion and metastasis. Gil et al. have shown that the macrophage recruitment in
tumour microenvironment is mediated by TF-FVIIa-dependent clot formation in
tumour cells [95]. They have further shown that these clot-mediated recruitments of
tumour-associated macrophages (TAMs) are essential for the survival of tumour
cells. These TAMs provide a suitable microenvironment for tumour growth, tumour
survival, motility, tumour cell invasion, intravasation and angiogenesis [96].
Previous reports claim that ablation of TAMs from the tumour mass results in the
inhibition of tumour progression and metastasis. Inflammation mediated by IL-6,
TNF-α, IFN-γ, IL-4, TGF-β, arginase-1, etc. in the tumour mass induces the recruit-
ment of macrophages [97, 98]. In turn these recruited macrophages secrete several
growth factors and cytokines which positively regulate the tumour cell division and
conversion of benign tumour to malignant one. Under the influence of CSF-1, TAMs
promote angiogenesis via production of VEGF [99]. Macrophages are also found to
promote neoangiogenesis in glioblastoma models [100]. Clot formations along with
recruitment of functional macrophages are needed to establish the premetastatic
niche in order to support tumour cell survival. TF expression is higher in cancer
cells and the surrounding stromal macrophages which lead to improved tumour
growth and metastasis. This is due to activation of signalling cascades via coagula-
tion proteases (FVIIa, FXa and thrombin).
The microenvironment of a tumour mass is a complicated heterogenous system
that supports the tumour cells and positively affects the process of cancer progres-
sion and propagation [101]. A number of leukocytes having role in both adaptive as
well as innate immune responses get recruited to the tumour microenvironment in
response to different stimuli. Among these recruited immune cell population, mac-
rophages are the most prevalent and remain present in the tumour vicinity in all
stages of tumour progression [102]. Monocytes are the universal precursors of mac-
rophages. These precursor cells circulate in the blood for immune surveillance of
the entire body. During any tissue damage, a number of various cytokines get
6 Role of Tissue Factor-FVIIa Blood Coagulation Initiation Complex in Cancer 111
Fig. 6.5 Classification of macrophage (M1 and M2) depending on its inflammatory nature. M1
and M2 type of macrophage behave differently in the human body due to differential expression of
different receptors and cytokines
released from the affected cells, and concomitantly a gradient of these chemoattrac-
tant gets established from the affected area to the blood. Sensing these chemicals,
monocytes become activated and cross the endothelial barrier and finally reach to
the affected area where they differentiate into macrophages and contribute to the
wound healing procedure. The morphology of these differentiated macrophages is
very much heterogenous, and moreover they perform diverse type of functions
[103]. These macrophages can be subdivided into two categories: classically acti-
vated or M1 class and alternatively activated or M2 class [104], as shown in Fig. 6.5.
The role of macrophages in the tumour microenvironment is ubiquitous. Being pro-
inflammatory, M1 category of macrophage is a key player in immune system for the
suppression of cancer. On contrary M2 macrophage is anti-inflammatory and proan-
giogenic in nature [105].
As shown in Fig. 6.6, due to TF-FVIIa proteolytic activity-mediated clot forma-
tion, TAMs get aligned along with the blood vessels to promote tumour cell intrava-
sation into the circulation, which is a vital phenotype of malignancy [106]. TAM
secretes epidermal growth factor (EGF) and other ligands of EGF family which
form a paracrine loop by interacting with CSF-1, synthesized by the tumour cell to
support directional tumour cell migration and invasion [107]. Abundant elevated
coagulation factors expression and macrophage infiltration have been observed in
various tumours. It was shown previously that FXIIa and TF-FVIIa treatment of
monocytic cell line THP-1 transform their phenotypic expression to M2-like pheno-
type of interleukin (IL)-4high, IL-10high, tumour necrosis factor (TNF)-αhigh and
112 A. Roy et al.
Fig. 6.6 TAMs-dependent tumour cell intravasation into the circulation. Interaction of tumour
cells and its neighbouring TAMs through TF-FVIIa-mediated blood clot, leads to tumour progres-
sion and metastasis
transforming growth factor (TGF)-βhigh. It has been elucidated recently that TAM
activation by coagulation factors could induce VEGF/MMP-9 expression, which
promotes the invasion of cancer cells. Studies assess that HUVEC cells cocultured
with TAM (PMA-treated THP-1 macrophages cocultured with cancer cells) express
higher levels of FXIIa [108]. In the current scenario, the understanding behind the
metastatic progression of cancer cells by TAM has emerged a focus of attention in
the field of cancer. Lastly, transformation of TAM-like cells by coagulation factors
facilitates cancer cell migration and invasion.
C (APC) also inhibits blood coagulation by neutralizing activated factor V and fac-
tor VIII [113, 114]. In spite of the presence of high amount of protein C, TF-mediated
tumour-induced coagulation activation leads in hypercoagulability, mostly in dis-
persed malignancies.
Cancer patients often suffer from bleeding disorders in advanced malignant
stage, and defect in coagulation is also associated with disseminated intravascular
coagulation deep venous thrombosis (DVT) linked with malignancies like acute
promyelocytic leukaemia and prostate cancer [115]. In thromboembolism, a large
fraction of patients suffering from acute myelogenous leukaemia; renal, ovarian,
pancreatic, gastric and lung cancer; and non-Hodgkin lymphoma also show
enhanced level of thrombotic complications. As a consequence of abnormal blood
coagulation, these patients have high risk of cerebrovascular disease, peripheral
arterial/venous thrombosis and acute coronary syndrome-associated haemorrhagic
complications. Another major consequence of abnormal coagulation disorder is dis-
seminated intravascular coagulation (DIC), which is responsible for many malig-
nant diseases in acute form. Cancer cells release high level of coagulation stimulatory
factors like VEGF which activates the process of coagulation in damaged cancer
blood vessels and surrounding tissues with concordant production of fibrinogen and
fibrin degradation products. This local consumption of platelets and fibrinogen in
fast-growing tumours lead to the systemic fibrinogen and platelet deficiency [116].
6.7 Conclusion
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Metalloproteases in Adaptative Cell
Responses 7
Pavel Montes de Oca Balderas
Abstract
Throughout evolution, cells have acquired molecular mechanisms that enable
them to interact with and adapt to the extracellular milieu. In the last decade,
ectodomain shedding (ES) has emerged as a critical sensing mechanism of the
environment that may remodel cell membrane molecular repertoire, eliciting
dynamic intracellular responses. ES is the proteolytic release of the extracellular
domain (ectodomain) from cell membrane molecules (CMM). This proteolysis is
mediated mainly by matrix metalloproteases (MMPs) or disintegrin and metal-
loproteases (ADAMs). Virtually, all functional categories of CMM are subject of
this proteolysis; therefore, ES is involved in most cellular processes including
proliferation, apoptosis, migration, and differentiation or pathologies such as
inflammation or cancer. ES releases membrane molecule’s ectodomain to the
extracellular space where it can play biological functions. ES of transmembrane
molecules also generates membrane-attached terminal fragments comprising
transmembrane and intracellular domains. These fragments may be further pro-
cessed by intramembrane-cleaving proteases (i-CLiPs), a mechanism known as
regulated intramembrane proteolysis (RIP), which releases molecule’s intracel-
lular domain (ICD). Contrary to the initial hypothesis, fragments that result from
ES and/or RIP are not necessarily in the pathway of degradation. Instead, they
may carry out specific functions that cannot be performed by full-length native
molecules. Thus, ES has emerged as a switch that unmasks multifunctional activ-
ities of CMM. In this chapter, the general mechanism of ES is reviewed, and
Keywords
Metalloproteases • MMP • ADAM • “Ectodomain shedding” • Signaling • RIP •
i-CLiPs • Transmembrane molecules
7.1 Introduction
The cell membrane has played a fundamental role in evolution because it is the
structure that maintains vital differences between the extracellular space and the
cytosol. However, this isolating role must also permit information exchange between
outside and inside of the cell. The flow of information at membrane level is itself
also critical for cell survival and its adaptation to the extracellular environment. This
function is carried out mainly by cell membrane molecules (CMM) that sense extra-
cellular space information and transduce it into intracellular signals that enable
dynamic adaptative cell responses to its environment. The messengers, receptors,
and intracellular transducers that mediate cell signaling have been the subject of
study through decades in cell biology. The work on this subject has resulted in the
identification and modus operandi of cell membrane receptors that belong to differ-
ent families, including the G protein-coupled receptors, the tyrosine kinase family
of receptors, or the tyrosine phosphatase family of receptors, to name a few. This
knowledge has even been translated into specific therapies for certain diseases.
However, and despite the advances in the field, the mechanisms that mediate cell
responses to the environment are far from being fully understood. One of the rea-
sons for this is the nonlinear, network-like complexity that cell signaling presents.
In this regard, the mechanism of ectodomain shedding (ES) has reached the arena of
cell signaling as an unexpected player that may help to understand the complexity
of this cell function.
Since the 1970s, CMM shedding was considered a mechanism that mediates
membrane molecule turnover, accounting it as an initial step in a degradative path-
way that concomitantly enables molecular release from cell membrane [1]. By then,
this mechanism was known to occur at higher rates in transformed, activated, or
proliferating cells in comparison with resting cells [1]. Shedding was known to be
an active metabolic process requiring respiration, protein synthesis, and energy,
although the responsible molecular mechanism was unknown [1]. Proteases that
mediate ES or sheddases started to be unmasked by the middle of the 1990s; since
then its number has augmented considerably, but more importantly, the number of
molecules that undergo ES in the cell has been growing incessantly [2–6]. Nowadays,
taking into account that all sort of CMM are subject to ES, it is considered that vir-
tually any CMM may undergo this proteolytic mechanism under certain circum-
stances. This assumption poses new challenges for the study of cell signaling and
7 Metalloproteases in Adaptative Cell Responses 123
cell responses since sheddases are part of a web with high redundancy (one CMM
may be cleaved by different sheddases) and promiscuity (one sheddase may cleave
a large portfolio of CMM). Furthermore, since ES may yield functional molecular
fragments capable of cell signaling, not only from receptors but from all sort of
CMM, this mechanism has called for a reconsideration of the paradigms of cell
signaling [7–9].
Fig. 7.1 General scheme of ectodomain shedding (ES) and regulated intramembrane prote-
olysis (RIP), players, and their regulators. Different classes of transmembrane molecules undergo
ES releasing their ectodomain to the extracellular milieu. This proteolytic cleavage is mediated by
sheddases that are soluble or attached to the cell membrane by a transmembrane domain or other
anchoring. Sheddases’ cleavage site (arrow A) is mainly located in the so-called stem region, <30
amino acids upstream the transmembrane domain. ES may be constitutive or induced by ligand
binding, PKC activation, Ca++ dynamics, reactive oxygen species (ROS), pH, and bacterial toxins or
inhibited by TIMPs. Target molecule posttranslational modifications such as ectodomain phosphor-
ylation (P) or glycosylation, as well as its structural conformation, modulate its ES. Lipid rafts, actin
dynamics, protein tyrosine kinases (PTK), and GTPase activity are also known to modulate
ES. Target molecule ES generates NTF and CTF that remain associated to cell membrane that may
further be subject to RIP mediated by an intramembrane-cleaving protease (i-CLiP). These prote-
ases cleave within the transmembrane domain or few amino acids downstream it (arrow B). This
second sequential proteolytic processing is regulated by intracellular posttranslational modifications
such as ubiquitination (Ub), palmitoylation, or phosphorylation (P). RIP releases target molecule
intracellular domain from cell membrane. Target molecule fragments generated by ES or RIP are
known to carry out extracellular, intracellular, or intranuclear signaling functions. Some reports
have demonstrated that multipass transmembrane molecules (MuPass) may also be sheddase or
i-CLiP targets. See text for details and references (Modified from Ref. [4])
7 Metalloproteases in Adaptative Cell Responses 125
(TIMPs), four related proteins expressed by a wide array of cells [29]. In addition,
it has been demonstrated that ADAM-17 transmembrane domain regulates its target
specificity that can also be regulated by additional associated molecules or other
proteases [30–32]. On the other hand, ADAM-10 intracellular domain (ICD) has
been found to mediate its dimerization proposed to regulate its activity [33, 34] and
control its constitutive activity [31]. Together, these regulatory mechanisms allow
the spatiotemporal regulation of these enzymes and their activities. For more details
on the expression, regulation, and activation of these proteases, please refer to the
selected reviews and references therein [6, 11, 23, 24].
Although there is some consensus about the sequence specificity that some
MMPs recognize, mainly a Pro residue at P3 position [35–37], there does not seem
to be a sequence conservation in ADAM or MMP cleavage sites. In the same study
by Turk et al. (2001), it was shown that MMPs have different amino acid prefer-
ences near P3 position. Nevertheless, in most cases of ES, cleavage occurs within
transmembrane molecule stem region, that is, <30 amino acids upstream the trans-
membrane domain, a feature that has been considered more important than sequence
itself [10, 38]. Sheddase loose sequence specificity provides their high promiscuity
and redundancy and has some important implications for cell physiology (discussed
below) [10]. It is credited that CMM are preferentially cleaved by a particular shed-
dase; however, it is well known that the same target molecule may be cleaved with
different efficiencies by alternative sheddases [2, 3, 6]. This may become experi-
mentally evident when the main associated sheddase is absent in a cell or may
depend upon the cell model, tissue analyzed, or stimulation paradigm employed.
The high promiscuity and diversity of sheddases, as well as their loose sequence
specificity shared with the intramembrane-cleaving proteases (i-CLiPs) (see below),
may be the evolutionary result of the fundamental role played by this proteolytic
system in extracellular information sensing that conveys signals into the cell. The
phylogenetic conservation from prokaryotic cells supports the notion of its funda-
mental role [39, 40].
ES may occur in a constitutive ligand-independent manner as has been described
for γ-protocadherins, TrkA, interleukin-2 receptor beta (IL-2Rβ), or others [41–43]
or in a ligand binding-dependent manner as described for Notch, ephrinB2, or oth-
ers (Fig. 7.1) [44, 45]. As mentioned above, for some time it was believed that ES
downregulated CMM function, since it was considered that it depended fully on the
ectodomain [10]. Therefore, it was assumed that the remaining fragment attached to
the cell membrane comprising the transmembrane and intracellular domains was
nonfunctional; thus, ES was believed to be the initial step of a degradation pathway
for molecular turnover [1]. However, studies on this proteolytic processing have
demonstrated that this is not necessarily the case, since it has been found that ES
enables extracellular (EC) and intracellular (IC) fragments to carry out additional
functions beyond those of full-length native molecules, revealing their multifunc-
tional nature [2, 4, 6]. Membrane-attached intracellular terminal fragments may be
carboxy-terminal (CTF) or N-terminal (NTF) depending if the native molecule is a
type I (with extracellular N-terminal) or type II (with intracellular N-terminal)
transmembrane molecule. The activities of these fragments depend upon their IC
126 P. Montes de Oca Balderas
On the other hand, nowadays it is accepted that ES is a prerequisite and the main
regulator for RIP, although some exceptions have been reported and rhomboid ser-
ine proteases are known to perform RIP without previous ES [46–48]. Similarly to
ES, target molecule posttranslational modifications beyond proteolysis such as
phosphorylation [44], palmitoylation [80], glycosylation [81], and ubiquitination
[82] have been reported to regulate RIP (Fig. 7.1). Likewise, there is loose sequence
specificity in cleavage site of those i-CLiPs analyzed, although it is commonly
located within the transmembrane domain or few amino acids downstream where
the only common motif are basic and hydrophobic amino acids [46, 47, 83]. Also, it
has been found that helix destabilization at cleavage site facilitates i-CLiP’s activity
[46, 47, 83]. In the case of PS, their target molecules are supposed to require short
(<30 amino acids) extracellular N-terminus tails generated after ES, apparently rec-
ognized by Nicastrin (also member of the γ-sec complex), although this is still dis-
puted [46, 49, 50, 84, 85].
Cellular compartments where ES occurs have been investigated with different
target molecules and include the pathway from the Golgi apparatus to the cell mem-
brane and endosomes [2, 3, 6, 10, 86, 87]. In agreement with the accepted temporal
sequence that exists between ES and RIP, the last has been reported to occur in the
pathway from cell membrane to endosomes, and indeed the substrate traffic has
been found to regulate its RIP [46, 47, 87, 88]. In addition, a role of lipid rafts as
regulators or platforms where ES and RIP occur has been reported [46, 87, 89–91].
Also, ES has been found to occur in exosomes, where MPs have been found and are
proposed to participate in the production of exosomes and in cell target activation
through ES among other functions [92–94].
After examination of ES and RIP reports, a general scheme that includes recent
findings of transmembrane molecule intracellular fate may be delineated (Fig. 7.2).
In this scheme a considerable number of a given type of transmembrane molecule
undergo ES in a constitutive or stimulated manner. From the total number of NTF
or CTFs generated, some may carry out different intracellular functions including
intracytoplasmic signaling [43] or intranuclear transcriptional regulation by a ret-
rograde membrane trafficking pathway [57]. Concomitantly, or perhaps alterna-
tively, NTFs or CTFs may follow the lysosomal pathway where they are degraded
[43] or enter the exosome pathway through multivesicular bodies [92–94], whereas
only a minor fraction undergoes RIP releasing their ICDs [55]. From endosomes,
some transmembrane molecules may be recycled back to the cell membrane by
recycling endosomes [95]. Seemingly, a large population of ICDs may follow the
proteosomal pathway or other alternative undescribed degradative pathway [51,
96]. ICDs may directly regulate signal transduction pathways either by associating
with kinases or regulating its RIP [44]. Ultimately, only a very small fraction of
ICDs is translocated into the nucleus where it is involved in gene transcriptional
regulation [48, 51]. Accordingly to the idea proposed by Anders et al., ICD’s short
life and low abundance reflect their important regulatory roles, as well as the low
requirement of these fragments to fulfill their intracellular and intranuclear signal-
ing functions [55].
128 P. Montes de Oca Balderas
Fig. 7.2 Transmembrane molecule intracellular fate after ES and/or RIP. A large diversity of
transmembrane molecules undergo ES and RIP in the cell membrane, although there are some
reports that indicate sheddase activity in the pathway from Golgi to cell membrane (arrow A). In
the cell membrane, these molecules are endocytosed and trafficked into the endosomal pathway
that is known to regulate RIP (arrow B). NTF and CTF generated by ES, as well as ICD generated
by RIP, may participate in cytoplasmic intracellular signaling by regulating i-CLiP (arrow B΄) or
kinase function (arrow B˝), directly as kinases or phosphatases or regulating molecular localiza-
tion. Concomitantly or perhaps alternatively, NTFs and CTFs may follow the multivesicular body
pathway (arrow C) and then degraded in the lysosomal pathway (arrow D) or secreted (arrow E)
in exosomes (Ex). From endosomes, some transmembrane molecules may be recycled back to the
7 Metalloproteases in Adaptative Cell Responses 129
As mentioned above, the spectra of CMM that undergo ES are wide and include all
sort of functional molecules. Receptors, growth factors, cytokines, receptor ligands,
cell adhesion molecules, and ion channels include members that have been reported
to undergo ES. Either with transmembrane domains or other membrane anchoring,
sheddases release extracellular soluble fragments from these molecules that in some
cases have been reported to carry out functions associated with signal regulation in
the EC milieu [4, 10]. Nevertheless, it must be considered that not all extracellular
fragments are released to the EC milieu, despite they may lack transmembrane
domain after processing. For instance, NMDAR subunit GluN1 extracellular frag-
ment generated by MMP’s activity was not found in the culture supernatant [97].
This observation suggests that after generation, it undergoes other fate, perhaps
remaining attached to the extracellular matrix or cell membrane through some
molecular interaction, or even internalized and degraded [98].
Despite the full number of molecules that undergo ER has not been evaluated, in
a 2010 review [4] of the molecules expressed in the central nervous system (CNS)
that are subject to ES and/or RIP (including only those with transmembrane
domains), 110 molecules were found as target of ES. Of these, 9 were type II mol-
ecules, 93 were type I molecules, and only 2 were molecules with more than one
transmembrane domain or multipass. These molecules belonged to different func-
tional categories: 48 receptors, 21 receptor ligands, 25 involved in adhesion, 4 with
adhesion-receptor function, 4 proteases, 2 involved in antigen presenting, 2 channel
subunits, 1 with channel-adhesion function, 1 enzyme, and 2 without specific func-
tion described. In the receptor group, 11 belonged to the tyrosine protease kinase
family, 3 to the protein tyrosine phosphatase family, 1 G protein-coupled receptor
(GPCR), and 1 with guanylate cyclase activity. Despite these numbers are not
updated, since new molecules have been discovered to undergo ER or other mole-
cules have been found expressed in the CNS, the examples above are useful to
substantiate the large diversity of molecules that are subject to ER, despite their
function and biochemical or physicochemical properties.
Fig. 7.2 (continued) cell membrane by recycling endosomes (arrow G) or by a retrograde mem-
brane trafficking pathway (arrow F), targeted to the endoplasmic reticulum (ER, arrow F΄) from
where they reach the nuclear envelope inner membrane (arrow F˝) and participate in gene tran-
scription regulation in association with transcription factors (TF). A minor fraction of RIP-
generated ICDs is translocated into the nucleus through nuclear pores (arrow H) and carries out
transcriptional regulatory functions (arrow I), whereas the major fraction follows the proteasomal
pathway and is degraded (arrow J). Some work has suggested that transmembrane molecules may
be cleaved without prior ES by presenilin or cleaved by ES-independent i-CLiPs (K) (See text for
details and references. Modified from [4])
130 P. Montes de Oca Balderas
In a more recent review [16], the authors counted 182 molecules as MMP’s tar-
gets. Nevertheless, this list included only 32 molecules reported as shedded
membrane-bound substrates that included receptors, receptor ligands, adhesion
molecules, and a plethora of molecules. Other categories of MMP’s target mole-
cules in this review included extracellular matrix molecules (38), soluble growth
factors and cytokines (12), cryptic factors (29), chemokines and cytokines (18),
immunity molecules (9), blood molecules (9), proteases and inhibitors (15), intra-
cellular proteins (13), and multitask or multilocated molecules (7).
Notably, in both reviews just a few multipass transmembrane molecules were
found to undergo ES, and none multipass transmembrane molecule has been
reported to undergo both ES and RIP. Indeed, multipass transmembrane molecules
ES and RIP are still under debate, despite some reports support this notion [6, 50].
These molecules are interesting in terms of ES and RIP because their processing
would implicate additional complexity not observed with single-pass transmem-
brane domains, since these molecules could yield a larger diversity of fragments.
This diversity and additional complexity would result from fragments with trans-
membrane domains with IC and EC loops, thus conferring the resulting fragments
with biochemical and biophysical properties that could result in specific functional
features different from those generated from single-pass transmembrane molecules.
In this regard, ES has been demonstrated for GluN1 (NR1) subunit of NMDAR that
is subject to activity-mediated ES in two different extracellular loops [97]. This
subunit is also target of ES by an exogenous MMP that regulates its function [99].
Moreover, in our recent reports, we have observed the generation of GluN1 frag-
ments that included different transmembrane domains and loops. Notably, in cul-
tured astrocytes this correlates with a metabotropic-like flux-independent NMDAR,
although causality was not established [100, 101]. In addition, GPCR PAR1 is a
MMP-1 target that generates Ca2+-dependent signals in breast cancer cells [102].
Also, GPCR brain angiogenesis inhibitor 1 (BAI1) has been reported to undergo ES
generating the anti-angiogenic molecule vasculostatin [103]. More recently, GPCR’s
GPR37, GPR124, and GPR56 have also been reported as targets of ES [19, 104,
105]. In addition, in recent proteomic studies, other multipass membrane molecules
have been identified as ES targets including GPCR, ion channel subunits, transport-
ers, and others [106, 107]. On the other hand, GluR3 subunit from AMPAR is a
multipass transmembrane molecule that apparently undergoes RIP independently of
ES [108], whereas CXCR4 has also been reported to undergo γ-sec cleavage [50].
MPs have been found in the cytosol or intracellular organelles such as the
nucleus, where their functions have been poorly studied and in some cases have
been related to apoptosis [2, 16, 20–22]. Likewise, it has been reported that some
nontraditional intracellular molecules are also targets of extracellular MPs, mainly
metabolic enzymes, cytoskeletal proteins, or chaperones. Despite it has been argued
that the extracellular presence of intracellular molecules is an artifact due to cell
lysis, independent degradomic approaches have identified this kind of molecules as
MMP’s targets [2, 16, 20]. The review of these non-common MP’s targets and loca-
tions is not the objective of the present chapter. For further information regarding
these topics, please refer to the reviews above cited and references therein.
7 Metalloproteases in Adaptative Cell Responses 131
Fig. 7.3 The putative role of molecular bundles or ensembles processed by ES and/or RIP
and their NTFs, CTFs, and ICDs in cell signaling. (a) Under basal conditions, bundles or
ensembles of CMM in full-length form are organized in cell membrane regions such as lipid rafts
(thick black line in the membrane). In these conditions, MPs and i-CLiPs are inactive, and ES/RIP
does not occur or occurs in low levels. Under these conditions, cytoplasmic Ca2+ is also low and
PKC is not activated. (b) After ligand binding to its receptor (arrow panel A) or some unspecific
stimulus that rise cytoplasmic Ca2+ activates PKC and/or initiates actin dynamics (black arrow on
the left panel B), MPs and i-CLiPs are activated (black arrow on the right panel B) and ES and RIP
are initiated. Then bundles or ensembles of CMM are processed, generating CTFs, NTFs, and
ICDs from them. The specific processing of these CMM depends upon their identity but also from
the MPs and i-CLiPs expressed by the cell, helping to carve the specific intracellular response to a
particular stimulus. Beyond the canonical IC transduction pathways (TP)(open arrow panel B)
associated with a particular receptor (JAK/STAT, NFκB, etc.), the fragments generated by ES and/
or RIP play a role in IC signaling, as explained in the text and Fig. 7.2. Together, IC TP and
molecular fragments generated from CMM by ES and/or RIP and their IC activities induce the
adaptative intracellular response to a given stimuli or condition that includes, for example, gene
regulation within the nucleus (See text for details and references)
7 Metalloproteases in Adaptative Cell Responses 133
moving in the membrane bilayer. Instead, molecules are grouped together, and their
movement is constrained by the interactions among them or by membrane biophysi-
cal properties that are given, for instance, by lipid rafts [111]. Moreover, this
arrangement into supramolecular entities seems to be a common strategy in the cell
that optimizes molecular interactions and therefore cellular responses [112–115].
Taking this conjecture further, the specific MPs and CMM expressed by each cell
would then suppose a unique supramolecular signaling entity of proteases, recep-
tors, their fragments, and activated signal transducers that would elicit specific
intracellular responses (Fig. 7.3). This conjecture could help to understand the old
fundamental question of cell biology regarding how different receptor-mediated
intracellular cascades are able to generate specific cellular responses, despite the
intracellular components are the same [114]. In addition, this may further account
for some of the variability, diversity, and redundancy observed in cell biology mod-
els; however, experiments are needed to test this idea.
Interestingly, complexity of this model would be increased because cleavage of
a given target molecule by more than one sheddase would open the possibility that
different cleavage sites are recognized and consequently that resulting fragments
generated have different N- or C- terminal sequences. These terminal sequence dif-
ferences may yield important distinct results, as they occur with α- and β-secretase
cleavage sites in amyloid precursor protein (APP) that differ by only two amino
acids. This generates extracellular fragments with different biochemical character-
istics that in one case result in pathological features. With this panorama, it is pos-
sible to conceive that this phenomenon may also occur with other shedded targets.
This may also occur with intracellular fragments, as demonstrated by ADAM-10-
CTFs generated by ADAM-9 or ADAM-15, which showed different intracellular
traffics, thus suggesting different intracellular functions [56]. Similarly, complexity
may still be increased since MP’s biochemical properties modify their function. In
this regard, it has been reported that the expression of membrane-tethered ADAM-9
or a soluble spliced variant has opposing functions in cancer cell migration [116].
At the same time, my unpublished observations suggest that intercellular contacts
cast additional intricacy to the ES mechanism that could be related to cell density
sensing, among other functions.
Thus, considering these observations, despite redundancy and promiscuity in
MP’s web, the set of sheddases expressed by a cell would be critical since shedded
molecules and resulting fragments size would vary accordingly (Fig. 7.3). This
would finally result into a specific response to a stimulus and/or localization and
fate of the molecular cluster and fragments. Moreover, involvement of different
sheddases in target molecule ES that depends upon cell type, tissue, or stimulus
analyzed together with their specific glycosylation pattern indicates that despite
their promiscuity and redundancy, sheddases display cell-type-specific specificity
as hypothesized by Arribas and Borroto [10]. It is possible that this cell-type-specific
specificity may provide the basis for the cell distinctive activation of intracellular
pathways that results in cell specialization or differentiation. This idea is further
supported by the spatial and temporal control in the expression of the sheddase, the
substrate, and the regulatory factors [38]. Moreover, consistently with this idea, ES
134 P. Montes de Oca Balderas
enters the cell nucleus through nuclear pores under particular cell circumstances
(present in 30% of cells in the experiments described above). Therefore, it seems
that nuclear entrance of proteins below 40 kDa is mediated by an unspecific-
regulated mechanism rather than a free diffusion mechanism. However, more exper-
iments are required to explain these observations and test this possibility. Strikingly,
Pcdh-γA3-ICD that is <200 aa (≈25 kDa) has a bipartite nuclear localization motif
[96]. An ICD of such size would be expected to enter the nucleus freely and would
not require a nuclear localization motif, unless it does so as part of a molecular
complex that impedes such free entrance. Interestingly, when ICD size of the mol-
ecules that potentially undergo ES and RIP is analyzed, most of them are under the
40 kDa cutoff [4], opening the possibility that these fragments may freely enter the
nucleus either by free diffusion or the putative unspecific-regulated mechanism.
7.5 Conclusions
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Proteases from Protozoa and Their Role
in Infection 8
Anupama Ghosh and Sanghamitra Raha
Abstract
One of the major classes of virulence factors acting in different host-pathogen
interaction systems is comprised of proteases. Pathogen-secreted or membrane-
associated proteases could be found to participate in different stages of establish-
ment of infection. They are explored as candidate drug targets due to their key
participation in the disease development process carried out by the pathogen. In
this chapter we present an extensive review of the proteases of different proto-
zoan parasites. Throughout the article we have made an effort to provide a com-
prehensive list of different proteases from various parasitic protozoa that have
been demonstrated to execute major functions in the respective infection pro-
cesses. Attempts have also been made to present their mode of action with respect
to host invasion and disease development.
Keywords
Protozoa • Parasite • Protease
A. Ghosh (*)
Division of Plant Biology, Bose Institute, Centenary Campus, Kolkata 700054, India
e-mail: ghosh.anupama@jcbose.ac.in; ghosh.anupama1982@gmail.com
S. Raha (*)
Integrated Science Education and Research Center and Department of Biotechnology,
Visva Bharati University, Shantiniketan 731235, India
e-mail: srr1987@gmail.com
8.1 Introduction
Fig. 8.1 Schematic representation of different roles played by protozoan proteases in para-
site pathogenesis. Protozoan proteases play varied roles during host invasion by the parasite.
Included among these is the degradation of the host extracellular matrix that aid in the passage of
the parasite to the host cell surface. Besides, protozoan proteases also participate actively in the
degradation of host immune molecules like immunoglobulins, thereby enabling the pathogen to
evade host immune responses. Other activities of the protozoan protease involve cytolysis and
phagocytosis of target cells
8 Proteases from Protozoa and Their Role in Infection 145
Parasitic protozoan cysteine proteases have been found to be involved in all the
major steps of disease establishment starting from invasion of host cells till immune
evasion [1–3]. It is due to their key role in virulence of the associated pathogen that
in some cases cysteine proteases have been investigated elaborately for their poten-
tial to become promising drug targets [4].
Among the best characterized cysteine proteases of the malaria parasite Plasmodium
are the family C1 (papain family) of clan CA cysteine proteases. Genome sequence
analysis of Plasmodium has revealed that the parasite harbours many members of
this particular family of cysteine proteases that include falcipains, dipetidyl pepti-
dases, proteins related to serine-rich antisera (SERA) and a calpain homologue [5].
There are four falcipain genes within the genome of the parasite, among which
the proteins encoded by falcipain2 and falcipain3 have been found to catalyse
hydrolysis of native haemoglobin and denatured globin [6]. Genetic studies involv-
ing RNAi-mediated downregulation of both falcipain1 and falcipain2 genes in
Plasmodium falciparum showed inhibited development of erythrocytic parasites
[7]. Haemoglobin hydrolysis is an important part of Plasmodium survival within the
bloodstream. During rapid asexual multiplication of the parasite within the erythro-
cyte, it takes up the erythrocyte cytosol to an acidic food vacuole through a special-
ized organelle called cytostome. It is within this acidic vacuole that the haemoglobin
is degraded [8]. Falcipains have a very intricate domain architecture that enables
them to specifically target erythrocyte haemoglobins for hydrolysis within the acidic
food vacuole. The primary structure of each of the four falcipains can be divided
into two domains, the N-terminal prodomain and the C-terminal mature domain.
While the N-terminal prodomain possesses a trafficking subdomain that guides the
localization of the protease to the acidic food vacuole, the C-terminal mature domain
comprises the haemoglobin-binding subdomain needed for substrate recognition
[9]. Due to their participation in one of the vital functions of plasmodium blood
stage cells that is absolutely essential for the survival of the pathogen within host
erythrocyte, falcipains are treated as promising candidates for drug targets against
malaria [10].
Among the other members of the clan CA cysteine proteases, besides falcipains,
dipeptidyl aminopeptidases (DPAP) are the ones that also catalyse haemoglobin
hydrolysis within the food vacuoles of host erythrocytes [11, 12]. Plasmodium
genome codes for three dpap genes. DPAP1 has been shown to be absolutely essen-
tial for the survival of the pathogen since viable forms of DPAP1 deletion strains
could never be generated by applying different genetic approaches [12]. However
DPAP2 deletion studies involving the effect of DPAP1 and DPAP2 inhibitors
ML4118S in both the human parasite P. falciparum and rodent parasite P. berghei
indicated an additional role of Plasmodium dipeptidyl aminopeptidases in the
146 A. Ghosh and S. Raha
transmission of the disease [13]. Besides, a forward chemical genetic study involv-
ing a library of 1200 covalent serine and cysteine protease inhibitors revealed a
possible role of DPAPs in the erythrocyte rupture by the parasite in coordination
with a subtilisin family serine protease PfSUB1 [14]. All these studies indicate a
vital contribution of Plasmodium DPAPs in different stages of the pathogenic devel-
opment of the malaria parasite. Suitable drugs that could target these aminopepti-
dases therefore pose great promise for controlling the spread of the disease. Another
very important target for drug development against malaria could be the only cal-
pain homologue of Plasmodium sp. Regulated knockdown studies of the respective
gene in Plasmodium falciparum revealed its necessity in the cell cycle progression
of the pathogen to maintain a proper disease cycle [15].
Serine-rich antigen (SERA) family of cysteine-like proteases is another group of
Plasmodium proteases with possible roles in maintaining the disease cycle of the
pathogen. Plasmodium genome codes for 9 SERA genes [16, 17], among which
several codes for proteins that contain an atypical serine residue at the active site
instead of a canonical cysteine residue. Studies have shown that SERA family mem-
bers act as substrates of PfSUB1 [18] and possibly play a role in PfSUB1 induced
release of Plasmodium cells from the erythrocytes (egress). Attempts to generate
SERA 1, SERA 4, SERA 5 and SERA 9 deletion strains of Plasmodium falciparum
revealed the lethal nature of ∆SERA 5 strains indicating an important blood stage
function of this particular SERA protein [19]. Moreover a twofold increase in
expression of SERA 5 in SERA 4 null strains is in support of the common notion of
functional redundancy among different members of SERA family of proteins.
Serine proteases are among the most abundant group of proteases in different organ-
isms. This class of proteases has gained tremendous functional diversity during the
course of evolution [46, 47]. Among the many biological functions exhibited by
parasite serine proteases, involvement in defining the pathogenicity of concerned
protozoan parasites is one. Serine proteases are found to play a significant role in the
pathogenicity of several parasitic protozoans including the genera Plasmodium and
Entamoeba.
8 Proteases from Protozoa and Their Role in Infection 149
Plasmodium spp. are not the only members of apicomplexan parasites that exhibit
ample use of serine proteases in their virulence mechanisms. Similarly, significant
contribution of serine proteases could be noticed in the molecular mechanisms of
host cell invasion by Toxoplasma gondii. Being an obligate intracellular pathogen,
T. gondii spends its entire lifecycle within a specialized parasitophorous vacuole
formed in the cytoplasm of infected cells [64]. During its survival within the vacu-
ole, the parasite produces many subtilisin-like serine proteases which play impor-
tant roles in the establishment of infection by the pathogen like TgSUB1. TgSUB1
is processed within the secretory pathway of the parasite and finally secreted as
smaller products by the microneme [65]. Secreted and processed TgSUB1 then par-
ticipate in the processing of various micronemal proteins, thereby regulating adhe-
sive properties of different cell surface macromolecular complexes that are involved
in host cell invasion [66]. Besides TgSUB1, T. gondii also possess other subtilisin-
like serine proteases like TgSUB2. A genetic approach to study the function of
TgSUB2 revealed its indispensible nature with respect to pathogen survival. The
protein undergoes autocatalytic processing during its passage through the pathogen
secretory pathway and localizes to rhoptries where it associates with ROP1. TgSUB2
therefore functions as a rhoptry protein maturase [67]. Among serine proteases
other than subtilisin-like family, rhomboid proteases of T. gondii also contribute
significantly to the host cell invasion by the pathogen. Rhomboid proteases are
intramembrane serine proteases. T. gondii genome codes for five nonmitochondrial
rhomboid proteases. However the expression of each of these rhomboid proteases is
dependent on the morphological form of the parasite. While TgROM1, TgROM4
8 Proteases from Protozoa and Their Role in Infection 151
and TgROM5 are expressed in the tachyzoite stage which is responsible for disease
development by the parasite, TgROM2 and TgROM3 are expressed in the oocyst
stage that is involved in transmission of the pathogen [68]. The key protease activity
necessary for host cell invasion by T. gondii is provided by TgROM5 that catalyses
the cleavage of MIC2 cell surface adhesin [68]. Although majority of the serine
proteases from T. gondii are needed during host cell invasion which is the primary
step of establishing infection, there are instances available in the literature that
shows important roles of these proteases in the intracellular survival of the pathogen
as well. For example, genetic studies revealed key functions of TgROM1 in the
intracellular growth of T. gondii [69]. Besides, TgROM4 has been demonstrated to
maintain the normal apical-posterior gradient of T. gondii cell surface adhesins
which is a prerequisite for efficient cell motility and successful host cell invasion by
the pathogen [70]. However despite their individual roles in adhesin cleavage, host
cell invasion and regulation of intracellular growth of T. gondii, none of the rhom-
boid proteases are indispensible for the pathogen lifecycle [71]. This gives us a
glimpse of the redundant pathways maintained by the pathogen comprising differ-
ent classes of proteases to ensure host invasion by the pathogen and its subsequent
survival within the host environment.
activity further adds on to the list of serine proteases essential for the survival of the
parasite. Genetic deletion strains of T. brucei for the abovementioned genes showed
defective growth both in vivo and in vitro [77].
One of the best characterized and explored protozoan aspartyl proteases playing
significant role in pathogen virulence includes plasmepsins from Plasmodium sp.
Primarily, the protease aids haemoglobin degradation within the parasite food vacu-
ole. The resulting degradation products that include mainly amino acids serve as
both nutrient and energy sources for the survival of the pathogen within intra-
erythrocytic environment [80]. Haemoglobin degradation function for plasmodial
plasmepsins however has been successfully assigned for only four of the total ten
members, namely, plasmepsins I, II, and IV and histoaspartic protease. Among
them a detailed study on the mechanism of trafficking of cytosolic plasmepsin II to
the acidic food vacuole of the parasite has been done. This particular study revealed
an initial transport of the protein through the secretory system to the cytostomal
vacuole. Within the cytostomal vacuole, the protease binds to its substrate haemo-
globin from where it is carried to the food vacuole where the actual degradation of
the haemoglobin takes place [81]. Moreover when it comes to the rest of the six
plasmepsins, the functions are mostly uncharacterized. Nevertheless a very unique
function could be demonstrated for one of the members, plasmepsin V by Goldberg
8 Proteases from Protozoa and Their Role in Infection 153
DE and colleagues [82]. According to their study, plasmepsin V plays a key role in
the cleavage of the PEXEL motif within the Plasmodium exported proteins while
they are still present within the parasite endoplasmic reticulum. PEXEL is an essen-
tial signature motif for plasmodial exported proteins [83]. These PEXEL proteins
thus processed are then exported to the erythrocyte cytoplasm through an ATP-
driven translocon channel. Plasmodium exportome thus translocated and deposited
to erythrocyte cytosol plays a key role in suitable orchestration of the host environ-
ment for subsequent survival of the pathogen. Anything interfering in the normal
protein export process of the parasite therefore might alter the virulence of the
organism. Plasmepsin V hence can be considered to play a significant contribution
towards the pathogenicity of Plasmodium falciparum.
It is due to their very important roles in the disease development process of the para-
sitic protozoans that the proteases could be targeted for chemotherapy of the con-
cerned disease. There are several instances available in the literature where one or
more of the key virulence-related proteases of the parasite are targeted to control the
respective disease. One such example of drug development against parasite prote-
ases dates back to 1996. During this period Erickson JW and his group designed a
series of low molecular weight compounds with potential to inhibit a key serine
protease of Plasmodium plasmepsin I that is involved in haemoglobin degradation
[84]. Further studies in this direction led to the discovery of an adaptive inhibitor for
the entire plasmepsin group of serine proteases of Plasmodium. Adaptive inhibitors
in general are targeted to and are specific to one of the members of a family of pro-
teins but have the flexibility to inhibit other members as well although with less
efficiency. This particular adaptive inhibitor against Plasmodium plasmepsin family
is targeted to plasmepsin II but can also inhibit plasmepsins IV and I and HAP with
almost equal efficiency [85]. Likewise aspartic acid proteases from other protozoan
parasites have also been evaluated extensively for their potential to become compe-
tent drug targets [86]. Cysteine proteases and serine proteases also serve as attrac-
tive targets for drug discovery against protozoan infection. However the greatest
hindrance in such drug development programmes involves specificity of the prote-
ase inhibitors used. In order to be accurate in imparting its inhibitory activities to
parasite proteases and not to similar family of host proteases, a drug needs to be
designed in accordance with the unique structure function relationship of the para-
site protease [87].
8.6 Conclusion
Protozoan proteases play key roles in the process of disease development by patho-
genic protozoa. These proteases participate in almost every essential step during the
infection of the host. Their contribution in the disease process starting from the
154 A. Ghosh and S. Raha
initial recognition of the host cell receptors to their final entry within the host cell is
well substantiated in the literature. This class of protozoan proteases however is not
limited to any specific family of protease, but almost all the major family of proteo-
lytically active enzymes are involved. These proteases therefore serve as promising
drug targets to control respective infection processes. However active research is
ongoing to identify more and more unique protease targets in different protozoan
parasite systems that can be used for development of suitable drugs against the
associated diseases.
Acknowledgements A.G. acknowledges the financial support of the Dept. of Science and
Technology, Govt. of India, for DST INSPIRE faculty fellowship.
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Regulation of Extracellular Matrix
Remodeling and Epithelial- 9
Mesenchymal Transition by Matrix
Metalloproteinases: Decisive Candidates
in Tumor Progression
Y. Rajesh and Mahitosh Mandal
Abstract
Tumor biology is intricate and multifaceted. The genetic and epigenetic altera-
tions accelerate normal cells to transform into aggressive malignant phenotype.
Molecular principles of invasion and metastasis are indeed indispensable for pro-
found understanding of tumorigenesis. The seeding pioneer cells from growing
tumor eventually discharge from the original clump of mutant cells, invading
adjacent tissues and mobilizing to distant sites. This attribute of cancer cells
reduces patient’s survival rate and prognosis. Inquisition of mechanistic approach
for metastasis is bestowed by two processes—extracellular matrix (ECM)
remodeling and epithelial-mesenchymal transition (EMT). Proteases pave the
way for invaders by breaking down the ECM and releasing pro-invasive factors
from cell surface and ECM. Indeed, highly conserved EMT program leads to
dissemination of single tumor cells from primary tumors. The zinc-dependent
matrix MMPs are the most important effectors in these processes and frequently
overexpressed in most of the tumors. Besides proteolysis, by activating or deac-
tivating several growth factors, MMPs affect tumor neoangiogenesis and prolif-
eration. The tissue inhibitors of metalloproteinases (TIMPs) play a central role in
complex regulation of MMPs. An apt equilibrium between TIMPs and MMPs is
significant in cell invasion and metastasis. These concepts are encouraged for
pursuing MMPs as a signature for predicting metastasis and also as therapeutic
target. A comprehensive understanding regarding enzyme-substrate interactions
and regulation and specific MMPs’ functionality in cancer addresses that MMP
inhibitors (MMPIs) should be specific in terms of MMP or degrading definite
Keywords
Invasion • Metastasis • Extracellular matrix (ECM) • Epithelial-mesenchymal
transition (EMT) • Matrix metalloproteinases (MMPs)
9.1 Introduction
The malfunctioning in cellular activities that are pivotal for growth, differentiation,
and tissue integrity leads to cancer. Failure in growth control results in amassing
cells and producing tumor. The key risk and underlying reason behind tumor-related
deaths are not primary tumors, but the secondary tumors, i.e., metastasis. The phe-
notypic and biochemical alterations in relation to growth factors, cell-cell adher-
ence, and genetic expression occur during metamorphosis of a normal cell into
invasive cell [1].The attributes acquired by a healthy cell in the course of transfor-
mation to malignant one are cell division in the absence of external growth stimula-
tory signals, growth in spite of exogenous growth inhibitory signals, evading
apoptosis, neoangiogenesis, potent immortalization, and invasion and metastasis
[2]. The molecular principles of invasion and metastasis are indispensable for pro-
found understanding of tumorigenesis. Furthermore, the issue is immensely signifi-
cant since 90% of all cancer deaths are attributed to metastasis. The conventional
therapeutic approaches target rapidly proliferating cells. New insights toward
molecular progression of invasion and metastasis might pave the way for new,
highly specific and potential tumor management strategies. The prerequisite for this
resolution is further research in the field, for the better comprehension of these
processes.
9.2.1 Invasion
deaths are attributed to this only. Cell invasion occurs as single cells or as collection
of cells in sheets or clusters on the basis of cell type and host tissue matrix. In cancer,
invasion occurs with less homogeneity. However, in leukemia, lymphoma, sarcomas,
and glioma, cells invade heterogeneously in pattern of single cells. But in tumors from
epithelial origin, collective cell configurations infiltrate poorly into structured clusters
or sheets. They expand, dedifferentiate (epithelial-mesenchymal transformation
(EMT)), and disseminate as single cells, ensuing metastasis and poor prognosis [3].
We will be focusing on single cell invasion (key mode of invasion in cancer). Cellular
invasion is associated with immunity, angiogenesis, and metastasis.
9.2.1.2 Angiogenesis
New vasculature sprouts by penetrating the tissue matrix and provides nutrients to
tissue in morphogenesis and regeneration. Angiogenesis in cancer occurs due to
deficit of nutrient diffusion and oxygen exchange [6], and this erratic signaling
forms new blood vessels with altered structure. The tumorigenic abnormalities lead-
ing to enhanced permeability and retention (EPR) effect are poorly aligned, and
irregular-shaped endothelial cells result in large fenestrations, leaky vasculature,
and deficient lymphatic drainage. This EPR effect could be exploited for delivering
macromolecular drugs [7]. Many anti-angiogenic strategies targeting endothelial
cell invasion are under clinical evaluation. The combinational approaches inhibiting
both endothelial and tumor cells’ invasion are on the horizon [8].
9.2.1.3 Lymphangiogenesis
Lymphangiogenesis refers to sprouting lymph vasculature for draining waste, dur-
ing morphogenesis and regeneration. It has a huge role in tumor progression and
metastasis and in tumors lacking sufficient lymphatic vessels causing EPR effect.
However, in lymph node metastasis (breast, colon, and prostate), the primary route
is lymphatic vasculature [9], and in some tumors, pro-lymphangiogenesis factors
promote lymph node metastasis [10]. Hence, lymphangiogenesis inhibitors might
effectively target tumor metastasis.
162 Y. Rajesh and M. Mandal
9.2.2 Metastasis
In cancer, metastasis is the foremost cause of mortality in patient. The scientific and
clinical drive has to be geared up to unravel the poorly understood mechanisms of
metastasis. The enhanced knowledge in genetic/cellular behavior and biological
proceedings in cancer progression has added some new prospects in the diagnosis,
prognosis, and treatment of metastasizing diseases. Fine understanding regarding
the barrier’s role and paracellular permeability allowed formulating a new path
toward regulation of trespassing cancer cells and invading cells. The EMT bio-
marker offers new opportunity in the field of both prognostic methods and therapeu-
tic target for the metastatic prospective of a primary tumor. Angiogenesis has been
already established as a significant area in cancer therapy. A method directed toward
the detection of organ-specific spreading of solid tumors may grant a new approach
for targeting metastatic tumor cells. The genetic and epigenetic basis of metastasis
and the acquisition of ability to complete a series of steps involved in metastasis
during emergence of secondary tumors has to be revealed. Enormous challenges
have to be sorted out to anticipate these lines of research into clinical practice [11].
Single-cell migration involves isolated and dispersed tumor cells in an adjacent tis-
sue, whereas in collective cell invasion, healthy adjacent cells are being displaced
by moving cancerous tissue. Actually, cancer cells migrate and invade through the
ECM as single cell in fibroblast-/leukocyte-like fashion.
For establishing secondary sites of tumor growth, cancer cells leave the primary
tumor by losing adhering potential and gaining migratory and invasive capability, to
9 Regulation of Extracellular Matrix Remodeling and Epithelial-Mesenchymal… 163
The protein families that play a role in invasion and metastasis are enlisted in
Table 9.1, where some proteins are products of promoter/suppressor genes or targets
of proteins encoded by these genes. For instance, DNA-binding HIF fails to degrade
upon tumor suppressor von Hippel-Lindau (VHL) protein’s mutation; subsequently,
CXCR4 gene (encoding motility factor receptor attracting metastatic cells) gets
constitutively activated. The key players in distinct activities of invasive cells are the
members of these protein families. The proteins crucial in homotypic and hetero-
typic cell-cell adhesion that counteract primary invasion and stimulate metastasis
are cadherins and IgCAMs [26]. Apart from mechanistic role, they extensively par-
ticipate in signal transduction through their association with cytoplasmic compo-
nents and undergo ectodomain shedding for regulating invasion by their soluble
fragments. Integrin receptors and their ECM protein ligands mechanistically regu-
late cell-matrix adhesion and de-adhesion from cell to matrix and vice versa. This
interaction arrests cells in the matrix, assists migration and motility factors + recep-
tors, and stimulates locomotory machinery of cancer cells through their invasion
pathways [27]. Proteases pave the way for invaders by breaking down the ECM and
releasing pro-invasive factors from cell surface and ECM. Proteases with inactive
precursors get activated by other proteases, whereas active forms are neutralized by
9 Regulation of Extracellular Matrix Remodeling and Epithelial-Mesenchymal… 165
specific inhibitors. Normally, cells are anchorage dependent, and when they are
released from their substratum, they undergo apoptosis. But, invasive cancer cells
evade apoptosis via activation of growth and survival pathways and inactivation of
death pathways. The abovementioned proteins participate in integrated invasion
programs by forming multi-protein complexes, such as β-catenin assist as invasion
suppressor in E-cadherin/catenin complex and as invasion promoter in APC/
GSK-3β complex. These types of networks mediate positive and negative invasion
signaling pathways [28] in terms of invasion factors binding to specific receptors,
implicated in invasion and metastasis.
The cancer biology is intricate and multifaceted. During genetic and epigenetic
changes, normal cells transform into aggressive malignant phenotype. The manipu-
lation of migrating behavior of tumor generally affects proliferation/apoptosis or
both. Rather than regulating the migrating behavior of diffuse tumor, envision of
interventions that specifically target the invasive phenotype should be addressed.
The distant settlements of cancer cells are a bad news, with significantly reduced
166 Y. Rajesh and M. Mandal
survival rate and poor prognosis. However, cancer cells depending on feedback and
paracrine signaling from other tumor cells and stromal cells have a profound influ-
ence on carcinogenesis and metastasis. It is well established that hallmarks of can-
cer include cell growth and metastasis facilitated by MMPs and TGF-β, which
remodel the ECM. Indeed, highly conserved EMT program gives rise to dissemina-
tion of single tumor cells from primary tumors. Thus, it would be valid saying that
inquisition of the mechanistic approach of distant settlements/metastasis is being
bestowed by two processes—ECM and EMT.
9.3.1 E
xtracellular Matrix (ECM) Remodeling in Tumor
Progression
Fig. 9.1 (a) ECM Remodeling – The chronic inflammation/ tissue injury, TGFβ, connective tissue
growth factor (CTGF), IL13 and other factors stimulate the chief ECM producer (fibroblasts and
myofibroblasts) for more ECM production. This ECM contributes towards positive feedback loop
by further stimulating fibroblasts for continuous production of ECM. Resultant fibrosis is a major
risk for developing cancer. Tumor cells induce fibroblast activation, endothelial cell proliferation
and leucocyte recruitment; in contact with stromal cells and effect of growth factors. Activated
fibroblasts and endothelial cells express MMPs and secrete growth factors/ ECM components. The
leucocytesdisplays activated phenotype under influence of MMPs, cytokines and chemokines. The
MMP derived from both the tumor and stromal cells hastens ECM degradation and growth factor
release, increasing stromal cell activation and remodeling process. The tumor cells promotecell
migration, invasion, survival, and proliferation. (b) EMT – Under the influence of EMT regulators
(Slug/Snail/Twist) stimulated growth factors and cytokines, the epithelial cells undergo mesenchy-
mal transition. This enhances the mesenchymal motility of the cells. They even attain the ability of
invading the vascular regions. The expression levels of mesenchymal markers (N Cadherin/
Vimentin/ Fibronectin) also get upregulated. (c) MMP Signaling Pathway – Various signals inte-
grate towards MMP’s promoter activation. This ultimately results in elevation in MMP’s expres-
sion level. Thus, influencing the cell’s migratory and invasive potential
9.3.2 E
pithelial-Mesenchymal Transition (EMT) in Tumor
Progression
EMT plays a critical role in the embryonic development, formation of body plan,
differentiation, and tissue repair. It is a highly conserved cellular program allowing
polarized, immotile epithelial cells to convert into motile mesenchymal cells.
Adversely, it promotes invasion and metastasis, induces stemness, averts apoptosis
and senescence, and contributes to immunosuppression.
9 Regulation of Extracellular Matrix Remodeling and Epithelial-Mesenchymal… 169
The link between loss of E-cadherin expression and EMT has been well established
[50]. Furthermore, in EMT, upon ectopic expression of E-cadherin containing
β-catenin binding site, epithelial cell adhesion complexes reorganize suppressing
cell proliferation. Such cells lose mesenchymal phenotype [45]. The mutations in
E-cadherin gene make EMT cells more susceptible to EMT and metastasis [51].
The actions of EMT-inducing transcription factors facilitating acquisition of
170 Y. Rajesh and M. Mandal
mesenchymal phenotype illustrate the central role played by E-cadherin loss [52]. A
correlation between loss of E-cadherin and Wnt signaling or high expression of
Snail in nucleus has been reported. The expression of Snail and E-cadherin is
inversely correlated with the prognosis of breast cancer and oral cancer patients [53,
54]. Some labs also report that MMP-3 facilitates genomic instability via Rac1b-
and ROS-induced EMT [55] and noncoding microRNA regulation of EMT program
(miR200 and miR205 inhibiting the E-cadherin expression repressors, ZEB1 and
ZEB2 maintaining epithelial cell phenotype) [56, 57]. A loss of miR200 in breast
cancer is correlated with increased expression of vimentin and decreased levels of
E-cadherin [58], but miR21 upregulation facilitates TGF-β-induced EMT [59].
Here, it is also important to record the association between proteases and ECM
network. Invasion is favored by Snail and Zeb that induce the metalloprotease
expression for basement membrane’s degradation. By increasing the ROS cellular
levels, MMP-3 triggers EMT by inducing Snail1 expression. FGF1-induced MMP-
13 and Eplysin via TGF-β also trigger EMT. In colon cancer, metastasis is being
promoted by overexpression of transmembrane serine protease TMPRSS4-induced
EMT through Zeb transcription and E-cadherin downregulation. EMT, invasion,
and metastasis are also being promoted by an ECM protein “Periostin” secreted by
osteoblasts via PI3K/Akt interaction with integrins [60].
The tumor cells invade and metastasize by breaching ECM and several tissue layers.
For this, activation of proteolytic enzymes is a must, and apart from ECM proteins,
other components such as glycosaminoglycans are also degraded. Perhaps zinc-
dependent MMPs are vital in this process and are frequently overexpressed in most
of the tumors. The endopeptidases are secreted by macrophages, mast cells, and
fibroblasts. The cellular substrates involved in MMP’s degradation are fibronectin,
collagen, laminin, and proteoglycans. Actually in ECM, soluble MMPs are secreted
as inactive proenzymes which are activated by other enzymes [61]. By activating or
deactivating several growth factors, MMPs also affect tumor neoangiogenesis and
proliferation. The tissue inhibitors of metalloproteinases (TIMPs) play a central role
in complex regulation of MMPs. An apt equilibrium between TIMPs and MMPs is
of essential relevance in cell invasion and metastasis.
Advanced cancer research dealing with intended role of proteolysis in tumor inva-
sion and metastasis analyzed the role of MMPs in tumor progression through identi-
fied members of MMP family (as secreted enzymes + ECM components as substrate)
and elevated expression of MMPs. This motivated many laboratories for designing
proof-of-principle experiments. Initial experiments justified that the endogenous
MMP inhibitors modulated the MMP activity by manipulating the levels of TIMPs.
9 Regulation of Extracellular Matrix Remodeling and Epithelial-Mesenchymal… 171
These advances encouraged for pursuing MMPs as therapeutic targets, but unlikely
the number of MMPs expanded to more than 20. Their expression pattern deter-
mines cell- and tissue-specificity. The expansion of MMPs’ role affecting angiogen-
esis and growth of both benign and malignant tumors, substantially the range of
potential relevant substrates, got broadened. The development of pharmaceutical
reagents and initiation of clinical trials targeting MMPs enhance the therapeutic
benefit to cancer patients [1]. Further understanding of MMP biology, their regula-
tion, expression pattern in different types of tumor, role in invasion, metastasis,
ECM remodeling, EMT and tumor growth, and biomarkers will finely tune our
knowledge in developing anticancer therapeutics by potentially inhibiting MMPs.
Since long it is understood that proteinase activity is essential for tumor cells to
invade and metastasize to distant sites, where the potentially invasive cells first
attach to BM via cell-surface receptors (integrin) and administer extracellular pro-
teolytic action, and cellular locomotive action is initiated, depending on chemo-
tactic factors. Since proteolysis of BM and ECM components is considered to be
an essential step in cancer invasion and metastasis, tumor proteases are consid-
ered to be accessible targets for therapeutic intervention. It is evident that MMP
activity contributes to early-stage tumorigenesis, angiogenesis, and later events of
invasion and metastasis. Let us now overview the role played by MMPs in tumor
progression.
The MMPs are abundantly expressed in malignant disease. A survey on tumor-
associated MMPs is shown in Table 9.2. Literature findings reveal that a positive
correlation exists between level of MMPs expression and tumor grade. Alongside,
research on levels of endogenously produced inhibitors reveals that elevated MMP
levels and reduced TIMP levels define the aggressiveness of a tumor [81–84]. These
findings led to a concept stating that aggressive, invasive, and metastatic potential of
Table 9.2 Survey on tumor-associated MMPs [77–80]
MMP Common name Structural class Role Type of cancer MMP inhibition: effect on tumors
MMP-1 Collagenase-1 Simple (1) Degrade major component of bone ECM Breast –
hemopexin (2) It is expressed by fibroblasts, keratinocytes, Colon
domain endothelial cells, monocytes, and macrophages
(3) MMP-1 was significantly downregulated, Gastrointestinal
while TIMP-1 levels were increased, in a time- Head and neck
and pressure-dependent manner in a smooth Prostate
muscle cell Esophagus
MMP-2 Gelatinase A, Gelatin binding (1) It is widely expressed in embryonic CNS Glioma Reduction in tumor-induced
72-kDa type IV angiogenesis and in experimental
collagenase metastasis
(2) Expression of MMP-2 and β-catenin loss has Bladder
a role in the pathogenesis and progression of ESC
(3) Decreased E-cadherin has an important role in Breast
the development of both ESC and EEC Cervical
Colon
Lung
Melanoma
Pancreas
Prostate
Skin
Stomach
(continued)
9 Regulation of Extracellular Matrix Remodeling and Epithelial-Mesenchymal…
173
174
9.4.5 M
MPs: Regulator Protein Family of ECM Remodeling
and EMT
Table 9.3 EMT traits and corresponding MMP expression in different cancer
Cancer model Specific EMT traits MMPs expressed References
Bladder cancer E-cadherin reorganization MMP-2 [111]
Breast cancer Lack of E-cadherin, vimentin and Activation of [112]
invasive potential MT1-MMP, MMP-2
Bronchial cancer Altered vimentin expression and MMP-3, MMP-9, and [113, 114]
migratory potential MMP-11
Prostate cancer Loss of E-cadherin, vimentin, MT-MMP [115]
and invasive potential
Cervical cancer Loss of E-cadherin, vimentin, Activation of [116]
and invasive potential MT1-MMP, MMP-2
Squamous cell Loss of E-cadherin, vimentin MMP-2 [117]
carcinoma
the suspicion of cancer would be effective in terms of both time and resources. The
serum MMP-9 levels are currently being employed as an accurate test for colon
cancer patients [119]. There are also evidences displaying the presence of MMP-2
and MMP-9 as a marker of bladder and prostate cancer [120, 121]. Additionally,
studies involving tissue fluids of different cancers assessed the diagnostic value of
MMP-2, MMP-7, and MMP-9 and/or TIMP-1 and TIMP-2 [77]. However, these
tests fail to demark the patients with malignant tumors and benign tumors or an
inflammatory disease. Similar genre of MMPs are upregulated in different types of
cancer as well as in inflammatory diseases, hence IHC is often employed in clinical
pathology as a differentiating tool for benign and malignant tumors and also among
different type of cancers. Recently, research has established MMP-11 to be more
effective in this context [122, 123].
The standard treatment procedure involves surgical abscission of tumor followed
by adjuvant therapy like radio/chemo/hormonal therapy or angiogenic/kinase inhib-
itors. The treatment procedure implemented might pose adverse effects, and inabil-
ity to identify patients with low risk of tumor recurrence might be overtreated.
Recent development of biomarkers predicting the chances of relapse has contrib-
uted toward treatment stratification. There are accumulated evidences addressing
the potential of MMPs and TIMPs as prognostic markers in different cancer.
However, owing to conflicting results, it is not that easy to draw general conclusions
concerning prognostic value of MMPs/TIMPs in cancer. The possible reasons
behind this are [77]:
• Since MMPs are multifunctional in nature and their specific role depends on the
acting substrate in a biological situation such as variation between patients,
organs, phases of tumorigenesis, and progression.
• TIMPs are multifaceted proteins and their MMP-independent roles are in start-
ing phase.
• Variations in the parameters considered for studying such as:
–– MMP/TIMP level in blood/urine or tissue samples/extracts
–– Enzymatic activity or total expression at transcription/translation level
–– Separating active enzymes and proenzymes or MMP/TIMP expressing cell
types
and severe adverse effects were also reported. The reasons behind the therapeutic
failure which helped in modifying the therapeutic strategy involving MMPs are:
• The patients enrolled in clinical trials were in advanced stage, and MMPs are
involved in early stages of tumorigenesis, and the drugs targeting MMPs might
be effective if applied in nonmetastatic patients.
• Preclinical mouse experiments involving MMPIs were generally successful, as
they were administered before developing metastasis.
• The first-generation MMPIs come under broad-spectrum inhibitors inhibiting
both tumor-promoting and tumor-repressing MMP activities.
• Level of MMP expression in patients enrolled for clinical trials should be checked
and administered as adjuvant therapy with conventional cytostatic drugs or radia-
tion [124].
These findings indicate that new MMPIs developed should be specific in terms of
MMP or degradation of certain substrates. This is challenging because the active
sites of MMPs are very similar among MMP family members. But different MMPs
have different subsites/pockets in active site clefts. The substrate specificity is
defined by the ability of the cleaving substrate to fit into these pockets [125]. Instead
of targeting the active site Zn ion, such subsites should be focused for designing
more specific MMPIs [126]. Additionally, targeting exosites or noncatalytic sites in
MMPs may inhibit detrimental effects of MMP, as it is known that large protein
substrates require cross talk between active site and noncatalytic domains for effi-
cient cleavage [127]. An example displaying exosite’s requirement is collagenases
(MMP-1) which aided cleavage of triple-helical collagen. The large triple-helical
cord is processed (unwinding hemopexin domain of collagenases acts as un-
helicase) to fit into the cleft of active site and hydrolyzed [128, 129]. Targeting
exosites of defined substrates might aid in inhibiting specific MMP functions only,
not their entire activity. However, comprehensive understanding regarding enzyme-
substrate interactions and regulation and role of specific MMPs in different cancers
will certainly aid in designing such drugs. Another possibility is targeting MMPs at
expression level [130, 131] or generating specific cytostatic drugs as prodrugs by
exploiting the advantage of elevated MMP expression in tumors. Prodrug approach
would release higher concentrations of active drug in the tumor environment after
being processed by cancer-associated MMP and successively reduce the adverse
effects in other tissues. The MMP synthesis could be usually inhibited by agents
which avert them from associating with the molecules mediating their activities to
the cell surface or impeding their enzymatic activity. The different approaches
inhibiting MMP gene transcription, targeting extracellular factor signal transduc-
tion pathways or nuclear factors that stimulate genetic expression and downregulate
MMP production, have been illustrated in Table 9.4.
Table 9.4 Different approaches inhibiting MMP gene transcription, targeting extracellular factors, signal transduction pathways, or nuclear factors that stimu-
late genetic expression and downregulate MMP’s production
MMP inhibition
strategy Approach involved Molecules Experimental model Observations References
Inhibiting MMP Antisense mRNA/ – Mouse models Reduced tumor burden or [132–134]
synthesis oligonucleotide-transfected metastasis by downregulating
cells MMP-7 or MMP-9
Ribozyme-targeted mRNA
directly inhibits MMP
synthesis
Inhibiting signal Inhibit tyrosine kinase Halofuginone Chickens Regulating MMP gene [135]
transduction receptor signaling (Coccidiostat) expression and experimental
pathways inducing cancer cell metastasis
MMP transcription
Inhibiting MMP and Inhibiting binding of – Animal models Specifically targets cancer- [136]
other protein’s MMP-2 to α-v-β3 integrin promoting function
interaction
MMP activity’s Fuses to MMP cleavage Recombinant proteins – Cell death [137]
manipulation site, upon activation by containing anthrax toxin Tumor treatment
MMP cleavage at cell
surface internalized to the
cell
(continued)
9 Regulation of Extracellular Matrix Remodeling and Epithelial-Mesenchymal…
181
182
Inhibiting proprotein Reduces processing of α1-PDX – Prevents tumor growth and [160, 161]
convertase pro-MMP-2 and prevents invasive potential
MT1-MMP activation
(continued)
183
184
9.6 Conclusion
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194 Y. Rajesh and M. Mandal
Abstract
From the development of the spermatozoa within the seminiferous tubule of tes-
tis to the fertilization events that occur in female reproductive tract, all the repro-
ductive processes in mammals are regulated by a highly orchestrated and
integrative mechanism. Several proteases and their protease inhibitors form an
important part of this mechanism. So far, vast arrays of proteases have been iden-
tified in the reproductive system of mammals playing critical role in the major
events associated with the several male reproductive processes. Several endoge-
nous inhibitors of these proteases are also produced in the male reproductive
tissues/fluids that cater to the role of regulating the protease production/degrada-
tion, activation/inactivation, etc. Thus, there exist a fine balance between the pro-
duction of these proteases and their regulators for maintaining the blood-testes
barrier and the gamete development. A disturbance in this equilibrium leads to
progression of reproductive failures including azoospermia, impaired sperm
functions, low fertilizing efficiency, etc. and culminates in infertility cases. This
chapter focuses on an account of such proteases and the protease inhibitors with
their role in mammalian male reproduction.
Keywords
Proteases • Protease inhibitors • Male • Reproduction • Fertility • Infertility
10.1 Introduction
Fig. 10.1 Classification of proteases: The proteases are classified based on the hydrolysis of
peptide bonds in proteins as exopeptidases and endopeptidases. The exopeptidases are further clas-
sified based on the identity of the liberated fragment as aminopeptidases, carboxypeptidases,
dipeptidyl peptidases, tripeptidyl peptidases, peptidyl dipeptidases, dipeptidases, tripeptidases,
omega peptidases, etc. and the endopeptidases based on the catalytic site present as serine prote-
ases, metzincins, threonine, cysteine, and aspartic proteases
attack only the peptide bonds confined at/or close to the amino- or carboxy-terminal
portion of peptide chains. The endopeptidases attack the internal peptide bonds in
the polypeptides. The exopeptidases can be classified further based on the size or
identity of the liberated fragment as aminopeptidases, carboxypeptidases, dipepti-
dyl peptidases, tripeptidyl peptidases, peptidyl dipeptidases, dipeptidases, tripepti-
dases, omega peptidases, etc. Dipeptidyl-peptidase IV purified from porcine seminal
plasma and carboxypeptidase C identified from human seminal plasma are exam-
ples for exopeptidases.
The endoproteases are divided into five subclasses depending on their catalytic
sites as metzincins, aspartic, cysteine, threonine, and serine proteases (Fig. 10.1). In
metzincins, zinc is present at their catalytic sites. They also exploit an activated
water molecule to attack the peptide bond in the substrate. Metzincins are subdi-
vided into four distinct families: matrixins, adamalysins, astacins, and serralysins.
Matrixins and adamalysin-related proteases play fundamental role in many physio-
logical processes. Matrix metalloproteinases (MMPs) or matrixins are extracellular
matrix (ECM)-digesting enzymes and are Ca2+- and Zn2+-dependent endopeptidases
198 V.S. Gurupriya and S.C. Roy
active at neutral pH. They use a metal ion to polarize a water molecule to hydrolyze
the peptide bond in the substrate [2]. MMPs are secreted as latent forms which can
be activated by chaotropic agents or by cleavage of the inhibitory propeptide by
MMP family of proteases or the plasminogen activator of the urokinase type. The
active MMPs have a relative molecular mass of about 10 kDa less than the latent/
pro-forms. MMPs are also involved in the release and activation of growth factors
and cytokines [3]. MMPs can be broadly classified into four groups: (i) collagenases
that are active against native collagen, (ii) gelatinases that have high activity against
gelatin and denatured and type IV collagens, (iii) stromelysins that degrade noncol-
lagen ECM components, and (iv) membrane-type MMPs (MT-MMPs) that are
transmembrane molecules mainly cleaving ECM components at the same time acti-
vating other MMPs also [4]. Adamalysin-related proteinases also known as ADAMs
(a disintegrin and metalloprotinease) contain a disintegrin domain having specific
role in cell adhesion and proteolytic processing. In mice and human, respectively, at
least 34 and 26 ADAM genes have been identified so far [5]. Unlike mammals,
avian genome lacks ADAM1–7 and ADAM30 genes [6]. ADAMTS (a disintegrin
and metalloproteinase with thrombospondin type 1 motifs) are the soluble counter-
parts of the ADAMs. They contain thrombospondin type 1 motifs that help in ECM
association, inflammation, angiogenesis, etc.
In aspartic proteases, an aspartate is present at the catalytic site to attack the
peptide bond linkage in the substrate. The digestive proteases like pepsin and
cathepsin H of the spermatozoa are some of the examples of aspartic proteases.
Cysteine proteases have at their catalytic sites Cys molecules that act as nucleo-
philes [7]. Cysteine proteases are common in plants and animals, act as lysosomal
enzymes, and exhibit tissue-specific expression for bone growth and lung function
[8, 9]. Many of the cathepsins, calpains I and II in seminal fluid belong to the class
of cysteine proteases [10, 11]. Threonine proteases are classes of proteases having
the catalytic site with Cys, Ser, or Thr to act as a nucleophile [7].
Serine proteases are the most common protease in the both insects and mammals
[12]. Serine proteases have a conserved catalytic triad of a His, Ser, and Asp to
coordinate a water molecule. Serine protease family can be subdivided into 16 sub-
families including plasminogen activators (PAs), type II transmembrane serine
proteases (TTSPs), kallikreins, and serine protease with trypsin-like specificity [7].
Plasminogen activators (PA) are trypsin-like proteases that help in the conversion of
plasminogen to plasmin and digestion of fibronectin, laminin, vitronectin, etc. [13].
They are physiological activators of pro-/latent metalloproteases (MMPs) for col-
lagen degradation [14]. Two classes of PAs are known in mammals: the tissue-type
plasminogen activator (t-PA) and the urokinase-type plasminogen activator (u-PA).
Both catalyze the activation of plasminogens. The u-PA is associated with the physi-
ological and pathological tissue remodeling, whereas t-PA is mainly involved in
thrombolysis and neurobiology. Type II transmembrane serine proteases (TTSPs)
interact with the cell surface and soluble or secreted proteins, cell matrix compo-
nents, and proteins on surrounding cells. TTSPs are synthesized as zymogens and
are activated by cleavage of arginine or lysine present at the highly conserved acti-
vation motif and remain as membrane bound after activation. Kallikrein family of
10 Proteases and Protease Inhibitors in Male Reproduction 199
Fig. 10.2 Classification of major protease inhibitors: Protease inhibitors are classified based
on the type of protease they inhibit as metalloproteinase inhibitors, serine protease inhibitors,
aspartic protease inhibitors, threonine protease inhibitors, and cysteine protease inhibitors. The
serine protease inhibitors include major proteases such as trypsin inhibitors, Kazal inhibitors, plas-
minogen activator inhibitors, etc.
proteases is present in many animal species including human, rat, mouse, etc. and
found to express in tissues including the prostate, breast, ovary, and testis. Plasma
and tissue kallikreins are the two categories coming under kallikrein family. The
kallikreins have a main role in regulation of blood pressure and semen liquefaction
[15]. Serine protease with trypsin-like specificity includes acrosin- and testes-
specific serine proteases [16]. Acrosin has an enzymatic activity for the limited pro-
teolysis of the zona pellucida and a lectin-like carbohydrate-binding activity for
binding of acrosome-reacted sperm to the zona [17, 18]. Testes-specific serine pro-
teases play different roles in spermatogenesis and are required for germ cell survival
during meiosis. They also help in sperm-oocyte interaction and penetration of the ZP.
An overactivity of proteases may cause premature activation of pathways in tis-
sues leading to tissue damage. Their action needs to be controlled by protease inhib-
itors by keeping the proteases in its pro-form or zymogen form so as to maintain the
tissue integrity, cell migration, cell signaling, cell surface and tissue remodeling,
tissue support, repair, growth and development, etc. [19]. More than 2% of human
genes belong to the class of proteases or protease inhibitors [7]. It shows the impor-
tance of balanced mechanism of proteolysis in mammalian system. The protease
inhibitors are broadly classified into five groups based on the type of protease they
inhibit: (i) metalloproteinase inhibitors, (ii) serine protease inhibitors, (iii) aspartic
protease inhibitors, (iv) threonine protease inhibitors, and (v) cysteine protease
inhibitors (Fig. 10.2). Among these, the metalloproteinase inhibitors and the serine
protease inhibitors are most commonly associated with the reproductive functions
of mammals. Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors
200 V.S. Gurupriya and S.C. Roy
10.3 R
ole of Proteases and Protease Inhibitors in Male
Reproduction
Fig. 10.3 Schematic depiction of the role of proteases and protease inhibitors: Different
classes of proteases are involved in several male reproductive processes like spermatogenesis,
sperm maturation, capacitation, acrosome reaction, and fertilization. Their action must be strictly
controlled by protease inhibitors by keeping the sperm proteases in its pro-form or zymogen form
to maintain the tissue integrity and reproductive roles of spermatozoa. Some of the examples of
proteases/protease inhibitors involved in various male reproductive processes have been indicated
in boxes
of maintaining the equilibrium between proteases, and their regulators are depicted
in Fig. 10.3 and Fig. 10.4.
In mammals, MMPs play vital roles in several physiological processes that are
associated with male reproduction. MMP-2 and MMP-9 have been demonstrated in
mouse fetal testes [35]. In human and mouse testes, the expressions of MMP-18,
MMP-23, MMP-26, and MMP-28 were detected [36–39]. In dogs, the expression of
metalloproteinase and semen parameters are positively correlated [20]. Latent forms
of the matrix metalloproteinases in semen are inversely correlated with sperm qual-
ity trait and ejaculate volume in dogs and humans [20, 40]. Both latent and active
forms of MMP-9 and MMP-2 are present in canine seminal fluid with predominant
expression of latent forms [41]. Expressions of MMP-2, MMP-9, TIMP-1, and
TIMP-2 were detected in the testes, epididymis, and ejaculated semen of dogs, and
MMP-2 was present in spermatozoa from all the developmental stages as well as in
ejaculated semen, and thus MMP-2 could be considered as a marker for predicting
the quality of semen [42]. MMP-2 has been detected in the inner acrosomal mem-
brane of bull, human, and mouse spermatozoa [43]. In mouse spermatozoa, MMP-2
202 V.S. Gurupriya and S.C. Roy
Fig. 10.4 Schematic depiction of imbalance between proteases and their inhibitors: The loss
of equilibrium between proteases and their inhibitors may cause premature activation of pathways
or tissue damage affecting blood-testes barrier, gamete development, epididymal sperm matura-
tion, sperm-egg binding during fertilization, etc. leading to reproductive disorders that culminate
in infertility cases
detected in the stereo-ciliated epithelial cells and in the lumen of the vas deferens of
mice [66]. There are evidences for urokinase PA expression in the seminal vesicles
and epididymis of mice and monkeys and t-PA mRNA in the epididymis of mon-
keys [59]. Rat and monkey epididymal epithelial cells also secrete both t-PA and
u-PA, and the PA activities (PAA) seemed to be diminished toward the cauda epi-
didymis [59]. This confirms the role of the PA in sperm maturation process.
The kallikreins have important role in motility, penetration, and migration of
sperm through the cervix and uterus [69]. Leydig cells are the unique site of expres-
sion of kallikrein-21, kallikrein-24, and kallikrein-27 in the testis [7, 70]. Kallikrein-
like proteases (KLKs) such as KLK-2, KLK-3, KLK-4, KLK-5, KLK-8, KLK-11,
KLK-12, KLK-14, and KLK-15 are also secreted by the prostate and help in semen
liquefaction [71]. KLK-14 has been recognized as a potential activator of KLK-3
(also known as prostate-specific antigen, PSA) from prostatic fluid. Further, its abil-
ity to degrade seminogelin I and II from seminal vesicle causes release of trapped
spermatozoa from the seminal clot/semen coagulum thereby allow sperm motility
and capacitation [72]. The semen liquefaction is activated within 5–20 min post-
ejaculation and regulated by the action of eppin (an epididymal protease inhibitor),
KLK-3, and other KLKs (kallikrein-like proteases) in the prostatic fluid. Eppin (or
SPINLW1) is a member of Kunitz-type and the whey acidic protein (WAP)-type
protease inhibitor gene family [73]. Human-ejaculated spermatozoa are coated with
eppin over the head and tail regions [73–75]. The C-terminal region of eppin (aa
75–133) binds a fragment of semenogelin (Sg, aa 164–283) secreted from seminal
vesicles and prevents the action of KLK-3 on Seminogelin [76]. Human epididymis
protein-4, commonly known as HE-4, is an epididymal protease inhibitor. HE-4 has
been detected in human seminal fluid [77]. The inhibitory nature of HE-4 on differ-
ent serine, aspartyl, and cysteine proteases has demonstrated it as a cross class pro-
tease inhibitor [77].
Acrosin is a serine protease present in acrosome of spermatozoa and is involved
in the recognition, binding, and penetration of the spermatozoa of/into the zona pel-
lucida of the oocyte. It is the most widely studied and well-characterized acrosomal
enzyme [78]. In mouse, rat, human, and swine, acrosin gene is localized on chromo-
somes 15, 7, 22, and 5, respectively [79–81]. Acrosin is present as an inactive pre-
cursor, namely, proacrosin in acrosome of freshly ejaculated sperm [82]. Upon
acrosome reaction, proacrosin is autoproteolyzed into most stable active forms by
an intrazymogen mechanism, and later it is delivered to the extracellular milieu [83,
84]. Acrosin is conserved among birds and mammals [85]. The presence of proac-
rosin/acrosin system was demonstrated in turkey spermatozoa [86]. Apart from
acrosin, several other enzymes with properties similar to acrosin have been identi-
fied in mammalian spermatozoa [87]. Acrosin II of molecular mass 30.9 kDa is an
acrosin-like protease similar to other avian acrosins [88]. The presence of trypsin-
like activity in turkey and chicken spermatozoa extract has been demonstrated [89].
Kotlowska et al. [90] detected the sperm amidase activity and acrosomal serine
proteases activity in turkey spermatozoa. Serine proteases of molecular weight 29
and 88 kDa have been detected in turkey seminal plasma [45]. In fresh and f rozen/
thawed dog spermatozoa, proacrosin, alpha-acrosin, and beta-acrosin with 40, 32,
10 Proteases and Protease Inhibitors in Male Reproduction 205
and 27 kDa bands, respectively, are identified by in vitro capacitation studies [91,
92]. The proacrosin to acrosin conversion during sperm-zona interaction has been
demonstrated in boar spermatozoa [93, 94]. The three molecular forms of 64, 38,
and 25 kDa human acrosin have been identified [95]. A direct correlation between
the levels of proacrosin/acrosin activity with the fertilizing potential was observed
in human spermatozoa [96]. The amino acid sequence of mouse proacrosin has a
high degree of homology with that of porcine, human, and rat [97–100]. Serine
protease of mouse sperm is different from other rodents and mammals. The amount
of acrosin and gelatinase activity has been shown to be less for mouse sperm when
compared with rat and hamster sperm [101]. The mouse proacrosin has an extra Cys
residue at positions 143 and 144 and may cause incorrect formation of a disulfide
bridge and prevent the acrosin activity. The crystal structures of pig and ram acro-
sins have been identified [16]. The presence of acrosin in ram epididymal fluids
suggested that it may be released from dead spermatozoa [44]. Both proacrosin and
acrosin have been purified from porcine, guinea pig, and human cauda epididymal
and ejaculated spermatozoa [102–104]. In porcine spermatozoa, proacrosin with
molecular weight 55 and 53 kDa and active forms with molecular sizes of 49, 43,
and 35 kDa have been identified [93, 98, 105]. Analysis N- and C-terminal sequence
of the five forms of boar proacrosin demonstrated that conversion of 55 kDa proac-
rosin to the 35 kDa mature acrosin occurs by removal of three C-terminal segments
and cleavage of a peptide bond near the N-terminus [98, 105]. This peptide bond
cleavage is responsible for the initiation of the protease activity of acrosin [98, 105].
Genomic and cDNA sequences of acrosins were characterized from human, bovine,
boar, and mouse [99, 106, 107]. Two forms of proacrosin with different molecular
masses (55 and 53 kDa) and three different forms of acrosin, named α (49 kDa), β
(35 kDa), and γ (25 kDa), have been identified in ejaculated boar spermatozoa
[108]. It has been suggested that the acrosin activity can be used as an index for
assessing the fertility check in males [109]. Serine proteases other than acrosins
have also been detected in mouse sperm acrosome [110].
The murine testis-specific serine proteases (TESP) are located as a gene cluster
on chromosome 9F2-F3. Prss42/Tessp-2 is expressed in sperm membrane and cyto-
plasm, Prss43/Tessp-3 in the sperm membranes, and Prss44/Tessp-4 in the cyto-
plasm of the secondary spermatocytes and spermatids. Thus, these serine proteases
play different roles in spermatogenesis and are required for germ cell survival dur-
ing meiosis [111]. In mouse spermatozoa, the testes-specific serine proteases,
namely, TESP1, TESP2, and TESP4, were identified in the acrosome, TESP3 from
the spermatogenic cells of the testis, and TESP5 on the cauda epididymal sperm
membranes. The 42 and 41 kDa isoforms of TESP5 were identified in mouse sperm
extract [112]. Since these isoforms are glycosylphosphatidylinositol (GPI) anchored
and are located in the lipid rafts of mouse sperm membranes, TESP5 is suggested to
play important role in signal transduction at the sperm surface that help in sperm-
zona binding [113, 114].
Two serine proteases, namely, BSp66 and its dimeric form BSp120, have been
identified in bovine and hamster spermatozoa [87, 115]. In hamster spermatozoa,
BSp66 is involved in sperm-oocyte interaction [87]. Proteases such as aspartate
206 V.S. Gurupriya and S.C. Roy
in mouse seminal vesicle [138]. SPINKL was demonstrated to inhibit sperm capaci-
tation and sperm motility in vitro [138]. SPINKL-bound spermatozoa were detected
from the uterus but not from the oviduct. In mouse, the SPINKL was demonstrated
to inhibit the capacitation-related signals such as cholesterol efflux, calcium influx,
and cAMPi in uterine spermatozoa [139]. In mouse, SPINK2 expression has been
demonstrated in spermatogenic cells and mature sperms in testes [140]. Mutation in
SPINK2 locus demonstrated impaired spermatogenesis, loss of testicular integrity,
and reduced sperm number and serine protease-mediated germ cell apoptosis.
SPINK3 expression has been demonstrated in seminal vesicle and plasma mem-
brane of apical hook of spermatozoa [141]. It is also known as Caltrin or calcium
transport inhibitor. In mouse, the SPINK3- spermatozoa binding reduced [Ca2+]i in
the head and suppressed the acrosome reaction in spermatozoa before encountering
egg. Release of sperm-bound SPINK3 by SPINK3-inhibiting trypsin-like activity
(SITA) in uterine luminal fluid demonstrated restoration of sperm fertilization abil-
ity [141]. Expressions of SPINK8, SPINK10, SPINK11, and SPINK12 have been
demonstrated in mouse epididymis [142]. In rat, specific expression of an androgen-
responsive SPINK13 has been demonstrated in epididymal tissues and spermatozoa
[23]. RNAi knockdown of SPINK13 gene in rat has demonstrated enhanced acroso-
mal exocytosis and fertility defects both in vivo and in vitro. SPINK13 is involved
in proteolytic processing of epididymal proteins required for sperm maturation.
SERPIN-E1 is commonly known as plasminogen activators inhibitors (PAIs).
PAIs play crucial role in spermatogenesis, spermatozoa capacitation, and fertiliza-
tion [62]. PAI-1 has been identified from rat, mouse, and monkey testes [68]. In rat,
the highest expression of PAI-1 mRNA has been detected in the germinal cells of
testes [143]. Sertoli cells have also demonstrated to secrete PAI-1 to the adluminal
compartment of seminiferous tubules [144]. PAI-1 is suggested to protect the Sertoli
cell barrier during passage of pre-leptotene spermatocytes across the blood-testes
barrier and prevents the release of developing spermatid in spermiation process. In
monkey, the increased expression of PAI-1 has been demonstrated in caput epithe-
lial cells compared to that of initial and caudal regions of the epididymis [59]. PAI-1
is suggested to play major role in regulation of epididymal sperm maturation. PAIs
were also detected in human-ejaculated spermatozoa and seminal plasma [145,
146]. PAI-1 was detected in the head, middle piece, and tail regions of human and
rhesus monkey spermatozoa [65].
SERPIN-E2 is generally known as protease nexin-1. SERPIN-E2 was identified
from mouse seminal vesicle. The mouse deficient in SERPIN-E2 genes demon-
strated fertility defects [147]. In mouse, SERPIN-E2 was detected in seminal vesic-
ular secretions, epithelium of the seminal vesicle, epididymis, and vas deferens
[148]. SERPIN-E2 was also detected on the acrosomal cap of testicular and epi-
didymal spermatozoa. In ejaculated spermatozoa, SERPIN-E2 was detected mostly
from the uncapacitated spermatozoa and was found to inhibit the sperm capacitation
signals and sperm-zona binding in vitro. The presence of SERPIN-E2 in uncapaci-
tated mouse spermatozoa suggested that it may act as a decapacitation factor in
fertilization [148].
208 V.S. Gurupriya and S.C. Roy
10.4 Conclusions
A vast array of proteases and protease inhibitors have been identified in the male
reproductive organs of mammals playing critical role in the major events associated
with the several male reproductive processes including spermatogenesis, epididy-
mal sperm maturation, capacitation, and fertilization. Protease inhibitors have
important role for regulating the protease production/degradation, activation/inacti-
vation, etc. There exists a fine balance between the protease and their regulators for
maintaining the above male reproductive processes including tissue integrity, blood-
testes barrier, and the gamete development. A disturbance in this equilibrium leads
to progression of reproductive failures such as azoospermia, impaired sperm func-
tions, low fertilizing efficiency, etc. that culminate in infertility cases. Although
there have been considerable knowledge in many aspects of mammalian male
reproductive health, a better understanding of the protease and protease inhibitors
may in turn help in developing the accurate methodologies or specific approaches
for improving the sperm function and male fertility.
Acknowledgments The authors thank the Director, National Institute of Animal Nutrition and
Physiology (NIANP), Bangalore, India for providing funds and facilities for carrying out the pres-
ent study. The first author was supported by Junior Research Fellowship from Indian Council of
Medical Research, New Delhi. The authors also thank the Director, Indian Veterinary Research
Institute (IVRI), Bareilly, India for providing an opportunity to the first author for conducting the
present study at NIANP, Bangalore.
10 Proteases and Protease Inhibitors in Male Reproduction 209
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Physiological and Pathological
Functions of Cysteine Cathepsins 11
Mansi Manchanda, Nishat Fatima,
and Shyam Singh Chauhan
Abstract
Cysteine cathepsins are lysosomal hydrolases that belong to the papain family of
cysteine proteases. This group comprises of 11 members and a majority of them
are endo-proteases. They are initially synthesized as inactive zymogens, which
are then processed into their active forms in the acidic and reducing environment
of the lysosomes. The most striking element of cysteine cathepsins is their active
site that contains a catalytic triad of a cysteine, histidine, and an asparagine resi-
due. Originally, turnover and degradation of intracellular proteins was consid-
ered the only function of cysteine cathepsins. However, substantial evidences
accumulated over the years have established their role in several physiological
and pathological processes. Tissue-specific distribution and gene knockout anal-
ysis of these housekeeping proteases established their several physiological
functions including antigen presentation, bone and tissue remodeling, keratino-
cyte differentiation, extracellular matrix degradation, cell cycle regulation, and
death. Expression and activity of these proteases are tightly regulated, and their
deregulation has been reported in a variety of pathological conditions such as
cancer, lung diseases, metabolic disorders, atherosclerosis, cardiomyopathy,
rheumatoid arthritis, osteoporosis, etc. These proteases have been proposed to be
potential drug targets, and some of their inhibitors are under phase I clinical trial.
This chapter presents an overview of the structure, synthesis, mode of action,
regulation of expression and activity, and physiological as well as pathological
role of lysosomal cysteine cathepsins.
Keywords
Cathepsins • Antigen presentation • Cell death • Angiogenesis • Extracellular
matrix degradation • Cancer • Lung disorders • Cardiovascular diseases •
Metabolic syndrome
Abbreviations
&: and
APCs: Antigen-presenting cells
BALF: Bronchoalveolar lavage fluid
bFGF: Basic fibroblast growth factor
CLIP: Class II-associated invariant chain
COPD: Chronic obstructive pulmonary disease
cTEC: Cortical thymus epithelial cells
DPPI: Dipeptidyl aminopeptidase I
ECM: Extracellular matrix
EMT: Epithelial-mesenchymal transition
ER: Endoplasmic reticulum
Ets: Erythroblast transformation-specific transcription factor
GAGs: Glycosaminoglycans
hCATL: human cathepsin L
HMWK: High-molecular-weight kininogens
IFN: Interferon
IL: Interleukin
IR: Insulin receptor
IRES: Internal ribosomal entry site
L: Left-hand domain
LMP: Lysosomal membrane permeabilization
LMWK: Low-molecular-weight kininogens
M6PR: Mannose-6-phosphate receptor
MMPs: Matrix metalloproteinases
MOMP: Mitochondrial outer membrane permeabilization
PAI: Plasminogen activator inhibitor
R: Right-hand domain
ROS: Reactive oxygen species
SMA: Smooth muscle actin
TGF: Transforming growth factor
TGN: Trans-Golgi network
TIMP: Tissue inhibitors of metalloproteinases
TNF: Tumor necrosis factor
UTR: Untranslated regions
VEGF: Vascular endothelial growth factor
XIAP: X-chromosome-linked inhibitor of apoptosis
11 Physiological and Pathological Functions of Cysteine Cathepsins 219
11.1 Introduction
Fig. 11.1 Classification of human proteases. Human degradome includes five distinct classes of
proteases. The number of members in each class is indicated in parenthesis
220 M. Manchanda et al.
Cathepsins are a large and diverse group of enzymes ubiquitously present in all organ-
isms ranging from prokaryotes to mammals. These peptidases were first discovered in
the acidic gastric juice by Willstätter and Bamann in the year 1929 and were named as
“cathepsin” which means to digest. Later in the year 1955, Christian de Duve identi-
fied the membranous sacs (lysosomes) enriched in acid hydrolases [12]. Lysosomal
enzymes were characterized in the due course of time, and it was discovered that
cysteine cathepsins play a major role in the degradation of proteins internalized by
endocytosis/phagocytosis and in autophagy. However, under certain physiological
and pathological conditions, cathepsins can also be secreted into the cytosolic and
extracellular compartments and retain their proteolytic activity [13]. Cellular function
and general properties of cysteine cathepsins have been summarized in Table 11.1.
Table 11.1 General properties and functions of cathepsins as revealed by knockout analysis
Phenotype of null
Cathepsin Catalytic activity Location mice References
Cathepsin B Endopeptidase, Ubiquitous Impaired prohormone [224, 225]
carboxydipeptidase thyroglobulin
processing and
thyroid liberation,
trypsinogen
activation in
pancreatitis
Cathepsin C Aminodipeptidase Ubiquitous Defects in activation [226]
(dipeptidyl of granzymes and
peptidase I) serine proteases
Cathepsin F Endopeptidase Ubiquitous Accumulation of [227]
lipofuscin in neurons
and
neurodegeneration
Cathepsin H Endopeptidase, Ubiquitous Impaired pulmonary [228]
aminopeptidase surfactant protein B
Cathepsin K Endopeptidase Mainly in Osteopetrosis, [91, 224]
(cathepsin O2) osteoclasts, deposition of bone
ovary matrix, exhibits
clinical features of
pycnodysostosis,
reduced
thyroglobulin
processing
(continued)
11 Physiological and Pathological Functions of Cysteine Cathepsins 221
Table 11.1 (continued)
Phenotype of null
Cathepsin Catalytic activity Location mice References
Cathepsin L Endopeptidase Ubiquitous Reduced CD4+ T [13, 224,
cell, epidermal 229, 230]
hyperplasia, periodic
hair loss, acanthosis,
hyperkeratosis,
dilated
cardiomyopathy,
impaired enkephalin
processing, reduced
thyroglobulin
processing, abnormal
bone development
Cathepsin O Endopeptidase Ubiquitous Normal [131]
Cathepsin S Endopeptidase Mainly in Impaired MHC class [131]
APC I and II and CD 1
antigen presentation
Cathepsin V Endopeptidase Thymus and Normal [13]
(cathepsin L2) testis,
cornea,
keratinocytes
Cathepsin X Endopeptidase, Ubiquitous Not reported [131]
(cathepsin Z) carboxymonopeptidase
Cathepsin W Endopeptidase Cytotoxic Immune defects [231]
(lymphomain) lymphocytes
The primary structure of all cysteine cathepsins consists of a signal peptide, propep-
tide, and catalytically active mature functional enzyme [3] (Fig. 11.2). Cathepsins
are synthesized on rough endoplasmic reticulum (ER) as pre-proenzymes and con-
tain a 10–20 amino acid-long signal peptide required for their entry into the lumen
of ER. The signal sequence is then proteolytically cleaved by the signal peptidases
in ER followed by the glycosylation of proenzymes in ER trans-Golgi network
(TGN) [3]. Proteolytically inactive procathepsins possess a variable N-terminal pro-
region which is 36-amino acid long in cathepsin X to 251 amino acids for cathepsin
F. Propeptide acts as a potent reversible auto-inhibitor of the mature enzyme and
prevents inappropriate activation of the catalytically functional cathepsins [14].
Maturation of zymogens can be a pH-dependent autocatalytic event or requires pro-
cessing by other proteases, namely, pepsin, legumain, and cathepsin D occurring in
acidic endo-/lysosomal compartments [15]. These released propeptides retain their
inhibitory function and are degraded by surrounding proteases after performing
their task [16]. Moreover, the propeptide acquires its structural confirmation prior to
the remaining part of the enzyme and thus acts as a chaperone for the proper folding
of active cathepsin. In addition, the prodomain also facilitates the endosomal/
222 M. Manchanda et al.
Fig. 11.2 Primary structure of cysteine cathepsins. The primary structure of all cysteine
cathepsins consists of a signal peptide, propeptide, and catalytically active mature functional
enzyme. Cysteine cathepsins have been classified in three categories (“L like,” “B like,” and “F
like”). The members in each category have been arranged in the increasing order of the length of
their proregion
Fig. 11.3 Schematic representation of substrate-binding subsites in the active-site cleft and
catalytic activities of different cysteine cathepsins. Structural features determine the enzyme-
substrate interaction and hence the catalytic activity of cysteine cathepsins. Substrate-binding sites
of endopeptidase cathepsins F, O, S, K, V, L, and W (top) and exopeptidase cathepsins B, C, H, and
X (left, a diaminopeptidase and, right, a dicarboxypeptidase) have been diagrammatically repre-
sented. Peptide-binding subsites within the active site of cathepsins have been denoted as S (S4 to
S3′). Each of these subsites interacts with seven different residues of the peptide/substrate, desig-
nated as P (P4 to P3′). Substrate-binding subsites N- terminus to the scissile peptide bond have
been labeled as S1–S4 (non-primed sites) and those located on the C-terminal side as S1′–S3′
(primed sites). Amino acid binding to these sites has been designated as P1–P4 and P1′–P3′,
respectively. In endopeptidases, all the subsites of active site accommodate the substrate amino
acid residues and are cleaved at the scissile bond marked by an arrow. Additional structural fea-
tures in exopeptidases (occluding loop of cathepsin B, mini-loop in cathepsin X, mini-chain in
cathepsin H, and exclusion domain of cathepsin C) restrict the access of the substrate to C- (car-
boxypeptidases) and N- terminal (aminopeptidases) subsites, respectively
positioned within the R-domain [16]. Catalytic triad consists of negatively charged
thiolate ion (Cys25), positively charged imidazolium ion (His159), and an aspara-
gine or aspartic acid residue (Asn175) required for the activation of imidazolium
ring [19]. All cysteine proteases perform proton-transfer catalysis using Cys residue
as a nucleophile and His residue as a proton donor [16]. Nucleophilic cysteine
attacks the carbonyl carbon atom of the peptide bond that generates the tetrahedral
thioester transition intermediate which bears the negative charge on the carbonyl
oxygen (oxyanion) [18]. Negatively charged ion pair is also stabilized by helix
microdipole formed by catalytic Cys25 located at the N-terminus of α-helix and
preceding Glu19. These two residues also stabilize the transient tetrahedral interme-
diate by H-bond interactions and form an oxyanion hole. After the collapse of tetra-
hedral intermediate, a proton is transferred from the positively charged His159 to
the amino group of the cleaved peptide bond which results in the release of amine
components [19].
Catalytic activity of cysteine cathepsins varies depending upon the enzyme-
substrate interaction. As shown in Fig. 11.3, the substrate-binding pocket of these
enzymes contains seven subsites, four on one side of the Cys25 and the remaining
224 M. Manchanda et al.
three on its other side. Amino acid residue of the substrate at the N-terminal of the
scissile bond and the subsites of enzyme to which they bind are referred to as non-
primed, while the amino acid residue of the substrate located on the C-terminal side
of the scissile bond and their corresponding binding sites in the active cleft of the
enzyme are termed as primed sites (Fig. 11.3). These binding subsites are denoted
as S (S4 to S3′) extending over a 25°A long domain of the protease that interacts
with seven residues of the peptide/substrate, designated as P (P4 to P3’) [20].
Structural analysis revealed that S2, S1, and S1′ are the well-characterized binding
sites in cysteine cathepsin where S2 site situated in the groove is occupied by hydro-
phobic and aromatic amino acids. Insights on the enzyme-inhibitor complexes
revealed that substrate residues P2, P1, and P1’ fit well into these binding sites and
hence act as major determinants of substrate selectivity. The binding area between
the substrate residues with the enzyme is further broadened by the presence of addi-
tional subsites S4, S3, S2′, and S3′ [21]. The substrate-binding site of endopepti-
dases along with two loops of L-domain binds specifically with the amino acid
residues at P3, P1, and P2 position of the peptide targeted for degradation. Also resi-
dues at P2 and P1’ positions of the substrate interact with amino acid residues of
R-domain loops of the enzyme [22]. The amino acid residue at the P2 position of the
substrate binds with both L- and R-domains and S2 pocket of the enzyme through
hydrogen bonding and therefore determines its ability to bind the active site [16].
Cysteine cathepsins have a highly structured extended propeptide which is held
in an opposite orientation over the substrate-binding cleft and thereby, physically
blocks enzyme-substrate interaction [19]. This propeptide interacts non-covalently
with the active site of all cysteine cathepsins except cathepsin X, where a disulfide
linkage between the cysteine residue in the active site and the proregion holds pro-
peptide over the substrate-binding pocket of the proenzyme [13]. Analysis of crystal
structures of endopeptidase cathepsins K, L, S, F, V, O, and W revealed that active-
site cleft is extended along the interface of L- and R- domains [21]. Substrate access
to the active-site cleft of exopeptidases is restricted due to the additional structural
elements (Fig. 11.3) such as a mini-loop of cathepsin X [23] and occluding loop of
cathepsin B [24]. These loops block the binding of substrate at the C-terminus
(primed side) of the active-site cleft limiting the access of substrate residues [25].
Dipeptidyl cathepsin B and monopeptidyl cathepsin X cleave the amino acid resi-
dues from the C-terminal of the substrate by using side-chain histidine residue(s) to
position the negatively charged carboxylic group of the peptidyl substrate at the
cleavage site. Cathepsin B contains an insertion of an 18-residue-long occluding
loop (Pro107, Asp124) and utilizes the adjacent imidazolium rings of His110 and
His111 to bind the carboxyl group of the C-terminal residue of the substrate [24].
However, the occluding loop of cathepsin B is very flexible as it gets displaced from
the substrate-binding site under the acidic pH, and therefore, it also exhibits endo-
peptidase activity [22]. The proregion of monocarboxypeptidase cathepsin X con-
tains three-residue-long “mini-loops” (between His23 and Tyr27) that extend toward
the active-site cleft and influence the access of substrate to the S’ binding region of
the protease. Binding of the carboxylate group of P1’ residue is favored by H-bond
interactions with His23, Tyr27, and Trp202 in the S1′ region of active-site cleft [23].
11 Physiological and Pathological Functions of Cysteine Cathepsins 225
11.4 Classification
Cathepsins have been classified on the basis of their structure, active site, and sub-
strate specificity into three different categories: (1) aspartic proteases (cathepsins D
and E), (2) serine proteases (cathepsins A and G), and (3) cysteine proteases consist-
ing of 11 lysosomal cathepsins (B, C, F, H, L, K, O, S, V, X, and W) [29]. Papain-
like proteases are the most abundant among the cysteine proteases belonging to the
C1A (clan CA) family [14]. Members of cysteine cathepsins exhibit diverse cata-
lytic activity, and a majority of them are endopeptidases (F, O, S, K, V, L, and W)
with wide variations in their substrate specificity. Additionally, cathepsins H and B
possess both endo- and exopeptidase activities, whereas cathepsins C and X are
exclusively amino- and carboxypeptidase, respectively [30]. Based on the length
and amino acid sequence of their proregion cysteine, cathepsins have been further
226 M. Manchanda et al.
D Lysosome
C Caveolae A Late
Mitochondria endosome
Early H+
endosome
B
TGN
E Leaky
Lysosome
RER
Cathepsin
Truncated Cathepsin
Man-6-P
Fig. 11.4 Synthesis, sorting, and subcellular localization of cathepsins. Cysteine cathepsins
are synthesized as pre-proenzymes having N-terminal signal peptide that guides entry of the poly-
peptide chain into the ER. Signal peptide is removed and three-dimensional structural confirmation
is attained with the help of the proregion in the ER. Procathepsin undergoes disulfide bond forma-
tion and glycosylation with high mannose glycans that are later targeted to the TGN network.
Procathepsins are then tagged with M6P, which is used to target the protein to the endosomal/
lysosomal compartment through M6PR. Activation of cathepsins occurs either by the low pH of
the endosomes or through proteolytic processing by other protease and is transformed into
disulfide-linked heavy and light chains in the lysosomes (A). Leakage of lysosomal proteases can
trigger the cell death pathways (B). A portion of the cathepsins that escape the phosphorylation of
mannose residues pass through the exocytosis into extracellular compartments. Cysteine cathep-
sins being intracellular proteases are usually but not exclusively localized in the acidic and reduc-
ing endo-lysosomal vesicles. Relatively small but a significant portion (approximately 5%) of
cathepsins is secreted as proenzyme from the TGN into the extracellular milieu (C). Cathepsins are
either expressed by the transformed cells on their cell surface (caveolae) or secreted into the extra-
cellular locations for tumor invasion and metastasis (D). Truncated cathepsins lacking the signal
peptide as a result of alternative splicing and exon skipping are also detected in the unusual loca-
tions such as the nucleus or mitochondrial matrix (E)
Secretion of active cathepsin from epithelial cells is required for proenzyme pro-
cessing and ECM remodeling in the pericellular environment [39, 40]. Cathepsins
are either expressed by the transformed cells on their cell surface or secreted into the
extracellular locations for tumor invasion and metastasis. Localization of cathepsin
228 M. Manchanda et al.
11.6 R
egulation of Expression and Activity of Cysteine
Cathepsins
The rate of transcription which plays a key role in the regulation of gene expression
is determined by the strength of the promoter. Promoters of both cathepsins L and
B have high G + C content, lack canonical TATA box, and contain binding sites for
several transcription factors including multiple Sp1 binding sites which resemble
the features of housekeeping genes [52–55]. Both these cathepsins are overex-
pressed in mouse and human tumors as a result of binding of Sp1 and Sp3 to the
GC-rich regions of their promoter [53, 55–57]. Furthermore, the binding sites for
NF-Y, Sp1, and Sp3 are essential for the transcription of the gene encoding human
cathepsin L in melanoma cells [58]. The expression of cathepsin L is upregulated by
several growth factors, proinflammatory cytokines, oncogenes, and tumor promot-
ers [59–61]. Angiogenesis-promoting vascular endothelial growth factor (VEGF)
also upregulates cathepsin L expression in glioblastoma cells. A 47 base pair region
containing Sp1 and AP4 motifs plays a critical role in conferring VEGF responsive-
ness to cathepsin L promoter [60]. Interestingly, treatment of peritoneal macro-
phages with interferon (IFN)-α decreases the expression of cathepsin L without
affecting the levels of cathepsin S [62]. The regulatory region of human cathepsin L
contains multiple CpG islands which are methylated in lymphoma cells, thereby
silencing its expression in this malignancy [58]. Consistent with this observation,
promoter methylation downregulates the expression of cathepsin L in chronic
myelogenous leukemia patients during accelerated phase/blast crisis [63].
Erythroblast transformation-specific (Ets) family transcription factors regulate the
transcription of cathepsins K and C during bone and breast cancer progression [52].
Cathepsins are encoded by multiple mRNAs that contain variable length of 5′
and 3′ untranslated regions (UTRs) as a result of alternate splicing [64]. For instance,
human cathepsin L (hCATL) is encoded by at least five mRNA species, namely,
hCATL A, AI, AII, and AIII and B [65]. Among them, the first four are generated by
the alternate splicing of the same primary transcript. The full length of exon I (280
nucleotides) is retained in the transcript variant hCATL A, whereas 27, 90, and 145
nucleotides are spliced out from the 3’ end of this exon to generate AI, AII, and AIII
variants, respectively. HCATL AIII, the shortest variant, is most efficiently trans-
lated, whereas the longer mRNA species exhibit lower translational efficiencies [45,
65]. The fifth mRNA species hCATL B is transcribed from an alternate promoter
located in the first intron of human cathepsin L gene. The alternate promoter like its
proximal counterpart also lacks a canonical TATA box. However, it contains puta-
tive binding sites for several transcription factors like AP1, AP4, GATA-1, Lmo2,
NF-kappa B, etc. [66]. The most efficiently translated cathepsin L transcript AIII is
abundantly expressed in cancers which may explain the elevated levels of the
230 M. Manchanda et al.
protease in malignancy [65]. In line with this, splice variant of cathepsin B lacking
Alu sequence containing exon 2 is translated more efficiently compared to other
variant(s) which may be responsible for overexpression of cathepsin B in malignan-
cies of the breast, colon, prostate, and brain [52, 67, 68]. Thus, alternative splicing
of cathepsin L and B pre-mRNA results in differential expression of these proteases
in different cell types. Hence, understanding of these molecular mechanisms is
essential to delineate the role of different mRNA variants in tumors.
Interestingly, the longest human cathepsin L splice variant A contains a func-
tional internal ribosomal entry site (IRES) which is involved in overexpression of
cathepsin L by hypoxia [69–71]. This IRES besides providing a translational con-
trol may also account for discrete mitochondrial and nuclear localization of the
cathepsin L which lacks the signal peptide at the N-terminus [13].
11.6.3 pH
Lysosomal cathepsins function optimally at low pH (pH < 4.5) and have narrow
functional pH, ranging from 4 to 6 [62]. Acidic pH of lysosomal compartments
(pH < 5) destabilizes the interaction of the inhibitory propeptide and the active site
and thus facilitates the movement of propeptide from the normal position [14].
Furthermore, proenzyme after conformational change in propeptide structure exhib-
its very less catalytic activity that is just enough for the activation of other proca-
thepsins and hence initiates the bimolecular chain reaction [73]. Cathepsins not
only exhibit enzymatic activity at acidic pH but several of them retain significant
activity over a wide range of pH [18]. For example, cathepsin S is stable at pH rang-
ing from 4.5 to 8.0 with pH optima of 6.0. This perhaps facilitates its role in antigen
processing and presentation within less acidic compartments [74]. Ubiquitously
expressed cathepsin B also exhibits stability at neutral pH 7 with half-life of 15 min
and optima in the range of 4–6 presumably accounting for its presence in distinct
locations [75], while other cathepsins such as L, H, K, V, and F retain only partial
activity at neutral pH and, therefore, are less active outside lysosomes [76].
11 Physiological and Pathological Functions of Cysteine Cathepsins 231
The most critical and preeminent control of cathepsins that has been spilled into the
cytosol is accomplished by their endogenous protein inhibitors including cystatins,
thyropins, and serpins [16]. These inhibitors based on their binding potencies and
physiological role have been divided into emergency and regulatory inhibitors [77].
Emergency inhibitors exhibit competitive and reversible binding and rapidly form a
complex with enzyme that remains associated for longer duration. This class of
inhibitors is more abundant than the enzymes and is localized in a distinct compart-
ment from that of the target enzyme [78]. Certain delayed inhibitors such as blood
plasma serpins are converted to emergency inhibitor by heparin. In contrast, regula-
tory type of inhibitors not only blocks but also modulates the proteolytic activity
[79]. Cystatins were first found in chicken egg white and exhibited their inhibitory
effects against only papain-related proteases [78]. These proteins have been well
characterized and belong to the MEROPS family I25. In humans, there are 12 mem-
bers in cystatin superfamily that have been classified into three families including
stefins (type I), cystatins (type II), and kininogen (type III) [15]. Cystatins are low-
molecular-weight (10–13 kDa) competitive inhibitors that interact reversibly with
the lysosomal cysteine proteases and prevent the inadvertent tissue damage by regu-
lating the misplaced cathepsins. Cystatins are not very selective, and the picomolar
amount is enough to inhibit the endopeptidases [80]. An inhibitory domain of cys-
tatin is composed of five antiparallel β-sheets enfolded around a central α-helix. The
binding groove of papain is blocked by the wedge shape formed by flexible
N-terminal and two β-hairpin loops of cystatin. The two hairpin loops of cystatins
dock with the “prime” subsites of the substrate-binding sites, whereas the elongated
N-terminus interacts with the “unprimed” subsites [78].
11.6.4.1 Stefins
Stefins are present in the cytosolic compartments along with the low physiological
concentration in serum and are potent intracellular inhibitors due to their stability in
a wide pH range [16]. They exist as non-glycosylated single chain of approximately
100 amino acid residues [77]. In humans, this class comprises of two members,
cystatins A and B. Human cystatin A has been localized in the skin epithelium and
blood cells. On the contrary, human cystatin B is widely distributed mainly in the
cell cytoplasm. Interestingly, this inhibitor has also been detected in the nucleus
where it regulates the cathepsin L-mediated processing of histone proteins [15].
11.6.4.2 Cystatins
Type II cystatins are the members of MEROPS subfamily I25B consisting of cys-
tatins C, D, E/M, F, G, S, SN, and SA [15]. Unlike the stefins, these inhibitors are
synthesized as pro-inhibitors with 20–26 amino acid residue signal sequence
required for their secretion into extracellular milieu [79]. Similar to type I cystatins,
they are usually non-glycosylated (except cystatins F and E/M), single-chain
polypeptides but larger in size having 120 amino acid residues. The most salient
feature is the presence of Pro-Trp and two conserved disulfide bonds at the
232 M. Manchanda et al.
C-terminal segment where the respective cysteine residues are held 10–20 amino
acids apart. The cystatins C, D, S, SA, and SN share >50% sequence homology,
while cystatins E/M, F, and G are only <35% identical in sequence [81]. They are
present in most biological fluids [77]. Cystatins S and SA are found in the saliva and
seminal fluids; cystatin SN is present in the saliva and tears [81]. Human cystatin E
is present in the amniotic fluid [15], and cystatin F also called as “leukocystatin” is
primarily expressed by the immune cells and the spleen [77]. Cystatin F-deficient
mice exhibit altered regulation of cysteine proteases due to impaired granule bio-
genesis in eosinophils resulting in defective immune response to combat the patho-
gens [82]. Cystatin G is mainly expressed in epididymal and spermatogenic cells
[15]. Cystatin C (also known as post-γ-globulin) is one of the most thoroughly stud-
ied human cystatins. It is secreted into all body fluids, and its concentration is par-
ticularly high in seminal plasma and cerebrospinal fluid [81]. Cystatin C displays
broad-spectrum selectivity against all the papain-like proteases and cannot distin-
guish endo- or exopeptidases. Human cystatin C is a potent “emergency inhibitor”
which rapidly neutralizes the activity of lysosomal cathepsins that escaped in the
extracellular milieu [15].
11.6.4.3 Kininogens
Type III cystatins belong to I25C subfamily and comprise of three members includ-
ing high (HMWK, approximately 120 kDa) and low (LMWK, approximately 60
kDa) molecular weight kininogens in humans [83]. However, kininogen T has only
been described in rats [81]. These glycosylated inhibitors are synthesized as pre-
proteins. Both HMWK and LMWK are synthesized in the liver and are encoded by
splice variants generated as a result of alternate splicing of the same primary tran-
script. Human kininogens contain N-terminal heavy and C-terminal light chain con-
nected by a disulfide bond. Three cystatin-like domains (D1–D3) are present in their
heavy chain. However, only pentapeptide (QVVAG)-containing domains D2 and
D3 are able to inhibit cysteine proteases [15]. Cathepsins L, S, and H are strongly
inhibited by type III human cystatins, whereas cathepsin B is only weakly inhibited.
Higher concentration of kininogen is found in hepatocytes, spleen, and dermato-
phytes [81]. Both human kininogens are present in equal amount in the blood and
thus act as a potent inhibitor of cysteine proteases in circulation [77].
Assigning the specific roles to the cathepsin has been a challenging task owing to
the redundancy in their functions. However, lysosomal cysteine cathepsins perform
various important physiological functions as their deficiency in mice results in vari-
ous hereditary disorders. Traditionally, these peptidases were only thought to par-
ticipate in nonspecific proteolysis within the lysosome. However, this view is
rapidly changing, and these proteases are now being implicated in specific biologi-
cal roles.
11 Physiological and Pathological Functions of Cysteine Cathepsins 233
ECM occupies the void space between the cells and provides the meshwork for
holding the cells embedded within the tissues. This noncellular component is also
an important mediator for cross talk between the cells, angiogenesis, wound heal-
ing, bone remodeling, and many other physiological processes. ECM is mainly
composed of fibrous proteins (elastin, collagen, laminins, and fibronectins), proteo-
glycans, water, and minerals. Deregulated synthesis and remodeling of ECM have
been attributed to fibro-proliferative disorders and cancers affecting almost every
organ of the body. In healthy tissue, ECM homeostasis is mainly monitored by
matrix-degrading proteases like MMPs, serine proteases, and cysteine cathepsins
[84]. ECM degradation is not necessarily restricted to the extracellular milieu, but a
number of ECM proteins are also acquired intracellularly by endocytosis [14]. ECM
proteins such as fibronectin; laminin; elastin collagen types I, IV, and XVII; and
tenascin C are well-characterized substrates for cysteine cathepsins [85]. Earlier, the
responsibility of bulk degradation of matrix proteins was solely assigned to MMPs.
However, this concept was challenged as treatment with MMP inhibitor does not
confer protection against ECM-related pathologies such as cancer, atherosclerosis,
and many other fibrotic conditions. Interestingly, pan-inhibitor E64D was able to
overcome the bone resorption by inhibiting cartilage degradation in osteoporosis
[14] and thus highlighted the specialized role of cathepsins in the maintenance of
tissue architecture. Collagen is the most abundant and viscoelastic structural com-
ponent of ECM that exists in right-handed triple superhelical confirmation. Highly
abundant forms of collagen, i.e., types I and II, are hydrolyzed only by specific
proteases such as MMP-1, MMP-8, MMP-13 and the cysteine protease, cathepsin K
[21]. The presence of additional C-terminal hemopexin domain in MMPs equips
them with the ability to unwind the collagen helical structure and hydrolyzes the
peptide bond in collagen helix into three-fourths and one-fourth telopeptides which
are in turn cleaved by other tissue proteinases [14]. In contrast, cathepsins lacking
such special structural features can only cleave the non-helical telopeptide regions
of collagens except for cathepsin K [86]. Cathepsin K contains additional GAG
binding site located opposite to the substrate-binding site which forms an oligo-
meric complex with ECM. Cathepsin K interaction with chondroitin sulfate resem-
bles “beads on a string-like arrangement” [87]. It cleaves within the collagen helix
at multiple sites as opposed to MMPs that specifically attack the peptide bond
between 775 and 776 amino acid residues [14]. Cathepsin V, on the other hand, has
the ability to form the complex with GAGs but lacks the collagenolytic activity.
Hence, the collagenolytic activity cannot alone be assigned to the formation of
cathepsin-GAG complexes but to its unique ability to accommodate proline resi-
dues in the P1 and P2 positions [88]. However, cathepsin K-like variant of cathepsin
L generated by mutating S2 subsite possessed the similar affinity for type I collagen
but lacked the collagenolytic activity supporting the critical role of both active-site
and GAG complex formation [89]. Cathepsin K along with cathepsins B, L, and S
degrades the telopeptide region of the collagen into monomers [21]. Cathepsin K is
overexpressed in osteoarthritis and rheumatoid arthritis patients, and its specific
234 M. Manchanda et al.
Cell death is a biological process by which unwanted cells are eliminated through
apoptosis, necrosis, necroptosis, and autophagy [104]. All these pathways some-
times work simultaneously or in tandem to decide the cell fate [105]. Lysosomal
damage and subsequent drainage of its components in the cytoplasm can trigger the
cell death pathway [106, 107]. Lysosomal membrane permeabilization (LMP) can
progress to apoptosis, necrosis, autophagy, or necroptosis depending upon the type
of cellular injury, leakage of the cathepsins, as well as the expression of their inhibi-
tors [80, 108]. LMP is triggered by various agents such as reactive oxygen species
(ROS), lipid metabolites, lysosomotropic compounds, as well as by proapoptotic
factors such as Bax [109]. ROS such as hydrogen peroxide enter the lysosomes and
get transformed into highly reactive hydrogen oxide free radical in a step that is
facilitated by lysosomal iron that disrupts the lysosomal cell membrane by lipid
peroxidation [108].
Apoptosis (derived from the Greek word meaning fall off) is a programmed cell
death characterized by ATP-dependent biochemical pathways, defined morphologi-
cal changes, and activation of executioner caspases in the dying cell. The apoptotic
response is induced by either an intrinsic or extrinsic pathway based upon the source
of cellular stress. Microinjecting cathepsins into the cytoplasm can experimentally
induce apoptosis [110]. LMP can either be an initiating event leading to the caspase
signaling cascade or it can just be a supportive event amplifying the death signals
independent of caspase [111]. Release of cathepsins as a result of LMP triggers the
further downstream events of an intrinsic apoptotic pathway. Cathepsins B, H, L, S,
and K have been shown to activate the Bcl-2 family member proapoptotic Bid to
truncated (t-Bid) form which then facilitates the oligomerization of Bax and Bak
proteins [80]. This proapoptotic complex then translocates to mitochondria and
induces mitochondrial outer membrane permeabilization (MOMP) by forming the
pores [108, 112]. Cysteine cathepsins further facilitate apoptosis by degrading anti-
apoptotic proteins such as Bcl-2 and Bcl-xL. They can also directly activate cas-
pase-3, caspase-7, and caspase-9 as well as degrade X-chromosome-linked inhibitor
of apoptosis (XIAP) and thereby expand the downstream apoptotic cascade [113].
The proapoptotic role of cathepsin B is further supported by the fact that genetic or
pharmacological inhibition of this protease reduced apoptosis in various experimen-
tal models of liver injury including tumor necrosis factor (TNF)-α-mediated hepa-
tocyte apoptosis [114–117], obstructive cholestasis [118], hepatic
ischemia-reperfusion injury [119], and lipotoxicity [120]. Treatment with cholesta-
sis causing toxic bile salt, glycochenodeoxycholate, induces LMP, cathepsin B
translocation, caspase activation, and cell death in rat hepatocytes. Caspase inhibi-
tors and overexpression of the cathepsin inhibitor cystatin A reduced this toxicity
[121]. Cathepsin B translocation from lysosomes to the cytosol causes mitochon-
drial dysfunction and cytochrome c release which in turn induce apoptosis [120].
Redistribution of cathepsins to cytosol may activate classical mitochondrial path-
way of apoptosis. This redistribution may also induce caspase-dependent or caspase-
independent apoptosis [80]. LMP occurs as an early event followed by the release
236 M. Manchanda et al.
Various cysteine cathepsins play important roles in MHC class II antigen presenta-
tion by degrading the antigenic peptides and processing of invariant chain (Ii) [130].
Specific cathepsins comprising L, S, F, and V are expressed by different types of
antigen-presenting cells (APCs) for the maturation of their antigenic complexes
[16]. Cathepsin S is expressed by most of the antigen-presenting cells including
dendritic cells, macrophages, and B cells. Spleen, lymph nodes, and vascular
smooth muscle cells also express high levels of cathepsin S [74]. It is the most
potent protease involved in immune response due to its activity over the broad pH
range. Interestingly, cathepsin S-null mice exhibit impairment in the invariant chain
(Ii) processing leading to deposition of MHC II complexes in the endosomes [2].
High levels of cathepsin S are secreted by the macrophages during various patho-
logical conditions like rheumatoid arthritis, atherosclerosis, and bronchial asthma
[62]. Due to the specific role of this protease in modulating the immune response, it
is considered a major therapeutic target, and its commercial inhibitor “celera” has
reached phase I clinical trial for the treatment of psoriasis [131]. Loss of cathepsin
L in mice results in impaired Ii processing to the class II-associated invariant chain
(CLIP) in the cortical thymic epithelial cells (cTEC), thereby confirming the role of
this protease in antigen presentation [2]. This incomplete processing of Ii fragment
in cathepsin L-deficient mice results in accumulation of I-Ab-bound p-12 and p18–
22 Ii fragments leading to the defect in thymic selection of CD4+ T cells, whereas
11 Physiological and Pathological Functions of Cysteine Cathepsins 237
the cathepsin L-deficient cTECs and splenic APCs do not show any such accumula-
tion of MHC II-bound Ii fragments [132]. However, cathepsin V that is exclusively
present in the thymus and testis performs the same function in humans [16]. The
p41 isoform of Ii specifically inhibits cathepsin L in APC where cathepsin S is the
major protease involved in antigen presentation [131]. Cathepsins X, B, and H may
also participate in antigen presentation, but they are not essential [133].
11.8.1 Cancer
with hormone receptor-positive breast cancer, and elevated levels of this protease
suggest the poor outcome of the disease [147]. Levels of cathepsin H are also high
in the serum and tissues of breast cancer patients [148]. Overexpression of cathep-
sins B and L has been reported in the serum and tissues of patients suffering from
ovarian cancer [149, 150]. Similarly, cathepsins B and L are also increased in atypi-
cal invasive and aggressive meningiomas [151], gliomas [152], and hepatocellular
carcinoma [139] compared to their benign counterparts and may serve as potential
diagnostic markers. Expression of cathepsins L and B increases in parallel with
histological grade of pancreatic adenocarcinoma and correlates with short overall
survival after the surgical resection [134]. Increased levels of cathepsins L and B in
pediatric acute myeloid leukemia patients are strong markers for poor prognosis of
the disease [153, 154]. However, cathepsins B and H and plasminogen activator
inhibitor (PAI)-1 are considered more sensitive biomarkers and have a major prog-
nostic value in colorectal cancer [155]. As in other cancers, cathepsins B, L, H, and
S are also upregulated in lung cancer patients [143]. Enhanced levels of cathepsin B
correlate with hematogenous and intrapulmonary metastases of lung cancer cells
[156]. Moreover, tumors and tumor cell-infiltrated lymph nodes have high cathepsin
B activity which may be used as the predictor of poor prognosis in lung carcinoma
[157]. Similarly, stromal fibroblasts in squamous cell carcinoma of the skin overex-
press cathepsin K, which in turn promotes tumor invasion and metastasis by ECM
degradation and vascularization [158]. Overexpression of cathepsins B and L is also
reported in gall bladder cancer patients [159].
Formation of new blood vessels enhances the tumor vascularization and helps
tumor cells to reach the bloodstream and metastasize to the secondary sites. During
angiogenesis, endothelial cells proliferate, migrate, and invade the surrounding
perivascular stroma, forming tube-like structures that give rise to neocapillary net-
work. Various cysteine cathepsins such as B, L, S, and X have been implicated in
angiogenesis [19, 134, 160]. Several pro-angiogenic factors and inhibitors are also
the substrates for these cathepsins [14]. The use of the broad-spectrum inhibitor of
11 Physiological and Pathological Functions of Cysteine Cathepsins 239
cysteine cathepsins in the Rip1-Tag2 mouse model established the role of these
proteases in angiogenesis, tumor growth, and invasiveness within pancreatic islet
tumors. In these mice, genetic inhibition of cystatin C increases the formation of
vascular networks, while cathepsin S deficiency leads to impaired tumor angiogen-
esis and invasion [103]. Cathepsin S-deficient mice display defective microvessel
development despite high levels of VEGF and basic fibroblast growth factor (bFGF)
and promote angiogenesis by the degradation of anti-angiogenic peptides canstatin
and arresten [14]. Si-RNA-mediated knockdown of cathepsin S expression reduces
cell proliferation, invasion, and angiogenesis in human hepatocellular carcinoma
[161]. Similar to the findings in Rip1-Tag2 pancreatic cancer mouse model, cathep-
sin S overexpressed and secreted by both tumor and tumor-associated cells mediates
tumor growth and vascularization in the syngeneic model of colorectal cancer. Loss
of cathepsin S in these mice abrogates the formation of new blood vessels, cell
growth, and viability and thus further establishes the pro-tumorigenic role of this
protease [162]. Compared to benign tumors, increased cathepsin B levels are dif-
fusely distributed in microvessel of neoplastic prostate cancer. Cell surface-
associated overexpression of this protease is seen in highly metastatic prostate
cancer [163]. Similarly, downregulation of cathepsin B reduces the aggressiveness
and angiogenesis in gliomas [164, 165]. Cathepsin H is required for the develop-
ment of blood vessels, tumor growth, and invasion in the mouse model of pancreatic
islet cancer [166]. Although cathepsin L expressed by endothelial progenitor cell is
required for ischemia-mediated neovascularization, its role in tumor angiogenesis is
still not clear as deletion of this protease had no significant impact on the angiogen-
esis in Rip-Tag2 mice [52]. However, cathepsins L and S favor the generation of
laminin-derived pro-angiogenic factor-gamma 2 and induce the neoplastic progres-
sion [156, 167]. Anti-angiogenic effects of cathepsins L and S are mediated by
cleaving C-terminal of collagen XVIII that leads to endostatin formation [168].
Cathepsin B also contributes to angiogenesis by degrading the TIMPs which leads
to increase in the angiogenesis-promoting MMP activity [169].
Cathepsin activity and expression are mainly localized at the invasive edges of
the tumors which have been attributed to the ectodomain shedding of E-cadherin,
transmembrane proteins, and other cell surface-associated molecules enhancing the
invasion and migration of cancer cells [170]. Invasiveness of highly metastatic mel-
anoma cells is assigned to cathepsins B and L, and their downregulation and phar-
macological inhibition impair the invasive potential of human melanoma cells in
matrigel invasion assays [143]. Furthermore, overexpression of cathepsin L confers
highly invasive phenotype to nonmetastatic melanoma cells. Similarly, reduction in
cathepsins B, L, H, and S expression/activity lowers the invasive ability of glioblas-
toma cells [143]. Likewise, cathepsin B facilitates the invasion of esophageal cancer
fibroblasts, and cathepsin H performs the same role in prostate cancer [160]. It has
been proposed that cathepsin H mediates the processing of talin (actin- and β-integrin
tail-binding protein) which promotes activation of integrins and consequently
migration of prostate cancer cells [171]. Cathepsin X removes C-terminal Tyr139 of
profilin1, which abolishes its tumor-suppressor function. This cleavage also abro-
gates its ability to bind clathrin and enhance prostate cancer cell migration and
240 M. Manchanda et al.
Despite the crucial role of cysteine cathepsins in the maintenance of lung microen-
vironment, their deregulated expression and activity have been implicated in several
lung pathologies such as fibrosis, asthma, bronchopulmonary dysplasia, chronic
obstructive pulmonary disease (COPD), and silicosis [15]. Cysteine cathepsins dis-
play distinct immunostaining patterns in normal human lung tissue. Bronchial and
alveolar epithelial cells display intense staining for cathepsin K [180]. Cathepsin S
is located on the surface of ciliated cells and may favor the motility of cilia by pre-
venting unspecific binding with circulating plasma-derived proteins [181].
Cathepsins B and L are predominantly expressed in bronchial epithelial cells and
protect against airborne foreign particles and microbes. However, significant
amounts of cathepsin H are detected in macrophages, bronchial epithelial cells, and
type II pneumocytes [180]. Type II alveolar epithelial cells also express cathepsin C,
while X and S are mainly present in macrophages [182].
11 Physiological and Pathological Functions of Cysteine Cathepsins 241
In lung fibrosis, the balance between repair and inflammatory pathway is regu-
lated by multifaceted cross talk between the cells and surrounding ECM [15].
Inflammatory cells (neutrophils and macrophages) in the airway get activated in
response to the lung damage and secrete proteases along with their inhibitors which
then disturb ECM homeostasis and alter the lung architecture [15]. Fibroblasts from
patients diagnosed with pulmonary fibrosis display higher-level expression and
activity of cathepsin K than the normal lung specimens. In line with this observa-
tion, cathepsin K expression is temporally upregulated in bleomycin-induced lung
fibrosis mouse model. Cathepsin K-null mice after bleomycin treatment display
aggravated fibrosis due to increase in the ECM deposition, upregulation of α-SMA
and vimentin, and decrease in collagen degradation by fibroblasts from these mice
[182]. Similarly, exposure of experimental animals to crystalline silica particles
induces pulmonary expression of cathepsin K [15]. The role of cathepsin K in pre-
venting silicosis is further strengthened by the observation that in response to silica
particles, silica-sensitive mouse strain (C57BL/6) expresses low levels of cathepsin
K mRNA in comparison with the resistant strain (BALB/c) [15]. The anti-fibrotic
role of cathepsin K is further confirmed by its ability to degrade fibrogenic cytokine
TGF-β1 and thus diminish the ECM accumulation [183]. Bronchoalveolar lavage
fluid (BALF) from silicosis patients has high levels of active cysteine cathepsins
such as B, H, K, L, and S [184]. Interestingly, loss of cathepsins B and L lowers the
expression of α-smooth muscle actin (SMA) in the fibroblasts from idiopathic pul-
monary fibrosis patients. In addition, TGF-β1-mediated transdifferentiation of
fibroblast is prolonged after treatment with cathepsin B inhibitor. This finding is
further confirmed by the observed increase in levels of cystatin C as opposed to
cathepsin B during TGF-β1-dependent differentiation of fibroblasts. Elevated cys-
tatin C levels inhibit cathepsin B and facilitate TGF-β1-mediated pulmonary fibro-
sis [185]. Cathepsin B is also upregulated in models and patients with interstitial
lung disease. Inflammation and progression of experimental pulmonary fibrosis can
be attenuated by the use of CA-074 Me, a specific inhibitor of cathepsin B [186]. On
the contrary, reduction in the extent of bleomycin-induced lung fibrosis by cur-
cumin is associated with the induction of cathepsins K and L expression [185].
COPD includes emphysema and chronic bronchitis mainly caused by cigarette
smoking and inhalation of particulate pollutants. Levels of cathepsins B, L, and S as
well as their endogenous inhibitor cystatin C are increased in smokers afflicted with
COPD [187]. The lungs of emphysema patients express high levels of cathepsin K
[15]. Similarly, cathepsins B, L, H, K, and S are upregulated in IL-13 and IFN-γ
transgenic mouse models of emphysema. Treatment of these transgenic mice with a
common inhibitor of cysteine cathepsins attenuates lung inflammation and emphy-
sema and hence establishes the pathogenic role of these proteases in COPD [180].
Ozone-induced hyperresponsiveness and inflammation in BALB/c mice are associ-
ated with increased BAL levels of cathepsin S. Treatment of these mice with cathep-
sin S inhibitor decreased the levels of proinflammatory cytokines IL-6 and TNF-α.
These results further confirmed the role of cathepsin S in the oxidative stress-
induced airway hyperresponsiveness and suggest its utility as a potential therapeutic
target [188]. It is possible that cathepsin S mediates proteolysis of pulmonary ECM,
242 M. Manchanda et al.
fibrosis, and apoptosis, which engage Akt/GSK-3β pathways that are inactivated by
cathepsin L [199]. The balance between cysteine cathepsins and their endogenous
inhibitor cystatin C plays a vital role in the normal myocardial ECM remodeling.
Consistent with this view, hypoxia-induced cardiac failure is associated with eleva-
tion of cystatin C levels, inhibition of cathepsin B activity, and myocardial deposi-
tion of collagen and fibronectin [200]. However, cathepsin B overexpressed in
myocardial necrotic zone has been implicated in cardiac cell death [201]. In contrast
to results in experimental models, levels of cathepsins B, L, S, and K are elevated in
human dilated and hypertrophic cardiomyopathies [13]. Elevated expression of
cathepsin L is seen in human abdominal aortic aneurysm and atheromata. High
serum levels of this cathepsin show strong positive correlation with arterial stenosis
suggesting its involvement in vascular diseases [202].
Atherosclerotic lesions involving arterial wall remodeling are also associated
with high activities of elastase, collagenase, and gelatinase [203]. Cysteine cathep-
sins due to their potent elastinolytic and collagenolytic activities lead to the genesis
and rupture of atherosclerotic plaques [136]. Apo E-deficient murine atherosclerotic
lesions exhibit high levels of cathepsins B, L, and S [194]. Cathepsins K, S, and L
are overexpressed in human atherosclerotic plaques. These proteases are mainly
localized in the macrophages, smooth muscle cells, and fibrotic and lipid-rich areas
of the plaque [137]. Expression of cathepsins K and S in the vascular wall facilitates
the elastin proteolysis which aids in smooth muscle cell migration and collagen
degradation of the fibrous cap [136]. Cathepsin K and S expression in the endothe-
lium of atherosclerotic coronary arteries positively correlates with the breaks in
elastic lamina, thus substantiating their role in neovascularization of atherosclerotic
lesions [204, 205]. Pro-inflammatory cytokines such as TGF-β, IFN-γ, and IL-1β
induce cathepsin L and S expression in human aortic smooth muscle cells and mac-
rophages [137, 202, 205]. The role of cysteine proteases in the development of
atherosclerosis has been studied in various knockout mouse models. Phenotypes
associated with the knockout of these proteases in the proatherogenic genetic back-
ground have been summarized in Table 11.3. These findings highlight the patho-
genic role of cysteine cathepsins in atherosclerosis.
Table 11.3 Role of cathepsins in atherosclerosis elucidated using experimental mouse model
Genotype of mouse model Vascular phenotype
Genetic defect
Cathepsin leading to Plaque Plaque Collagen Elastin
deletion atherosclerosis size progression content breaks Macrophages Reference
K Apo E ↓ ↓ ↑ ↓ ↑ [239–241]
K LDLR = ↓ ↓ ↑ [242]
L LDLR ↓ ↓ ↓ ↓ [243]
S LDLR ↓ ↓ ↓ ↓ ↓ [241]
S Apo E ↓ ↓ ↓ [244]
244 M. Manchanda et al.
Diabetes and obesity are the most common human metabolic disorders. Apart from
other factors, cysteine proteases such as cathepsins L, S, and K have been impli-
cated in their pathogenesis [206–208]. Diabetes-prone C57BL/6J mice demon-
strated differential expression of cathepsin L in their muscle tissues compared to the
resistant strain of mice. Cathepsin L gene expression in muscle tissues inversely
correlates with plasma glucose in these mice implying its association with glucose
intolerance [207]. Interestingly, basal cathepsin L mRNA levels were found to be
comparable in the muscle biopsies from monozygotic twin pairs discordant for type
2 diabetes and control subjects. However, cathepsin L mRNA levels were reduced
by post-insulin clamp in diabetic twins that correlated with insulin-mediated glu-
cose transport [207]. Cathepsin L deficiency confers protection against the insulitis
and autoimmune type 1 diabetes (T1D) in diabetes-susceptible NOD mice [209].
Protective effects of cathepsin L deficiency seen in NOD mice have been attributed
to increase in the ratio of T regulatory (Treg) cells that attenuate autoimmune
response [208]. Similarly, deletion of cathepsins S and B conferred only partial
protection against T1D [209]. In addition to protection by Treg cells, cathepsin L
inhibition curtails the CD8+ T cells cytotoxicity by inhibiting granzyme B and hin-
ders the development of T1D [210]. Cathepsin L degrades fibronectin, insulin
receptor (IR), and insulin-like growth factor-1 receptor and plays an important role
in fat deposition and glucose tolerance [211]. Cathepsin L knockout NOD mice
have leaner phenotype with lower levels of serum glucose and insulin but accumu-
late IR-β subunits, glucose transporter, and fibronectin in their muscles accounting
for enhanced insulin sensitivity and glucose utilization [211]. High cathepsin L lev-
els in obese and diabetic patients further supported its involvement in the metabolic
disorders [211]. Inhibition of the cysteine cathepsins B and L results in impaired
autophagy, and accumulation of cathepsins B, D, and L pro-forms and triggers the
caspase-dependent β-cell apoptosis cultured in hyperglycemic conditions [212].
Cathepsin S and H levels strongly correlate with pro-inflammatory cytokines in
the tear glands of NOD mouse model of Sjögren’s syndrome [213]. Compared to
healthy subjects, patients with type 2 diabetes have increased serum cathepsin S
levels [214]. This finding has been further corroborated by Jobs and coworkers, who
reported that elevation in serum cathepsin S levels leads to decreased insulin sensi-
tivity and higher susceptibility to develop type 2 diabetes [215]. Cathepsin S is
overexpressed in adipose tissue of obese patients and therefore may serve as a bio-
marker for adiposity [216]. Its role as a biomarker is further corroborated by the
observation that weight loss and amelioration of glycemic status following bariatric
surgery lead to reduced serum cathepsin S levels [217]. Diet-induced obesity in
cathepsin S −/− mice is associated with low blood and hepatic glucose and better
glucose tolerance compared to wild-type littermates [218]. Therefore, cathepsin S
inhibition may be of relevance in delaying the onset of diabetic phenotype.
Cathepsin K also is implicated in adipocyte differentiation and glucose metabo-
lism [219]. White adipose tissue of obese db/db mice expresses higher cathepsin K
11 Physiological and Pathological Functions of Cysteine Cathepsins 245
Cathepsins were long thought to remain functional only within the confines of
acidic lysosomal compartments, which implied that their role was limited to the
intracellular protein degradation and turnover. On the contrary, a number of studies
emphasized the role of cathepsin-mediated proteolysis in extracellular matrix deg-
radation. Rampant expression, activity, or cytosolic escape of the lysosomal
enzymes results in number of human pathologies. Cathepsins are usually overex-
pressed in cancer, and a majority of them are either secreted into the extracellular
tumor microenvironment or targeted to the surface of malignant cells. Cathepsins B,
L, and S have been used for diagnosis and predicting the outcome of chemotherapy
in various malignancies. Several clinical conditions such as disorders of the lungs,
heart, and kidney are caused due the overexpression and dysregulated ECM degra-
dation. Specific inhibitors of these proteases may potentially be used to protect
against the detrimental effects of their nonspecific proteolysis. However, targeting
them to the specific site remains a challenge.
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Role of Serine Proteases and Inhibitors
in Cancer 12
Nitesh Kumar Poddar, Sanjeev Kumar Maurya,
and Vanshika Saxena
Abstract
Serine proteases, the largest human protease family, are found in many key
developmental and physiological processes in the biological system. Protease
signalling pathways are stringently controlled, and deregulation of proteolytic
activity results in the degradation of extracellular matrix which plays a major
role in cancer progression. The Type II transmembrane serine protease, hepsin,
matriptase-2 and TMPRSS4, and secreted serine protease, urokinase plasmino-
gen activator (uPA), kallikreins and HtrA, are closely related to cancer-associated
proteases and also involved in perturbation of uPA plasminogen system, matrix
metalloproteases (MMPs), upregulation of adhesion molecules like integrin fam-
ily, activation of intracellular signalling cascade, inhibition of apoptosis pathway
in various types of cancers which causes cell proliferation, invasion and metasta-
sis. Serpin, an endogenous serine protease inhibitor, regulates the homeostasis by
maintaining a delicate balance with the serine protease and prevents the process
of invasion and metastasis of cancer cells thus inhibiting tumour growth. This
chapter focuses on the role of serine proteases and their inhibitors in different
types of tumours associated with cancer prognostication and therapy.
Keywords
Type II transmembrane serine protease • Extracellular matrix • Matrix metallo-
proteases (MMPs) • Secreted serine proteases • Therapy • Tumour • Serpin •
Urokinase plasminogen activator (uPA) • Plasminogen system
12.1 Introduction
Proteases occupy a pivotal position among biological molecules required for the
physiological roles in living systems and commercial biotechnology markets and
medical fields. They are called proteolytic enzymes or systemic enzymes, and their
catalytic function is to hydrolyse the peptide bond that links amino acids together in
a polypeptide chain. These are also called peptidase or proteinase (Fig. 12.1).
A large variety of proteases are found in intracellular or extracellular space in all
eukaryotic and prokaryotic cells. They are mainly located in different organelles of
eukaryotic cells such as the cytosol, mitochondria, vacuoles, lysosomes and endo-
plasmic reticulum. These intracellular proteases are involved in many important
functions such as regulating synthesis, activation and proteolysis of proteins. The
extracellular proteases are mostly secreted in the gastrointestinal tract of animals or
involve in the blood coagulation and complement cascade events. Consequently,
different organisms or different tissues have different sets of proteases.
Fig. 12.1 Hydrolysis of peptide bond of protein by protease in the presence of water
12 Role of Serine Proteases and Inhibitors in Cancer 259
According to the MEROPS database, about 33% are serine proteases which are
categorised into 40 families and 13 clans in both eukaryotes and prokaryotes [7, 8].
Usually, the family name is derived from the nucleophilic Ser present in the
active site of the enzyme. The Ser amino acid of the active site cleaves the carbonyl
terminus of the peptide bond to form an acyl-enzyme intermediate [9].
Thus, serine proteases (or serine endopeptidases) prefers serine at the active site
for the hydrolysis of the peptide bond in proteins.
260 N.K. Poddar et al.
Usually, they are found in the form of zymogens (digestive enzymes are released
in inactive forms) to regulate the enzyme activities by controlling the specific acti-
vation of proteolysis.
The main division of serine proteases is based on the site of cleavage of specific
amino acids of the peptide bonds:
1. Trypsin such as serine peptidases prefers to cleave the peptide bonds which have
lysine and arginine at the cleavage sites.
2. Chymotrypsin such as serine peptidases prefers aromatic amino acids (phenyl-
alanine, tyrosine or tryptophan) at the cleavage site for the digestion of the pep-
tide bond.
3. Elastase such as serine peptidases prefers to cleave amino acids with short side
chain groups such as alanine in their cleavage site.
The prime contributors of amino acids for the catalytic mechanism of serine prote-
ase classes such as chymotrypsin (in eukaryote) and subtilisin (in prokaryote)
enzymes are their catalytic triad (Fig. 12.2). This triad is found in the active site of
enzyme and conserved in all serine proteases. The triad comprises of three amino
acids, namely, His57, Ser195 and Asp102, bonded in a network fashion (Fig. 12.2).
The position of each amino acid of the triad is far from one another in the primary
structure of the protein, but once folded, they will be in close proximity to the
12 Role of Serine Proteases and Inhibitors in Cancer 261
Fig. 12.2 Catalytic triad of serine protease consists of aspartic acid (Asp-102), histidine (His-57)
and serine (Ser-195)
enzyme. This explicit tertiary structure of the triad members is vital for the specific
catalysis of the enzyme.
Serine proteases follow ping-pong catalysis mechanism, and this involves forma-
tion of unstable enzyme-peptide intermediate by covalent catalysis mechanism, and
finally the intermediate is stabilised, and consequently the peptide fragment is
released [10].
The serine protease mechanism can be summarised in the following two steps:
acylation followed by de-acylation process in which a nucleophilic attack takes
place on the intermediate by water, which leads to the hydrolysis of the protein
(Fig. 12.3). The overall process of the reaction mechanism utilises the catalytic triad
(Asp-102-His-57-Ser-195) of serine protease. In this process, the serine-OH acts as
a nucleophile, while histidine-NH acts as a base catalyst to activate the serine but
later on it acts as an acid catalyst, whereas aspartate plays a supportive role by sta-
bilising the histidine in the whole reactions.
The detailed process is given in the following steps:
1. The peptide binds to the active site of the enzyme, in such a way that the sessile
bond of the protein (indicated by –N - C-) is placed into the active site (catalytic
262 N.K. Poddar et al.
Fig. 12.3 Mechanism of a serine protease. In the acylation step, (a) substrate binds to active site
of enzyme. (b) Tetrahedral intermediate is formed due to nucleophilic attack of serine on carbonyl
part of peptide. (c) Acyl-enzyme intermediate is formed by breakage of peptide bond of the sub-
strate. In the de-acylation step, (d–e) water acting as a nucleophile stabilises the cleavage peptide
of carbonyl carbon and gives rise to a new tetrahedral intermediate with the nitrogen of the histi-
dine. (f) Regeneration of the active site by releasing the product (Redrawn based on figure by Pratt
CW, Cornely K (2012) Essential Biochemistry, 3rd edn. Wiley, New York, p 170)
triad) of the enzyme and the carbonyl-C part of peptide is present close to the
nucleophilic serine [Fig. 12.3a].
2. First, the electron-rich –N atom of the histidine activates the serine residue of the
catalytic triad by extracting the –H atom from serine-OH and make it more
nucleophilic, and thus the serine-OH is more likely to attack the electron sink of
the carbonyl-C of the peptide. Consequently, a tetrahedral intermediate is gener-
ated in which a newly covalent bond is formed between the carbonyl-C of the
peptide and –O atom of serine, whereas the hydrogen part of serine is covalently
bond to –N atom of histidine as well as a pair of electrons from the double bond
of the –C = O moves to the –O atom of carbonyl part of peptide, creating a nega-
tive charge on the –O atom, and this causes an unstable carbonyl anion of the
peptide [Fig. 12.3b]. Moreover, the histidine residue carries a positive charge due
to the newly covalent bond with the serine-OH which is stabilised by the hydro-
gen bond of aspartic acid of the catalytic triad.
12 Role of Serine Proteases and Inhibitors in Cancer 263
3. Because of the positive charge in the histidine, the histidine donate the –H atom
to the –N atom of scissile bond of the peptide which results in the breaking of the
sessile bond in which the sessile bond is now covalently bond with the –H atom
of histidine. The negative charge on the oxygen atom that formed previously on
the –C = O moves back to recreate a double bond. Thus, the peptide bond break
results in the release of N-terminus part of the peptide, and C-terminus part of
the peptide is covalently attached to serine residue, generating an acyl-enzyme
intermediate. Now, the histidine residue of catalytic triad is back into the original
form as an acid catalyst [Fig. 12.3c].
4. After that, water comes to play an active role in this catalysis reaction. The
electron-rich nitrogen atom of histidine residue acts as a nucleophile and extracts
the proton of water, and this allows the –OH part of water to act as a nucleophile,
and because of this, it attacks the electron sink of –C = O part of the peptide. This
is exactly the same step as in 1. Now, the new -N-H bond is formed, and histidine
again carries a positive charge which is stabilised by the hydrogen bond of aspar-
tic acid of the catalytic triad. Once again, the electron pairs from the –C = O of
the substrate move back to the oxygen making it negative charge, as the bond
between the –OH of water and the carbonyl-C of the substrate is formed. Overall,
this generates other tetrahedral intermediate results in an unstable carbonyl anion
of peptide [Fig. 12.3d, e].
5. Finally, in order to neutralise the positive charge of histidine, the covalent bond
of -N-H of histidine residue is now breaking, and new covalent bond is formed
between -H atom of -N-H bond of histidine and –O atom of serine residue by
breaking the bond between the carbonyl-C of the peptide and oxygen atom of
serine. Now, the electron-deficient carbonyl carbon of the peptide regains the
previous double bond with the oxygen. Consequently the C-terminus of the pep-
tide is now released along the formation of new –OH group of water [Fig. 12.3f].
In a nutshell, the peptide is hydrolysed with the help of protease by adding –H
atom to N-terminus, and –OH atom is attached to C-terminus of the peptide bond
of the substrate.
1 . Secreted type
2. Membrane-anchored type
Fig. 12.4 Classification of serine proteases: (1) secreted serine protease type ((i) chymotrypsin
type, (ii) role of uPA enzyme in pericellular proteolysis by binding to specific cell-surface recep-
tors uPAR (GPI-anchored type)) and (2) membrane-anchored type: (i) The human GPI-anchored
serine proteases, prostasin and testisin, (ii) Type 1 transmembrane serine protease, Tryptase γ1,
(iii) The Type II transmembrane serine proteases (TTSPs): (a) the human airway trypsin-like
protease expressed in squamous cell carcinoma (HAT/DESC) subfamily; (b) hepsin/TMPRSS
subfamily which consists of SR, SEA and LDLA domains; (c) Matriptase subfamily, particu-
larly, Matriptase-2, which consists of SEA, two CUB and three LDLA domains; and (d) the
corin subfamily which consists of two FD domains, eight LDRA domains and one SR domain
(abbreviations: SR scavenger receptor domain (group A), SPD serine protease domain consist of
three catalytic residues histidine, aspartate and serine, FD Frizzled domain, LDLA LDL receptor
class A)
On the basis of their structural features, they can be divided into subgroups and
are anchored to the membrane by three ways: (1) a carboxyl-terminal transmem-
brane domain through a GPI (glycosyl phosphatidylinositol) linkage which is added
post-translationally, (2) a carboxy-terminal transmembrane domain (Type I) and (3)
an amino-terminal transmembrane domain with a cytoplasmic extension (Type II
transmembrane serine proteases – TTSPs) [13, 14]. Type I serine proteases, Tryptase
γ1 and the GPI-anchored serine proteases, prostasin and testisin contain a carboxy-
terminal hydrophobic extension that serves as a transmembrane domain (ranging
from 310 to 370 amino acids). GPI anchors have been known to modify C-terminal
domain of prostatin and testisin post-transcriptionally [15–17] (Fig. 12.4).
TTSPs are the group of membrane-anchored serine proteases with 17 members
and 19 members of humans and mice, respectively. TTSPs are trypsin-like (family
S1) proteases and have the potential to be linked to cellular membranes via a hydro-
phobic stretch at their amino terminus. These proteases have two parts, one with a
cytoplasmic amino-terminal signal anchor of variable length (20 to 160 amino
acids) and the other with a catalytic serine protease domain at the carboxyl-terminus.
All the membrane-anchored serine proteases are structurally conserved catalytic
domains and belong to S1 peptidase family. These serine proteases usually exist as
zymogens (inactive form); and their autoactivation cleavage occurs after a basic
amino acid residues present in a highly conserved activation motif producing a two-
chain form with their chains bonded by a disulphide bridge, eventually, separating
the pro- and catalytic domains with the catalytic domain remaining membrane
bound. Some examples of TTSPs are TMPRSS2, matriptase, hepsin and TMPRSS4
[18]. Therefore, they represent enzymes whose peptide bond cleaving activities are
specifically targeted to cellular membranes. They have been phylogenetically cate-
gorised into four subfamilies on the basis of C-terminal transmembrane domain: (1)
the human airway trypsin-like (HAT)/differentially expressed in squamous cell car-
cinoma gene (DESC) subfamily, (2) the hepsin/transmembrane protease serine
(TMPRSS) subfamily, (3) the matriptase subfamily, and (4) the corin subfamily
(Fig. 12.4). In compare with the GPI-anchored and Type I serine proteases, which
consist of SPD (serine protease domain) and membrane anchor, the TTSPs possess
a stem region which is C-terminal to SPD having a variety of modular structural
accessory domains (SEA, CUB, FD, SR) that are involved in protease activation,
localisation and substrate recognition to maintain the homeostasis of pericellular
microenvironment (Fig. 12.4).
Dysregulation of pericellular and extracellular proteolysis that involve the
membrane-anchored and secreted serine proteases, respectively, are the hallmarks
in various clinical disorders. Reports have shown that proteolytic breakdown of the
extracellular matrix (ECM) is the key step in spreading tumour cell [19, 20]. The
series of activities of proteases involved in tumour progression is collectively called
as the cancer ‘degredom’. A positive cooperativity between the aggressiveness of
tumour and the overexpression of many proteases has been detected [20]. In the
series of events in cancer progression, serine proteases may be involved in any of
the fundamental processes of tumorigenesis with unique specifications [13]. In nor-
mal physiological conditions, an endogenous anti-serine protease system known as
266 N.K. Poddar et al.
serpins regulate the serine protease activity and maintain the balance between pro-
teases and their inhibitors in the organism. An imbalance between the proteolytic
and antiproteolytic may be of major significance in the cancer development. For
example, hepsin, a cell surface serine protease, and maspin, a serine protease inhibi-
tor, are both showing highly upregulated and downregulated, respectively, in pros-
tate cancer, and this causes an imbalance in cellular homeostasis which are believed
to promote tumour growth, invasion and metastasis. This shows that the improper
function of serine protease leads to cancer which will suggest the need of therapeu-
tic agents against the serine protease to prevent tumour progression and metastasis.
As a tumour biomarker, serine proteases are important in detecting certain cancers
at an earlier stage. For example, determination of coagulation factor levels and
serum prostate-specific antigen can be used for detecting thrombotic and prostate
cancer patients. Furthermore, targeting and modulation of overexpressed proteases
are the efficient selective approaches for the development of antitumour therapies
[21]. Due to the ever-increasing, newly found roles of serine proteases in cancer,
there has been increasing attention in the specific roles of members of serine prote-
ases and their inhibitors in array of diverse cancer progression. In this chapter, a
review of the role of these members of serine proteases (secreted type and mem-
brane anchored of TTSPs family) and their inhibitors in tumour progression has
been discussed in order to understand their therapeutic applications.
There are about 175 predicted serine proteases in humans. Most of them are found
to be secretory in nature that has major roles in a multiple metabolic functions in
maintaining the tissues homeostasis. For example, the uPA (urokinase plasminogen
activator) and kallikrein system participate in a range of physiological function
from cell growth, cell signalling to tissue remodelling process. However, dysregula-
tion expression of serine proteases leads to tumour invasion and cancer. In this sec-
tion, the main focus is on particular secreted serine proteases, which are reported to
be the vital causes of cancer progression and metastasis.
the inactive zymogen, plasminogen, which can break down most extracellular pro-
teins. However, tPA mainly acts as a fibrin-dependent pathway for blood clot dis-
solution process [23]. While uPA performs through fibrin-independent pathway and
largely acts on the cell surface receptor-bound plasminogen activator like uPAR
which controls the pericellular proteolysis of the system, this involves in the degra-
dation of ECM and causes invasion and cancer metastasis [24] (Fig. 12.4). uPA and
uPAR are observed to be highly expressed in various human cancers in contrast to
the corresponding normal tissue. In this regard, uPAR is a highly glycosylated cell
surface protein which do not contain transmembrane and intracellular domains but
are attached to the cell membrane by a GPI anchor (Fig. 12.4).
uPA is a small trypsin-like protease having molecular weight of 53 kDa. It per-
forms the catalysis of zymogen, plasminogen, into its active form plasmin that facil-
itates the degradation of various ECM proteins such as fibronectin (FN), vitronectin
(VN) and fibrin which results in the loss of interactions between cells, leading to the
invasion of cancer cells [25]. In addition, it is also able to activate the inactivated
forms of various metalloproteases (MMPs) [26]. In this regard, uPA in combination
with uPAR plays a pivotal role in inducing the proteolytic cascade reactions that
promote tumour growth through the process of metastasis of cancer cells [27]. The
effect of uPAR on cancer cell migration is characterised on the basis of whether it is
protease dependent or not. The protease-dependent function is catalysed by uPAR-
bound uPA. Since uPAR has no transmembrane structure, its non-protease function
depends on the interaction with VN, integrin family, G-protein-coupled receptors
and growth factor receptors to relay its downstream signals [28]. Signalling through
uPAR activates Tyr kinases, Src, the serine kinase Raf, FAK and extracellular signal-
regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway which
results in the broad modulation of cell proliferation, metastasis and cell-cell interac-
tions [29]. The universal functions of uPAR are proteolytic extracellular matrix deg-
radation for the progression of cancer, angiogenesis, modulation of cAMP levels for
downstream signalling and cell interaction with integrins, tyrosine kinases and ser-
ine/threonine kinases [30].
uPA, uPAR and also PAI-1 are constitutively expressed in human breast cancer.
In most of the cancers especially breast cancer, high levels of uPA and uPAR are
overexpressed, proposing the enhanced role in ECM degradation, migration and
adhesion and cancer invasion [31]. PAI-1 and uPA are the first novel tumour bio-
logical predictive factors found, in evidence with their clinical utility for breast
cancer [32]. uPA has also been observed to be a predictive marker in many types of
organ cancers such as cancers of the lung [33], bladder [34], stomach [35], etc.
Several studies suggest that uPA binds with uPAR that facilitate the cell migra-
tion process through diverse cell signalling pathways. In this regard, integrins are
crucial uPAR signalling co-receptors, and activation of integrin stimulates the focal
adhesion kinase (FAK) and, thereby also, activates Src/MEK/ERK-dependent sig-
nalling pathways, resulting in transcriptional activation of the uPA promoter, which
promotes tumour cell proliferation and tumour invasion (Fig. 12.5). Similarly, the
p38 MAPK and myosin light-chain kinase (MLCK) pathways are involved in
268 N.K. Poddar et al.
Fig. 12.5 uPA system in cancer malignancy. In the plasma membrane, uPA binds to uPAR which
promotes uPA activation resulting in the catalysis of plasminogen into active form, plasmin.
Plasmin can subsequently activate MMPs in the pericellular environment that results in invasion
and metastasis via ECM degradation. Intracellular activation of uPA/uPAR along with integrins
activates FAK, PI3/AKT and p38-MAPK signalling pathways, which finally leads to pathophysi-
ological events, such as metastasis and inhibition of apoptosis pathway
12.2.2.2 Kallikreins
In 1930, Kraut and colleagues coined the term kallikrein (kallikreas is the Greek
word for pancreas) from an identified substance (human kallikrein 1) that was pres-
ent at significant concentration in the pancreas. Human tissue kallikreins (hKs) are
secreted serine proteases that convert high molecular weight proteins into biologi-
cally active peptides known as kinins. There are two families of kallikreins, the tis-
sue and plasma kallikreins. Human plasma kallikrein cleaves high molecular weight
kininogen into a bradykinin which is a potent vasodilator nonapeptide (Fig. 12.6).
The only enzyme which has been found with appreciable kallikrein activity is kal-
likrein I (hK1, pancreatic-renal kallikrein) in human tissue kallikreins. There are a
12 Role of Serine Proteases and Inhibitors in Cancer 269
Fig. 12.6 Kallikrein’s signalling pathway system. Kallikreins activate the uPA system resulting in
the catalysis of plasminogen into active plasmin which in turn activates the pro-KLK proteins lead-
ing to the breakdown of various downstream targets, such as latent MMPs. Kallikreins take part in
ECM remodelling directly and/or indirectly via activation of pro-MMPs. Active kallikrein is
involved in the conversion of kininogen into kinin fragment which induces angiogenesis and other
pathological processes via activation of the cAMP, Akt/PKB and VEGF pathway
cascade by various hK-family members further widens their various routes in can-
cer progression. Thus, many members of proteolytic network especially kallikrein
family is involved in cascading reactions of tumour progression [38].
Angiogenesis is a process of differentiation of new capillary blood vessels from
pre-existing vessels. Angiogenesis is mainly controlled by the ratio of pro- and anti-
angiogenic growth factors exist in the blood. The increased ratio of pro-angiogenic
stimuli and inhibitory regulators activates or switches on the angiogenic process.
Human tissue kallikrein possesses potent angiogenic effects by processing many
elements of the extracellular matrix. It has been classified as a pleiotropic angio-
genic agent which catalyses the inactive form of kininogen into active form of kinin
peptides which in turn activate cAMP, Akt/PKB and VEGF (vascular endothelial
growth factor) pathways, and this promotes the process of angiogenesis [40]
(Fig. 12.6). KLKs may also participate in remodelling of ECM indirectly through
the MMPs, uPA and kinin signalling pathways [41–43]. The kallikrein family such
as KLK2, KLK4 and KLK12 activates the uPA system, resulting in plasmin forma-
tion, and this activated plasmin causes the breakdown of a number of ECM proteins,
for example, fibronectin, proteoglycans and fibrin [28, 44–46] (Fig. 12.6). Similarly,
KLK1 and KLK12 catalyse the conversion of kininogen to active kinin peptides and
bradykinin, and this promotes angiogenesis and metastasis through the activation of
downstream signalling pathways, for example, basic fibroblast growth factor
(bFGF) cAMP, Akt/PKB and VEGF pathways [45, 47, 48] (Fig. 12.6).
mammalian system, and their structural homology does not linked to a common
biochemical function. They are mostly participating in either hormone or growth
factor activation or in the initiation of proteolytic cascades. This suggests that they
are maintaining basic homeostasis by activating or deactivating the signalling mol-
ecules involve in the biochemical reactions.
Recently, great attention has been paid to the members of TTSP family such as
hepsin, matriptase-2 and TMPRSS4 for their vital physiological roles, role in
tumourigenic activity and distinctive regulatory mechanisms. These TTSPs are
being increasingly documented for their important roles in regulating the pericel-
lular microenvironment and thus providing new insights of their mechanisms of
mammalian health and diseases.
12.2.3.1 M
embrane-Anchored Type II Serine Proteases (TTSP)
and Its Role in Cancer
Hepsin
In the United States, about 2 lakhs of new cases of prostate cancer of adult men and
40,000 deaths were observed in 1995. The occurrence of prostate cancer is more
prevalent in the later age of 60 years and above, and about 80% of prostate cancers
are diagnosed of this age group [77].
Hepsin (TMPRSS1) is one of the members of Type II transmembrane serine
proteases and is expressed in prostate cancer [78]. Hepsin is mostly present in the
liver but is also found at trace amount in tissues of the stomach, kidney, prostate,
thyroid and inner ear. This subfamily is composed of seven members in human and
mice. It possesses only one additional domain in its stem region, a group A scaven-
ger receptor domain (SR) in addition to serine protease domain (SPD) (Fig. 12.4).
It can cleave and activate pro-uPA, pro-HGF, Laminin332 and pro-MSP [78]. It
activates pro-hepatocyte growth factor and is inhibited by hepatocyte growth factor
activator inhibitor-1B (HAI-1B) and hepatocyte growth factor activator inhibitor-2
(HAI-2) [79]. It is involved in the activation of various proteolytic cascades espe-
cially the activation of non-active proteases which leads to breakdown of the extra-
cellular matrix proteins. Moreover, this membrane-associated serine protease helps
in the blood coagulation pathway by converting factor VII to VIIa resulting in the
formation of thrombin, deposition of pericellular fibrin as well as the activation of
PAR-1 (protease activated receptor) [80]. Hepsin was also to be highly expressed in
ovarian cancers [81–83].
The TMPRSS2 and TMPRSS4 are the Type II serine proteases that are highly
expressed in prostate and pancreatic cancers, respectively, and activate proteolytic
cascades which lead to metastasis events [84].
TMPRSS4
TMPRSS4 is a member of Type II transmembrane serine protease and is found to be
overexpressed mostly in the pancreas, the thyroid and cancer tissues. As compared
to hepsin, it possesses an additional low-density lipoprotein receptor class A
(LDLA) domain which is N-terminal to the SR and SPD domains (Fig. 12.4). The
12 Role of Serine Proteases and Inhibitors in Cancer 273
Fig. 12.7 Effect of overexpression of TMPRSS4 in cancer malignancy. It helps in the activation
of the intracellular pathways through phosphorylation of ERK, JNK, Akt, Src, FAK and Rac1
which in turn upregulates integrin (ITG-α5) and transcription factors such as Sip1/Zeb2 (a repres-
sor of E-cadherin) resulting in invasiveness and EMT. In the cell membrane, TMPRSS4 converts
the precursor of uPA (pro-uPA) to its active form which accelerate the invasiveness
TMPRSS4 activates uPA by two ways, one through increased gene expression
(JNK and transcription factors Sp1 and Sp3 and AP-1 pathway) and another by
activating pro-uPA, and this leads to enhanced invasion [92] (Fig. 12.7).
Matriptase-2
Matriptase-2 or TMPRSS6 (80–90-kDa cell surface glycoprotein) belongs to a Type
II transmembrane serine protease family [93, 94]. Matriptase-2 comprises of a short
N-terminal cytoplasmic tail, a transmembrane domain, an extracellular stem region
containing a SEA domain (a single sea urchin sperm protein), two CUB domains
(urchin embryonic growth factor), three LDLA repeats and a C-terminal trypsin-
like SPD domain [93, 94] (Fig. 12.4). It was mostly found in breast and prostate
cancers [95, 96].
Matriptase-2 is a hepatic membrane serine protease and is expressed as zymogen
on the cell surface, and this inactive proenzyme undergoes shedding to a single
chain form followed by autoactivation by cleavage at conserved site represented as
RIVGG between the pro-domain and the catalytic domain, and the activated prote-
ase domain fragment remains on the membrane via a single disulphide bond linking
the pro- and catalytic domains [97, 98]. Matriptase-2 shows high homology in terms
of structure as well as its function with matriptase-1 [99], which is found to be over-
expressed in epithelial cells, and in various cancers [100]. Matriptase-2 is primarily
found to be expressed in human liver that shows connection with the dissolving of
extracellular matrix proteins including laminin and fibronectin [94]. It was estab-
lished that the degradome components such as hepsin, MTSP1, MMP26, plasmino-
gen activator inhibitor-1 (PAI-1), uPAR, MMP15, TIMP3, TIMP4, maspin and
RECK are associated with cancer progression in human prostatic tissues [101].
The protease activity can be controlled by its pericellular environment in various
ways. In our living system, it was found that several proteases can be activated in an
acidic environment such as cathepsins in lysosomes and pepsinogen in the stomach
[102–104]. It was also observed that the activity of matriptase is firmly controlled
by the chemical environment of the cell [105]. Like other secreted or lysosomal
proteases which are activated by an acidic pH, matriptase is also activated in the
same way but is unique in the sense that it is attached onto the surface of cells [106].
Matriptase is released as a zymogen and its autoactivation activity depends on
intrinsic activity of matriptase zymogen, non-catalytic domains of the enzyme and
post-translational modifications [107, 108].
This protease is mainly co-expressed with hepatocyte growth factor activator
inhibitor-1 (HAI-1) in the normal epithelial components of tissues, suggesting that
the protease activity of matriptase is tightly regulated [100, 109, 110].
Reports suggested that an imbalance of matriptase and HAI-1 ratio is the key
factor for the indication of a cancer-related proteolytic events. It is being shown that
the ratio of matriptase and HAI-1 has been increased in many cancers such as in
breast and prostate [111, 112]. Although the proper mechanism of the dysregulation
of matriptase activity is still not known, it may directly affect the cellular microen-
vironment via the activation or inactivation of downstream signalling molecules
leading to the breakdown of ECM components and cell-cell adhesion [113].
12 Role of Serine Proteases and Inhibitors in Cancer 275
12.3.1 Serpin
Ischemic stroke is a single largest cause of stroke and accounts to be the second
largest contributor to mortality in the world [144]. It is due to the obstruction of a
certain cerebral artery resulting in an absence of blood flow to artery and brain tis-
sue, and this could induce an energy metabolism disorder which in turn perturbs the
ion gradients and an excessive release of excitotoxic neurotransmitters such as
dopamine and glutamate which ultimately leads to neuronal death [78, 145]. The
effective treatment for acute ischemic stroke is the administration of tPA within
3 hours on the onset of stroke. Meanwhile, tPA is capable of activating matrix
MMPs and converting plasminogen to plasmin which results in the blood brain
degradation [145, 146]. This extra administration of extravascular tPA beyond its
therapeutic window (hours) causes a more deleterious effect on the brain. However,
the adjuvant treatment with neuroserpin along with tPA is found to increase the
therapeutic window, and this could have better treatment for cerebral ischemia.
Thus, the neuroprotective effect of neuroserpin for the treatment of cerebral isch-
emia is dependent on the balanced expression of tPA which in turn regulates the
recanalisation of the occluded vessel [147]. It was observed that neuroserpin gene
is a cancer-associated gene and acts as a tissue-specific tumour suppressor gene in
the brain [148]. Like other members of serpin family such as maspin and pancpin,
this tissue-specific tumour-suppressive gene is found to be downregulated in brain
tumour and even absent in brain cancer cells. As mentioned before, tPA converts
inactive plasminogen into active plasmin which results in the breakdown of the
extracellular matrix that facilitates the invasion of cancer cells and enables tumour
migration. It was observed that neuroserpin functions in the inhibitory process of
tPA, and its absence in the CNS causes brain tumorigenesis. Based on this finding,
it may be suggested that neuroserpin may represent a new approach for cancer ther-
apy. Additionally, neuroserpin is a tPA-independent mediator of neurite such as out-
growth, cell-cell adhesion and N-cadherin and NFκB expression. The
tPA-independent regulatory effect of neuroserpin participates in tumorigenesis as
well as emotional and cognitive processes. These new finding of the multifaceted
roles of neuroserpin and its polymers will be helpful in designing better methods for
treating cancer-like diseases.
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12 Role of Serine Proteases and Inhibitors in Cancer 287
Abstract
Proteases catalyze the breakdown of proteins by hydrolysis of peptide bonds.
These enzymes are involved in a number of pathophysiological processes rang-
ing from the cellular to organism level. These processes include cell growth,
homeostasis, remodeling, renewal, division, metabolic pathways, tumor growth,
metastasis, etc. A number of proteases are found to be involved in mediating the
biochemical pathogenesis of metabolic syndrome such as diabetes and cardio-
vascular diseases. This chapter summarizes types of proteases, classification, and
their proteolytic function in diabetes-associated complications in the kidney, eye,
liver, heart, and lung. Understanding the role of proteases will provide insights
into the development of preventive and therapeutic modalities for diabetes and
diabetic complications.
Keywords
Proteases • Diabetes • Diabetic complications
13.1 Introduction
the entry of infectious agents into target cells and tissues and release membrane-
bound molecules facilitating the activation of signaling pathways in cells [1].
Extracellular proteases hydrolyze large proteins into amino acids for subsequent
absorption by the cell, whereas intracellular proteases play a vital role in regulation
of cell metabolism. Various proteases are shown to be involved in the pathogenesis
of metabolic disorders including diabetes. However, the information about their
mechanism of action is limited. Hence, this chapter summarizes types of proteases
and their mechanism of action in the context of diabetes and diabetic
complications.
Based on their site of action, proteases are subdivided into endopeptidases and exo-
peptidases. Endopeptidases cleave the peptide bonds that are away from the amino
or carboxy termini of the substrate, while exopeptidases cleave the peptide bonds
that are close to the termini of the substrate. Endopeptidases are classified into four
major groups based on the presence of functional group at the active site—aspartic
proteases, cysteine proteases, metalloproteases, and serine proteases [2].
These proteases are commonly referred to as acidic proteases and have aspartic acid
(Asp) in their active site situated in “Asp-Xaa-Gly” motif, where Xaa can be Thr or
Ser. They have isoelectric points in the pH range of 3–4.5 and show optimal activity
at acidic pH (pH 3 to 4). Their molecular mass ranges from 30 kDa to 45 kDa. The
substrate-binding cleft can accommodate seven amino acid polypeptides [3, 6]. The
mechanism of action involves removal of a proton by aspartate leading to the activa-
tion of the water molecule and attacking the scissile bond to produce a tetrahedral
intermediate. Scissile amide’s protonation following the rearrangement reaction of
the intermediate results in the breakdown of the substrate into peptides [5].
Serine proteases are one of the most studied proteases with nearly 800 structures
recorded in the protein data bank (PDB). One-third of these structures are of trypsin
and thrombin [3]. Serine proteases have serine, histidine, and aspartate residues
forming the catalytic triad in their active site. These are found in both endopepti-
dases and exopeptidases. Serine proteases show optimum activity ranging from the
neutral pH (pH 7) to the alkaline pH (pH 11) and have isoelectric points ranging
from pH 4 to pH 6. Their molecular mass ranges from 18 kDa to 35 kDa (except the
protease found in the Blakeslea trispora with 126 kDa molecular mass) [4]. These
13 Role of Proteases in Diabetes and Diabetic Complications 291
Cysteine proteases are found in both eukaryotic and prokaryotic organisms. They
have cysteine and histidine residues forming the catalytic dyad in their active site.
Cysteine proteases show maximum activity at the neutral pH, except lysosomal
proteases that are maximally active at acidic pH. The proteolytic action of these
proteases requires reducing agents, such as HCN or cysteine [8–10]. Similar to ser-
ine proteases, most cysteine proteases have a relatively shallow active site that can
accommodate short substrate segments such as a strand or protein loops (e.g.,
endogenous inhibitors, cystatins). Sulfur in cysteine as opposed to the serine in
serine proteases is used as the nucleophile for carrying out the proteolytic action on
the substrates.
13.2.4 Metalloproteases
The most diverse among the proteases require a divalent metal ion (Zn2+) for their
activity. Approximately, 30 families are identified. Of them, 12 contained exopepti-
dases and 17 contained endopeptidases, while one of them contained both endo- and
exopeptidases. Water molecule hydrogen bonded to a glutamate, and the three donor
groups of the enzyme coordinate with the Zn2+ while carrying out the nucleophilic
attack during the proteolysis [3].
13.3 R
ole of Proteases in Diabetes and Diabetic
Complications
Proteases likely arose as simple destructive enzymes in protein evolution that are
necessary for the generation of amino acids through protein catabolism in primitive
organisms. For many years, studies on proteases focused only on their role in pro-
tein catabolism. However, recent research have shed light on other functions of the
proteases; proteases act as site-specific scissors and catalyze specific proteolytic
reactions to produce new protein products. Proteases can be detrimental or benefi-
cial in the inflammatory process depending on the biological contexts, such as dis-
ease state, cell type, location, substrate availability, and inhibitors.
292 P.V. Ravindra and T.K. Girish
13.3.1 Kidney
Among all the diabetic complications, diabetic nephropathy (DN) is the leading
cause of end-stage kidney disease worldwide. Moreover, the progressive decline in
kidney function in diabetic patients is positively correlated with all-cause mortality
and severe cardiovascular complications [11].
The kidney size is increased in the early phase of type I diabetes in human or in
experimental diabetes in rats. This increase in size is mainly due deposition of pro-
tein as a result of decreased protein catabolism and/or increased protein synthesis.
Both protein synthesis and degradation are energy dependent processes, which may
be regulated by various proteases. The lysosomes play a pivotal role in the break-
down of proteins in mammalian cells which involves sequestration of these proteins
in autophagic vacuoles, fusion of these vacuoles with primary lysosomes, and deg-
radation of proteins within the newly formed secondary lysosomes by highly active
proteases such as cathepsins B and cathepsin L.
The activities of these cathepsins were measured in kidneys from streptozotocin
(STZ)-induced diabetic rats by Olbricht et al. (1992) [12]. Cathepsins’ activities
decreased with an increase in kidney weight, which indicates renal hypertrophy in
STZ-injected rats. They also found decreased cathepsin activity in proximal tubule
segments and kidney cortex. Liver was a positive control in this study, and it was
found that liver weight and activities of these cathepsins were found elevated unlike
in the kidney. This indicates that diabetes might be associated with decreased
cathepsin activity independent of organ hypertrophy [12].
The clouding of the eye lens causes development of eye cataracts. Cataracted eye
contributes majorly for the vision loss observed in people over 40 years of age.
Diabetes is one of the primary contributing risk factor for the development of cata-
racts. The eye lens is made of specialized proteins, called crystallins. These include
α-, β-, and γ-crystallins, which account for nearly 90% of the lens proteins.
α-Crystallin is a predominant lens protein composed of αA and αB subunits with
chaperone-like activity, and β- and γ-crystallins function as structural proteins.
Diabetes induces the activation of polyol pathway and increases oxidative stress and
nonenzymatic glycation of these lens proteins, which subsequently aggregate lead-
ing to the cataract development [13].
Various peptidases and proteases have been identified or isolated from lens and
lens epithelial cells. These include acyl-peptide hydrolase, aminopeptidase III, cal-
pains, dipeptidase, caspases [3, 6, 7], cathepsin (B, D), matrix metalloproteases,
leucine aminopeptidase, serine-type protease, and trypsin-like protease. Lens pro-
teins are degraded into amino acids by subsequent hydrolysis by proteases and ami-
nopeptidases. Literature suggests both beneficial and detrimental effects of protease
activity in the lens. Peptide chaperones such as αA and αB released following the
cleavage of α-crystallin prevent aggregation and precipitation of unfolding proteins,
13 Role of Proteases in Diabetes and Diabetic Complications 293
similar to the full-length α-crystallin. On the other hand, αA-66-80 peptide gener-
ated following the proteolysis of αA-crystallin has been found to promote the for-
mation of protein aggregates [13, 14]. Further, αA-66-80 peptides are resistant to
downstream aminopeptidases and can suppress the degradation of other peptides.
Incomplete hydrolysis of peptides leads to protein aggregation in lens leading to
cataract formation [13–15].
13.3.3 Liver
Research findings on understanding the role of various proteases in the diabetic liver
are limited. Lysosome proteases are the main digestive enzymes in autophagic vacu-
oles in hepatocytes. Recent works on liver autophagy focus on glycogen digestion
and lipid digestion (lipophagy) [16–18]. Few studies have been done on the role of
lysosomal proteases in the diabetic liver [19] and has shown decrease in cysteine
proteases’ activity, especially cathepsin B in STZ-induced rat liver [19]. This
decrease in specific activity was attributed decreased expression levels of cathepsin
B and in the diabetic rat liver [19]. In another study by Uchimura et al. (2014) found
that serine protease prostasin ameliorates hepatic insulin insensitivity found in the
type 2 diabetes by decreasing the activation of toll-like receptor (TLR4) signaling
pathway [20].
13.3.4 Heart
13.3.5 Lung
13.4 Conclusions
Acknowledgement Authors are thankful to Prof. Ram Rajasekharan, Director, CSIR-CFTRI, for
his interest and valuable suggestions. Dr. Ravindra P. V. thanks the Department of Biotechnology,
New Delhi, for funding in the form of Ramalingaswami fellowship.
References
1. Dunn BM (2010) Introduction to the aspartic proteinase family. Aspartic Acid Proteases as
Therapeutic Targets:1–21
2. Rao MB, Tanksale AM, Ghatge MS et al (1998) Molecular and biotechnological aspects of
microbial proteases. Microbiol Mol Biol Rev 62:597–635
3. Tyndall JD, Nall T, Fairlie DP (2005) Proteases universally recognize beta strands in their
active sites. Chem Rev 105:973–1000
4. Govind NS, Mehta B, Sharma M et al (1981) Protease and carotenogenesis in Blakeslea tris-
pora. Phytochemistry 20:2483–2485
5. Erez E, Fass D, Bibi E (2009) How intramembrane proteases bury hydrolytic reactions in the
membrane. Nature 459:371–378
6. Polgár L (1987) The mechanism of action of aspartic proteases involves ‘push-pull’catalysis.
FEBS Lett 219:1–4
7. Barrett AJ (1994) Proteolytic enzymes: serine and cysteine peptidases. Academic Press
244:1–765
8. Kotler MOSHE, Katz RA, Skalka AM (1988) Activity of avian retroviral protease expressed in
Escherichia coli. J Virol 62:2696–2700
9. Kubo M, Imanaka T (1988) Cloning and nucleotide sequence of the highly thermostable neu-
tral protease gene from Bacillus Stearothermophilus. Microbiology 134:1883–1892
10. Koszelak S, Ng JD, Day J et al (1997) The crystallographic structure of the subtilisin protease
from Penicillium cyclopium. Biochemistry 36:6597–6604
11. Musante L, Tataruch D, Gu D et al (2015) Proteases and protease inhibitors of urinary extracel-
lular vesicles in diabetic nephropathy. Journal of diabetes research 2015:1–15
12. Olbricht CJ, Geissinger B, Gutjahr E (1992) Renal hypertrophy in streptozotocin diabetic rats:
role of proteolytic lysosomal enzymes. Kidney Int 41:966–972
13. Raju M, Santhoshkumar P, Sharma KK (2016) Alpha-crystallin-derived peptides as therapeu-
tic chaperones. Biochimica et Biophysica Acta (BBA)-General Subjects 1860:246–251
14. Santhoshkumar P, Kannan R, Sharma KK (2015) Proteases in lens and cataract. In: Babizhayev
MA, Li DW-C, Jacobi AK et al (eds) Studies on the cornea and lens. Springer, New York,
pp 221–238
15. Hariharapura R, Santhoshkumar P, Sharma KK (2013) Profiling of lens protease involved in
generation of αA-66-80 crystallin peptide using an internally quenched protease substrate. Exp
Eye Res 109:51–59
16. Singh R, Kaushik S, Wang Y et al (2009) Autophagy regulates lipid metabolism. Nature
458:1131–1135
17. Papackova Z, Palenickova E, Dankova H et al (2012) Kupffer cells ameliorate hepatic insulin
resistance induced by high-fat diet rich in monounsaturated fatty acids: the evidence for the
involvement of alternatively activated macrophages. Nutrition & metabolism 9:1–15
18. Kalamidas SA, Kondomerkos DJ (2010) Autophagosomal glycogen-degrading activity and
its relationship to the general autophagic activity in newborn rat hepatocytes: the effects of
parenteral glucose administration. Microsc Res Tech 73:495–502
19. Peres GB, Juliano MA, Aguiar JAK et al (2014) Streptozotocin-induced diabetes mellitus
affects lysosomal enzymes in rat liver. Braz J Med Biol Res 47:452–460
20. Uchimura K, Hayata M, Mizumoto T et al (2014) The serine protease prostasin regulates
hepatic insulin sensitivity by modulating TLR4 signalling. Nat Commun 5:1–13
296 P.V. Ravindra and T.K. Girish
Abstract
Proteases are ubiquitously present in several organisms including plants. In
plants, one of the rich sources of protease is latex. Over 110 latices of different
plant families are known to contain at least one proteolytic enzyme. The primary
role of proteases in latices is defense against pests/insects. Apart from the defen-
sive role in plants, latices are pharmacologically important and are integral com-
ponents in herbal management of wounds, where it is extensively used in
traditional medicines to stop bleeding and to promote healing of wounds. Plant
latex proteases exhibit both clot-inducing and clot-hydrolyzing properties. Clot
formation is vital for hemostasis, the initial phase of wound healing, whereas clot
hydrolysis is a prerequisite for the events of regenerative phase. Overall, the
plant latex proteases provide optimal conditions for physiological wound healing
by complementing the endogenous proteases in hemostasis, wound debridement,
microbial attenuation, cell proliferation, and angiogenesis. Further, complete
functional characterization of purified proteases from latex along with physico-
chemical characterization is very crucial to strengthen the existing knowledge
and will be pivotal in developing latex protease-based wound care supplements
with minimal side effects.
Keywords
Ethnopharmacology • Hemostasis • Plasmin-like proteases • Thrombin-like
proteases Wound healing
14.1 Introduction
Proteases are the major class of hydrolytic enzymes which cleave the peptide bond
in the protein to give peptides and amino acids [1]. They are abundantly present in
the human system evidenced by human genome sequencing which revealed that
more than 2% of the genes encodes for proteases. Proteases mediate versatile and
complex array of functions apart from the primary roles in food digestion and intra-
cellular protein turnover [1–3]. Proteases are involved in the regulation of a large
number of key physiological processes such as hemostasis (coagulation), tissue
remodeling, wound healing, DNA replication, cell-cycle progression, cell prolifera-
tion, cell death, and immune response [2, 3]. In view of their ability to regulate key
physiological processes, proteases have been employed for treating specified clini-
cal conditions. Till date, the predominant use of proteases was restricted to cardio-
vascular disease [3, 4]. Of late, they are also emerging as useful therapeutic agents
in the treatment and management of debilitating conditions including sepsis, chronic
inflammatory disorders, cystic fibrosis, retinal disorders, and psoriasis [3, 5].
Although proteases are involved in the regulation of many key physiological
processes, they have not been typically considered as a drug class despite their
application in the clinic over the last several decades. In recent past, proteases have
emerged as an expanding class of drugs that hold great promise and are used for
treating a wide range of clinical conditions [3, 6]. In addition to the endogenous
proteases, exogenous sources of proteases have been widely studied and employed
for their therapeutic efficacies.
Microorganisms, insects, invertebrates, vertebrates, and plants constitute the
major sources of proteases [7–12]. Among these sources, microbial, animal (venom),
and plant latex proteases are widely employed for their therapeutic applications.
Although there are few studies on therapeutic applications, still there is significant
scope for exploring newer and potent proteases with therapeutic efficacy. Well-
characterized plant latex proteases like papain, chymopapain, and ficin are used for
treating wounds, cancer, digestive and viral disorders [13, 14]. In light of these
evidences and the recent findings pertaining to latex proteases, the present chapter
focuses on the role of plant latex proteases as wound healers and provides an over-
view of their application in wound healing.
The presence of proteolytic enzymes in plant latex was first reported in 1940 [15].
Over 110 latices of different plant families are known to contain at least one proteo-
lytic enzyme [16]. More than 70 proteases have been purified from latices of differ-
ent plants belonging to various families, and the crystal structures of few proteases
are known, which are listed in Tables 14.1 and 14.2, respectively. The majority of
proteases found in latices belong to the cysteine and serine protease family; only
one is a member of the aspartate and metalloprotease family, and none of the prote-
ases reported till date belong to threonine, glutamic, and asparagine protease.
Table 14.1 List of purified plant latex proteases with reported characteristics and molecular weight (NR – no reports)
Functional characteristics
Protease Plant Purified protease Crude latex Molecular weight References
1. Aspartate proteases
A. Moraceae
Ficins Ficus racemosa NR Used for curing 44.5 ± 05 [17, 18]
hemorrhoids, boils,
edema, and chronic
infected wounds
2. Cysteine proteases
A. Apocynaceae
Cg24-I Cryptostegia grandiflora Antifungal Antioxidant 24,1 [19, 20]
Ervatamin A, B, C Ervatamia coronaria Papain-like Gelatinolytic 27.6, 26, 23 [21–25]
Funastrain CII Funastrum clausum Papain-like NR 23.636 [26]
Heynein Ervatamia heyneana NR NR 23 [23]
Pergularain e I Pergularia extensa Thrombin-like Plasmin- and 23.356 [27, 28]
14 Plant Latex Proteases: Natural Wound Healers
thrombin-like
Peruvianin-I Thevetia peruviana Germin-like Antifungal and 120 [25, 29, 30]
gelatinolytic
Philibertain g I and g II Philiberta gilliesii Papain-like NR 23.530 [31, 32]
23,9
Plumerin-R Plumeria rubra Thrombin-like, Antioxidant 81.85 [19, 33]
plasmin-like, anti-
inflammatory, and
excision wound healer
B. Asclepiadaceae
Araujiain H I, H II, H III Araujia hortorum NR NR 24.03, 23.718, [34, 35]
23.546
Asclepains A3, B5 Asclepias syriaca NR NR 23, 21 [39]
299
(continued)
Table 14.1 (continued)
300
Functional characteristics
Protease Plant Purified protease Crude latex Molecular weight References
Asclepain C I, C II Asclepias curassavica Papain-like Antifungal, plasmin-like, 23.2 [27, 37, 38]
and thrombin-like
Asclepain F Asclepias fruticosa Papain-like NR 23.652 [39]
Asclepain G (ten forms) Asclepias glaucescens NR NR Ag3 22.6, [40]
Ag6 23.5,
Ag7 23,
Ag8 23.5
Asclepain S Asclepias speciosa Milk coagulant NR NR [15]
Calotropins DI, DII Calotropis gigantea NR Gelatinolytic, plasmin- 23.8, 24.2 [25, 27, 41–43]
and thrombin-like, and
wound healer
Morrenain BI, BII Morrenia brachystephana Papain-like NR 23.205, 25.5 [44, 45]
Morrenain OII Morrenia odorata NR NR 25.8 [44]
Procerain, procerain B, Calotropis procera Milk coagulant and Anti-inflammatory and 28.8 [25, 46–48]
CpCP-1, 2, and 3 thrombin-like gelatinolytic
C. Caricaceae
Caricain, chymopapain, Carica papaya Factor XIIIa-like, Antifungal and 23.429, 23.280, [16, 25, 49–51]
glycyl endopeptidases, thrombin-like, and gelatinolytic 23.650, 23.313
papain wound healer
Endopeptidase, CCI, Carica candamarcensis Mitogenic and Angiogenic, cell 23–28.6 [16, 52–58]
CCII, CCIII,CCIV, papain-like proliferant, gastric ulcer,
CC23, CC28, and wound healer
chymopapain isoform II,
CMS1MS2, CMS2MS2
Mexicain Jacaratia mexicana NR NR 23.8 [59, 60]
Quercifoliain I, VQ-VII Vasconcellea quercifolia Milk coagulant and NR 24.2, 23.98 [61, 62]
papain-like
A.P. Urs et al.
Functional characteristics
Protease Plant Purified protease Crude latex Molecular weight References
D. Euphorbiaceae
Nivulian-I, Nivulian-II, Euphorbia nivulia Milk coagulant Gelatinolytic and 31.4, 43.6, and [25, 63–65]
and Nivulian-III procoagulant 52.8
Pedilanthin Pedilanthus tithymaloides NR Gelatinolytic and 63.1 [25, 65, 66]
procoagulant
E. Moraceae
Ficain Ficus glabrata NR NR NR [16]
Ficins A, B, C, D, E, F, Ficus carica NR Activation of human 24–26 [67–71]
G, H, I, J, S coagulation factor X and
antimicrobial
Ficain P I Ficus pumila NR NR 28.6 [16]
Microcarpain Ficus microcarpa NR NR 20 [72]
Protease Ficus hispida NR Gelatinolytic NR [25, 73]
3. Metalloprotease
A. Euphorbiaceae
Cotinifolin Euphorbia cotinifolia NR NR 79.76 [74]
14 Plant Latex Proteases: Natural Wound Healers
4. Serine proteases
A. Amaryllidaceae
Crinumin Crinum asiaticum Antiplatelet, NR 67.7 [75, 76]
chymotrypsin-like, and
thrombolytic
B. Apocynaceae
Wrightin Wrightia tinctoria Trypsin-like Collagenolytic, 57.9 [77, 78]
gelatinolytic and wound
healer
C. Asclepiadaceae
Cryptolepain Cryptolepis buchananii NR NR 79.5 [79]
D. Asteraceae
Parthenain Parthenium argentatum NR NR 63 [80]
301
(continued)
302
Table 14.1 (continued)
Functional characteristics
Protease Plant Purified protease Crude latex Molecular weight References
Taraxalisin Taraxacum officinale Subtilisin-like NR 65 [81]
E. Convolvulaceae
Carnein Ipomoea carnea NR Gelatinolytic 80.236 [25, 82]
F. Euphorbiaceae
Euphorbains D1, D2 Elaeophorbia drupifera NR NR 117, 65 [16]
Euphorbain L Euphorbia lathyris NR Gelatinolytic 43 [83]
Euphorbains la1, la2, la3 Euphorbia lactea NR NR 66, 44, 33 [84]
Euphorbain lc Euphorbia lactea cristata NR NR 70 [84]
Euphorbain P Euphorbia pulcherrima NR NR 74 [79]
Euphorbains T1, T2, Euphorbia tirucalli Trypsin-like Antioxidant 74, 74, 74, 74 [19, 85]
T3,T4
Euphorbains Y1, Y2, Y3 Euphorbia cyparissias NR NR 67, 33, 67 [86]
Hevains A, B, L Hevea brasiliensis Antifungal Increases vascular 69, 58, 80 [87–91]
permeability,
angiogenesis, and wound
healing
Hirtin Euphorbia hirta Fibrino(geno)lytic Antimicrobial and 34 [25, 92, 93]
gelatinolytic
Latex glycoprotein Synadenium grantii Fibrino(geno)lytic and Gelatinolytic and 34.4, 76 ± 2 [25, 65, 94, 95]
(LGP), proteases procoagulant procoagulant
Milin Euphorbia milii NR Gelatinolytic 51.4 [25, 96]
Protease Euphorbia pseudochamaesyce NR NR 82 [16]
Protease Euphorbia supine NR NR 80 [97]
Protease Euphorbia heterophylla NR NR 77.2 [98]
Prunifoline Euphorbia prunifolia Milk coagulant Gelatinolytic 57.44 [25, 99]
A.P. Urs et al.
Functional characteristics
Protease Plant Purified protease Crude latex Molecular weight References
G. Moraceae
Amp48 Artocarpus heterophyllus Antimicrobial and NR 48 [100, 101]
fibrino(geno)lytic
Artocarpin Artocarpus heterophyllus NR NR 79.5 [102]
Benghalensin Ficus benghalensis NR NR 47 [103]
Ficin E Ficus elastica NR NR 50 [16]
Macluralisin Maclura pomifera Milk coagulant NR 65 [104]
Protease Ficus carica Collagenolytic and Activation of human 41 [25, 105]
gelatinolytic coagulation factor X,
antimicrobial, and
gelatinolytic
Religiosin Ficus religiosa Milk coagulant Gelatinolytic 43.4 [25, 106]
5. Type of protease not determined
Curcain Jatropha curcas Wound healer Antimicrobial 22 [107, 108]
14 Plant Latex Proteases: Natural Wound Healers
303
304 A.P. Urs et al.
The primary role of proteases in latices is plant defense against pests/insects. Many
of these proteases are cysteine proteases, although few insecticidal m etalloproteases
and serine proteases have also been reported. The targets of protease toxicity range
from the insect midgut to the hemocoel (body cavity) to the cuticle [9, 120]. Apart
from the defensive role in plants, they are extensively used in industries as they are
active over a range of temperature and pH (frequently used in food processing,
tenderization of meat, brewing, cheese elaboration, bread manufacture, and in the
leather and textile industries) [16, 121]. On the other hand, therapeutically, plant
latex is one of the most preferred materials in traditional medicine for treating
bleeding and wounds [16].
Wounds are the inevitable events encountered during the lifetime of an individual
resulting from mechanical, chemical, or surgical damage, microbial infection, or an
underlying pathological condition [122]. The host aptly responds to a wound
through a series of events as a damage control response to restore the tissue integrity
and function [123]. This complex physiological response, termed wound healing, is
controlled and coordinated by immune cells, extracellular matrix (ECM), and com-
ponents of hemostatic system [124]. In spite of the efficient host response, some
14 Plant Latex Proteases: Natural Wound Healers 305
14.4 R
ole of Plant Latex Proteases on Different Phases
of Wound Healing
Table 14.3 The list of latex-bearing plants used by tribal populations, folk medicinal practitio-
ners, and researchers to promote wound healing and related disorders
Family Plant names Type of wounds References
1. Altingiaceae Liquidambar orientalis Excision wounds [131]
2. Amaranthaceae Achyranthes aspera Wounds [131]
3. Anacardiaceae Mangifera indica Cracks, cuts, ulcer, and [132, 133]
wounds
Pistacia atlantica Wounds [134]
Semecarpus anacardium Cuts and wounds [135]
Spondias pinnata Cuts and wounds [136]
4. Apiaceae Ferula assafoetida Ulcers [137]
5. Apocynaceae Allamanda cathartica Excision and incision wounds [138]
Alstonia angustiloba Abscesses, boils, and skin [139]
sores
Alstonia scholaris Boils, burns, and wounds [131, 140]
Alstonia venenata Cuts and wounds [141]
Carissa carandas Wounds [135]
Holarrhena pubescens All types of wounds, aphthae [133, 138,
and, ulcer in intestine 142]
Ichnocarpus frutescens Bleeding wounds [143, 144]
Plumeria obtusa Ulcers [139, 145]
Plumeria rubra Excision wounds, gingival [33, 133]
wounds, and ulcers
Rauvolfia serpentina Cuts, otorrhea, and wounds [133, 146]
Strophanthus Wounds [131]
sarmentosus
Tabernaemontana Cuts [133]
divaricata
Tabernaemontana Cuts and wounds [133]
heyneana
Vallaris solanacea Old wounds and sores [147]
Voacanga thouarsii Wounds [131]
Wrightia tinctoria Excision and fresh wounds [77, 131]
(continued)
14 Plant Latex Proteases: Natural Wound Healers 307
Table 14.3 (continued)
Family Plant names Type of wounds References
6. Asclepiadaceae Araujia sericifera Warts [148]
Asclepias curassavica Wounds [143]
Calotropis gigantea Corns, cuts, excision wounds, [43, 129,
gingival wounds, incision 133]
wounds, otorrhea, and thorn
wounds
Calotropis procera Excision wounds [138]
Ceropegia juncea Ulcers [149]
Cryptolepis buchananii Cuts and wounds [133]
Cynanchum acutum Ulcers [149]
Cynanchum callialatum Ulcers [149]
Gymnema sylvestre Cuts and wounds [149]
Hemidesmus indicus Wounds [143]
Holostemma ada kodien Blisters [131]
Schultes
Hoya lanceolata Boils [150]
Oxystelma esculentum Ulcers [149]
Pergularia extensa Wounds [131, 138]
Sarcostemma acidum Burns, chronic ulcers, and [142, 149]
wounds
Sarcostemma viminale Bleeding wounds [151]
(hemorrhage)
Tylophora indica Ulcers and wounds [149]
Tylophora fasciculata Wounds [143]
7. Asteraceae Calendula officinalis Chronic ulcers and wounds [152]
Cichorium intybus Ulcers and wounds [153]
Sonchus arvensis Burns [154]
Taraxacum officinale Corns, stomach ulcers, warts, [152, 155]
and wounds
Tragopogon dubius Heel wounds [131]
8. Campanulaceae Asyneuma rigidum Burns [134]
9. Caricaceae Carica candamarcensis Gastric ulcers and wounds [58, 131]
Carica papaya Burns and wounds [50]
10. Convolvulaceae Argyreia speciosa Boils, ulcers, and wounds [156]
Ipomoea pes-caprae Wounds [157]
Ipomoea pes-tigridis Cuts and wounds [144]
11. Dipterocarpaceae Vateria indica Wounds [131, 143]
(continued)
308 A.P. Urs et al.
Table 14.3 (continued)
Family Plant names Type of wounds References
12. Euphorbiaceae Croton bonplandianum Cuts and wounds [158, 159]
Croton macrostachyus Sores, warts, and wounds [160]
Croton megalocarpus Wounds [160]
Euphorbia agraria Wounds [134]
Euphorbia antiquorum Burns and wounds [129]
Euphorbia armena Inflamed wounds [134]
Euphorbia caducifolia Bleeding wounds and [131]
cutaneous eruptions
Euphorbia candelabrum Warts and wounds [160]
Euphorbia cuneata Sores, warts, and wounds [131]
Euphorbia grantii Bleeding open wounds and [131]
tissue healing
Euphorbia helioscopia Skin eruptions [131]
Euphorbia hirta Warts and wounds [129, 161]
Euphorbia macroclada Warts [162]
Euphorbia neriifolia Wounds [143]
Euphorbia nivulia Ulcers and wounds [142, 163]
Euphorbia pallens Skin injuries and wounds [157]
Euphorbia pilosa Wounds [160]
Euphorbia primulifolia Syphilitic sores and warts [131]
Euphorbia prostrata Abscesses and warts [164]
Euphorbia pseudograntii Abscesses, warts, and wounds [164]
Euphorbia retusa Eczema and wounds [165]
Euphorbia rothiana Acne and boils [166]
Euphorbia seguieriana Inflamed wounds [134]
Euphorbia tirucalli Warts and wounds [160, 164]
Euphorbia thymifolia Scabies, warts, and wounds [143, 164]
Euphorbia unispina Leprosy sores [164]
Euphorbia virgata Bleeding wounds [183]
Excoecaria benthamiana Warts [164]
Excoecaria grahamii Worm sores [164]
Hevea brasiliensis Dermal and lip ulcers and [87, 168]
wounds
Jatropha chevalieri Abscesses, bleeding wounds, [169]
and boils
Jatropha curcas Cuts, ulcers, whitlow, and [107, 133]
wounds
Jatropha glandulifera Bleeding cuts and mouth ulcers [142]
Jatropha gossypifolia Ulcers and wounds [131]
Jatropha heynei Burns and cuts [142]
Jatropha mollissima Wounds [170]
Jatropha multifida Ulcers and wounds [169]
Pedilanthus tithymaloides Wounds [168]
Phyllanthus emblica Wounds with maggots in cattle [133]
Ricinus communis Wounds [168]
(continued)
14 Plant Latex Proteases: Natural Wound Healers 309
Table 14.3 (continued)
Family Plant names Type of wounds References
13. Moraceae Artocarpus heterophyllus Burns, dog bite, and wounds [133, 172]
Ficus auriculata Cuts and wounds [171]
Ficus benghalensis Cracks [132]
Ficus benjamina Boils [172]
Ficus carica Boils, inflamed wound [134]
Ficus hirta Wounds [172]
Ficus hispida Bleeding, dead space, and [173]
excision and incision wounds
Ficus lacor Blisters, boils, and ulcers [172]
Ficus neriifolia Boils [172]
Ficus palmata Thorn wounds [136]
Ficus racemosa Aphthae, blisters, boils, [133, 172]
cracks, cuts, and ulcers
Ficus religiosa Cuts and wounds [135, 172]
Ficus sarmentosa Boils [172]
Ficus semicordata Boils and ulcers [172]
Ficus sycomorus Boils and scabies [160]
Ficus virens Boils [146]
Milicia excelsa Excision wounds [174]
Morus nigra Oral wounds [162]
14. Papaveraceae Argemone Mexicana Blisters, dead space, and [138, 168]
excision and incision wounds
Chelidonium majus Warts [175]
15. Phyllanthaceae Phyllanthus niruri Offensive sores and ulcers [161]
16. Plumbaginaceae Plumbago zeylanica Scabies [131]
17. Sapotaceae Bassia longifolia Wounds [143]
Madhuca indica Aphthae, cuts, ulcers in [133]
intestine, and wounds
Mimusops elengi Wounds [143]
18. Solanaceae Datura stramonium Wounds [168]
19. Thymelaeceae Aquilaria agallocha Edema, ulcers, and wounds [176, 177]
20. Urticaceae Cecropia peltata Warts and wounds [178]
14.4.1 Hemostasis
Hemostasis is the physiological process which maintains the flowing blood in fluid
state within the blood vessel, while it aims at providing thrombotic response follow-
ing injury to limit the blood loss [179]. As an exogenous hemostatic agent, latex is
an important plant-based component, which is widely employed in traditional sys-
tem of medicine because of its ability to stop bleeding from fresh wounds [180].
The use of topical hemostatic agents from natural sources is gaining importance in
wound care and management, owing to the efficacy and safety of naturally derived
hemostatic agents [181]. The property of latex to stop bleeding is mostly attributed
to proteases [182]. The majority of latex proteases exhibits procoagulant effect irre-
spective of the nature of proteases. Calotropis gigantea latex protease has been
310 A.P. Urs et al.
Fig. 14.1 Physiological events during the four phases of wound healing and the role of plant latex
proteases (PLPs). PLPs exhibiting specific function are given in Table 14.1
Clinically, inflammation is the second stage of wound healing that shows character-
istic symptoms such as erythema, swelling, warmth, discomfort, and often associ-
ated with pain and functional disturbance [183]. The inflammatory phase ensures
that the injury-causing agent is attenuated, diluted, and neutralized. Collectively,
14 Plant Latex Proteases: Natural Wound Healers 311
Fig. 14.2 Purified latex proteases interfere in the coagulation cascade mainly in the common
pathway by activating factor X and exhibiting factor XIIIa-like, thrombin-like, and plasmin-like
activities
during this phase, the injured region is prepared for healing [184]. Although there is
fine balance between inflammatory mediators regulating inflammatory phase of
wound healing, latex proteases have been reported to mediate the inflammatory
phase and its smooth transition to repair phase. Few antimicrobial (Amp48 from
Artocarpus heterophyllus, Cg24-I from Cryptostegia grandiflora, and hevains from
Hevea brasiliensis) and anti-inflammatory (plumerin-R from Plumeria rubra) pro-
teases have been isolated from latices which help in attenuating the infection-
causing microbes. And mainly latex proteases have fibrinolytic (Amp48 from
Artocarpus heterophyllus, crinumin from Crinum asiaticum, hirtin from Euphorbia
hirta, LGP from Synadenium grantii, and plumerin-R from Plumeria rubra), gelati-
nolytic (serine protease from Ficus carica), and collagenolytic (serine protease
from Ficus carica) activities which help in wound debridement (Fig. 14.3a and
Table 14.1). Wound debridement is an important event during natural wound heal-
ing process and one of the vital aspects of successful wound management strategy.
It is the removal of nonviable/dead, contaminated tissue and foreign material from
the wound site, promoting the formation of granulation tissue and facilitating the
progression of ordered wound healing [5, 185]. The presence of necrotic tissue in
wound site mimics signs of infection and provides a suitable substrate for infecting
microbes [186]. It also slows down the vital repair events including matrix forma-
tion, angiogenesis, granulation, tissue formation, and epidermal resurfacing [186].
312 A.P. Urs et al.
Fig. 14.3 Plant latex proteases aid (A) inflammatory, (B) proliferative, and (C) remodeling phases
of wound healing
During the phase of repair, fresh tissue is layered in place of evacuated necrotic tis-
sue [188]. The way for the formation of fresh tissue will be paved by the process of
debridement of dead tissues by latex proteases in conjunction with endogenous pro-
teases which are prerequisite for proliferative phase. Plumerin-R isolated from
Plumeria rubra latex enhances wound healing by increasing the collagen formation
(Fig. 14.3b). Collagen plays a central role in the healing of wounds as it is a princi-
pal component of connective tissue and provides a structural framework for regen-
erating tissue [33].
Angiogenesis, revascularization, and enhanced vascular permeability are crucial
events to wound repair as they rescue tissues from ischemia. These events allow a
variety of cytokines, growth factors, and nutrients to reach the damaged tissue and
are also important for metabolite clearance. Further, proliferation of cells is impor-
tant to replace the damaged tissues. Few latex proteases from Carica candamarcen-
sis and Hevea brasiliensis have been shown with angiogenic and cell proliferative
properties (mitogenic) along with the ability to increase vascular permeability and
to activate the extracellular signal-regulated protein kinase (ERK) (Fig. 14.3b) [87,
189]. The ERK signaling cascade is a central MAPK pathway that plays a role in the
regulation of cellular processes such as proliferation, differentiation, and develop-
ment [190].
The events of remodeling phase include the deposition of matrix and its subsequent
changes including the alignment of ECM molecules along the tension lines [191].
Tissue remodeling occurs throughout the wound repair process and can overlap
with the repair phase as it can begin as early as 1 week after injury and can last as
long as 2 years, depending on the extent of wound [192]. Endogenous matrix metal-
loprotease expression is very important in the remodeling of collagen and non-
collagen extracellular matrix (ECM) components [193]. In similar lines,
collagenolytic and other ECM-degrading proteases of latex may also mediate the
14 Plant Latex Proteases: Natural Wound Healers 313
14.5 E
xperimental Validation for Plant Latex Proteases
as Wound Healers
Wound healing potential of latex proteases is studied mostly in rodents (mice, rat,
rabbit, and guinea pig) by excision, incision, and burn models. In situ, wound
healing activity is assessed by cell proliferation assay in various cell lines. Purified
proteases from latex of few plants have been shown to promote wound healing in
experimental animals, and few are being used clinically as approved wound care
supplements. Papain and chymopapain either singly or in combination with essen-
tial factors are being used in management of wound-related complications [13,
195]. Papain is widely used for clinical debridement in case of chronic wounds
with overgranulated tissue. Few proteases from plant latices, namely, curcain
(Jatropha curcas) and plumerin-R (Plumeria rubra), have been reported to pro-
mote wound healing in experimental animals. Although proteases are extensively
used for debridement, some are hemostatic (ficins, Pergularain e I, LGP) or anti-
microbial (Amp48 and Cg24-I) or promote the collagenization and fibrosis pro-
cess (plumerin-R) [33, 107, 163]. Apart from the common mechanisms mentioned,
few latex proteases exhibit mitogenic activity (CMS2MS2) evaluated in fibroblast
cell culture and show increased neovascularization and epithelialization (P1G10)
[52–54] (Table 14.1).
The reports from our lab also validate the use of latex for treating bleeding and
wounds by traditional practitioners. The benefits of latex proteases in the first phase
of wound healing, i.e., hemostasis, are due to procoagulant nature and thrombin-like
activity of proteases. And they also have beneficial effects in second phase of wound
healing, i.e., inflammatory phase, because of their fibrinolytic (plasmin-like activ-
ity), gelatinolytic, and collagenolytic activity which helps in wound debridement.
These results were also evaluated using purified proteases in mice model giving
substantial evidence [27, 28, 77, 94, 182, 196, 197]. Currently, we are engaged in
the elucidation of molecular mechanisms of plant latex proteases in hemostasis and
wound healing at the level of genus and species of latex-producing families.
314 A.P. Urs et al.
Acknowledgements APU and RGV thank University Grants Commission (UGC), New Delhi
and MVN thank IOE [funded by Ministry of Human Resource Development (MHRD), Government
of India], UOM, Mysuru for fellowships. Authors also thank UGC-SAP, DST-PURSE and VGST
for financial assistance to Department of Studies in Biochemistry, UOM and Vijnana Bhavan,
UOM.
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Emerging Roles of Mitochondrial Serine
Protease HtrA2 in Neurodegeneration 15
Ajay R. Wagh and Kakoli Bose
Abstract
High temperature requirement mitochondrial serine protease A2 (HtrA2), com-
monly known as Omi/PRSS25, is primarily known for its protein quality control
function. Loss of this prime function of the protein results in neurodegenerative
diseases such as Parkinson’s, Alzheimer’s, Huntington’s and certain forms of
epilepsy. HtrA2 belongs to the family of evolutionarily conserved proteases and
is one of the core determinants of mitochondrial quality control. HtrA2 helps
maintain normal mitochondrial functions during organelle biogenesis, metabolic
remodelling and stress. It has been very well established that under stressful
condition, HtrA2 is released from the mitochondria into the cytosol and facili-
tates apoptosis by binding to several members of the inhibitors of apoptosis pro-
tein (IAP) family. On the contrary, in vivo knockout studies showed a phenotype
similar to Parkinson’s suggesting its involvement in neurodegeneration along
with maintenance of mitochondrial homeostasis. Therefore, presence of different
cellular pathways and its unique multitasking ability makes HtrA2 a potential
therapeutic target. This chapter discusses different facets of HtrA2 with main
focus on its role as a quality control protease and its association with neurode-
generative disorders.
Keywords
Mitochondrial protein quality control • HtrA2 • Neurodegenerative disorders
15.1 Introduction
Folding Aggregation
Properly folded protein unfolded Aggregation of unfolded
protein
Nucleus
Diseased cell
Fig. 15.1 Perturbations in protein quality control. Cellular stress and lack of protein quality
control lead to aggregation of properly folded proteins in the mitochondrial membrane resulting in
a diseased cell
15 Emerging Roles of Mitochondrial Serine Protease HtrA2 in Neurodegeneration 327
Human HtrA1
(Full length)
1 22 33 100 98 157 206 364 382 473 480
Human HtrA2
(Full length)
Bacterial HtrA
(Deg P)
Bacterial HtrA
(Deg S)
Fig. 15.2 Domain organization of HtrA family members. Domain organization of different
HtrA protease family members. The numbers adjacent to each protein represent the size of each
protease and their individual domains. The main structural characteristics of HtrA family member
proteins are represented as boxes of different colours according to the key at the bottom of the
figure
All HtrA family proteins are involved in a protein quality control [22]. One of the
family members, DegP that is present in Escherichia coli (E. coli), has an additional
role of a chaperone that stabilizes specific proteins present in the cell [23]. In addi-
tion, HtrA proteases can activate or regulate various signalling pathways. This is
reflected in the multifaceted cellular functions with which they are associated such
as bacterial virulence, maintenance of the photosynthetic apparatus, proliferation,
cell migration and cell fate [22, 24, 25]. In mammals, several diseases are associated
with loss protease activity of HtrAs that include arthritis, cancer, age-related macu-
lar degeneration as well as Parkinson’s and Alzheimer’s diseases [26–30].
The variety of cellular functions that HtrAs are associated with primarily depends
on their cellular localization. Most HtrAs are localized extra-cytoplasmically; for
example, while in Gram-negative bacteria they reside in the cell envelope, in case of
Gram-positive bacteria, they are found in the extracellular space. Similarly, in
eukaryotes, they reside in mitochondria, chloroplasts, nucleus and extracellular
matrix.
Bacterial HtrAs are typically involved in various aspects of protein quality con-
trol, including the cellular response to protein folding stress and the degradation of
misfolded and mislocalized cell envelope proteins [22]. Likewise, eukaryotic HtrAs
15 Emerging Roles of Mitochondrial Serine Protease HtrA2 in Neurodegeneration 329
A)
Inter membrane space
(IMS)
PARL protease
Matrix
m-AAA protease
(AFG3L2/ paraplegin)
Matrix
ClpXP Lon
B) TM TM
TM
IBM
TM TM TM
PARL MTS 1 2 3 4 5 6
Six-TM protease
TM TM TM
Fig. 15.3 Schematic representation of different mitochondrial quality control proteases. (a)
Figure depicting localization of quality control proteases in mitochondria. (b) Cartoon represent-
ing domain organization of these mitochondrial proteases. Description of each one of these prote-
ases has been provided in the text (Abbreviations: MTS, mitochondrial targeting sequence; TM,
transmembrane domain; AAA, triple-A domain (ATPase associated with various cellular activi-
ties); M41 protease, metal binding proteolytic domain; CC, coiled coil; IBM, inhibitor of apoptosis
(IAP)-binding motif; S1 protease, trypsin-like protease domain; PDZ, postsynaptic of 95 kDa, disc
large, zonula occludens; IMS; intermembrane space; IM, inner membrane)
15 Emerging Roles of Mitochondrial Serine Protease HtrA2 in Neurodegeneration 331
2p13.1
Chromosome 2
HTRA2
Fig. 15.4 Localization and schematic organization of the HTRA2 gene. Illustration of HTRA2
gene on chromosome 2p13.1. The chromosomal localization has been zoomed below to show the
exons present in HTRA2 (dark yellow)
The nuclear-encoded HtrA2 gene with eight exons has its chromosomal localization
at 2p13.1 (Fig. 15.4). The gene codes for a 49 kDa polypeptide of 458 amino acid
residues. After complete translation, the full-length protein is targeted primarily to
the intermembrane space (IMS) [57], where it is attached through its N-terminal
transmembrane anchor to the inner membrane of mitochondria. During maturation,
the first 133 amino acids from the N-terminus get cleaved, and upon apoptotic stim-
ulation, it is released from IMS into the cytosol as a 36 kDa mature protease [51–53,
57] (Fig. 15.5). This cleavage exposes an internal tetrapeptide motif (AVPS) that
binds to inhibitors of apoptotic proteins (IAPs) such as XIAP, cIAP, etc. and relieves
their inhibition on caspases, thus promoting apoptosis.
The structure of mature form of human HtrA2 in a substrate-unbound form has been
solved by X-ray crystallography at 2.1 Å (Fig. 15.6) [58] that provides a wide over-
view of the global structural organization of the inactive protease. It has a trimeric
pyramidal architecture with the short N-terminal region at the top and PDZ domains
residing at the base of the pyramid. Each protease domain comprises 7α-helices and
19β-strands, which fold into a compact globular structure. Apart from the β-strands,
the protease domain contains several loops, which are named according to the chy-
motrypsin nomenclature—LA (residues 170–174), L1 (302–306), L2 (323–329),
L3 (275–295) and LD (259–266). These regulatory loops harbour active site pocket
as well as accommodate the catalytic triad residues (Ser306, His198 and Asp228) in
the hydrophobic core of the serine protease domain. Therefore these dynamic
332
IMS
TM
IBM
Mitochondrial
Matrix 1 458
IMM
AVPS
H198 D228 S306
IBM
458
133
Transporter Processed (Mature) form of HtrA2 (325 amino acid long, 36 kDa)
OMM
Mitochondrion Cytosol
Fig. 15.5 Schematic representation of full-length HtrA2 and its maturation process. Full-length HtrA2 (1–458 aas), comprising a mitochondrial localiza-
tion signal and a transmembrane domain (1–133 aas), is localized in the mitochondria. Maturation occurs as a result of cellular stress that removes first 133
amino acids and exposes a tetrapeptide IBM motif (AVPS) in its N-terminus. The catalytic triad comprising His 198, D228 and S306 has been shown in the
figure. The full-length HtrA2 has been shown as light blue ‘coil-like’ structure attached to mitochondrial inner membrane. Here, amino acids are referred to as
‘aas’. (Abbreviations: MLS, mitochondrial localization sequence; TM, transmembrane domain; IBM, IAP-binding motif; PDZ, postsynaptic of 95 kDa, disc
large, zonula occludens)
A.R. Wagh and K. Bose
15 Emerging Roles of Mitochondrial Serine Protease HtrA2 in Neurodegeneration 333
A
I
NTD
Trimerization
SPD III II
PDZ
Monomeric HtrA2
Tyr 147
Phe 149
Phe 256
B SPD
NTD
PDZ
Monomeric HtrA2
Ser306
His198
Asp228
Fig. 15.6 Structure of mature HtrA2. (a) Crystal structure of mature inactive (S306A) HtrA2
(PDB: 1LCY) protein with 7α helix and 19β-strands [58]. The trimerization residues have been
highlighted at the bottom of the figure. (b) Mature form of HtrA2 protein with catalytic triad (His
65, Asp 98, Ser 173). Different domains have been labelled and coloured differently. Images have
been created using PyMol, Delano Scientific, USA
334 A.R. Wagh and K. Bose
Cytosol
N-
terminal
P38 kinase Mitochondria
domain
pink1
HtrA2
Inactive HtrA2
PDZ
P P
Cleaved
linker Substrate
Substrate
Active site
P Phosphate
Fig. 15.7 Model for HtrA2 activation during stress. Binding of N-/C-terminal substrates result in intricate intermolecular PDZ-protease interactions and
subtle conformational rearrangements in the regulatory loops of HtrA2 protease. This, along with phosphorylation by p38 and PINK1 kinases, leads to forma-
tion of the active enzyme
A.R. Wagh and K. Bose
15 Emerging Roles of Mitochondrial Serine Protease HtrA2 in Neurodegeneration 337
[65]. Furthermore, it has been shown that binding of certain peptides to the PDZ
domain leads to enhanced HtrA2 activity. Ligand specificity at PDZ domain was
determined to characterize its binding properties using peptide libraries fused to the
C- or N-terminus of a phage coat protein. Series of peptides binding to the isolated
PDZ domain were selected, and it was found that the peptide GQYYFV (termed
PDZopt), which binds efficiently to PDZ, was able to stimulate HtrA2 activity that
was quantitatively determined using synthetic substrate peptides [66]. Thus, engage-
ment of binding partners with the PDZ domains results in opening up of access to
the catalytic site.
However, recent studies suggest a dual regulatory switch in HtrA2, since apart
from the classical mode of allosteric propagation, activation through N-terminal
‘AVPS’ tetrapeptide binding has also been observed, which adds complexity to its
overall mode of action [60]. However, further studies are needed to follow the com-
plex allosteric pathway at the molecular level and delineate the biological signifi-
cance of this tight regulation on HtrA2 activity.
Perturbation in the dynamic functions of HtrA2 protein, which include cell death
and cellular protein quality control, leads to distinctive defects in neurons and is
recognized as a key player in neurodegeneration. Two decades earlier, Gray and his
collaborators demonstrated the interaction between HtrA2 and presenilin-1 using a
yeast two-hybrid system, thus establishing its link with neurodegeneration [39].
Presenilin-1 is a catalytic component of γ-secretase enzyme that is implicated in the
inherited forms of early-onset Alzheimer’s disease. This interaction was later on
validated in vitro along with characterization of presenilin-mediated regulation of
HtrA2 protease activity [65]. Park et al. where in an in vitro system, demonstrated
interaction between HtrA2 and AD-associated amyloid β [71]. Similarly, in a break-
through in vivo study done by Jones et al. [55], a homozygous loss-of-function
15 Emerging Roles of Mitochondrial Serine Protease HtrA2 in Neurodegeneration 339
mutation (S276C) in the HtrA2 gene was identified in mnd2 mice leading to neuro-
degeneration, muscle wasting and death by 40 days of age. In another study, Martins
and co-workers generated HtrA2 knockout mice to emphasize the physiological
relevance of its protease activity. It was a clear observation that HtrA2 knockout
mice displayed neurodegenerative phenotype with Parkinsonian features [56].
Taken together, the similarity with the activities of bacterial HtrA2 homologues and
the fact that the loss of proteolytic activity in both mnd2 and HtrA2 knockout mice
result in enhanced sensitivity to stress, it can be said that this protease might play an
essential role in the mitochondria-related stress signals.
In the following sections, we summarize the available information pertaining to
the involvement of HtrA2 in the onset and progression of neurodegenerative dis-
eases, namely, Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s
disease (HD).
Alzheimer’s disease (AD) is one of the major health problems in the world. It is a
progressive and irreversible neurodegenerative disorder that is biochemically char-
acterized by the occurrence of various pathological features such as formation of
neurofibrillary tangles within neurons, progressive neuronal loss and accumulation
of amyloid (Aβ) in the walls of the blood vessels and senile plaques. This represents
the most common cause of dementia worldwide [72]. Mutations either in the pre-
cursor protein for Aβ (the β-amyloid precursor protein, APP) or in presenilin-1 (PS-
1) or presenilin-2 (PS-2) are the exclusive reasons for causing familial early-onset
forms of AD.
Amyloid beta (Aβ or Abeta) refers to a peptide comprising residues 36–43
(~4 kDa) of amyloid precursor protein (APP) that acts as the main component of the
amyloid plaques, primarily found in the brains of Alzheimer’s patients. APP protein
is found in many tissues and organs, including the brain and spinal cord (central
nervous system), with mostly unknown functions. Researchers hypothesize that it
may be associated with other proteins on the surface of cells or help cells attach to
one another. Recent studies suggest that it helps direct the movement of nerve cells
during early brain development.
According to the amyloid cascade hypothesis, APP is cleaved by successive
actions ofα, β- and γ-secretases, a family of proteolytic enzymes that process sev-
eral transmembrane proteins including APP at the transmembrane region. While
cleavage by α-secretase does not lead to Aβ formation, β- and γ-secretases cleave at
N- and C-termini of APP, respectively, and are key players in Aβ deposition.
γ-secretase attacks the C-terminal end of the Aβ peptide, cleaves within the trans-
membrane region of APP and generates a number of isoforms of 39–43 amino acid
residues in length (Fig. 15.8) [73].
The most common isoforms are Aβ40 and Aβ42. The Aβ40 form is the more
common of the two, but Aβ42 is the more fibrillogenic and toxic in nature due to its
high aggregation property and is thus associated with disease conditions. Mutations
340
sAPP β
A
C- terminal end Aβ
Monomeric Aβ Oligomeric Aβ
Extracellular
β-secretase
Aβ Aβ
-secretase
APP Intracellular space
N - terminal end
B AICD
β α Y40 Y42
Fig. 15.8 Molecular mechanism of Aβ peptide formation. (a) Processing of the membrane-bound amyloid precursor protein (APP) by β- and γ-secretases
leads to the formation of Aβ peptides. The cleavage products are finally secreted outside the cell. Cleavage by γ-secretase results in either Aβ 40 (soluble non-
toxic) or Aβ 42 (fibrillar, toxic) peptides, the latter being responsible for production of amyloid plaques. (b) Amyloid beta sequence with α, β and γ-secretase
cleavage sites (abbreviations: sAPPβ, soluble β-secretase producing APP; AICD, amyloid intracellular domain)
A.R. Wagh and K. Bose
15 Emerging Roles of Mitochondrial Serine Protease HtrA2 in Neurodegeneration 341
in APP associated with early-onset Alzheimer’s have been noted to increase the
relative production of Aβ42, and thus one suggested outcome of Alzheimer’s ther-
apy involves regulating the activity of β- and γ- secretases to produce mainly Aβ40
[74].
increased death of neural cells by apoptosis and necrosis. This interaction has been
hypothesized to regulate HtrA2 activity and determines its release from the mito-
chondria during apoptosis [39] [65]. Moreover, it has been demonstrated that HtrA2
selectively interacts with and disaggregates more neurotoxic oligomeric Aβ42
rather than its less toxic monomeric (Aβ42) form. Therefore, this interaction not
only protects neurons from the neurotoxic Aβ accumulation but also aids in the
decrease in proapoptotic activity of HtrA2, thus preventing death of neural cells.
This phenomenon has been aptly termed as ‘mutual detoxification’ [93].
Thus, HtrA2 aids in reducing the toxic effects of oligomeric Aβ, which makes it
a potential therapeutic target in neurodegenerative diseases.
Parkinson’s disease (PD) is the most common movement disorder and the second
most common neurodegenerative disease after Alzheimer’s that affects 1% of the
population over the age of 65. It is pathologically characterized by an exclusive
degeneration of dopamine releasing neurons of the substantia nigra pars compacta
in the brain and the presence of characteristic proteinaceous intracytoplasmic inclu-
sions, known as Lewy bodies in the affected brain areas [97]. Several reports pro-
posed that environmental factors, genetic sensitivity and ageing are important
components, which lead to the progression of this disorder [98–100].
Basically, the whole understanding of the molecular events in PD pathogenesis
has been greatly advanced by the identification and analysis of PD-associated genes
[101] that provided insights into the cellular mechanisms underlying PD
15 Emerging Roles of Mitochondrial Serine Protease HtrA2 in Neurodegeneration 343
pathogenesis [102, 103]. Over the past 20 years, mutations in several genes have
been definitively shown to mediate familial PD. Till date, 18 nuclear PD-related
genes (PARK) and some of their mutants have been implicated in the pathogenesis
of PD [104–111]. The PARK genes and their loci have been elaborated in Table 15.1.
Likewise, mutations in SNCA that encodes α-synuclein protein (PARK1) [106]
(a major component of Lewy bodies), PARK4 [105] and leucine-rich repeat kinase 2
(LRRK2)/dardarin (PARK8) [108, 109] have been found to be associated with
autosomal-dominant form of the disease. However, mutations in PARKIN (PARK2)
[101, 104], DJ-1 (PARK7) [107] and PTEN-induced kinase 1 (PINK1, PARK6)
[112] that are mitochondrially associated proteins are mainly involved in autosomal
recessive forms of PD. Mutations in ATP13A2 (PARK9), which encodes a lyso-
somal ATPase, have also been found in an atypical, autosomal recessive Parkinsonism
[111]. More recently, mutations in two other PARK genes VPS35 (vacuolar protein
sorting-associated protein 35) (PARK17) [113, 114] and EIF4G1 (eukaryotic trans-
lation initiation factor 4 gamma 1) (PARK18) [115] have been reported to cause
autosomal dominant form of PD.
HTRA2 (PARK13) [68] has also been implicated in autosomal recessive PD post
identification and characterization of two mutations that interfere with its protease
activity [116]. The first connection between HtrA2 dysfunction and PD came from
the characterization of mnd2 mutant mice as described in Sect. 3. The mnd2 muta-
tion, leading to neurodegeneration, muscle wasting, involution of the spleen and
thymus and death by 40 days of age, was identified in the HTRA2 gene [55].
Moreover, a neurodegenerative phenotype with Parkinsonian features has been
described in the HTRA2 knockout mice [56]. A loss-of-HTRA2 study on the mouse
344 A.R. Wagh and K. Bose
A SA
AVPSPPP ASP RSQYNFIADV VEKTAPAVVY IEILDRHPFL GREVPISNGS GFVVAADGLI VTNAHVVADR RRVRVRLLSG DTYEAVVTAV
20 30 40 50 60 70 80 90
A141S P143A
DPVADIATLR IQTKEPLPTL PLGRSADVRQ GEFVVAMGSP FALQNTITSG IVSSAQRPAR DLGLPQTNVE YIQTDAAIDF GNSGGPLVNL
100 110 120 130 140 150 160 170 180
S
DGEVIGVNTM KVTAGISFAI PSDRLREFLH RGEKKNSSSG ISGSQRRYIG VMMLTLSPSI LAELQLREPS FPDVQHGVLI HKVIL GSPAH
190 200 210 220 230 240 250 260 270
W G266S
R AGLRPGDVI LAIGEQMVQN AEDVYEAVRT QSQLAVQIRR GRETLTLYVT PEVTELE
280 290 300 310 320 327
R404W
G266S
R404W
A141S
P143A
P P
S A S W
B
Human HtrA2
AVPSPPPASPRSQYN LIHKVILGSPAHRAG
Amino Terminus PDZ Domain
Fig. 15.9 Pathogenic mutations and putative phosphorylation sites in HtrA2. (a) Locations
of PD-linked mutations A141S, G399S and R404W indicated in ‘red’ on the wild-type sequence
as well as on the crystal structure of HtrA2 (PDB: 1LCY). A141, G399 and R404 are surface
exposed residues. (b) The relative positions of phosphorylation sites in human HtrA2 are indicated
by circles above the serine residues
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Functional Relevance of Deubiquitinases
in Life and Disease 16
Julia M. Fraile, Carlos López-Otín, and José M.P. Freije
Abstract
Deubiquitinases (DUBs) are critical regulators of ubiquitin-mediated signaling
pathways through their ability to cleave the isopeptide bond that links ubiquitin
to target proteins. The human genome encodes at least 100 DUBs, grouped into
six families depending on sequence and structural properties. These proteolytic
enzymes have pivotal roles in ubiquitin homeostasis and control of protein stabil-
ity, and, consequently, their activities are tightly regulated by different mecha-
nisms. Due to their wide diversity, DUBs are involved in multiple biological and
pathological processes, including cancer. Accordingly, over the last years, many
mutations in DUB genes or changes in their expression levels have been related
to human malignancies. This chapter will focus on the description of the func-
tional complexity of these enzymes in physiological and pathological conditions.
We will review the involvement of DUBs in numerous biological processes and
highlight their critical implications in cancer. Finally, we will discuss the grow-
ing relevance of DUBs for the development of novel therapeutic strategies
against cancer.
Keywords
Ubiquitin • Deubiquitinases • DUB genes • Cancer
16.1 Introduction
Fig. 16.1 Classification of human DUBs. Human DUBs are classified into six families repre-
sented separately and in different colors: USPs (red), UCHs (orange), OTUs (green), MJDs (light
green), JAMMs (blue), and MCPIPs (purple). The catalytic core is indicated in plain light red if the
DUB is active and stripped if inactive. Additional domains are shown in different colors. DUBs are
represented with their N-termini oriented toward the center of the circle
358 J.M. Fraile et al.
DUBs have fundamental roles in ubiquitin homeostasis and protein stability. In this
regard, DUBs generate free ubiquitin through the ability to process ubiquitin pre-
cursors and also promote the stability of ubiquitinated proteins by preventing both
lysosomal and proteasomal degradation, depending on the nature of the ubiquitin
linkage [14]. Consequently, an additional level of regulation of DUB function
comes from the specificity for the ubiquitin chain linkages they processed (Fig. 16.2).
USPs and UCHs cleave indiscriminately most ubiquitin chain types, being Lys48-
or Lys63-linked polyubiquitin chains their most frequent targets. Interestingly, com-
plex mechanisms of action have also been described for some members of the
family. Thus, USP14 suppresses protein degradation by removing ubiquitin chains
en bloc, independently of chain linkage type [15]. On the other hand, JAMMs and
ATXN3 show a restricted specificity for Lys63-linked chains [12]. Similarly, most
human OTUs show intrinsic linkage specificity, preferring one or a small defined
subset of ubiquitin linkage types [7]. Although A20 only depolymerizes Lys63-
linked polyubiquitin, its function in inflammation is critically regulated by linear
ubiquitination levels [16]. Finally, OTULIN was described to exquisitely hydrolyze
linear ubiquitin chains [17].
Due to their importance in protein homeostasis, DUB activities are regulated
through a number of different mechanisms, such as transcriptional control of gene
expression, posttranscriptional modifications, changes in subcellular localization,
and activation mediated by interacting proteins [18]. Examples of transcriptional
regulation affect DUB-1, DUB-2, and DUB-3 [19–21], whose expression is induced
by inflammatory cytokines. Moreover, CYLD is induced by the activation of the
nuclear factor-κB (NF-κB) and MAPK kinase 3/6 (MKK3/6)-p38 pathways [22].
16 Functional Relevance of Deubiquitinases in Life and Disease 359
Fig. 16.2 Ubiquitin chain types processed by DUBs. Representation of the specificity of DUB
families for different types of polyubiquitinated proteins and their cellular functions
Bcl10 promotes A20 expression through the activation of its promoter, which con-
tains NF-κB-binding sites [23], and Snail1 mediates the transcriptional repression
of Cezanne2 in hepatocellular carcinoma [24]. Furthermore, miR-26b and miR-
4295 regulate USP9X and USP28, respectively, at posttranscriptional level [25, 26].
Many DUB functions are also regulated by posttranslational modifications.
Phosphorylation inhibits CYLD and USP8, while it activates A20, OTUD5, USP1,
USP4, USP7, USP15, USP16, USP19, USP28, USP34, and USP37 [27–29].
Furthermore, ubiquitin and ubiquitin-like modifications also modulate DUB func-
tion. Thus, ATXN3 and UCHL1 activities are modulated by ubiquitination [30, 31],
whereas USP25, USP28, and CYLD are regulated by SUMO conjugation [32–34].
Reactive oxygen or nitrogen species are also involved in regulating DUB activities
[35, 36]. Finally, USP1 and ATXN3 are inactivated by autoproteolytic cleavage,
whereas CYLD and A20 are inhibited through interaction with other proteases
[37–39].
As mentioned above, DUB activity can also be regulated by changes in subcel-
lular localization, which can facilitate the interaction with specific substrates.
USP36 localization in the nucleolus regulates its structure and function [40],
whereas USP30 modulates the morphological properties of mitochondria [41].
Moreover, USP1 contains two nuclear localization signals that mediate nuclear
import of the USP1/UAF1 complex [42]. The presence of ubiquitin-binding and
ubiquitin-like domains also contributes to regulate the activity and specificity of
several DUBs [2].
360 J.M. Fraile et al.
Finally, the regulation of many DUBs is mediated by the association with their
interaction partners. Thus, USP1 plays a role in DNA damage repair by interacting
with UAF1 [43]. Similarly, interaction of ASXL1 and ASXL2 with BAP1 is required
for ubiquitin binding and H2A deubiquitination [44, 45], and valosin-containing
protein (VCP/p97) interacts with ATXN3 to specify the cellular fate of its targets
[46]. In addition, guanosine5’-monophosphate synthase and USP7 form a deubiqui-
tination complex that is required to stabilize p53 [47]. Moreover, yeast DUB,
Ubp10, possesses multiple binding modules that regulate protein interaction and are
critical for ribosome biogenesis. Interestingly, its human homologue, USP36, con-
tains the same regions flanking its catalytic domain [48]. Several DUBs have been
reported to be integrated within large macromolecular complexes, such as protea-
some or COP9 signalosome, to become active [49]. Finally, a global proteomics
approach aimed at identifying proteins interacting with 75 DUBs uncovered a com-
plex landscape of more than 770 putatively associated proteins, thus reflecting the
complexity in the regulation of DUB functions [50].
The wide functional diversity of DUBs has a profound impact on the regulation of
multiple biological processes such as cell cycle regulation, DNA damage repair,
chromatin remodeling, and several signaling pathways, which are frequently altered
in human diseases, especially in cancer [3]. In this section, we will discuss the cur-
rent knowledge of DUB functions within each of these biological processes.
Several members of the DUB superfamily are critical components of the core cell
cycle machinery and cell cycle checkpoint. Functional analysis links USP28,
USP36, and USP37 with the stability of c-Myc, a central modulator of cell growth,
proliferation, and apoptosis [51–53]. By contrast, USP10 antagonizes the transcrip-
tional activity of c-Myc and inhibits cell cycle proliferation through the stabilization
of SIRT6 [54]. USP4 regulates the mono-ubiquitination of PDK1, a master growth
factor signaling kinase that plays a critical role in cell proliferation and metabolism
[55], whereas USP7 has an essential role in cell proliferation through the regulation
of phosphatase and tensin homologue (PTEN) and FOXO localization [56, 57].
Furthermore, PTEN transcription is regulated by ATXN3 [58], and USP13 and
OTUD3 regulate PTEN protein levels by deubiquitination [59, 60]. Similarly,
UCHL5 deubiquitinating activity modulates the transcriptional activity of E2F1, an
important transcription factor involved in cell cycle progression, DNA repair, and
apoptosis response [61].
Many DUBs are crucial regulators of events occurring in mitosis, such as CYLD
that modulates mitotic spindle orientation and regulates polo-like kinase 1 [62].
USP13 and USP37 antagonize anaphase-promoting complex (APC/C)-Cdh1
16 Functional Relevance of Deubiquitinases in Life and Disease 361
Several DUBs are essential components of DNA repair mechanisms, which are fre-
quently altered in human malignancies. USP1 deubiquitinates Fanconi’s anemia
protein (FANCD2), stabilizes CHK1, regulates the ubiquitination levels of prolifer-
ating cell nuclear antigen (PCNA), and interacts with UAF1 to promote double-
strand break repair through homologous recombination [37, 43, 81, 82]. Moreover,
USP3, USP16, USP26, USP37, USP44, OTUB1, BRCC36, and POH1 modulate the
RNF8c/168 pathway of double-strand break (DSB) repair [83]. POH1 and BAP1
facilitate homologous recombination repair through loading RAD51 at DNA dam-
age sites [84, 85], and USP11 and USP28 regulate the cellular response to mitomy-
cin C-induced DNA damage within the BRCA2 pathway and CHK2-p53-PUMA
pathway, respectively [86, 87]. USP9X regulates DNA damage responses through
the stabilization of CLASPIN [88], whereas USP17L2 and USP51 control H2AX
ubiquitination [89, 90]. Interestingly, Nishi and collaborators have demonstrated the
role of UCHL5 in DNA damage repair by inhibiting NF-κB degradation [91].
Another important DUB with critical functions in DNA damage repair is USP7,
which is a critical regulator of RAD18 protein levels [92] and suppresses oxidative
stress-induced PCNA ubiquitination and mutagenesis [93]. Furthermore, ATM-
dependent downregulation of USP7 by the phosphatase PPM1G leads to a
p53-dependent DNA damage response [94] and similarly to USP1, USP7 also
362 J.M. Fraile et al.
During the last decades, many DUBs have been described to regulate different sig-
naling pathways, which are frequently altered in cancer. In this section, we will
discuss the implication of DUBs in the regulation of several cancer-relevant path-
ways, such as those involving p53, NF-κB, receptor tyrosine kinases (RTKs), Wnt,
and transforming growth factor-β (TGF-β) (Table 16.1).
p53 is a tumor suppressor gene whose function is essential for preventing cancer
formation. So far, USP2, USP4, USP7, USP10, USP15, USP22, USP24, USP29,
USP42, and OTUB1 are known to participate in p53 regulation. USP7 has a dual
16 Functional Relevance of Deubiquitinases in Life and Disease 363
role in regulating the stability of p53 since it can deubiquitinate both p53 and
MDM2, an ubiquitin ligase that targets p53 for proteasomal degradation [111, 112].
USP2 and USP15 stabilize MDM2 but not p53 [113, 114], and OTUB1 directly
suppresses MDM2-mediated p53 ubiquitination [115]. Moreover, USP10 stabilizes
both mutated and wild-type p53, thus having a dual role in tumorigenesis depending
on p53 status [116]. By contrast, USP4 promotes p53 degradation through the deu-
biquitination of ARF-BP1 ubiquitin ligase and the histone deacetylase HDAC2
[117, 118]. Furthermore, USP22 inhibits p53 transcriptional activation through the
deubiquitination of SIRT1, leading to decreased levels of p53 acetylation and sup-
pressing p53-mediated functions [119]. USP24 and USP29 deubiquitinate p53 and
protect genomic stability by regulating UV damage and oxidative stress responses,
respectively [120, 121]. Finally, USP42 forms a direct complex with and deubiqui-
tinates p53 during the early phase of the response to a range of stress signals [122].
Nuclear factor-κB is a well-known modulator of innate and adaptive immune
responses that is frequently deregulated in cancer [123]. A20 and CYLD act as
tumor suppressors by inhibiting NF-κB signaling through the regulation of several
components of the pathway [124]. Thus, both of them deubiquitinate TRAF6,
whereas CYLD also controls the ubiquitin levels of TGF-β-activated kinase 1
(TAK1) [125], B-cell CLL/lymphoma 3 (Bcl3) [126], and mitogen-activated protein
kinases [127]. On the other hand, A20 promotes the degradation of TRAF2 in lyso-
somes by means of its own E3 ligase activity [128] and is recruited into a TNF
receptor signaling complex containing linear ubiquitin chain assembly complex
(LUBAC) and IκB kinase (IKK) [129]. Additionally, A20 removes Lys63-linked
ubiquitin of RIPK1 through its OTU domain and promotes its proteasomal degrada-
tion by Lys48 polyubiquitination [130].
RIPK1 ubiquitin levels can also be modulated by CYLD, USP4, USP7, and
USP21 [131–134]. Furthermore, Cezanne deubiquitinates RIPK1 signaling inter-
mediaries [135] and regulates noncanonical NF-κB signaling through inhibition of
TRAF3 degradation [136]. Similarly, OTUD5 removes Lys63-linked ubiquitin of
TRAF3, resulting in diminished type I interferon and IL-10 responses [137] and
364 J.M. Fraile et al.
Over the last few decades, several studies have demonstrated the involvement of
DUBs in other physiological processes that play additional roles in cancer progres-
sion, such as epithelial-to-mesenchymal transition (EMT), cell migration, apopto-
sis, autophagy, and stem cell maintenance and differentiation. UCHL1 acts as a
potent oncogene and regulates prostate cancer progression and metastasis by induc-
ing EMT [184], whereas USP17 has a critical role in cell migration through the
modulation of the subcellular localization of GTPases, which are essential for cell
motility [185]. CYLD and USP9X also regulate cell migration through the GTPase
Rac1 and the stabilization of the E3 ubiquitin ligase SMURF1, respectively [186,
187].
366 J.M. Fraile et al.
DUBs have dual and complex roles in the regulation of apoptotic processes,
either promoting (USP2, USP4, USP7, USP8, USP9X, USP15, USP16, USP17,
USP27, USP28, USP41, CYLD, A20, UCHL1, and ATXN3) or suppressing apop-
tosis (USP2, USP9X, USP18, UCHL3, and A20) [133, 188]. USP2 inhibits apopto-
sis in prostate cancer cells by stabilizing fatty acid synthase [189] and promotes cell
death by deubiquitination and stabilization of the truncated form of the apoptosis-
inducing factor, AIF [190]. As described above, USP4 and USP7 induce TNFα-
mediated apoptosis by negatively regulating RIPK1 ubiquitination [133, 134],
whereas USP7 deubiquitinates and stabilizes TIP60, an essential acetyl transferase
required for p53-dependent induction of apoptosis [191]. USP9X leads to cell sur-
vival by deubiquitinating MCL1 [192] but also promotes apoptosis by stabilizing
apoptosis signal-regulating kinase 1 [193]. Likewise, USP17 promotes apoptosis in
cervical carcinoma through the regulation of the histone deacetylase activity of
SDS3 [188]. Furthermore, USP27 deubiquitinates BH3-only protein and enhances
its levels, counteracting the antiapoptotic effects of ERK activity [194]. Although
A20 has been considered a tumor suppressor DUB-promoting cell death, it also acts
as a potent prosurvival gene by inhibiting apoptosis in gliomas and breast carcino-
mas [195].
Several DUBs are also involved in autophagy, a critical intracellular catabolic
mechanism that mediates the degradation of cytoplasmic proteins and organelles
[196]. Thus, USP19 is considered a positive regulator of autophagy but a negative
regulator of type I interferon signaling because it deubiquitinates Beclin-1 [197].
Ubiquitin levels of Beclin-1 are also modulated by A20, USP10, and USP13 [198,
199]. USP33 promotes autophagosome formation through the deubiquitination of
the mono-ubiquitinated RAS-like GTPase RALB, providing the switch for the dual
functions of RALB in autophagy and innate immune responses [200]. Moreover,
USP36 modulates the selective autophagic degradation of protein aggregates [201].
Mitophagy, the degradation of mitochondria by autophagy, is also regulated by sev-
eral DUBs. Thus, USP30 antagonizes mitophagy driven by the ubiquitin ligase par-
kin and protein kinase PINK1, which are encoded by two genes associated with
Parkinson’s disease [202]. Interestingly, USP8 and USP15, which are not located
within mitochondria, have also been involved in promoting and inhibiting parkin-
mediated mitophagy, respectively [203, 204].
Over the last years, the role of DUBs in stem cell maintenance and cellular repro-
gramming has been widely studied [205]. So far, several DUBs, such as USP7,
USP16, USP22, USP28, USP44, POH1, and MYSM1, are involved in regulating
stem cell biology. USP7 prevents neural stem/progenitor cell differentiation by the
deubiquitination and stabilization of the repressor element 1-silencing transcription
factor [206]. Furthermore, USP22 is required for c-Myc-induced transcription [103]
and functions as a critical regulator of the transition from self-renewal to differentia-
tion through the repression of the Sox2 locus [207]. Moreover, USP28 stabilizes the
chromatin modulator LSD1, which has a critical role in cellular pluripotency [208],
whereas USP44 function regulates stem cell differentiation by inhibiting the mono-
ubiquitination of H2B [209]. In addition, POH1 is required to maintain embryonic
stem cell (ESC) self-renewal and pluripotency [210], and USP16 regulates ESC
16 Functional Relevance of Deubiquitinases in Life and Disease 367
There is a growing list of human malignancies in which several DUBs are mutated
and behave as oncogenes or tumor suppressors [3]. Moreover, malignant tumors are
frequently associated with profound changes in the expression levels of many
DUBs. Furthermore, DUBs may function as tumor suppressors or oncogenes
depending on the cellular context and the target affected by their regulation.
Mutations in CYLD have been described in familiar cylindromatosis, character-
ized by a predisposition to develop multiple head and neck skin tumors [216].
Chromosomal deletions and inactivating mutations in A20 gene have been described
in several lymphoma subtypes [217, 218]. Furthermore, point mutations and dele-
tions in BAP1 have been found in breast, lung, and clear cell renal cell carcinomas
[219–221], as well as in metastasizing uveal melanomas, in malignant pleural
mesotheliomas, and in myelodysplastic syndromes [220, 222, 223]. Finally, inacti-
vating mutations of USP9X have been found in pancreatic ductal adenocarcinoma
[224]. Regarding DUBs with pro-tumorigenic roles, somatic mutations in USP6 and
USP28 are present in different human cancers. Thus, the fusion between the osteo-
blast cadherin 11 gene (CDH11) promoters and USP6, created by chromosomal
rearrangement, leads to the overexpression of USP6 in aneurismal bone cysts [225].
Furthermore, mutations in USP6 and USP28 have been reported in lobular breast
cancer [226]. Finally, dominant mutations of USP8 were found in four of ten corti-
cotroph adenomas of the pituitary, thus causing Cushing’s disease [227].
Many DUBs are linked to cancer through changes in their expression patterns.
Thus, UCHL1, JOSD1, CSN5, and USP9X are overexpressed in non-small cell lung
carcinomas, whereas USP10, USP11, USP22, USP48, and CSN5 are overexpressed
in malignant melanoma, correlating high levels of USP10, USP11, and USP22 with
a more aggressive and invasive phenotype [228]. Moreover, CSN5 is overexpressed
in early hepatocellular carcinoma [229], and USP22 is upregulated in colorectal
carcinomas and belongs to a set of marker genes that can predict metastatic poten-
tial and therapeutic outcome in human cancer [230]. Moreover, USP22 and OTUB1
overexpression correlates with poor prognosis in invasive breast [231] and ovarian
cancer [232], respectively. OTUB1 and USP4 are overexpressed in colorectal cancer
tissues and are associated with tumor size, differentiation, distant metastasis, and
poor survival [233, 234]. Additionally, USP4 is overexpressed in other human
tumors [117, 235], but it is downregulated in small cell lung cancer cell lines [236].
Furthermore, USP1 expression correlates with early steps of transformation in
368 J.M. Fraile et al.
gastric cancer [228], whereas USP2 is upregulated in ovarian and prostate carcino-
mas and is associated with lesions of poor prognosis [237]. In agreement with these
results, overexpression of USP2 protects prostate cancer cells from apoptosis and
endows them with resistance to chemotherapeutic agents by promoting p53 degra-
dation [238]. By contrast, USP2 is downregulated in breast cancer, suggesting that
this enzyme acts as an oncogene or tumor suppressor in a time- and tissue-specific
manner [141]. USP7 overexpression has also been associated with tumor aggres-
siveness in prostate cancer [56], whereas its expression and activity are repressed by
STAT3 in colon cancer [239]. Additionally, overexpression of USP17 has been
found in primary lung, colon, esophagus, and cervix tumor biopsies [240], and aber-
rant expression of USP14 has been found in a variety of cancers, such as multiple
myeloma and colorectal, lung, and epithelial ovarian cancers [241, 242]. USP39
overexpression correlates with poor prognosis in prostate cancer patients [243],
whereas USP44 expression is elevated in human T-cell leukemia and is associated
with chromosomal instability [244]. By contrast, USP44 is downregulated in lung
adenocarcinomas and associates with poor prognosis [80]. Furthermore, USP44 is
epigenetically inactivated in colorectal adenomas, representing an early event in this
pathology [245]. Likewise, USP15 is downregulated in paclitaxel-resistant ovarian
cancer [246] and CYLD in melanoma, hepatocellular carcinoma, and other malig-
nant tumors [34, 247–249]. USP18 expression is reduced in human leiomyosarco-
mas, and, accordingly, mutant mice deficient for this USP develop these tumors
spontaneously [250]. Finally, USP46 is downregulated in colorectal cancer samples
[251] and A20 in some types of lymphoma [252].
Due to their wide functional diversity and critical implication in human pathologies,
DUBs have emerged as appealing targets in the development of new specific thera-
pies, especially against human malignancies [253]. Cyclopentenone prostaglandins
were the first DUB inhibitors that targeted active sites [254]. So far, several small-
molecule inhibitors against DUBs, mainly USPs and UCHLs, have been described.
Thus, two different classes of inhibitors have been used to neutralized UCHL1:
isatin O-acyl oximes and 3-amino-2-oxo-7H-thieno[2,3-b]pyridine-6-one deriva-
tives [255]. Competitive inhibitors with similar dihydro-pyrrole skeletons and sev-
eral isatin derivatives have been described to inhibit UCHL3 [3]. LS1 is another
UCHL3 inhibitor identified in a FRET-based screen [253]. Regarding USPs, pimo-
zide, GW7647, and ML323 are potent USP1-UAF1 inhibitors [13, 256], whereas
2-cyano-pyrimidine and triazine derivatives have been described to block USP2
function [257]. Moreover, HBX 41,108, HBX 19,818, HBX 28,258, P022077,
P5091, and spongiacidin C have been described as selective USP7 inhibitors. In this
sense, a more potent analog of P5091 inhibits USP7 and USP47 [253, 258]. HBX
90,397, HBX 90,659, and 9-oxo-9-H-indeno [1,2-b]pyrazine-2,3-dicarbonitrile and
analogs selectively inhibit USP8 activity [257, 259]. Finally, mitoxantrone targets
16 Functional Relevance of Deubiquitinases in Life and Disease 369
USP11 and impacts pancreatic ductal adenocarcinoma cell survival [260], whereas
IU1 inhibits USP14 and enhances proteasome activity [261].
There are also broad-spectrum inhibitors that target several DUBs. WP1130
blocks the activity of USP5, USP9X, USP14, and UCHL5 [253]. Furthermore, nat-
ural-occurring isothiocyanates exert anticancer effects by inhibiting DUBs, includ-
ing USP9X and UCHL5 [262]. Similarly, small-molecule inhibitors against
proteasome-associated DUBs have been described, such as RA-9, auranofin (Aur),
4-arylidene curcumin analog (AC17), and the chalcone derivative b-AP-15. RA-9
and Aur inhibit USP14 and UCHL5 and diminish tumor growth in vitro and in vivo
[253, 263]. RA-9 displays more broad range activity and also inhibits UCHL1,
UCHL3, USP2, and USP8 [263, 264]. AC17 also inhibits the DUB activity of 19S
regulatory particles, resulting in inhibition of NF-κB signaling and reactivation of
p53 [253], whereas b-AP15 blocks the activity of USP14 and UCHL5 and induces
apoptosis that is insensitive to p53 status [265]. Furthermore, betulinic acid is a pan-
DUB inhibitor that selectively kills prostate cancer but not normal cells [266].
Finally, gambogic acid inhibits cellular DUB activity, inducing the accumulation of
polyubiquitinated proteins [253], and PR-619, another broad specificity DUB inhib-
itor, activates autophagic pathways [267].
Due to the increased cellular stresses produced by cancer, tumor cells might
exhibit an exacerbated dependence on the normal function of certain DUBs [268].
In this scenario, DUBs will be essential for human malignancies, even without
undergoing activating mutations. This concept is referred to as non-oncogene addic-
tion and opens a new window of therapeutic opportunities for the treatment of
human pathologies. Thus, the inhibition of USP2 has been described as an effective
approach to suppress growth in cancer cells addicted to cyclin D1 expression [72].
Moreover, downregulation of USP11 leads to spontaneous DNA damage repair acti-
vation and hypersensitivity to PARP inhibition, ionizing radiation and other sources
of genotoxic stress [269]. Furthermore, gemcitabine disrupts the interaction of
USP9X and MCL1 and sensitizes cells to ABT-737 treatment, inducing caspase-
dependent apoptosis [270]. Additionally, depletion of USP4 diminishes TGF-β-
induced EMT and metastasis [29], and USP15 downregulation decreases TGF-β
activity and oncogenesis [169]. Moreover, USP6 acts as an oncogene by positively
regulating NF-κB, whose activity is essential for USP6-mediated tumorigenesis
[147]. Finally, inhibition of USP37 blocks the proliferation of lung cancer cells by
reducing c-Myc levels [52], and depletion of USP1-UAF1 overcomes the resistance
of cancer cells to cisplatin [43] (Fig. 16.3).
As discussed above, multiple DUBs present tumor-protective properties, and,
consequently, effective cancer therapies should spare their activity or even be
designed to potentiate DUB activities lost as a consequence of malignant transfor-
mation. In this sense, demethylating drugs, in case of loss of expression by promoter
hypermethylation, or exogenous compounds that compensate DUB activity could
emerge as effective strategies to overcome DUB deficiency. As notable examples of
this possibility, anti-inflammatory drugs, inhibition of tropomyosin kinase TRK,
and downregulation of Snail have been proposed as strategies for CYLD inactivating
mutations [271–273].
370 J.M. Fraile et al.
Fig. 16.3 Non-oncogene addiction of USPs. Several examples of non-oncogene addiction phe-
nomena involving USPs and their functional interaction with different proteins in cancer
The human genome encodes at least 100 DUBs that, despite sharing a core domain,
exhibit a wide structural and functional diversity. For this reason, DUBs are impli-
cated in numerous physiological and pathological conditions, including cancer. In
this sense, there is a broad spectrum of DUBs with critical functions at several
stages of cancer development and progression. In fact, the number of DUBs known
to be mutated that behave as oncogenes or tumor suppressor genes has kept growing
over the last years. Moreover, transcriptional expression of several DUBs is also
altered in many human malignancies. On this basis, DUBs emerge as appealing
targets in the development of therapies against cancer, although further understand-
ing of the activity and regulation of these proteolytic enzymes will be required for
their functional and clinical validation as drugs targets. In this regard, the generation
of gain- or loss-of-function mouse models for DUBs will help to understand their
individual relevance in normal physiology, as well as their contribution to tumori-
genic progression. Hopefully, these studies will contribute to clarify the functional
16 Functional Relevance of Deubiquitinases in Life and Disease 371
complexity of this superfamily of proteases and pave the way for the development
of new anticancer therapies based on DUB-targeting approaches.
Acknowledgments We thank Dr. Víctor Quesada for helpful comments and assistance. Our work
is supported by grants from Ministerio de Economía y Competitividad, Instituto de Salud Carlos
III (RTICC), and Principado de Asturias, Spain. The Instituto Universitario de Oncología is sup-
ported by Fundación Bancaria Caja de Ahorros de Asturias.
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Part II
General Aspects of Proteases
Submitochondrial Calpains
in Pathophysiological Consequences 17
Pulak Kar, Krishna Samanta, Tapati Chakraborti,
Md Nur Alam, and Sajal Chakraborti
Abstract
It has now been well established that different mitochondrial compartments con-
tain varieties of calpains. The expression levels of these calpains are tissue and
cell type specific. Although, mitochondrial compartments contain different types
of calpains, the precise location within mitochondria and their functions remain
imprecise. The aim of the present review is to confer information concerning the
localization of calpains in different mitochondrial compartments affiliated with
their function, particularly in pathophysiological conditions. For instance, mito-
chondrial μ-calpain is located within the inner membrane, intermembrane space,
and mitochondrial matrix depending on cell types. μ-Calpain activity facilitates
cleavage of apoptosis-inducing factor (AIF) within inner membrane and inter-
membrane space, while the activated μ-calpain within matrix is associated with
cleavage of complex I subunits and metabolic enzymes. Understandably, inhibi-
tion of the μ-calpain could be a potential strategy to ameliorate ischemia–
reperfusion-associated injuries.
Keywords
Submitochondria • Calcium • Calpain • Apoptosis • Ischemia–reperfusion •
Disease
P. Kar • K. Samanta
Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford, UK
e-mail: pulak_2006@yahoo.com; pulak_kar@rediffmail.com
T. Chakraborti • M.N. Alam • S. Chakraborti (*)
Department of Biochemistry and Biophysics, University of Kalyani,
Kalyani 741235, West Bengal, India
e-mail: saj_chakra@rediffmail.com
17.1 Introduction
Calpains are known to be cytoplasmic enzymes, but we and other investigators have
shown that calpains also localized within mitochondria [4–7]. Mitochondrial com-
partmentalization of calpains is tissue specific and cell type dependent. Mitochondrial
μ-calpain was initially localized within the liver mitochondrial intermembrane
space [6]. Our previous study showed that μ-calpain is also present in the inner
membrane of the isolated pulmonary artery smooth muscle mitochondria [9]. We
also reported that the pulmonary smooth muscle mitochondria contain calpastatin
[9]. Kar et al. [9] also demonstrated that μ-calpain–calpastatin association exists in
the inner mitochondrial membrane of pulmonary artery smooth muscle under physi-
ological Ca2+ level. Chen et al. [10] reported that μ-calpain immunoactivity was
detected in a component including inner membrane and matrix in cardiac mitochon-
dria. The large subunit of μ-calpain contains the leader sequence in its N-terminus,
which plays a critical role in importing its corresponding small regulatory subunit
[7]. The biochemical characteristics of mitochondrial μ-calpain are similar to cyto-
solic μ-calpain with an 80 kDa large catalytic subunit as well as a 28 kDa regulatory
small subunit [5].
17 Submitochondrial Calpains in Pathophysiological Consequences 387
mitochondrial OM is different from cytosolic one [22]. Ozaki et al. [22] have dem-
onstrated that mitochondrial μ-calpain associates with ERp57, but cytosolic
μ-calpain and m-calpain do not. Their studies also suggested that ERp57 is involved
in μ-calpain large subunit refolding, by forming disulfide bonds to shape up func-
tional conformations. Not only ERp57, but IMS proteins also are implicated for
refolding and formation of disulfide bonds by protein oxidation in the IMS [23].
Therefore, the association between mitochondrial μ-calpain with ERp57 may pro-
vide important clues in understanding mitochondrial μ-calpain functions [22]. In
addition to μ-calpain, studies have indicated m-calpain’s association with ERp75 as
well. Importantly, ERp75 is an important molecular chaperone belonging to the heat
shock protein 70 family, which releases tAIF (truncated apoptosis-inducing factor)
from mitochondria [24, 25]. It has multiple functions including the import of pro-
teins into mitochondria and the proper folding of newly synthesized chromosomal
(nuclear) and extrachromosomal (mitochondrial) encoded proteins. ERp75 response
17 Submitochondrial Calpains in Pathophysiological Consequences 389
is associated with many forms of stress including depleted glucose levels, oxidative
injury, and ultraviolet irradiation [26].
Calcium concentration is increased to approximately 1 μM and 2.6 μM at the end
of 25 min and 30 min reperfusion, respectively [4]. This calcium overload is believed
to be associated with the activation of calpain 10; however, some isoforms of calpain
10 are not exclusively calcium dependent [13]. Although phosphorylation and post-
translational modifications may affect calpain 10 activity, the actual mechanisms
associated with the regulation of calpain 10 are still not clear [27]. Calpain activity is
not only dependent on endogenous regulatory mechanisms; it is also affected by
other different physiological conditions. Under acidic conditions the activities of cal-
pains are decreased [4, 28], albeit different calpains show varied sensitivity toward
acidification. A number of synthetic and nature-derived calpain inhibitors are avail-
able to study and manipulate calpain activity under pathophysiological conditions
and in experimental models. MDL-28170, for example, a typical synthetic inhibitor
of calpain, is used to inhibit both μ- and m-calpains [4].
the calcium-mediated AIF cleavage [6]. These aforesaid results support the fact that
activation of mitochondrial calpain leads to AIF cleavage. Even though μ-calpain is
identified in the intermembrane space of cardiac mitochondria [10] and implicated
in the cleavage and activation of AIF in heart mitochondria, reports show that
μ-calpain is not involved in AIF cleavage in brain mitochondria [33]. Therefore,
complementation of AIF cleavage with mitochondrial μ-calpain activation may be
tissue dependent. The translocation of the cleaved AIF from the intermembrane
space to the nucleus is eased by the permeation of the outer mitochondrial mem-
brane [6], which is accomplished by the activation of mitochondrial m-calpain by
cleaving VDAC and induction of Bax on the outer membrane in isolated liver mito-
chondria [6] (Fig. 17.1).The aforementioned phenomenon induced by m-calpain is
not universal; rather it seems to be species specific [10, 34]. Ischemia–reperfusion
is known to increase the outer membrane permeability by opening of the mitochon-
drial permeability transition pore (MPTP) [35]. Not only that, but in ischemic con-
dition, induction of imbalance between anti-apoptotic and proapoptotic bcl-2 family
proteins also increases the permeability of the outer membrane [4]. The aforesaid
observations assure that AIF release is controlled by both mitochondrial μ-calpain
and mitochondrial m-calpain. Conceivably, inhibition of mitochondrial calpains
could prove useful as a therapeutic measure in preventing many disorders such as
retinitis pigmentosa/retinal degeneration and cerebral ischemia [24].
Cytosolic calpain activation can cleave bid to t-bid, which in turn can elevate outer
mitochondrial membrane permeability [36]. μ-Calpain is also localized on the endo-
plasmic reticulum (ER) [37, 38]. So its activation can be associated with calcium
overload through ER stress [37, 38], and induction of intracellular calcium overload
and subsequent mitochondrial calcium overload can be associated with MPTP sensi-
tivity and opening via activation of cytosolic calpains. In ischemic cardiac mitochon-
dria, μ-calpain activity is increased; conversely calpain inhibition decreased the
MPTP opening [4]. In isolated perfused heart, calpain inhibitors have been shown to
ameliorate ischemia–reperfusion injury by improving oxidative phosphorylation [4].
Chen et al. [4] proposed that μ-calpain within the mitochondrial matrix damages
complex I. Not only μ-calpain, but m-calpain is also implicated in complex I damage
and MPTP permeation (Fig. 17.1). Thus, multiple studies converge to support the
pivotal role of mitochondrial calpain in disruption of mitochondrial metabolism and
cell death. Not only ETC, but ischemia–reperfusion impairs metabolic enzymes in
the tricarboxylic acid (TCA) cycle as well [4, 39, 40]. More precise proteomic stud-
ies show that ischemia–reperfusion leads to degradation of several metabolic
enzymes including pyruvate dehydrogenase (PDH), malate dehydrogenase (MDH),
and succinate dehydrogenase (SDH) in rat heart mitochondria [4, 39, 40].
Along with μ- and m-calpain, calpain 10 is also localized within mitochondrial
matrix, which has recently gained importance because of its role in type 2 diabetes
[5]. In renal mitochondria, calpain 10 activity is associated with ETC impairment by
17 Submitochondrial Calpains in Pathophysiological Consequences 391
17.5 M
itochondrial Oxidative Stress and Submitochondrial
Calpain in Pathophysiology
In aerobic cells mitochondria represent the key site of molecular oxygen consump-
tion and ROS production [43]. Reactive oxygen species (ROS) production is associ-
ated with stress and can lead to cell death, if not detoxified. ROS produced by the
mitochondria can oxidize proteins and induce lipid peroxidation, which in turn
compromise the versatility of membrane properties of the mitochondria. Not only
proteins and lipids, but mitochondrial DNA (mtDNA) is also susceptible to ROS-
mediated damage. ROS-mediated damage of mtDNA is highly plausible as they are
devoid of protective histones and localize in close proximity to the respiratory chain.
It is further substantiated by the studies that tell oxidative modification in mtDNA
bases is 10- to 20-fold higher than nuclear DNA [44]. Some mitochondrial proteins
are encoded by its DNA, which are essential mainly for the respiratory chain and
ATP synthesis by oxidative phosphorylation. ROS production is not only associated
with direct oxidation and damages. It also augments cellular deterioration via induc-
tion of Ca2+-mediated pathways. ROS-mediated sustained Ca2+ activates mitochon-
drial intermembrane space, and inner mitochondrial membrane localized μ-calpain
which then cleaves the apoptosis-inducing factor (AIF) [5, 45, 46] (Fig. 17.1).
Mitochondrial ROS mediates oxidative modification of AIF which further trigger
proteolysis and also carbonylation of the protein, thus increasing susceptibility to
calpain cleavage [47]. Apoptosis-inducing factor (AIF) is a 62 kDa flavoprotein and
is anchored to the inner mitochondrial membrane (IMM) in the close proximity of
complex I. AIF needs to be cleaved from the anchor peptide so that the 57 kDa pro-
apoptotic fragment can be released into the cytosol for further translocation into the
nucleus, where it promotes large-scale DNA damage. Therefore, oxidative stress,
ROS generation, Ca2+ elevation, calpain activation, release and translocation of AIF,
and DNA fragmentation are associated with cell death [46, 48].
Ischemia–reperfusion and associated injury have often been implicated with
mitochondrial permeability transition, and pretreatment with cyclosporine A (CsA)
has been found to exert a protective effect. Therefore the paradigm suggests that
PTP formation is the cause of the damage [45]. The precise molecular mechanism
by which activation of the mitochondrial calpains sensitizes the MPTP opening in
cardiac mitochondria is not clear. p53 is a tumor suppressor protein, and its content
is normally maintained at a low level by interaction with Mdm2 (mouse double
minute 2 homolog). During oxidative stress Mdm2 dissociates from the p53-Mdm2
complex by phosphorylation, thus rapidly increasing its level in cytosol and nucleus.
The phosphorylated p53 translocates to and accumulates within mitochondria to
392 P. Kar et al.
form a complex with cyclophilin D, which in turn increases MPTP opening [4]. The
translocation of p53 from cytosol to brain mitochondria is aided by the activation of
cytosolic calpain [4], implicating cytosolic calpain’s possible role in induction of
MPTP opening (Fig. 17.1). Complex I and cyclophilin D are associated with MPTP
opening via activation of mitochondria-localized μ-calpain or m-calpains [4, 34].
Cyclophilin D, which is located within the matrix of the mitochondria, can be
accessed by the mitochondrial μ-calpain and plays a decisive role in the regulation
of MPTP opening. Genetic inhibition of complex I leads to increased protein acety-
lation of MPTP [4]. During ischemia–reperfusion, calpain-mediated MPTP opening
eventually damages complex I, and that can be attenuated by calpain inhibitors [4,
34]. Therefore, activation of mitochondrial μ-calpain corroborates with the increase
in MPTP opening, albeit by impairing complex I in the respiratory chain or by
facilitating translocation of cyclophilin D from the matrix to the inner mitochon-
drial membrane [4] (Fig. 17.1).
Mitochondrial μ-calpain also plays a critical role in cardiomyopathic changes in
type 1 diabetes (T1D). In cardiomyocytes of transgenic mice, capn4 deletion or
injection of streptozotocin in the wild types induces T1D [49].
An increase in the expression of μ-calpain in mitochondria has been observed in
diabetic mouse hearts and that augments ROS levels leading to a reduction in ATP
synthase (ATP5A1) activity. The μ-calpain has been shown to be induced in both
type 1 and type 2 diabetic heart mitochondria. In some [49], but not all [50, 51],
system genetic inhibition of μ-calpain increases the ATP synthase activity due to a
marked attenuation of mitochondrial ROS generation with concomitant decrease in
damage of diabetic heart.
Upon activation, calpain contributes to myocardial dysfunction in diabetes by
triggering proteolysis of some cytosolic proteins such as protein kinase C and
nuclear factor-kB [52, 53], myofibrils, and intracellular Ca2+ regulatory proteins
[49, 54, 55].
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17 Submitochondrial Calpains in Pathophysiological Consequences 395
Abstract
The complement system plays a crucial role in host defense against pathogen
infections and in the recognition and removal of damaged or altered self-
components. Complement system activation can be initiated by three different
pathways—classical, alternative, and lectin pathways—resulting in a proteolytic
cascade, which culminates in multiple biological processes including opsoniza-
tion and phagocytosis of intruders, inflammation, cell lysis, and removal of
immune complexes and apoptotic cells. Furthermore, it also functions as a link
between the innate and adaptive immune responses. The lectin pathway (LP)
activation is mediated by serine proteases, termed mannan-binding lectin (MBL)-
associated serine proteases (MASPs), which are associated with the pattern rec-
ognition molecules (PRMs) that recognize carbohydrates or acetylated
compounds on surfaces of pathogens or apoptotic cells. These result in the pro-
teolysis of complement C2 and C4 generating C3 convertase (C4b2a), which
carries forward the activation cascade of complements, culminating in the elimi-
nation of foreign molecules. This chapter presents an overview of the comple-
ment system focusing on the characterization of MASPs and its genes, as well as
its functions in the immune response.
Keywords
Serine proteases • Complement system • Lectin pathway
Fig. 18.1 Complement activation by the classical pathway (CP), lectin pathway (LP), and alterna-
tive pathway (AP). CP typically requires an antigen-antibody complex on pathogen surface and
binding to C1 complex (C1q, C1r, and C1s) for its activation. LP recognizes mannose-terminating
glycan or acetylated residues on pathogens leading to MBL/ficolins/collectins-MASP complex
activation. Both pathways induce the formation of C3 convertase, C4b2a. AP is permanently acti-
vated at a low level by spontaneous hydrolysis of C3 into C3(H2O). Lack of complement inhibitors
on pathogens induces AP activation by the C3bBb assembly. Complement activation leads to opso-
nization and phagocytosis of pathogens owing to C3b and C4b deposition, bacterial lysis by C5b-9
complex formation, and inflammation by C4a, C3a, and C5a, leading to recruitment of immune
cells, endothelial and epithelial cell activation, and platelet activation
400 F.A. Andrade et al.
AP, a more active C3 convertase (C3bBb) is formed instead, which is further stabi-
lized by properdin. In contrast to other pathways, AP functions as an amplification
loop providing a strong positive feedback activation of C3, thereby increasing the
production of pro-inflammatory mediators [18]. In fact, 80–90% of pathological
complement activation in disease is driven by the AP [19]. Furthermore, the alterna-
tive convertase assembly may also be initiated by non-covalent attachment of pro-
perdin to some target surfaces (Fig. 18.1) [20, 21].
The existence of the LP was first discovered in the 1970s when the plant lectin
mannose-binding protein (concanavalin A) was found to activate the complement
system [22]. This pathway was further characterized by using proteins isolated from
rabbit liver and serum; however, its function remained unclear initially [23, 24]. In
1992, Matsushita and Fujita reported that MBL and MASPs activated the LP, which
was a landmark study on the mechanism of LP activation [25]. Thus far, 6 different
PRMs that initiate the activation of the LP have been identified: 3 ficolins (M-ficolin,
L-ficolin, and H-ficolin, also known as ficolin-1, ficolin-2, and ficolin-3, respec-
tively), and 3 collectins (MBL, collectin 11 or collectin kidney-1 or CL-K1, and
collectin 10 or colletin-L1or CL-L1). Similar to AP, the LP may be activated in the
absence of immune complexes by the binding of PRMs to carbohydrates or acety-
lated compounds on the surfaces of pathogens (PAMPs) or apoptotic cells (DAMPs)
(Fig. 18.2). The PRMs form complexes with the serine proteases, MASPs (MASPs
Fig. 18.2 The lectin pathway of complement activation. MBL and ficolins undergo conforma-
tional changes upon interaction with PAMPs and DAMPs by binding MBL and ficolin, respec-
tively. This activates MASP-1, followed by MASP-2, which initiates a cleavage cascade of
complement factors, with anaphylatoxins C4a, C3a, and C5a playing important roles in the inflam-
matory process
402 F.A. Andrade et al.
Fig. 18.3 Membrane attack complex (MAC) formation and the resultant consequences in target
cell. Newly formed C5b reacts with C6 to form the stable C5b6 complex. Binding of C7 results in
a hydrophobic complex that targets the membrane (mC5b-7). Membrane insertion is initiated upon
binding of C8 (C5b-8) after which 12–18 copies of C9 polymerize to form the pore-forming ring
structure to induce lysis of microbial membranes
18.4 M
BL-Associated Serine Proteases (MASPs) of the Lectin
Pathway
There are three serine proteases associated with the LP PRMs, MASP-1, MASP-2,
and MASP-3, in addition to two nonenzymatic proteins MAp19 and MAp44 [1].
The three MASP enzymes have an identical domain organization (Fig. 18.4), which
is also similar to that of the two classical pathway serine proteases, C1r and C1s.
The regulatory domain (A-chain) is composed of C1r/C1s, Uegf, and bone morpho-
genetic protein 1 (CUB1), followed by the epidermal growth factor (EGF), a second
CUB domain (CUB2), and two contiguous complement control proteins (CCPs) 1
and 2 [33, 34]. The regulatory domain is responsible for dimerization of MASP
polypeptides and binding to PRMs [35–37]. The regulatory domain is followed by
the module with the catalytic activity (B-chain), the serine protease (SP) domain
[33, 34]. The CCP2 and SP domains are connected through a linker peptide (also
termed the activation peptide), where an Arg-Ile bond is cleaved through autolysis
when MASP/PRM complexes bind to pathogens, linking the A- and B-chain con-
nected via a disulfide bond [33].
All MASPs are generated from two genes. MASP-1, MASP-3, and MAp44 are
encoded by the MASP1 gene through an alternative splicing process [38, 39], while
MASP-2 and MAp19 are alternatively spliced products of MASP2 gene [40].
Fig. 18.4 MASP1 gene and transcripts. The primary transcript can be spliced into three different
mRNAs encoding the MASP-1, MASP-3, and MAp44 proteins. Blue boxes indicate the translated
part of the exons. MASP: mannose-binding lectin associated serine protease. MAp44: mannose-
binding lectin-associated protein of 44 kDa. CUB, C1r/C1s, Uegf, and bone morphogenetic pro-
tein; EGF, epidermal growth factor; CCP, complement control protein. Exons are drawn to scale
and introns are truncated
404 F.A. Andrade et al.
The MASP1 gene contains 18 exons and is located on chromosome 3q27–q28 span-
ning 76 kb (Fig. 18.4) [41, 42]. The gene encodes a primary pre-mRNA transcript,
which is spliced differentially to yield three distinct mRNAs encoding the MASP-1,
MASP-3, and MAp44 (also termed MAp 1) proteins [38, 39]. MASP-1 and MASP-3
regulatory domains (CUB1-EGF-CUB2-CCP1-CCP2) are encoded by exons 2–8
and exons 10 and 11, while the SP domain is encoded by exons 13–18 and exon
12 in MASP-1 and MASP-3, respectively. MAp44 lacks the SP domain but shares
the first four domains (CUB1-EGF-CUB2-CCP) with MASP-1 and MASP-3 that
are encoded by exons 2–8. Exon 9 is unique to MAp44 [39, 43]. The mRNA encod-
ing MASP-1 is largely observed in the liver, while mRNA for MASP-3 is primarily
observed in the liver and cervix, followed by bladder, brain, colon prostate, and
placenta [39]. The highest expression of MAp44 is observed in the heart; it was
weakly expressed in cervix, colon, and liver [39].
Some MASP1 gene polymorphisms are associated with the serum levels of
MASP-1, MASP-3, and MAp44 (Table 18.2); most associations were observed in
healthy individuals. In Danish blood donors, heterozygotes of rs190590338 (G > A)
lead to increase in MASP-1 median concentration, while the minor allele of
rs7625133 (A > C) decreased MAp44 concentration. The minor alleles of SNPs
rs3774275 (A > G), rs698090 (T > C), and rs67143992 (G > A) result in an increase
in MASP-1 and MAp44 and a decrease in MASP-3 serum concentrations; SNPs
rs72549154 (G > T) and rs35089177 (T > A) showed the opposite effect—the minor
alleles result in an increase of MASP-3 and a decrease of MASP-1 and MAp44 [44].
The additive effect of some MASP1 SNPs in haplotypes on MASP-1, MASP-3, and
MAp44 serum concentrations has also been described. The MASP1 TGAG haplo-
type (rs35089177 (T > A), rs62292785 (G > A), rs7625133 (A > C), and rs72549254
(G > A)), for example, leads to an increase in MASP-1 and MAp44 and decrease in
MASP-3 concentration in healthy blood donors [44].
In patients with cystic fibrosis homozygous (A/A) and heterozygous (G/A)
alleles, SNP rs850312 (G > A) was associated with the earlier onset of Pseudomonas
aeruginosa colonization [45]. These same genotypes were associated with higher
on-admission MASP-3 levels in critically ill children, exhibiting a protective effect,
as higher MASP-3 levels are related to a better outcome [46]. The T/T genotype of
rs710469 (C > T) was also considered a protective genotype in critically ill children
by increasing on-admission MASP-3 levels, although the genotype was equally dis-
tributed among controls and patients [46]. A non-synonymous polymorphism
(rs38343199) in exon 10 (G > A) located in the MASP-1 and MASP-3 CCP2
domain was evaluated in systemic lupus erythematosus (SLE), systemic inflamma-
tory response syndrome (SIRS), and/or sepsis patients. However, no association
was found between this amino acid substitution and the diseases [47]. Some muta-
tions in MASP1 gene are also related to the autosomal-recessive 3MC syndrome
(Carnevale, Mingarelli, Malpuech, and Michels) [48–50].
Table 18.2 MASP1 gene polymorphisms associated with MASP-1, MASP-3, and MAp44 concentration and diseases
Gene Amino acid Protein
dbSNP Allele MAF Gene region position position region Serum levelsa Disease association
rs190590338 G > A <1% Promoter −2464 n.a. n.a. G/A: Increase –
MASP-1 levels
rs7625133 A > C 3% Promoter −961 n.a. n.a. A/C, C/C: Decrease –
MAp44 levels
rs35089177 T > A 28% Promoter −1418 n.a. n.a. T/A, AA: Decrease –
MASP-1 and
MAp44 levels
rs75284004 A > G 1% Promoter −1479 n.a. n.a. A/G decrease –
MASP-3 levels
rs62292785 G > A 10% Promoter −1251 n.a. n.a. G/A: Decrease –
MASP-1 levels
rs72549254 G > A 17% Intron 1 9 n.a. n.a. A/G: Increase –
MASP-3 levels
AG, AA: Decrease
MAp44 levels
rs710469 C > T 49% Intron 2 24,903 n.a. n.a. T/T: Higher Protective effect on
on-admission critically ill
MASP-3 levels in children [46]
critically ill children
rs3774275 A > G 24% Intron 8 44,153 n.a. n.a. A/G, G/G: Increase –
18 Serine Proteases in the Lectin Pathway of the Complement System
MASP-1 and
MAp44 and
decrease MASP-3
levels
(continued)
405
Table 18.2 (continued)
406
18.4.2 MASP-1
MASP-1 was characterized by Matsushita and Fujita (1992) as the first serine pro-
tease C1s-like and was designated as mannose-binding protein (MBP)-associated
serine protease (MASP). This serine protease plays a central role in the initiation of
the LP, by carrying out the activation of MASP-2. It is considered a promiscuous
protease since its substrate binding groove is wide and resembles that of trypsin
rather than early complement proteases [51].
Recent findings supported MASP-1 as an essential component of the LP, whose
concentration is 20-fold higher than MASP-2 in the plasma. MASP-1 undergoes
autoactivation to subsequently activate MASP-2 efficiently—acting in a manner
analogous to that of C1r and C1s in the CP, being responsible for 60% of the C2
cleaved and C3 convertase formation [52, 53]. MASP-1 autoactivation seems to
control the initiation of the LP [54], but does not cleave C4, being not capable of
generating C3 convertase by itself, although direct activation of C3 by MASP-1 can
occur at a relatively low efficiency [55, 56].
MASP-1 is primarily expressed in the liver, with mean plasma levels of 11 μg/ml
(range 4–30 μg/ml) [57], and significantly contributes to the development of the
inflammatory reaction by proteolytic activity. MASP-1 induces Ca2+ signaling,
NF-κB and p38 MAPK pathways in endothelial cells through protease-activated
receptor 4 (PAR4) [58]. This activity leads to the release of IL-6 and IL-8, activating
the chemotaxis of neutrophil granulocytes [59]. MASP-1 is also able to modulate
the immune response by the release of pro-inflammatory bradykinin from high-
molecular-weight kininogen [60].
MASP-1 is immediately activated after microbial infection by the binding of
PRM complexes to targets leading to opsonization, cell lysis, release of anaphyla-
toxins, chemotaxis of neutrophils, and inflammation. In fact, MASP-1 plasma levels
have been associated with some inflammatory disorders, and the activity of MBL/
MASP-1 complex has been associated to disease severity in post-streptococcal
acute glomerulonephritis and hepatitis C virus (HCV) infection, leading to glomer-
ular fibrinogen deposits and sustained hematuria [61], and liver fibrosis [62], respec-
tively. In addition, MASP-1 plasma levels were also higher in patients who suffered
myocardial infarction and lower in patients with acute ischemic stroke [63]. High
levels of MASP-1 were also observed in patients with type 1 diabetes mellitus [64].
In autoimmune diseases, high plasma levels of MASP-1 were associated with
SLE [65]. In contrast, MASP-1 levels were reduced in patients with hereditary
angioedema in response to the degree of complement C4 consumption, which was
expected to contribute to the pathophysiology and severity of the disease [66].
Furthermore, MASP-1 was shown to play a role in coagulation, cleaving factor
XIII and fibrinogen and mediating the formation of cross-linked fibrin, although
with lower catalytic efficiency compared to thrombin [67]. In fact, antithrombin in
the presence of heparin is a more potent inhibitor of MASP-1 then C1 inhibitor. The
ancient origin of MASP-1 and its thrombin-like activity suggests its involvement in
a coagulation-based defense mechanism in the early evolution of innate immunity
[68]. Interestingly, components of the coagulation cascade amplify complement
408 F.A. Andrade et al.
activation in such a manner that both complement and coagulation cascade are
interconnected through an important crosstalk [9]. In addition, MASP-1 was associ-
ated with thrombus formation in a mouse model of arterial injury [69], and in
patients with diabetes, contributing to an enhanced thrombotic environment and
consequent vascular complications [64].
18.4.3 MASP-3
18.4.4 MAp44
MAp44 is an alternative splice product of the MASP1 gene, which lacks the SP
domain and consequently, its functional activity. The polypeptide was named
MAp44 due to its molecular mass of 44 kDa. MAp44 is mainly expressed in the
heart and skeletal muscle, with a mean serum concentration of 1.7 μg/ml (range
0.8–3.2 μg/ml) [39, 43].
18 Serine Proteases in the Lectin Pathway of the Complement System 409
Although MAp44 does not contain the SP domain, the other domains interact
with MBL or ficolins, thereby competing with MASP-1, MASP-2, and MASP-3
and resulting in the inhibition of C4 deposition and consequently the inhibition of
downstream complement activation [39, 43, 77]. In addition to inhibiting the incor-
poration of MASPs into MBL/ficolin complexes, MAp44 was shown to prevent
MBL deposition on MBL ligands and restricting complement activation and C3
deposition [78].
MAp44 has been associated with cardioprotective effects, preserving cardiac
function, decreasing infarct area, and preventing thrombogenesis in murine models
of ischemia/reperfusion injury by inhibiting MBL and C3 deposition [69, 78]. Due
to its protective effects on cardiovascular system, MAp44 has been suggested to be
used in a therapeutic approach for the treatment of myocardial ischemia/reperfusion
injury and thrombogenesis [78]. In contrast, Frauenknecht et al. demonstrated that
MAp44 levels were not directly related to the pathophysiology of cardio- and cere-
brovascular diseases, but instead was associated with cardiovascular risk factors
such as dyslipidemia, obesity, and hypertension [63].
Fig. 18.5 MASP2 gene and transcripts. Alternative splicing of primary transcript gives rise to two
different mRNAs encoding MASP-2 and MAp19 proteins. Blue boxes indicate translated part of
the exons. MASP: mannose-binding lectin-associated serine protease. MAp19: mannose-binding
lectin-associated protein of 19 kDa. CUB, C1r/C1s, Uegf, and bone morphogenetic protein; EGF,
epidermal growth factor; CCP, complement control protein. Exons are numbered and drawn to
scale; introns are truncated
exon 12, were found to be associated with the modulation of serum levels [84–87].
Some of them were associated with the susceptibility to leprosy [87], hepatitis C
[88], malaria [89], bacterial infections after orthotopic liver transplantation [90],
Chagas disease [91], rheumatoid arthritis [92], tuberculosis [93], rheumatic fever
[94], and endemic pemphigus foliaceus [95].
18.4.6 MASP-2
The second MASP was identified in 1997 by Thiel et al., which showed notable
homology with the first reported MASP (MASP-1) and the serine proteases, C1s
and C1r, of the CP. Nevertheless, subsequent analysis demonstrated that despite the
homology, MASP-2 was entirely different in assembly and function to C1s [56, 68,
96] with a 1000-fold higher catalytic activity and could be inhibited by C1-inhibitor
50-fold more rapidly [97]. The almost identical substrate specificity of MASP-2 and
C1s is mediated through different group of enzyme-substrate interactions, and it is
very probable that the major functional difference between them is reflected in the
different loop structures of the two enzymes [96].
MASP-2 is synthesized as single-chain proenzyme, and its activation proceeds
through the cleavage of a single Arg-Ile bond, generating the two disulfide-linked
Table 18.3 MASP2 gene polymorphisms associated with MASP-2 and MAp19 concentration and diseases
Gene Amino acid Protein
dbSNP Allele MAF Gene region position position region Serum levelsa Disease associations
rs7548659 G > T 43% Promoter −175 n.a. n.a. High MASP-2 and Susceptibility to
low MAp19 leprosy [87]
concentration
rs72550870 T > C 1% Exon 3 599 p.D120G CUB1 Low MASP-2 and Endemic pemphigus
MAp19 foliaceus [95],
concentration rheumatic fever [94],
persistent inflammatory
disease, and severe
pneumococcal
pneumonia [82]
rs61735600 C > T 2% Exon 3 537 p.R99Q CUB1 High MASP-2 Susceptibility to
concentration leprosy [87]
rs56392418 C > T 4% Exon 3 620 p.P126L CUB1 Low MASP-2 –
concentration
rs2273343 T > C 1% Exon 4 1689 p.H155R EGF Low MASP-2 –
concentration
rs2273344 C > T 16% Intron 4 2143 n.a. n.a. High MASP-2 and –
low MAp19
concentration
rs9430347 G > A 15% Intron 5 2420 n.a. n.a. High MASP-2 and –
low MAp19
18 Serine Proteases in the Lectin Pathway of the Complement System
concentration
rs17409276 G > A 16% Intron 9 16,060 n.a. n.a. High MASP-2 and –
low MAp19
concentration
rs2273346 A > G 12% Exon 10 16,368 p.V377A CCP2 Low MASP-2 Susceptibility to
concentration tuberculosis [93],
rheumatic fever [94]
411
(continued)
Table 18.3 (continued)
412
18.4.7 MAp19
18.5 Conclusions
This chapter discussed several aspects and research findings that point out the
importance of serine proteases of the LP and its gene polymorphisms in the human
physiology and pathology. The activation of complement by complex PRMs/
MASP-1/2 has been associated not only with immune response but also with other
biological processes, such as coagulation and embryonic development. However,
future studies are required in order to clarify the role of MASP-3, MAp19, and
MAp44 proteins in the activation of the LP.
18 Serine Proteases in the Lectin Pathway of the Complement System 415
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Pups, SAMPs, and Prokaryotic
Proteasomes 19
Subrata Ganguli and C. Ratna Prabha
Abstract
In eukaryotes, barrel-shaped proteases known as 26S proteasomes are responsi-
ble for removing misfolded proteins and regulatory proteins after they serve their
function. 26S proteasomes are ATP-dependent proteases with three different pro-
teolytic activities. The proteolytic active sites are segregated into the inner com-
partment of proteasome to prevent nonspecific degradation of cytosolic proteins.
Eukaryotic cells tag the proteins with ubiquitin, in order to selectively target
them for degradation by proteasomes. The presence of proteasomes in some spe-
cies of actinobacteria and archaea is known for more than two decades. However,
the details of the molecules used as tags and the mechanism of tagging are com-
ing to light only in the recent times. In actinobacteria prokaryotic ubiquitin-like
molecules (Pups) and in archaea small archaeal modifier proteins (SAMPs) are
used as tags. Though prokaryotic proteasomes show homology to their eukary-
otic counterparts, the prokaryotic tagging mechanism is vastly different suggest-
ing convergent evolution. The structure of prokaryotic proteasomes, Pups, and
SAMPs and the tagging mechanisms are presented here in detail, and the simi-
larities and differences with eukaryotic system are highlighted. The possible
applications of the knowledge generated in this area to the treatment of tubercu-
losis are underscored at the end of the chapter.
S. Ganguli
R&D, Consortium for Training, Research, and Development, AE 641, Salt Lake,
Kolkata 700064, India
C. Ratna Prabha (*)
Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of
Baroda, Vadodara 390002, India
e-mail: chivukula_r@yahoo.com
Keywords
Prokaryotic ubiquitin-like protein • Pup • Small archaeal modifier proteins •
SAMP1 • SAMP2 • Prokaryotic proteasomes
19.1 Introduction
regulatory subunits present at one or both ends of the particle. The 20S proteasome
is a hollow cylindrical structure formed by four stacked rings comprising of seven
protein subunits each [9]. Each of the inner two rings of the complex is made up of
seven -subunits, whereas the outer two rings are made up of seven α-subunits each.
There are three protease active sites in the β-ring, located on the interior surface of
the central chamber. The protease activities associated with proteasome are
described as trypsin-like, chymotrypsin-like, and caspase-like based on the cleavage
pattern. The outer two rings control the gate through which proteins must enter the
inner chamber for proteolysis. The entrance to the complex could be as narrow as
13 Å with the interior chamber at most 53 Å wide and is 150 Å by 115 Å in case of
mammals. In eukaryotes the 20S core proteasome is essential for survival.
19.3 U
biquitin: The Protein Tag Used for Posttranslational
Modification of Eukaryotic Proteins
In the late 1970s, studies with reticulocytes lacking lysosomes led to identifica-
tion of a novel ATP-dependent intracellular proteolytic mechanism [10, 11].
Further studies identified several protein chains making up the protease system. It
was also observed that modification of lysine residues in substrate proteins is a
prerequisite for proteolytic degradation by this novel mechanism. Finally the
entire system was described as the ubiquitin-mediated protein degradation path-
way, wherein proteins are degraded by the 26S proteasome after being tagged by
ubiquitin. This arrangement is referred to as the ubiquitin-proteasome system
(UPS) in the eukaryotes. The Nobel Prize in Chemistry for the year 2004 was
awarded to Aaron Ciechanover, Avram Hershko, and Irwin Rose for the discovery
of this novel proteolytic pathway.
Ubiquitin is a small protein modifier, about 8.5 KDa in molecular size, and is
ubiquitously found in eukaryotic cells ranging from single-celled microorganisms
to plants and mammals [12]. Early work on ubiquitin identified it as ATP-dependent
proteolysis factor or APF1 [10, 11]. Eukaryotic cells conjugate the protein ubiquitin
to modify other proteins in a process called ubiquitination [7, 8]. Conjugation of
ubiquitin to substrate proteins is achieved by formation of a covalent isopeptide
bond between the carboxy terminus of ubiquitin and lysine side chain of the target
protein. Ubiquitination is catalyzed by the serial action of three enzymes: the
ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiqui-
tin ligase (E3). A molecule of ubiquitin is conjugated to substrate protein in monou-
biquitination, or ubiquitin molecules can form chains on the protein by
polyubiquitination. Ubiquitination serves many different functions inside the cell;
however one of the most important functions is guiding substrate proteins to protea-
somes for degradation [10]. Ubiquitination of substrate protein is a prerequisite for
degradation by ubiquitin-proteasome system (UPS). The deregulation of ubiquitin
pathway has been implicated in Alzheimer’s disease, Parkinson’s disease, cancers,
and several other genetic disorders [13].
424 S. Ganguli and C. Ratna Prabha
archaea as well as in eukaryotes. It was observed that the second residue from the
C-terminus of the ATPases is aromatic, and it sits in a groove in the α-ring, bringing
about a conformational change in the latter leading to opening of the obstructive
gate formed by N-termini of the α-chains [30, 31]. The assembly of purified ATPase
ring and 20S proteasome could not be demonstrated with bacteria, in spite of the
aromatic residue as the penultimate residue in the C-terminus of ATPase chains and
the similarity in the architecture of proteasome with that of archaea [32, 33]. The
possible reasons for this failure are speculated to be either conformational changes
introduced into the subunits or the loss of accessory factors which may be required
for the assembly, during the process of purification.
Substrate proteins are recruited to bacterial proteasome using the mechanism analo-
gous to ubiquitination found in eukaryotes. Here the protein substrate is conjugated
to a protein known as prokaryotic ubiquitin-like protein (Pup) [34, 35]. A screen
setup to find proteins associated with Mpa-proteasome using bacterial two-hybrid
system identified Pup as a protein bound to Mpa [35]. The process of tagging the
substrate with Pup protein is called pupylation. Pupylation was initially observed in
Mtb and Mycobacterium smegmatis (Msm), and later it was concluded that all the
actinobacterial species which show proteasome-mediated degradation use the same
mechanism for marking proteins. Pup is a small protein of 60 to 70 residues in
length [36]. However, its sequence and structure do not bear any homology to ubiq-
uitin [37–39]. The C-terminal residue in Pup is glutamate or glutamine, which is
preceded by diglycine. Pup displays disordered structure in unbound state. The
C-terminal region takes up coiled coil conformation, showing a weak propensity for
attaining helical conformation.
using pull-down assays identified two proteins, PafA and Dop [40]. Later on it was
found with Dop knockouts that Dop catalyzes the deamidation reaction on Pup in
mycobacteria, and in its absence the conjugation between PupQ and substrate pro-
teins fails to occur leading to accumulation of proteasomal substrates [41, 42].
Several other actinobacteria, which synthesize PupE with glutamate as the last resi-
due, could carry out pupylation even in the absence of Dop.
On the other hand, PafA acts as Pup ligase catalyzing conjugation of PupE to
substrate proteins (Fig. 19.2). In Mtb strains carrying pafA deletion, pupylated pro-
teins were not found and proteasomal substrates were accumulated, establishing
PafA’s role as Pup ligase [35, 43]. PafA catalyzes the reaction in two steps. PafA
initially binds ATP and activates PupE to phosphorylated PupE, by transferring the
phosphate group from ATP [44]. While ADP remains bound to PafA, PafA cata-
lyzes the second step in which activated Pup protein is transferred to substrate
releasing phosphate.
Only a single Pup ligase and/or monopupylation has been identified so far [40].
There is no experimental evidence suggesting the possibility for existence of more
than one Pup ligase or polypupylation.
19.6 D
op Catalyzes Depupylation to Maintain Protein
Homeostasis
However, the presence of Dop even in those bacteria which produce PupE instead
of PupQ suggested another, not so far identified function [40]. Studies on these
organisms revealed that Dop acts as depupylase (Fig. 19.3), removing Pup from
substrate proteins [45, 46]. Dop uses ATP as a cofactor for both deamidase and
depupylase reactions. In Dop-catalyzed reactions, ATP can be replaced by ADP suc-
cessfully as there is no energy requirement [40].
19 Pups, SAMPs, and Prokaryotic Proteasomes 427
19.7 B
inding of Pupylated Substrate to Mpa and Its
Subsequent Degradation by Proteasomes
Mpa ring undergoes pupylation at its C-terminal on Lys591 residue [49–51]. After
pupylation the Mpa ring is capable of binding to pupylated proteins and unfolding
them. However, it will not be able to interact with 20S proteasome, which makes
degradation of proteins impossible [52]. Depupylation by Dop can nullify the effect
of pupylation of Mpa [52]. Hence, while pupylation of proteins directs them for
degradation, pupylation of Mpa prevents protein degradation.
19.9 P
up-Proteasome System in Relation to Pathogenesis
of Mycobacteria
In most of the actinobacteria, proteasomes are not essential under normal conditions
of growth as there are other compartmentalized proteases such as Clp proteases,
Lon protease, and FtsH [53–55]. Mycobacterium tuberculosis (Mtb) does not
require proteasomes for its in vitro growth, while it cannot survive without Clp
protease genes. However, Mtb requires proteasomes for its persistence in the mac-
rophages inside the host [55–59], which may be attributed to the observed absence
of Lon protease [60]. Infected macrophages produce NO and reactive nitrogen
intermediates (RNI) with the help of inducible nitric oxide synthase (iNOS) induced
428 S. Ganguli and C. Ratna Prabha
The 20S proteasomes of actinobacteria, archaea, and eukaryotes are clearly homol-
ogous. 20S proteasomes in all these organisms are threonine proteases. There is an
ongoing debate on the origin of 20S proteasomes. Many evolutionary biologists are
of the view that proteasomes originated in actinobacteria and were passed on to
archaea as actinobacterial ancestors gave rise to archaea [67], while others believe
that actinobacteria came into possession of proteasomal genes through horizontal
gene transfer from either archaea or eukaryotes [68, 69]. Interestingly, the hexa-
meric ATPase rings present in all three lineages also show strong homology. The
basic structural plan of heptameric barrel-shaped protease associating with hexa-
meric ATPase ring is also uniformly found in all these organisms.
Even though tagging substrate proteins is common to both PPS and UPS, Pup
and ubiquitin do not bear any homology either in their sequence or structure. While
ubiquitin is a globular protein with β-grasp fold, Pup is an unstructured protein.
The process of ubiquitination and pupylation also is analogous. A cascade of
three enzymes transfers ubiquitin to substrate protein. Activation of ubiquitin
involves ubiquitin adenylate. There is a single enzyme PafA which catalyzes conju-
gation of Pup to substrate protein. Though the reaction is ATP driven, the intermedi-
ate formed in the reaction is phosphorylated Pup. Pup has glutamate or glutamine as
the C-terminal residue, whereas diglycine is found at the C-terminal of ubiquitin.
The isopeptide bond formed by Pup is between the γ-carboxyl of glutamate and
lysine of substrate, and in the case of ubiquitin, the isopeptide bond is formed
19 Pups, SAMPs, and Prokaryotic Proteasomes 429
between α-carboxyl of glycine and lysine of substrate. There is a single Pup ligase,
and only monopupylation occurs on substrate in contrast to ubiquitination, where
more than six hundred ubiquitin ligases and polyubiquitination with different
branching patterns are present. Another interesting difference is the sequences of
PafA and DopA that are homologous to glutamine synthetase [70, 71].
19.13 Conclusions
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Role of Proteases in Photo-aging
of the Skin 20
Rita Ghosh
Abstract
R. Ghosh (*)
Department of Biochemistry & Biophysics, University of Kalyani,
Kalyani 741235, West Bengal, India
e-mail: ritadg2001@yahoo.co.in
pounds that inhibit the molecular pathways that result in expression of the prote-
ases have proved to be useful in prevention/reversal of skin aging.
Keywords
Photo-aging • Ultraviolet radiation • Skin • Proteases • Prevention
20.1 Introduction
Aging is an inevitable process common to all living organisms that result from
molecular damage over time. The skin is the largest of all organs of the human body
that constitutes about 16% of the total body weight. It maintains body temperature
and hydration; it also has roles in sensory perception and immune surveillance. It
acts as a barrier between the inner body parts and the environment [1]. Being the
most exposed part of the body, different environmental aggressors leave their mark
on the skin. The skin is thus a good model for studying the consequences of aging.
Skin aging consists of two clinically and biologically independent processes – the
intrinsic chronological aging and the other is due to aging through extrinsic factors.
The intrinsic aging process proceeds at a genetically determined pace due to buildup
of damaging products from cellular metabolism and is similar in all parts of the
body [2]. Among the extrinsic causative agents, the most significant is the exposure
to solar radiations that produce biological damages to the cells. UV exposure has
been associated with photo-carcinogenesis, photo-aging, and photo-
immunosuppression [3, 4]. Photo-aging adds up to the effects of chronological
aging.
The ultraviolet (UV) radiations from the solar spectra are primarily responsible for
the photo-aging of the skin. The UV spectrum has been divided into three ranges –
UVA (400–320 nm), UVB (320–280 nm), and UVC (280–200 nm). The UVC is
readily absorbed by the most important biomolecules like cellular and mitochon-
drial DNA and proteins, but the atmosphere blocks out this range of radiation, and
therefore, it does not contribute toward photoaging. UVB is also damaging to differ-
ent cellular components, but the ozone layer is capable of blocking this range of
radiation from reaching the earth’s surface. However, there is a serious concern
about exposure to solar UVB due to the thinning of the ozone layer. UVB rays can
only penetrate up to the basal layer of the epidermis of the skin. Ninety six percent
of the solar radiation that reaches the earth surface is UVA. It penetrates the skin
more deeply than UVB through the epidermis and dermis and contributes most
significantly toward photo-aging [5].
20 Role of Proteases in Photo-aging of the Skin 437
The process of photo-aging of the skin is multifactorial and complex; it affects vari-
ous layers of the skin with the major damage being observed in the connective tis-
sues of the dermis. The skin is composed of three layers – the outer epidermis, the
thin basement membrane (BM), and the thicker inner layer, called the dermis. The
epidermis is about a tenth the thickness of the dermis. The epidermis is made up of
three layers. The stratum corneum is the outermost layer that is made up of a few
layers of nonnucleated dead cells. It is followed by the granular layer that consists
of granulated interconnected cells. They have a granular appearance due to deposi-
tion of keratin. The next Malpighian layer contains the squamous epithelial cells
and the dendritic melanocytes. The melanocytes produce the melanin that is trans-
ported outward through these dendrites [6].
The basement membrane is important for maintaining a healthy skin. The BM is
present at the junction of the dermis and epidermis to provide mechanical support to
the outer protective epidermis. It also prevents contact between the two layers and
determines the polarity of the epidermis. The proliferating cells of the epidermis
remain attached to it, while the daughter cells migrate outward. It also consists of
three different layers – the lamina lucida, the lamina densa, and the lamina fibrore-
ticularis [7].
The dermis consists of the connective tissues. Both the dermis and the BM con-
tain various types of collagens, elastic fibers, and glycosaminoglycans in the extra-
cellular matrix (ECM). The ECM is most important for the skin structure and
elasticity. The ECM contains different collagens of which about 85% is type I pro-
collagen [8]. The elastic fiber network constitutes about 2–4% of the ECM and is a
key component of the dermal connective tissue that provides elasticity to the skin.
Morphologically, it consists of two distinct components – the elastin and the micro-
fibrils [9]. The elastic fiber network accounts for 2–4% of the ECM in sun-protected
skin and provides elasticity to the skin [10]. The glycosaminoglycan and the proteo-
glycans comprise only 0.1–0.35 of the dry weight of the skin. Their role is in hydrat-
ing the skin and biological signaling [11].
Both chronological and photoaged skin show alteration in skin microrelief. Deep
furrows appear in the skin leading to wrinkles. Intrinsically aged skin is thin, and
there is laxity of the skin due to reduced elasticity [12]. There are no pigmentary
changes or deep wrinkles. In contrast, the photoaged skin appears with fine and
deep coarse wrinkles, shallowness, uneven blotchy pigmentation, and rough and
leathery skin texture with increased fragility. There is loss of mature collagen with
a distinctive basophilic appearance called basophilic degeneration [13]. No vascular
damage is observed in intrinsically aged skin. Both acute and chronic exposures to
UV radiation increase skin vascularization and angiogenesis [14]. Emerging
438 R. Ghosh
information has revealed that though phenotypically it differs extensively from the
intrinsic chronological aging [15], they share some fundamental molecular path-
ways [16].
Collagen is a major structural protein found in the dermal ECM that provides
stability and tensile strength to the dermis [17]. Procollagens are synthesized in the
dermal fibroblasts; these are secreted into the extracellular space where these are
enzymatically processed to mature collagen. The mature collagen spontaneously
forms fibrils that are responsible for the strength and resilience of the skin. These
fibrils are stabilized by cross-linkages. These collagen fibrils have a half-life of 17
years. The accumulation of fragmented collagen has a lasting consequence on the
structure of the skin that contributes to the chronological aging. UV radiation also
alters the dermal collagen. It promotes the breakdown of collagen and also inhibits
the biosynthesis of procollagen. It has been observed that exposure to UV radiation
can result in complete loss of procollagen synthesis for 24 hours [18]. Both colla-
gens I and III decreased on UV exposure, but between types I and III collagen pres-
ent in the ECM, the relative amount of collagen I was more [19]. Collagen VII
contains the anchoring fibrils, and they contribute toward stabilization of the dermis-
epidermis junction (DEJ). Collagen VII is also severely reduced in photoaged skin.
Loss of collagen VII and elastic fibers was found in fibroblasts. Wrinkling is due to
the loss of collagen fibers and deposition of the abnormal degenerative elastic mate-
rial [13]. In vitro UVA induces elafin in fibroblasts. It forms the elafin-elastin com-
plex that inhibits the binding of elastase to elastin. Formation of this complex
prevents elastic fibers from elastolytic degradation resulting in accumulation of
elastic fibers. Elafin is thus believed to be integral to actinic elastosis [5].
Elastic fibers constitute the structural elements of the connective tissues that have
a central core of amorphous, cross-linked hydrophobic elastin surrounded by
fibrillin-rich microfibril. The elastic fiber network extends from the DEJ into the
dermis. Disruption and reduplication of the BM at the DEJ are observed in sun-
exposed skin [7]. Loss of elasticity is an important clinical feature of photo-aging.
A leathery weather-beaten appearance known as solar elastosis is the result of elas-
tic fiber degradation and is a hallmark of photo-aging.
Tenascin is a large glycoprotein that is found just below the DEJ. In sun-exposed
skin, this protein is increased both in the dermis and epidermis; while its increase is
small in the dermis, there is an appreciable increase of this protein in the epidermis
of the skin [20]. The increase in deposition of glycosaminoglycans and dystrophic
elastosis was revealed through immune-positive staining of the severely disorga-
nized tropoelastin and its associated microfibrillar component, fibrillin. Fibrillin is
truncated and depleted in the upper dermis and DEJ of photoaged skin.
Plasminogen activators (PAs) are produced by different cell types including the
epidermal keratinocytes. They degrade the zymogen, plasminogen, to plasmin
directly and also by activating other proteases. Activity of urokinase-type plasmino-
gen activator (uPA) was found in the stratum corneum and in the basal layer after
disruption of the barrier, which is responsible for the delay in recovery from photo-
aging [7].
20 Role of Proteases in Photo-aging of the Skin 439
UV, MMPs may be involved in processes other than ECM degradation; higher
doses of irradiation can only lead to immediate changes associated with ECM
degradation by neutrophil [29, 32]. The cumulative damage to ECM by the MMPs
through chronic exposure was proposed by Fisher et al. [37, 38]. They have pro-
posed that skin damage is only repaired partially and the cumulative effects of
such damages are involved in photo-aging by MMP-1, MMP-3, and MMP-9.
Neutrophil-derived elastase is responsible for photoaging [39]. At erythemogenic
doses of UV, the elastase induced is linked to neutrophils, while at sub-erythemo-
genic doses, MMPs may be derived from the keratinocytes and fibroblasts.
Therefore, in the pathogenesis of photo-aging, the contribution from direct dam-
age induced to ECM by the generated reactive oxygen species (ROS), neutrophil-
derived proteases, as well as the MMPs secreted by keratinocytes and fibroblasts
may be involved.
Various physiological processes generate a low level of ROS that contribute toward
chronological aging. Both UVB and UVA are known to generate ROS [40]. UVB is
directly absorbed by DNA, while other chromophores present in the skin, like trans-
urocanic acid present in the epidermis, absorb UVA [41]. These excited photosensi-
tizers subsequently react with oxygen and produce ROS like superoxide
anions (Ȯ 2̅ ) and singlet oxygen (1O2). UV light also increases the NADPH oxidase
and xanthine oxidase activity, which is also involved in the generation of ROS [42].
Neutrophils also produce Ȯ ̅2 and 1O2. The Ȯ ̅2 is converted to hydrogen peroxide
(H2O2) by superoxide dismutase. The H2O2 produced crosses the cell membrane and
generates the highly toxic hydroxyl radical (OḢ) through Fenton reaction with Fe
(II). 1O2 and OḢ initiate lipid peroxidation of cellular membranes. OḢ and interme-
diates of lipid peroxidation are also involved in induction of MMP-1 and MMP-3 by
UVB [22]. UVA-generated 1O2 initiates the c-Jun N-terminal kinase (JNK) and p38
MAPK leading to expression of MMPs [13]. UVB-induced OḢ and lipid peroxida-
tion products also stimulated ERK, p38 MAPK, and subsequently induced JNK2.
UV light upregulates c-Jun and c-Fos that increases the activation of transcription
factor AP-1, required for the expression of the MMPs [43].
ROS plays an important role in collagen metabolism both directly and indirectly.
ROS not only destroys the interstitial collagen but also inactivates tissue inhibitors
of matrix metalloproteases (TIMPs) and induces the synthesis and activation of the
matrix metalloproteases (MMPs) [44]. It has been demonstrated through different
approaches that 1O2 and H2O2 are involved in the induction of MMP-1, MMP-2, and
MMP-3 by UVA.
ROS have been implicated as a causative agent for intrinsic aging. Mutations in
mitochondrial DNA impair the function of mitochondria leading to defects in elec-
tron transport and oxidative phosphorylation, which in turn enhances the generation
of ROS. It has also been demonstrated that 1O2 generated by UVA causes the same
442 R. Ghosh
mutations in the mitochondrial DNA that are involved in aging [45]. ROS also
results in shortening of the telomere [46]. Shortening of telomere length provides
signal for replicative senescence. Thus, there exists some overlap in the causes of
photo-aging with that of intrinsic aging through ROS.
MMPs are a family of zinc proteinases that consist of 28 members of which 24 are
expressed in mammals. They are classified in relation to their substrate specificities
and whether they are bound to the cell membranes or secreted as soluble proteins.
The different classes of MMPs are collagenases (MMP-1, MMP-8, and MMP-13),
gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, MMP-10, and MMP-11),
matrilysin (MMP-7 and MMP-26), membrane-type MMPs (MMP-15, MMP-16,
MMP-17, MMP-24, and MMP-25), and other types (MMP-12,MMP-19, MMP-20,
MMP-21, MMP-22, MMP-23, and MMP28) [47]. In normal healthy, sun-protected,
adult human skin, the basal mRNA expression levels of the MMP family members
were found to be extremely low, MMP-14 being the only exception. While the tran-
scripts for MMP-8, MMP-10, MMP-12, MMP-20, and MMP-26 were not detected
at all, the transcripts for the remaining MMPs were almost near the level of detec-
tion, being almost thousand-fold lower than the housekeeping gene, 36B4, that was
used as internal control. Basal expression of MMP-14 mRNA was approximately
35-fold higher than that of other detectable MMPs. It has been demonstrated that
there is induction of MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, and
MMP-12 in response to UVA and UVB irradiation. In an in vivo study where expres-
sion of 19 MMPs was observed in normal human skin, only three MMPs – MMP-1,
MMP-3, and MMP-9 – were significantly induced in response to UV irradiation.
There was several thousand-fold increase in the induction of MMP-1 and MMP-3
mRNA, whereas MMP-9 was only modestly induced by six-fold 24 hours after
irradiation. MMP-1 initiates the cleavage at a single site within its central triple
helix of type I and III fibrillar collagen in the skin [21]. Once cleaved by MMP-1,
collagen can be further degraded by elevated levels of MMP-3 and MMP-9. In con-
trast, following irradiation, MMP-14 was reduced nearly 80% by 8 hours and
remained so till 24 hours [23, 47]. The physiological function of MMP-14 in human
skin, however, remains to be determined. MMP-8 (neutrophil collagenase) and
MMP-12 (macrophage elastase) proteins were also present in human skin 24 hours
after UV irradiation.
UV irradiation of organ culture resulted in collagen fragmentation, together with
alteration in the structure and the organization of collagen fibrils in the dermis
through MMP-1 that resembled changes observed in photo-aged skin. Different
studies suggested that MMP-1, MMP-3, and MMP-9 are the primary UV-inducible
collagenolytic enzymes, MMP-1 being the major protease that is capable of initiat-
ing the degradation of native fibrillar collagens in human skin in vivo [21, 36].
MMP-2 degrades the elastin as well as the basement membrane molecules like col-
lagen IV and VII [48]. MMP-3 has broader substrate specificity for collagen IV,
20 Role of Proteases in Photo-aging of the Skin 443
Some bacterial and insect products too have a role in preventing photoaging.
Lipoteichoic acid from Lactobacillus sakei inhibits the pro-inflammatory cytokine,
TNFα, and blocks the phosphorylation of MAPK family in THP-1 cells. It also
decreased the expression of MMP-1. Lipoteichoic acid has the potential to suppress
UVA-induced damage and act as anti-photo-aging agent [63]. A 9-mer peptide
CopA3 that was synthesized from a natural peptide, coprisin, isolated from dung
beetle also has proven activity in prevention and treatment of skin aging [69].
The loss of structural and functional stability of the skin is an inevitable conse-
quence of aging that is caused by both intrinsic and extrinsic factors. Although it is
almost impossible to prevent aging completely, premature aging and damage from
photo-aging can be delayed. As there is considerable overlap between the features
of chronological and photo-induced aging as well as in the general mechanism of
both processes, understanding photo-aging is important as it would help to elucidate
aging in general. Such knowledge would help in development of strategies for pre-
vention as well as treatment protocols for reversal of the aging symptoms.
Considering the increase in the aged population worldwide, it is of prime
significance.
Most intervention methods developed so far have been from in vitro studies that
have to be proved in vivo. The depletion of stratospheric ozone layer and its impli-
cations in photo-aging needs to be highlighted. Emphasis on preventive strategies is
imperative; this can help to enhance the rationale for development of photo-
protective agents. Oxidative damages through generation of ROS have a prime role
in photoinduced aging process. For effective protection, therefore, many antioxi-
dant compounds have been found to be useful. While some have proven benefits
when applied topically or taken through diet, in some other cases, their role has
been uncertain [70]. Sunscreens are known to provide protection from sun-induced
damages, but their mode of action is different from antioxidants. In principle they
might act synergistically with antioxidants to prevent photo-aging, but research in
this area is needed as this has not been tested. Treatments with antioxidants are often
associated with side effects [71, 72]; this aspect therefore needs critical evaluation.
UV exposure is involved in the etiology of skin cancer. In photoaged skin various
signal pathways are triggered that turn the cells resistant to apoptosis [73]. Thus,
deterrence of photo-aging may also help to avoid cancer. Thus, an in-depth under-
standing of the physiological basis of skin aging is needed particularly in relation to
photo-induced carcinogenesis. It can help for the advancement in treatment of the
unwelcome consequences of both cosmetic and pathogenic skin aging. Development
of novel preventive as well as therapeutic approaches would not only be beneficial
for cosmetic science, but it would also enhance the overall quality of life.
Acknowledgments The author acknowledges assistance from the University of Kalyani, Kalyani,
and DST-PURSE, Government of India, for supporting the work.
446 R. Ghosh
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Insect Proteases: Structural-Functional
Outlook 21
Shounak Jagdale, Sneha Bansode, and Rakesh Joshi
Abstract
Proteases perform various activation and effector functions in development,
growth, and survival in insects. In this chapter, we have focused on the catalogue
of insect proteases and their structural and functional aspects. We have reviewed
the proteases involved in insect’s vital life processes like reproduction, develop-
ment, immunity, and defence. The indispensability of seminal fluid and egg pro-
tease during fertilization highlights their evolutionary primitiveness. Furthermore,
various cellular proteases like cathepsins and caspases take over the earlier one’s
functions. The role of cellular proteases is well documented in the developmental
process like embryogenesis, metamorphosis, moulting, and eclosion. Cellular
proteases are further supported by haemolymph and digestive proteases to facili-
tate the growth and survival of the insect. Apart from developmental cathepsin
and caspases, haemolymph contains a diverse pool of proteases that serves a
pivotal role in immunity against various pathogens. Amongst various insect pro-
teases, digestive proteases show highest structural and functional variability
according to developmental stage, food content and stress level. This chapter
provides an insight of structural-functional aspects of insect proteases and their
role in insect physiology.
Keywords
Insect • Development • Digestion • Immunity • Protease
21.1 Introduction
Table 21.1 (continued)
Sr.
No. Name Type Function References
Developmental and cellular proteases
12 Cathepsin L Cysteine protease Degradation of cuticle, [16, 21–22]
epidermis and labial gland
Differentiation of
imaginal discs
Granulocyte to
macrogranulocyte
transformation
Histolysis of fat bodies
and silk gland
Normal expression of
apoptosis effector gene
caspase 1
Autophagy of degraded
fat body cells
13 Cathepsin F Cysteine protease Required for cuticle and [16]
14 Cathepsin O epidermis layer
degradation during
moulting
15 Cathepsin B Cysteine protease Metamorphosis from [16, 23–27]
pupa to adult
Histolysis of fat bodies
and silk gland
Degradation of labial
gland
DNA fragmentation in fat
bodies
Immune response against
parasitic infection
16 Cathepsin D Cysteine protease Degradation of larval fat [16, 25, 26]
bodies
DNA fragmentation in
larval midgut cells
Immune response against
parasitic infection
17 Cocoonase Trypsin protease Softening insect cocoons [6]
to permit the escape of
adult moths
18 Dronc Caspases Stress-induced apoptosis [28–44>]
19 Dredd
20 Strica
21 Crice
22 DCP-1
23 Decay
24 Damm
(continued)
454 S. Jagdale et al.
Table 21.1 (continued)
Sr.
No. Name Type Function References
25 DM1-MMP Matrix metalloprotease Remodelling of [45–47]
extracellular matrix
26 DM2-MMP Matrix metalloprotease Development of the [48–50
central nervous system
and tracheae
27 ADAMs Metalloprotease Dendrite reshaping in [5152–56]
optic lobes of the brain
and eye imaginal discs
28 Neprilysin-like Metalloprotease Axon extension in [57]
nervous system
development
Activation of signalling
pathway resulting in
patterning of imaginal
discs
Proteolytic cleavage of
active signalling peptides
in cardiovascular,
nervous, inflammatory
and immune functions
Haemolymph proteases
29 Modular serine Serine protease Toll signalling pathway [58]
protease
30 Grass Toll signalling pathway [58]
31 PAPs Melanization, [59–63]
32 Sp22D degradation of tracheal
epithelium and larval
hypodermis during
pupation
Digestive proteases
33 Trypsin Serine proteases Digestion of proteins to [2, 64]
oligopeptides
34 Chymotrypsin Digestion of elastin
35 Elastase
36 Cathepsin L Cysteine proteases Digestion of [65]
oligopeptides with
hydrophobic amino acids
37 Cathepsin D Aspartic proteases Killing the ingested [66]
bacteria
38 Aminopeptidase N Aminopeptidases Hydrolysis of [64]
39 Aminopeptidase A oligopeptides into
40 Carboxypeptidase A Carboxypeptidase dipeptides
Hydrolysis of single [66]
41 Carboxypeptidase B amino acids from the
42 Dipeptidases C-terminus
Hydrolysis ofof dipeptides
the peptide [67–69]
chain
21 Insect Proteases: Structural-Functional Outlook 455
Reproduction is the most important activity for the survival of any species. In
insects, eggs produced in the female insect body get fertilized by sperm, and eventu-
ally the fertilized eggs are delivered outside the body, where they hatch. Recent
molecular understanding has suggested that proteases have a major role to play in
the process of insect reproduction [70–73]. Interestingly, the male and female repro-
ductive tract proteases and protease homologs show adaptive evolution, indicating
their intersexual co-evolution [71, 74–78].
The seminal fluid of insects contain various proteases that get transferred to the eggs
during fertilization. These proteases are expressed in almost all the tissues of the
male reproductive system. In Drosophila sp., the ejaculate has about 21 seminal
fluid proteases (SFPs), 17 protease inhibitors (PIs) and 10 putative serine protease
homologs [79, 80, 10, 81]. Most insect SFPs are conserved at structural and func-
tional levels across different taxa. SFPs have been identified in insects like Anopheles
gambiae, Ceratitis capitata, Heliconius sp., Bombyx mori, Tribolium castaneum,
Gryllus sp. and Amblyomma hebraeum as well [82–85, 11, 86–88]. ‘Seminase’, a
trypsin-like serine protease from the male gets transferred to the female body via the
seminal fluid after mating. It enhances the levels of oogenesis and the egg-laying
capacity of female insects (Fig. 21.1). Additionally, seminase interacts with the
other SFPs which leads to the binding of a sex peptide to the sperm [10, 89, 90].
This interaction is required for the efficient release of sperm from the spermathecae
and also for post-mating maintenance of egg-laying [91]. Females mated with semi-
nase knockdown mutants were observed to have a significant reduction in their egg-
laying capacity over time. In such a case, it has been illustrated that the males can
store the sperm normally, but in females, it remains stored in the seminal receptacle
and spermathecae even 10 days post fertilization [10]. Seminase has been observed
to induce the proteolytic activation of a metalloprotease CG11864, which is required
for the downstream processing of other SFPs like Acp36De and an ovulation pro-
hormone ‘ovulin’ [12]. This shared activity of SFPs in male and female insects
suggests their crosstalk during the evolutionary course of insect sex.
In males, trypsin-like serine proteases are essential for the activation of sperm
motility [92] and sperm activation; for example, in B. mori, a serine protease known
as ‘initiatorin’ has been identified to be necessary for sperm activation and has been
found to be conserved in various classes of insects from phylogenetic and evolution-
ary analysis (Fig. 21.1) [11]. In female insects, metallo-SFPs like CG6168 have
been found to protect females from systemic bacterial infections transmitted through
the seminal fluid [13]. The function of SFPs has been further complemented with
eggs and embryonic proteases. Various other studies have identified numerous
SFPs, but their role still remains to be investigated.
456 S. Jagdale et al.
Fig. 21.1 Diversity of insect proteases: Cladogram of insect proteases was generated by MEGA7:
Molecular Evolutionary Genetics Analysis Version 7.0 using a neighbour-joining algorithm with
1000 bootstrap iterations and p-distance amino acid substitution method. Structures of proteases
were retrieved from PDB. PDB IDs are 2FP3 (Dronc), 1JQG (carboxypeptidase), 1EQ9 (chymo-
trypsin) and 2XXL (Grass). Other proteases were modelled by using Modeller 9.15. All the
sequences were retrieved from NCBI protein database
cysteine proteases that participate in the degradation of yolk protein during embryo-
genesis such as, a papain-like C1 family of cysteine proteases which assists the
major serine proteases in degradation pathways in eggs. In B. mori eggs, a 47 kD
cysteine protease similar to cathepsin L is expressed as a zymogen in the ovary and
the fat bodies, and it helps in vitellin degradation [15], while in insects like
Helicoverpa armigera and Antheraea pernyi, vitellin degradation is carried out by
an acid protease similar to cathepsin B. The expression and activity of the cathepsin
B-like protease gradually decrease as the embryo development progresses [16]. A
cysteine protease similar to cathepsin B has been recognized as an enzyme that
digests egg yolk proteins in D. melanogaster and Musca domestica and is activated
by a serine protease, after proteolytic processing (Fig. 21.1) [96, 97]. Several other
serine proteases play a crucial role in embryogenesis and embryonic patterning as
follows.
21.3.1 Cathepsins
Cathepsins (Cath) are cellular proteases active at acidic pH and play a crucial role
in metamorphosis, moulting, eclosion and other related developmental processes
[16]. They display differential expression in various tissues and developmental
stages. Insects undergo metamorphosis through four life cycle stages: eggs, larvae,
pupae, and moths. During the process of moulting, insects transit from neonates to
458 S. Jagdale et al.
Fig. 21.2 (A) Protease cascade involved in the dorsoventral patterning of Drosophila embryo:
protease cascade which is activated by Nudel which leads to initiation of dorsal signalling path-
way. (B) Protease cascade involved in the insect’s immune response: antigens activate the protease
cascade which leads to activation of AMPs by Toll signalling pathway. Melanization pathway is
activated by prophenoloxidase-activating proteins which are clip serine proteases
six larval instars and resynthesize the exoskeleton by replacing the older one. Insect
metamorphosis and moulting, and thus the tissue remodelling of fat bodies and
haemolymph, are dominated by Cath degradation.
Lepidopterans show maximum expression of Cath L during moulting and pre-
pupation which suggest its role in the degradation of cuticle and epidermis during
moulting. During larva-pupa metamorphosis, Cath L transforms granulocytes to
macrogranulocytes which travel back to the fat bodies and release Cath L at an early
pupal stage for further utilization. Cath L proteolytically degrades and releases fat
body cells into the haemolymph. In a D. melanogaster haemocyte expressed cell
line, Cath L-like protease helps degrade phagocytosed material [19]. In Sarcophaga
peregrina, Cath L is involved in the differentiation of imaginal discs [20, 21]. In
dipteran insects like Delia radicum, Cath L functions in the metamorphosis of the
midgut, as is indicated by its higher expression in the midgut, at the beginning of the
late third instar [105].
Cath F and Cath O which are expressed in the midgut and the haemolymph,
respectively, are known to supplement the activity of Cath L in lepidopterans [16].
Another cathepsin, Cath B, is expressed in granulocytes and plasmatocytes at the
sixth instar stage in most insects, implying its role in larval to pupal transition. In B.
mori, Cath B and Cath L play an important role in the histolysis of the silk gland and
fat bodies, during pupation. Cath B is reported to be involved in the metamorphosis
from pupa to adult and is found active in the fat bodies of both adult males and
females, indicating its involvement in the ageing process. Cath B and Cath D are
highly expressed in the fifth instar larval fat bodies and the pupal gut and work
together to degrade fat bodies. Cath D is overexpressed in the larval midgut and
remains so until eclosion, aiding the formation of adult fat bodies [16].
21 Insect Proteases: Structural-Functional Outlook 459
With respect to tissue remodelling, some trypsin-like serine proteases are involved
in the softening of insect cocoons to permit the escape of adult moths, with the help
of the enzyme ‘cocoonase’. This enzyme, which had initially been identified and
characterized from B. mori, is secreted from a specialized mouth part called Galea
in its zymogen form – ‘prococoonase’ – and is then activated by proteolytic cleav-
age [6]. Homologues of cocoonase such as trypsin proteases are found in many
other lepidopterans and dipterans.
21.3.3 Caspases
The role of caspases has been widely studied in D. melanogaster and A. aegypti.
Caspases are usually present in the cytoplasm in an inactive state until the initiation
of the cell death signalling pathway. Activation of the initiator caspases triggers the
effector caspases, resulting in morphological and biochemical changes associated
with apoptosis, such as DNA fragmentation and plasma membrane blebbing [29].
Caspases are also involved in the autophagic activity and immune response [30]. It
is reported that the inhibitor of apoptosis (IAP) regulates the pro-apoptotic regula-
tion of caspases and its removal is responsible for the activation of caspases. In D.
melanogaster, seven caspases have been reported, namely, death regulator Nedd2-
like caspase (Dronc), death-related ced-3/Nedd2-like caspase (Dredd), Ser-/Thr-
rich caspase (Strica), death-related ICE-like caspase (Drice), decapping protein
1(DCP-1), death executioner caspase-related to Apopain/Yama (Decay) and death-
associated molecule related to MCH-2 (Damm). Initiation of apoptosis depends on
the death signals that converge on the initiator caspases, namely, Dredd, Dronc and
Strica (Fig. 21.1). Dredd has a long prodomain with two death-inducing domains
(DID) which interact with Fas-associated death domain-containing protein (dFADD)
[31]. Dredd is also involved in the innate immune response and is activated upon
interacting with Toll receptors. Once it binds to Toll, Dredd proteolytically cleaves
Relish, a transcription factor that regulates expression of antimicrobial peptides and
is homologous to mammalian NF-κB [32]. It has been reported that Dredd mutants
show reduced immune response against Gram-negative bacteria [33].
Another apoptosis caspase is ‘Dronc’. It has an N-terminal CARD domain that
interacts with death-associated APAF1-related killer (DARK), an adaptor protein
which activates Dronc [34]. Dronc is expressed in all body tissues and is required
for apoptosis in larval tissues in response to toxic agents and X- and γ-radiations
[35–37]. Dronc mutants show inhibition of IAP-induced apoptosis [38]. Zygotic
Dronc mutants showed reduced levels of apoptosis and resulting in delayed histoly-
sis of salivary glands [39]. The next caspase involved in apoptosis is Strica, which
has a long prodomain, rich in serine and threonine. It plays a crucial role in oogen-
esis and is also required for the removal of interommatidial cells of pupal retina and
salivary glands [40, 41]. In vitro studies show that Strica is essential for Hid-
mediated apoptosis, but its mechanism of action is still unknown [41]. Strica and
Dronc double mutants show significant defects in egg chamber during mid- and late
oogenesis indicating their redundancy in PCD during oogenesis [42].
Initiator caspases activate effector caspases like Drice, Dcp-1, Decay and Damm
which lead to the transmission of cell death signals. Drice is the primary target of
Dronc and acts downstream of cell death activators like Reaper and Hid which are
required for apoptosis [32, 35, 38–40]. It has been observed that Drice mutants
show reduced apoptosis in the pupal retina, embryonic nervous system, and adult
wings. Another effector caspase ‘Dcp-1’ – the first caspase to be discovered in
insects – acts similar to Drice, downstream of Reaper and Hid [43]. In Dcp-1
mutants, ecdysone-induced apoptosis is reduced while in some cases, Dcp-1 can
substitute for Drice [44]. The structural homologue of Drice and Dcp-1 is Decay,
and it is also involved in Hid-mediated apoptosis [35, 44]. Decay mutant shows
activation of cell death pathways which suggest that its function is redundant
21 Insect Proteases: Structural-Functional Outlook 461
21.3.4 Metalloproteases
Amongst the many diverse extracellular proteases, those involved in insect immune
response are the serine proteases. A cascade of serine proteases invokes a rapid
response to infection and wounding. When faced with a microbial challenge,
Drosophila activates a series of antimicrobial peptides (AMP) in the fat bodies.
AMP activation is brought about by the Toll signalling pathway, where effector
molecules are expressed upon the activation of several serine proteases and the pro-
cessing of Spätzle by Easter (Fig. 21.2B). Several clip domains (SPE) and modular
serine proteases (ModSPs) are involved upstream of the Drosophila Toll cascade
[58, 115]. Serine proteases such as Grass (Fig. 21.1) and Spirit have been identified
to play a role in connecting ModSP and SPE in terms of their activities and func-
tions albeit their limited biochemical functional analysis. Persephone (Psh) which is
a clip serine protease identifies virulence factors that can trigger the Toll pathway,
such as proteases from Gram-positive bacteria and fungi. It has been hypothesized
that the Psh zymogen plays a sensory role in the detection of these proteolytic
virulent factors.
Usually, a response to wounds or infection leads to activation of the melanization
pathway. In this pathway, prophenoloxidase (proPO) is proteolytically processed by
haemolymph serine proteases called prophenoloxidase-activating proteinases or
PAPs, to phenoloxidase, which oxidizes phenol to produce quinine. Quinines
besides being lethal for most pathogens can also polymerize into melanin and seal
wounds. In M. sexta, PAP1 has a single regulatory clip domain at its amino-terminal,
whereas PAP2 and PAP3 have two clip domains. PAP1 is a 44 kD protein with two
peptide chains of 31 kD and 13 kD. Apart from the haemolymph, PAP1 is also pres-
ent in the fat bodies and the cuticle of the insect and is highly expressed in haemo-
cytes and fat bodies during bacterial infection [59]. PAP2 and PAP3 are found in fat
bodies and haemolymph [60]. PAPs require serine protease homologs (SPH) 1 and
2 in order to cleave proPO. Several other PAPs have been identified, characterized
and purified from other insects like Holotrichia diomphalia and B. mori [61, 62].
M. sexta PAP1 shows structural homology to H. diomphalia PPAF-I, whereas
M. sexta PAP2 and PAP3 show structural homology to B. mori PPAE.
In M. sexta several haemolymph serine proteases (HP1–HP22) have been identi-
fied, and most of these HPs contain regulatory clip domain(s) at their amino-
terminal. HP1, HP2, HP6, HP8, HP13, HP17, HP18, HP21, and HP22 have a single
21 Insect Proteases: Structural-Functional Outlook 463
clip domain, while HP12 and HP15 have two clip domains. Increased levels of HP2,
HP7, HP9, HP10, and HP12–HP22 are seen in case of bacterial infection. This indi-
cates the presence of a complex serine protease network in M. sexta haemolymph
which responds to bacterial infection and wounding [116]. Other than immune
response proteases, some chitin-binding proteases are also present in the haemo-
lymph. In mosquitoes, a modular serine protease Sp22D (serine protease from chro-
mosomal division 22D) is expressed in various tissues with the highest expression
in haemocytes and is terminally secreted in the haemolymph [63], during early and
late embryonic development. It has a trypsin-like domain linked to a low-density
lipoprotein receptor-like domain (LDLr), scavenger receptor cysteine-rich (SRCR)
domain, mucin-like repeats and two chitin-binding domains (CBDs). Sp22D is
expressed in early and late embryonic development. It exhibits slow expression in
larval development, high expression during pupal development and moderate
expression in adults. This protein is spatially expressed, showing abundance in the
head, thorax, and abdomen. It is expressed in haemocytes and shows high chitin-
binding activity. It has been noted that exposure of chitin to haemolymph results in
mechanical disruption causing tissue remodelling during development. In pupae,
chitin is in direct contact with haemolymph, which causes the degradation of tra-
cheal epithelium and larval hypodermis. Chitin-binding activity of Sp22D and its
high expression during pupal development suggests that the primary role of Sp22D
is to detect the exposed chitin and then trigger the appropriate physiological
response.
After the completion of developmental cascade, there are a series of growth
cycles, which are primarily driven by physiological processes like feeding, diges-
tion, etc. Proteases involved in digestion are well known and characterized and will
be discussed in the next section in greater detail.
21.5.1 Trypsins
Trypsins are ubiquitous digestive proteases that cleave peptide bonds at the carboxyl-
terminal of basic amino acids, displaying a preference for Arg over Lys. In insects,
both anionic and cationic trypsins are found. Anionic trypsins were first reported in
the larvae of Tineola bisselliella [117]. From midgut extracts of T. molitor and
Locusta migratoria, three anionic and one cationic trypsinogens have been
464 S. Jagdale et al.
identified [118]. In highly evolved insects, trypsin subsites are more hydrophobic
[119], the binding strength of which varies in different insects.
On the basis of sequence conservation, trypsin-like enzymes are characterized by
conserved N-terminal residues – IVGG, a conserved catalytic triad of serine pro-
teinase (His57, Asp102, and Ser195) and Asp189. The active form of trypsin con-
tains single polypeptide chain which forms two β-barrels connected by a disordered
loop which contains the catalytic residues [120]. The catalytic mechanism of trypsin
has been reported and is well characterized.
Variation in sequences and the number of trypsin genes harbour the key molecu-
lar mechanisms of differential digestive activity in insects. Various insects have pool
serine protease, which usually exists as an array of diverse protease isoforms. In
many lepidopterans and hemipterans, digestive flexibility has been recorded to be
related to development, food content, and growth [2]. While adapting to specific
diets, multiple mutations in structural genes, regulatory genes and adaptive expres-
sion patterns contribute to the organism’s fitness. Variation and positive selection of
trypsin genes from multigene families lead to new gene functions along with new
tissue, development and environment-specific regulatory regimes [2, 64]. This
results in enhanced physiological flexibility and an improved matching of digestion
and heterogeneous food environment with the developmental changes in an insect’s
nutritional needs [2, 64].
21.5.2 Chymotrypsins
21.5.3 Elastases
described in many other insects. Many of the heteropteran insects are zoophytopha-
gous. The bug, Lygus hesperus which is a notorious crop pest and a capable predator
for a variety of insects, shows the presence of elastase in its salivary gland and gut.
The average elastase activity of gut elastase, however, is only 18–25% of the activ-
ity of salivary gland elastase. Elastase production is induced in response to the pres-
ence of specific food components, i.e. elastin which is present in specific plants and
is also a key component of the extracellular matrix of various insects on which the
L. hesperus feeds [124]. More research needs to be carried out in order to ascertain
the exact role elastases play in other insect species.
Cysteine proteases are found in the insect midgut in heteropterans and coleopterans.
Their widespread occurrence in the heteropteran midgut is probably attributed to
the loss of serine proteases after adaptation to sap feeding. In coleopterans, insects
belonging to the infra-order Cucujiformia show the presence of cysteine proteases,
as they are adapted to feed on seeds rich in naturally occurring trypsin inhibitors.
Intestinal cysteine proteases are endopeptidases. Cath L is a true endopeptidase
that specifically cleaves peptide bonds that have hydrophobic amino acid residues.
Most of the insect midgut cysteine proteases are Cath L-like enzymes. These prote-
ases have been isolated from Diabrotica virgifera, Acyrthosiphon pisum, and T.
molitor. They have pH optima of 5–6 and a mass of 20–40 kD. Their zymogens
possess an N-terminal peptide that is cleaved to activate the enzyme, along with the
catalytic triad (Cys25, His169, and Asn175) and the ERFNIN motif [120, 65].
Though these enzymes are very important in insect digestion processes, rela-
tively less information is available about them.
Aspartic proteases in insects were first reported in 1955 by Greenberg and Paretsky
in Musca domestica. Aspartic proteases are Cath D-like enzymes that are present in
the midgut of insects with acidic pH optima in the range 2.5–3.5 and cleave internal
peptide bonds in proteins. In M. domestica, three Cath D-like (CAD) enzymes are
expressed. CAD1 is expressed throughout the body, whereas CAD2 and CAD3 are
expressed only in the midgut of the insect. They all show the presence of catalytic
Asp33 and Asp229 [125]. The significance of acidic proteases in Diptera stems
from the larvae ingesting food rich in bacteria, such as decaying organic matter.
Bacteria once ingested are killed in the middle midgut by the combined action of
low pH and acidic proteases. Aspartic proteases similar to Cath D are found in many
coleopterans and hemipterans [66].
466 S. Jagdale et al.
21.5.6 Aminopeptidases
21.5.7 Carboxypeptidases
21.5.8 Dipeptidases
Dipeptidases hydrolyze those dipeptides that are produced due to the action of ami-
nopeptidases. The first true dipeptidase was isolated from the midgut of a hemip-
teran R. prolixus [67]. In M. domestica, dipeptidases are only found in the microvillar
membrane and show substrate specificity to Gly-Leu [68]. In S. frugiperda, two
21 Insect Proteases: Structural-Functional Outlook 467
dipeptidases are found in the midgut cells which are specific to Gly-Leu [69]. In R.
americana, three dipeptidases are present [132]. Two of them are soluble while one
is membrane bound. Soluble dipeptidases hydrolyse Gly-Leu and Pro-Gly but show
different efficiencies for the two dipeptides. The substrate specificity of membrane-
bound dipeptidase in still unknown, leaving dipeptidases to be the least studied
insect proteases, providing scope for further research.
It is evident that the insect proteases are involved in most of the cellular, physiologi-
cal and biochemical processes. The role of seminal and egg proteases in the repro-
ductive processes is very crucial; however, detailed structural studies need to be
done for functional understanding of these proteases. Seminal and egg proteases
show interdependent functionality; thus, high-throughput analysis at gene and pro-
tein level can provide comprehensive understanding of the co-evolution of these
proteases. The physiological and biochemical role of cathepsins and caspases in the
development processes is well studied. Although, due to limited structural informa-
tion on insect cathepsins and caspases, it is difficult to understand their mechanistic
action as activator or effectors. Insect metalloproteases show similarity across the
invertebrates as well as the vertebrates. The role of metalloproteases in the CNS
development can provide a framework for the study of these proteins across differ-
ent model systems. The digestive proteases show high variability in different insects
on basis of diet content, development and stress level. These enzymes are very well
studied at biochemical level; still significant structural and molecular information
are unavailable. Thus, detailed understanding of insect proteases at molecular level
can be utilized to provide new arena for basic research and also to be applicable in
the insect control field. The insect proteases can be used as new targets for develop-
ing effective and sustainable insect control strategies.
Acknowledgements Funding under the Divisional Research and Development Program (DRDP)
to the Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune,
India, is greatly acknowledged. Dr. Sneha acknowledges the Royal Society, UK, for its financial
support under the Newton International Fellowship scheme. Editorial assistance by Mr. Aniruddha
Agnihotri and Ms. Shriya Lele is highly acknowledged. The authors declare no conflict of
interest.
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Protease-Antiprotease Interactions:
An Overview of the Process from an 22
“In Silico” Perspective
Angshuman Bagchi
Abstract
Most if not all of the cellular processes involve protein-protein interactions
(PPIs). The detailed information of the amino acid residues involved in PPIs
may, therefore, be used in many important aspects like drug development, eluci-
dation of molecular pathways, generation of protein mimetic, understanding of
disease mechanisms, and development of docking methodologies to build struc-
tural models of protein complexes. Among the different physiological PPIs,
protease-antiprotease interactions play a significant role. An imbalance between
proteases and antiproteases is involved in many pathogenic reactions. This spe-
cial class of PPI, therefore, needs a thorough scrutiny. There are different PPIs
determining experimental tools. However, these tools are time-consuming and
expensive. In response to these difficulties, a number of bioinformatic software
tool have been developed. The algorithms are meant for prediction of three-
dimensional structures of proteins as well as protein complexes. The structure
prediction methods involve homology modeling, threading, and ab initio model-
ing. These methods have nearly 75%–80% overall accuracies. The other method
is molecular docking which is meant to generate the three-dimensional confor-
mations of protein complexes. The docking methods can broadly be classified as
rigid body docking and flexible docking. In this chapter, the different aspects of
experimental and computational modeling and docking strategies will be dis-
cussed. The basic terminologies will be revisited. This chapter is aimed at pro-
viding a firsthand knowledge on protein interaction methods using
protease-antiprotease interactions as an example.
A. Bagchi (*)
Department of Biochemistry & Biophysics, University of Kalyani,
Kalyani 741235, West Bengal, India
e-mail: angshu@klyuniv.ac.in; angshuman_bagchi@yahoo.com
Keywords
Protease • Antiprotease • PPI • Bioinformatics
22.1 Introduction
In the biological systems, almost all the biochemical reactions are the outcomes of
different forms of protein-protein interactions (PPIs). Proteins not only react among
themselves but also with other biomolecules like nucleic acids, organic or inorganic
cofactors, etc. [1–10]. However, one of such PPIs having a huge physiological sig-
nificance is protease-antiprotease interactions. Such interactions are at the heart of
many different diseases like chronic neutrophilic lung disease [11–20]. It has there-
fore become essential to physiologists to have a good understanding of PPIs. Thus,
various experimental and computational approaches have constantly being invented
by different scientific groups to predict the PPIs. The PPI identification technologies
have various degrees of accuracies. The experimental tools to study PPIs include
X-ray crystallography, nuclear magnetic resonance imaging, electron microscopy,
microarray analysis, co-immunoprecipitation techniques, etc. These techniques are
fairly accurate and produce good results. But the major drawbacks of these tech-
niques are cost and time. The failure rates are quite high too for these techniques
[20–30]. In order to have an alternative strategy to identify PPIs, different computa-
tional algorithms have constantly been being proposed. These computational tech-
niques have varying degrees and accuracies. These computational tools though not
as accurate as the experimental tools come up with fairly good predictive models of
PPIs. The computational approaches may, therefore, be considered to be the start
point of PPI prediction methodologies [31–40]. The present chapter is aimed to give
a firsthand knowledge of different computational PPI prediction methodologies tak-
ing protease-antiprotease interactions as a reference. However, before going into the
technical details of the PPI prediction methods, the basic definitions need to be
revisited.
22.2.1 Protease
• Serine proteases – serine proteases have a functional serine residue and cleave a
peptide linkage with the side chain of the serine.
• Cysteine proteases – cysteine proteases have a functional cysteine residue and
cleave a peptide linkage with the side chain of the histidine.
• Threonine proteases – threonine proteases have a functional threonine residue
and cleave a peptide linkage with the side chain of the threonine.
• Aspartic proteases – aspartic proteases have a functional aspartate residue and
cleave a peptide linkage with the side chain of the aspartate.
• Glutamic proteases – glutamic proteases have a functional glutamate residue and
cleave a peptide linkage with the side chain of the glutamate
• Metalloproteases – generally these types of proteases function using a metal ion-
like zinc.
• Asparagine peptide lyases – such proteases use an asparagine to perform and do
not require water for the hydrolysis of the peptide linkages [44, 45].
22.2.2 Antiprotease
These are inhibitors of proteases. The protease inhibitors can be another protein or
any other molecule that can inhibit the function of a protease. In physiology, a pro-
tease inhibitor is often used interchangeably with the enzyme alpha-1 antitrypsin
(A1AT). This protease inhibitor is often found to be associated with the disease
alpha-1 antitrypsin deficiency [41–45].
PPI interface refers to the area between the two protein chains. If the two protein
chains have the same amino acid compositions, the interface is called homomeric
interface otherwise heteromeric interface. The PPI interface has the following
characteristics:
• Surface area of interface: For heterodimer, the surface area is generally around
600Å2. For a homodimer, it is even larger.
• Shape of the PPI interface: In general, the PPI interface is nearly flat. A PPI inter-
face has two separate zones, viz., the core which is buried in the interface and the
rim which is solvent accessible.
• Composition of amino acids at the PPI interface: In general, it is observed that
PPI interface has an abundance of aromatic amino acid residues and Arg.
However, Cys is not generally found at the PPI interfaces.
• Secondary structural distribution at the PPI interface: Generally a PPI interface
is made up of beta sheet regions [46–55].
478 A. Bagchi
The PPIs can broadly be classified into several different classes based on the nature
of the interacting partners, stability of the PPI complexes, the life-span of the inter-
actions between the protein partners, and the nature of the PPI interface between the
proteins.
• Nature of interacting protein partners: If the interacting protein partners have the
same amino acid compositions, they form homo-oligomers, with structural sym-
metry. On the other hand, nonidentical protein partners form hetero-oligomers.
Hemoglobin is a homo-tetramer and a protease-antiprotease complex is a hetero-
mer [46–50].
• Stability of interacting protein complexes: If the individual protein partners
forming the PPI complex cannot exist in free state and are stable only in multi-
meric association, they are called obligate oligomers (homo-obligomers and/or
hetero-obligomers), like the Arc repressor dimer where dimerization is essential
for DNA binding. On the other hand, when the protein partners can exist in free
states on their own, they are called non-obligate partners like antigen-antibody
complex [46–52].
• Lifetime of PPI: When an association between the protein partners is highly
stable and needs external agencies to break them, they are called permanent com-
plexes. Hetero-trimeric G protein (Gα, Gβγ, and GDP) forms this type of PPI. In
contrast, the interacting partners of sperm lysin, a homodimer, exist in a dynamic
equilibrium consisting of association and dissociation of oligomeric forms. This
type of PPI is named as transient complex [46–55].
• Nature of the interaction interface: When the individual protein partners in a PPI
use the same interacting interface to join each other, they are called isologous
complexes. On the other hand, in heterologous assembly, the individual protein
partners in a PPI complex use different interfaces to form PPI without any closed
symmetry [46–55].
In order to interact, two protein molecules must be in close proximity. There are
mainly non-covalent forces responsible for the PPIs. However, the only covalent
binding interaction is disulfide linkage. The most important non-covalent interac-
tion found in PPIs is the hydrogen bonding between the polar atoms in proteins. The
hydrogen bonding involves both the main and side chain atoms of the amino acids
in proteins. The second type of non-covalent interaction leading to the formation of
PPIs is the ion pair formation. This occurs mainly between the side chains of an
acidic amino acid with that of the basic amino acid. There are other interactions like
stacking interactions, between the nonpolar hydrophobic side chains of the amino
22 Protease-Antiprotease Interactions: An Overview of the Process… 479
acids in proteins, and cation-pi interactions, between the aromatic side chains of
Phe, Tyr, and Trp with the positively charged side chains of Lys and Arg. However,
the binding interactions are specific for a protein complex. The biomolecular mech-
anisms of protease-antiprotease interactions follow some specific rules. The prote-
ase inhibitors are produced by different organisms to counteract the exogenous
proteases. As mentioned earlier, there are several classes of protease inhibitors.
Among the different protease inhibitors, the serine proteases are the best studied
ones. They are mostly 379–390 amino acid residue long proteins. These proteases
generally bind adjacent to the S-H-D complex present in the active site regions of
the proteases [46–56].
However, there is another class of protease inhibitor called the high molecular
weight macroglobulins. The characteristic of such inhibitors is that these inhibitors
can exhibit their functionalities irrespective of the catalytic mechanisms of the pro-
teases. These protease inhibitors exhibit their effects by forming covalent bonds
with the proteases. This covalent interaction would lead to some kind of conforma-
tional changes in the inhibitors, and the inhibitors can entrap the proteases. This
type of binding interactions between the protease inhibitors with proteases would
leave active site of the protease free to interact with the low molecular weight sub-
strates but not with high molecular weight substrates [46–56].
It is an already established fact that more than 80% of proteins function in associa-
tion rather in isolation. Therefore, studying and understanding of PPI are becoming
more and more relevant for fields like system biology, molecular medicine, etc. The
study of PPI is required to understand the molecular mechanisms behind mutations
which lead to disease onset. PPI detection methodologies can broadly be classified
as experimental and computational. As mentioned earlier in this chapter, experi-
mental tools give authentic results, but they are often very time-consuming and
expensive. The failure rates are also very high for experimental PPI prediction
methods. On the other hand, in order to have a firsthand knowledge about PPIs for
which experimental findings are not yet available, computational tools are devel-
oped. Thus, it has become a common practice to perform computational analyses of
PPIs before going into the experiments. In this chapter, the various aspects of PPI
predictions using computational tools are discussed [57–71].
The various computational algorithms for prediction of PPI can broadly be clas-
sified mainly into the following categories:
These methods are dependent on a function derived from the amino acid residues of
the protein chain under investigation. The different kinds of information collected
involve the details of secondary structure, sequence conservation, solvent accessi-
bility, physicochemical characteristics of the amino acids, etc. These different sets
of information are used to build a training model which has information about the
PPI and non-PPI amino acid residues. The training model may then be used to
detect the nature of a user-defined input. The possible sources of the information are
the Protein Data Bank (PDB) and literature mining. This method is used in support
vector machines (SVMs), random forests, etc. The method is heavily dependent on
the accuracy of the training dataset. And depending on the nature of the training
dataset, the method can produce a result with around 80% accuracy. However, the
most difficult problem is to find a suitable negative dataset [57–71].
The method uses the information of known protein complexes and then builds a
model from such information. The method uses sequence conservation data from
the protein complexes to first build the model. Then structural information like sec-
ondary structures, solvent accessibility, etc. are incorporated into the model. This
method is by far the most accurate method. However, the severe drawback of the
method is the presence of less number of protein complexes [57–71].
22 Protease-Antiprotease Interactions: An Overview of the Process… 481
This method is somewhat similar to the previous method that the method uses infor-
mation of characteristic sequence motifs in proteins. Each motif has a special affin-
ity toward binding another specific motif. The method uses this information to
search for interacting protein partners. The method uses a log odd score which is
calculated as log2(Pij/PiPj), where Pij is the observed frequency of motifs i and j
occurring together in one protein pair and Pi and Pj are the background frequencies
of motifs i and j in the data. Those interactions are considered positive for which the
log odd score is positive [57–71].
Acknowledgment The author would like to acknowledge the help rendered by the DBT-
sponsored Bioinformatics Infrastructural Facility of the University of Kalyani. The author would
also like to thank the Department of Biotechnology (DBT, India) for the financial support (SAN
No. 102/IFD/SAN/1824/2015-2016). The author is grateful to the Virologie et Immunologie
Moléculaires, INRA. UR892, Domaine de Vilvert 78352 Jouy-en-Josas, France, for the infrastruc-
tural support. The infrastructural help from the Department of Biochemistry and Biophysics,
University of Kalyani, is duly acknowledged.
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Snake Venom Proteinases as Toxins
and Tools 23
K.N. Suvilesh, A.N. Nanjaraj Urs, M.N. Savitha,
M.D. Milan Gowda, and B.S. Vishwanath
Abstract
Proteinases from snake venoms have long been fascinating targets due to their
structural, functional, and domain architectural diversities. Depending on these
differences, snake venom proteinases are broadly classified as snake venom
serine proteinases (SVSPs) and snake venom metalloproteinases (SVMPs).
Unlike SVSPs, additional domains along with catalytic domain present in
SVMPs are responsible for the subclassification. Non-catalytic domains of
SVMPs direct catalytic domain to site-specific target and thereby assist to
amplify the toxicities associated. The presence of additional domains along
with catalytic domain renders SVMPs more toxic than SVSPs. Though non-
catalytic domains function to facilitate the site-specific action of SVMPs, cata-
lytic domain with the metal ion zinc in the active site is critical in eliciting the
toxic action. Despite having a lot of reports regarding the toxic action of SVMPs
and SVSPs, they prove to be promising tools when studied individually. In
many cases their isolation and characterization have led to pharmacologically
active drugs or research/diagnostic tool. This chapter initially describes the
SVMP-induced local tissue damage such as hemorrhage and its neutralization
by employing a novel strategy; zinc specific chelation therapy. Secondly, venom
proteinase-induced systemic alterations such as perturbations in the comple-
ment and hemostatic system along with their applications as tools in the similar
area are discussed. Finally, diagnostic applications of both SVSPs and SVMPs
in coagulation laboratories and also their use in the identification of the snake
species responsible for bite are discussed.
K.N. Suvilesh • A.N. Nanjaraj Urs • M.N. Savitha • M.D. Milan Gowda • B.S. Vishwanath (*)
Department of Studies in Biochemistry, University of Mysore,
Manasagangotri, Mysore 570 006, Karnataka, India
e-mail: vishmy@yahoo.co.uk
Keywords
Chelators • Coagulation cascade • Complement system • Diagnostic tools • Local
tissue damage • Proteinases
Abbreviations
BM Basement membrane
CS Complement system
EC Endothelial cells
GPIb Glycoprotein Ib
SVMPs Snake venom metalloproteinases
SVSPs Snake venom serine proteinases
SVTL snake venom thrombin-like enzymes
vWF von Willebrand factor
α2β1 alpha2beta1
23.1 Introduction
Snake venoms have motivated interests and curiosity in many aspects of research
including pharmacy and medicine since ancient times. The endeavor for better
understanding of snake venom components and their effects extrapolated to application
has drawn commercial attention too [1]. Fundamentally snake venom is produced
by the venom gland and secreted to capture, kill, and digest the prey, which is executed
by the complex pool of proteins (90% of the dry weight) present [2, 3]. Further, the
protein composition of the venom can be broadly classified as enzymatic and non-
enzymatic (Fig. 23.1); among these, proteins with enzymatic activity are thought to
be the principal component in killing and digesting the prey [4].
In spite of the existing structural and functional differences among the myriad
blends of enzymes present in snake venom, they all are secretory in nature and
follow typical eukaryotic secretory protein synthesis mechanism involving mRNA,
ribosomes, endoplasmic reticulum, and Golgi apparatus [10]. Before secretion,
individual enzymes of snake venom undergo variable posttranslational modifica-
tions (PTMs), and proteinases being the high molecular weight toxins show the
higher variability in terms of structure, function, and PTMs [11]. This variability
makes snake venom proteinase a fascinating target, and to date more than 85
sequences of venom proteinases are available at UniProt database.
Further, proteinases present in snake venoms by themselves are diverse collection
of proteins with a broad array of molecular weights ranging between 15 and 380 kDa
and are generally classified as serine and metalloproteinases [12, 13]. Snake venom
serine proteinases (SVSPs), like other venom enzymes, are synthesized and secreted
23 Snake Venom Proteinases as Toxins and Tools 487
Fig. 23.1 General classification of snake venom proteins/toxins. NGF-nerve growth factor and
VEGF-vascular endothelial growth factor [5–9]
with simple PTMs [14], whereas snake venom metalloproteinases (SVMPs) are
synthesized as large precursors with different domains and undergo extensive PTMs.
SVMPs are synthesized in the secretory cells of the venom gland as a nascent
structure comprising constantly pre, pro, M (Metalloproteinase) with or without
D/DL (Disintegrin/Disintegrin-Like), and C (Cysteine-rich) domains in which pre
is a signal peptide through which the protein gets transported to the endoplasmic
reticulum where it undergoes sequential posttranslational processing like removal
of signal peptide followed by disulfide bond formation, glycosylation, and in some
specific circumstances dimerization. Final proteolytic processing of pro-forms of
SVMPs occurs in the Golgi apparatus to convert them as active proteins and thereby
get secreted in vesicles to the lumen. This proteolytic processing along with PTMs
results in different final products which leads to the classification of SVMPs
(Fig. 23.2).
Based on the presence or absence of D/DL, C domains as observed through
mRNA transcripts, or cDNA and proteins isolated from the venom, SVMPs are
categorized into three classes: P-I to P-III, where P-II and P-III are further divided
into subclasses. P-I SVMPs being the simplest class are composed of only M
domain; P-II SVMPs in continuation to M domain contain a D domain to the
carboxyl terminal which is in most cases posttranslationally and proteolytically
processed; based on which P-II class is further divided into P-IIa to P-IIe subclasses.
Further, P-III class along with M domain, instead of D, contains DL domain
followed by C domain. Based on posttranslational processing such as dimerization,
proteolysis, and attachment of C-type lectin-like (CTL) domain, P-III class is fur-
ther subdivided into P-IIIa to PIII-d. Presence of additional CTL domain along with
M, DL, and C domains is evident in PIII-d class of SVMPs. Earlier, even lectin-like
488 K.N. Suvilesh et al.
Fig. 23.2 Schematic of SVMPs classification. SVMPs are classified into three major classes and
subclasses depending on their domain architecture. A dashed arrowhead on the left-hand side
points the cleavage site of nascent protein. The final processed products are depicted on the right-
hand side
23 Snake Venom Proteinases as Toxins and Tools 489
domains were thought to be coded along with the other three domains, and SVMPs
with this domain were considered under separate class P-IV. To date, no P-IV
mRNA transcript has been observed and probably represents another PTM of the
P-III structure thereby merged into P-III class as P-IIId [3, 11, 15, 16]. Table 23.1
lists the examples of unprocessed proteinase(s) and their processed domains in each
different class of SVMPs.
Further, SVMPs as symbolized by their name are metal-/zinc-dependent proteo-
lytic enzymes and contain a consensus zinc-binding sequence HEXXHXXGXXH
(single-letter code of amino acids; X, any residue) in M domain [30].
Commonly associated systemic toxicities with SVMPs and SVSPs are fibrin(ogen)
olytic, platelet aggregation or inhibition, and blood coagulation factor activation/deple-
tion leading to consumption coagulopathy and intravascular bleeding. Few SVSPs and
SVMPs attack the complement system leading to rapid generation of excess anaphyla-
toxins [31]. However, these systemic toxicities can be effectively managed by the
administration of commercially available anti-snake venom (ASV) [32].
Additional to the above said complications, majority of SVMPs induces local
toxicities such as hemorrhage by degrading the components of the basement mem-
brane (BM) underlying the endothelial cells (EC) of the capillaries allowing the
circulating blood to enter the extracellular space which in turn leads to hypoxia,
eventually resulting in tissue necrosis [33], and administration of ASV generally
fails to neutralize this pathological effect [32].
In general, additional domains (D, DL, C, and CTL) of SVMPs have been shown
to direct the M domain (catalytic subunit) to specific substrates at relevant targets by
facilitating their backbone flexibility in specific surface regions. For instance, P-II
and P-III SVMPs have been reported to preferentially bind to the microvasculature
leading to co-localization with type IV collagen and perlecan thereby causing
intense local tissue damage [34–36]. In contrast, P-I SVMPs, devoid of these addi-
tional domains, show a widespread localization in the extracellular matrix and are
considered to be less toxic than P-II or P-III SVMPs [37]. In continuation, due to the
presence of D, DL, C, and CTL domains in P-II and P-III SVMPs, they escape the
trap mechanism adopted by α2 macroglobulin (a plasma proteinase inhibitor) to
inhibit endogenous proteinases, whereas P-I SVMPs are readily inhibited by α2
macroglobulin [38, 39].
In the past, different approaches such as immunohistochemistry of tissue sec-
tions, immunoblot from tissue homogenates, and proteomic analysis of exudates
collected in the vicinity of affected tissue have provided novel clues for understand-
ing the pathogenesis of SVMP-induced hemorrhage [40–42]. Some observations
demonstrate the disruption and formation of gaps between the EC at the site of
envenomation suggesting direct catalytic activity of SVMPs in hydrolyzing the key
substrates (laminin, nidogen, perlecan, type IV collagen) at the BM surrounding EC
in capillaries/microvessel thereby damaging the subendothelial structural integrity
Table 23.1 Examples for proteinases and their processed domains
490
23.3 A
New Approach Toward the Neutralization of Local
Tissue Damage Induced by SVMPs: Chelating Agents
The inability of ASV therapy to neutralize the local tissue damage induced by
SVMPs has urged scientists and medical fraternities to develop an alternate therapy.
Despite the involvement of non-catalytic domains in the above mentioned mecha-
nisms underlying extensive hemorrhage caused by SVMPs, the role of catalytic
domain is a must, and any SVMP with truncated catalytic domain will not cause
hemorrhage or severe local tissue damage. Thus, inhibition of catalytically active M
domain is a sensible and vital strategy for limiting the progression of local tissue
damage induced by SVMPs. As M domain requires zinc (Zn2+) for its catalytic
activity, use of chelation therapy (employing chelating agents) will be a better
approach to counteract the debilitating action of SVMPs.
In view of these structural and functional aspects of SVMPs, our research group
used pharmacologically approved chelating agents such as ethylenediaminetet-
raacetic acid (EDTA) and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-
tetraacetic acid (EGTA) to study the inhibitory effects of these compounds against
Trimeresurus malabaricus venom-induced local tissue damage [50]. Later on, other
492 K.N. Suvilesh et al.
report also came up with respect to chelation therapy against snake venom-induced
hemorrhage and myotoxicity [33]. The aforesaid divalent metal ion chelators were
shown to be highly effective in neutralizing the activity of SVMPs in vitro. However,
their nonspecific binding with physiologically vital divalent metal ions, particularly
calcium (Ca2+), poses an obstacle for their in vivo applications or pharmacological
use [51]. Thus, pharmacological use of specific Zn2+ chelators rather than nonspe-
cific divalent metal chelators might be effective in the management of local toxicity
as they can serve as adjunctive therapeutic molecules to aid ASV therapy by limiting
local tissue destruction in treatment mode [52].
Keeping the above mentioned aspects as background, recently our research
group designed a study using three Zn2+-specific chelators TPEN (N,N,N′,N′-
tetrakis (2-pyridylmethyl) ethane-1,2-diamine), DTPA (diethylene triamine penta-
acetic acid), and TTD (tetraethyl thiuram disulfide) in which their inhibitory
potential was evaluated against Echis carinatus venom metalloproteinase(s) in vitro.
Further, the results obtained were extended to the animal model to evaluate the
inhibitory potential, where upon challenging experiments, the selected chelators
were very efficient in inhibiting the Echis carinatus venom-induced local tissue
damage, and unlike EDTA and EGTA, they were nontoxic at minimal dose required
for inhibition studies [52].
All the abovementioned toxic symptoms associated with snake venom protein-
ases are cumulative effects, observable when they act together. However, individu-
ally they can be exploited to study many different unknown aspects of Biochemistry
and Pharmacology owing to their stringent specific action.
Different biochemical processes occurring in the body under apt regulation to main-
tain homeostasis are reported to be disturbed upon envenomation by snake venom
proteinases, and one such susceptible area is the complement system (CS) [53].
Both SVMPs and SVSPs have been shown to affect complement pathway leading
to inappropriate complement inhibition or activation which in most cases leads to
complement consumption [54, 55]. Further, CS is a central part of both innate and
adaptive immune responses and mediates a series of functions ranging from the
modulation of local inflammatory responses to the promotion of phagocytosis and
the lysis of pathogens [56]. The human CS is composed of more than 40 plasma and
cell surface proteins that participate in balanced activation and regulation of it [57].
The complement activation is triggered by a series of proteolytic activities arbitrated
by serine proteinases similar to blood coagulation cascade. The CS consists of and
can be activated by any of the three activation pathways, i.e., classical, alternative,
and mannose-binding lectin (MBL) pathways, which merge at the proteolytic
activation step of C3 resulting in the formation of C3 convertases, a central compo-
nent of the system. C3 convertase will cleave C3 to yield C3a and C3b; thus,
494 K.N. Suvilesh et al.
Cobrin Naja Metallo 67.66 C3 P-III Cleaves human C3 to form C3o and C3p which are different [64, 65]
siamensis from physiologically derived fragments C3b and C3a
C-SVMP Bothrops Metallo 23.14 C3, C4, P-I Activates complement pathway by cleaving the α chain [54]
pirajai and C5 of C3, C4, and C5
Flavoxobin Trimeresurus Serine 26.7 C3 NA Cleaves human C3 at the same site where C3 convertase [55]
flavoviridis cleaves and acts as a novel heterologous C3 convertase
F5 Crotalus Metallo 27 C3 ND C3 product generated was able to mediate cell lysis [66, 67]
scutulatus
M5 Crotalus Metallo 25 C2, C3, M5 completely hydrolyzed C2, C3, and C4. Hydrolysis of [68]
molossus and C4 C3 by M5 generated a fragment of ≈ 130 kDa, indicating
a different cleavage site from that of C3 convertases
Oxiagin Naja oxiana Metallo 49.8 C4b2a P-III Only reprolysin-lacking proteinase activity inhibits [69]
complex complement pathway through its lectin-like domain
rFII Agkistrodon Metallo 26.71 C5, C6, P-III Inhibition of MAC formation [70, 71]
acutus and C9
495
Notes: NA not applicable and ND not determined. Molecular weight of proteinases are retrieved from UniProt database
Fig. 23.4 Schematic of complement pathway and snake venom proteinases acting on differ-
ent components of complement system: Complement system (CS) can be activated by any of the
three pathways, viz., classical, alternative, and MBL pathway. Classical pathway involves the par-
ticipation of complement components C1, C2, and C4 and is triggered by the activation of C1.
Activated C1 in turn activates C4 and C2 resulting in the formation of C4b, C4a, C2b, and C2a of
which C4b and C2a bind to form the classical pathway C3 convertase (C4b2a). MBL pathway is
activated by MASPs (MBL-associated serine proteinases) the MASP-1 and MASP-2, which then
activate C4 and C2, to form the C3 convertase, C4b2a complex. Alternative pathway is just an
amplification of classical and MBL pathways in which traces of C3b present will bind to factor B
to form C3B. Later, factor D will activate B in C3B complex resulting in the removal of Ba and
leading to the formation of C3bBb, alternative pathway C3 convertase. C3 convertases formed will
activate C3 to yield C3b plus C3a, of which C3b binds to C3 convertases C4b2a and C3bBb to
form C5 convertases C4b2aC3b and C3bBbC3b, respectively. C5 convertases will act on C5 to
release C5b and C5a, of which C5b gets attached to the target surface and recruits C6, C7, C8, and
multiples of C9 to form MAC which mediates the lytic pathway [72, 73]. The dashed arrows with
numbers pointing toward the complement protein(s) scattered all along the pathway indicate the
targets of snake venom proteinases, and the numbers are in alphabetical order: (1) atrase B, (2)
Bap-1, (3) cobrin, (4) C-SVMP, (5) flavoxobin, (6) F5, (7) M5, (8) oxiagin, and (9) rFII
23 Snake Venom Proteinases as Toxins and Tools 497
Fig. 23.5 Action of endogenous C3 convertase and cobrin on human complement C3 in
comparison with cobra venom factor (CVF). (a) C3 convertase (CC) cleaves C3 α chain between
residues 726 and 727 to acquiesce C3a and C3b. After the targeted function of C3b, excess C3b
produced will be degraded by factor I as a regulatory step. First by using two cleavage sites (des-
ignated here as I1 and I2), factor I cleaves between residues 1281 and 1282 and 1298 and 1299 to
remove C3f and yield iC3b. Later, using the third cleavage site (designated here as I3), factor I
cleaves between residues 932 and 933 to remove C3dg fragment and yield C3c. The released C3dg
fragment is further hydrolyzed by tryptic-like enzymes to produce C3g (yellow) and C3d (magenta)
[75]. (b) Cobrin cleaves complement C3 through its three cleavage sites (designated here as C1,
C2, and C3) between the residues 736 and 737, 967 and 968, and 1331 and 1332 to yield C3o, C3p,
and C3a-like fragments. (c) Speculated action of cobrin on pro-CVF is synthesized in the venom
gland before secretion to yield active CVF. N and C represent the amino terminus and carboxyl
terminus of the polypeptide chains, respectively. The numbers used represent the terminal amino
acid residues in the whole protein and cleavage products
Another major target for snake venom proteinases is the blood coagulation process
which includes both thrombosis and hemostasis [77, 78]. Blood coagulation is a
complex network involving cellular and protein compartments which can be broadly
divided as primary and secondary hemostasis [79]. When circulating platelets are
exposed to the BM proteins such as von Willebrand factor (vWF) and collagen at
the site of injury, interaction takes place between the receptor(s) on platelet mem-
brane and the exposed proteins to form platelet plug; the process is called as pri-
mary hemostasis or thrombosis [80].
Snake venom proteinases can activate or inhibit primary hemostasis which can
be accomplished by catalytic and non-catalytic mechanisms [81]. SVMPs using
their non-catalytic D, DL, C, and CTL domains inhibit platelet aggregation. The
non-catalytic domains bind to the receptors on the platelet membranes, thereby
blocking the interaction of platelets with the physiological ligands such as fibrino-
gen, vWF, thrombin, and collagen eventually resulting in platelet aggregation
inhibition [82–84]. Observations on platelet activation/inhibition mediated by snake
venom proteinases through catalytic mechanism rather than non-catalytic provide
insight into various aspects of thrombosis.
It is a well-known fact that binding of membrane glycoproteins GPIbα, GPVI,
and α2β1 to their ligands vWF and collagen, respectively, leads to platelet activation
and chain of downstream reactions such as phosphorylation of tyrosine residues,
secretion of aggregation inducers, and so on, which culminate at platelet aggrega-
tion [85]. Still there are certain unknown aspects underlying thrombosis, and these
gaps in fact can be filled by exploiting differential catalytic mechanisms of snake
venom proteinases on platelet aggregation.
Some SVMPs mediate platelet aggregation inhibition by catalytically destruct-
ing the platelet membrane glycoprotein(s) (e.g., GPIbα and GPVI) receptors or oth-
erwise hydrolyzing the ligands required for thrombosis (Table 23.3). To take a few
examples, Barnettlysin-I, a metalloproteinase isolated from the venom of Bothrops
barnetti, selectively inhibits collagen and vWF-induced platelet aggregation, while
thrombin-induced aggregation is not affected. Barnettlysin-I degrades high molecu-
lar weight vWF into low molecular weight fragments which lose the function to
aggregate platelets, and at the same time vWF A1 domain which contains the stretch
of amino acid to bind GPIb is also degraded. On the other hand, Barnettlysin-I
degrades the receptor GPIb into two fragments making it further unable to bind
vWF. Conversely, Barnettlysin-I inhibits collagen-induced platelet aggregation by
Table 23.3 SVMPs acting on platelet membrane receptors and ligands
Name Molecular Target receptors and
of SVMP Snake species Class weight in kDa ligands Remarks References
Acurhagin Deinagkistrodon P-III 68.54 Collagen and vWF Impairs collagen and ristocetin- [93]
acutus induced platelet aggregation by
cleaving collagen and vWF
Alborhagin Trimeresurus P-III ND GP VI Induces platelet aggregation by [92]
albolabris ectodomain shedding of GP VI
Barnettlysin-I Bothrops barnetti P-I 23.38 vWF, GPIb, and α2β1 Cleaves peptide bond between basic [86]
residues Lys 413-Leu 414 and Arg
696-Leu 697
Jararhagin Bothrops jararaca P-III 63.98 α2β1 and vWF Binds to α2 subunit to degrade β1 [87, 88]
chain
Kistomin Calloselasma P-I 25 GPVI, GPIb, and Differentially cleaves GPVI and [89, 94]
rhodostoma vWF GPIb
Mocarhagin Naja mossambica P-III 68.17 GPIb Removes a peptide with highly [90]
23 Snake Venom Proteinases as Toxins and Tools
Fig. 23.6 Action of SVMPs and SVSPS on different ligands and platelet membrane recep-
tors: Numbers with the dashed arrows pointing either ligand or receptor indicate action of particu-
lar SVMP toward it, and SVSPs with dashed arrow pointing protease-activated receptor (PAR)
symbolize the only target. Numbers used to represent the name of SVMPs in alphabetical order are
(1) acurhagin, (2) alborhagin, (3) Barnettlysin-I, (4) jararhagin, (5) kistomin, (6) mocarhagin, (7)
natrahagin, (8) kaouthiagin, and (9) triflamp. Abbreviations used: glycoprotein1b (GP1b), glyco-
protein VI (GPVI), α2β1 (integrin α2β1 or glycoprotein Ia/IIa complex)
PA-BJ, serine proteinase isolated from the venom of Bothrops jararaca, induces
platelet aggregation through activation of PAR1 and PAR4. The cleavage occurs at
Arg41-Ser42 (like thrombin cleavage), and Arg46-Asn47 a site different and extra
compared to thrombin [104]. Determination of site-specific cleavage of these SVSPs
in comparison to thrombin will potentially help to fill the gap in the existing knowl-
edge on PAR-directed platelet aggregation.
In continuation, secondary hemostasis is a process which occurs by the combined
action of intrinsic, extrinsic, and common pathways of blood coagulation involving
series of serine proteinases activating the successive one culminating in conversion
of prothrombin to thrombin which converts fibrinogen to fibrin to hold and stabilize
the platelet plug formed as a result of primary hemostasis [105]. Both SVMPs and
SVSPs disturb blood coagulation pathways by functioning as thrombin-like enzymes;
prothrombin activators; fibrino(geno)lytic enzymes; blood coagulation factor V, VII,
and X activators; protein C activators; and plasminogen activators [106].
Briefly, SVTLEs are SVSPs and, like thrombin, able to clot fibrinogen by cleav-
ing from the N-terminal end. Based on catalytic action, they are divided into three
groups: SVTLE-A cleaves off fibrinopeptide A from fibrinogen Aα chain, SVTLE-B
cleaves off fibrinopeptide B from Bβ, and SVTLE-AB cleaves off both fibrinopep-
tides A and B. Commonly, majority of SVTLEs cleave off either fibrinopeptide A or
B, but rarely both fibrinopeptides as thrombin [107].
502 K.N. Suvilesh et al.
As a result of acting at only one position, SVTLEs lead to the formation of loose
clots but not stable ones like thrombin. These exceptional functional features allow
their clinical use as defibrinogenating agents, for example, ancrod (Arvin®; from
Calloselasma rhodostoma) and batroxobin (Defibrase®; from Bothrops moojeni)
[31]. Further, both SVMPs and SVSPs are reported to be prothrombin activators and
are divided into four groups A, B, C and D based on the mechanism of activation
and cofactor requirement. Prothrombin activators belonging to group A and B are
SVMPs in which proteinases belonging to group A function without cofactor
requirement, while group B activators require Ca2+ as cofactor. Group A prothrom-
bin activators belong to either P-I or P-III (a-c) class SVMPs, whereas Group B
activators are P-IIId class SVMPs. Irrespective of the class, both group A and B
activators cleave the Arg320-Ile321 bond in human prothrombin to produce
meizothrombin, which is then converted to α thrombin (active thrombin) by autoly-
sis. Group C and D prothrombin activators are SVSPs in which group C activators
require Ca2+ and negatively charged phospholipids for activity, whereas group D
activators requires Ca2+, phospholipids, and activated blood coagulation factor V
(FVa) [108].
Apart from having thrombin-like and prothrombin-activating properties, both
SVMPs and SVSPs possess fibrino(geno)lytic activities, but in contrast to SVTLEs
and thrombin, these fibrinogenases cleave fibrinogen at abrupt sites preferentially
from C-terminal end producing truncated form which is no longer a functional
fibrinogen [109]. Venom proteinases which degrade fibrinogen may be classified as
either α or β chain fibrinogenases depending on the chain they degrade. Only one or
few venom proteinase(s) which activate blood coagulation factor V, VII, and X,
protein C, and plasminogen have been isolated and studied [110–115].
As discussed, upon envenomation SVSPs or SVMPs exert action individually or
in combination resulting in pathological conditions such as disseminated intravas-
cular coagulopathy (DIC), consumption coagulopathy, and excess bleeding eventu-
ally resulting in hypoxia and death of the victim. Nevertheless, on individual
consideration, these proteinases have application in coagulation laboratories and in
diagnosis of the snake species responsible for bite.
Evident from the above discussion, for almost every factor involved in blood coagu-
lation, there is a venom proteinase that can activate or inhibit it. Many of these
venom proteinase(s) are resistant to endogenous inhibitors, and this property extends
their application to diagnose problems pertaining to coagulation abnormalities in
hematology laboratories and also aid in identification of the snake species respon-
sible for bite. Batroxobin, a SVTLE, belongs to group A and is used in diagnosing
the problems related to dysfibrinogenemia or hypofibrinogenemia under the name
23 Snake Venom Proteinases as Toxins and Tools 503
Recent updates show the existence of more than 600 venomous snake species
around the world, and all of them belong to any of the Colubridae, Viperidae, or
Elapidae families [136]. In India more than 200 snakes have been identified, and
only 52 of them are reported to be venomous. Among the 52 venomous snakes, Naja
naja, Bungarus caeruleus, Daboia russelii, and Echis carinatus are accountable for
the majority of envenomations reported and are collectively termed as “BIG FOUR”
venomous snakes of India [137, 138]. Naja naja and Bungarus caeruleus belong to
Elapidae family, whereas Daboia russelii and Echis carinatus belong to Viperidae
family.
Snake bite is considered as an occupational health hazard, and survey reports
show an annual estimated death of 1300 to 50,000 in India [139, 140]. The only
available treatment strategy against snake bite in India is the administration of
Table 23.4 Snake venom proteinases as tools in diagnostics
Type of
Name proteinase Snake species Trade name Manufacturer Remarks References
Batroxobin Serine Bothrops atrox Pefakit® Pentapharm, SVTLE-A, used for the determination of fibrinogen [143]
Reptilase® Basel, polymerization disorders and other clinical conditions
time Switzerland connected with the last phase of coagulation
Ecarin Metallo Echis carinatus Ecarin® Pentapharm, Group A prothrombin activator, used in determination of [119]
Basel, prothrombin levels in patients undergoing anticoagulant
Switzerland therapy
Carinactivase Metallo Echis carinatus Not yet NA Group B prothrombin activator, measures only normal [144]
(CA-1) available in thrombin but not des-carboxy thrombin as evident with
market patients undergoing warfarin therapy
Textarin Serine Pseudonaja Textarin® Pentapharm, Group D prothrombin activator, used in combination with [124]
textilis Basel, Ecarin to detect delay in clotting time due to the presence
Switzerland of lupus anticoagulants (LA)
RVV-V Serine Daboia RVV-V® Pentapharm, RVV-V is used to destabilize and selectively inactivate FV [145]
23 Snake Venom Proteinases as Toxins and Tools
siamensis Basel, in plasma and thus to prepare a routine reagent for the FV
Switzerland determination
RVV-X Metallo Daboia RVV-X® Pentapharm, Used for the detection of FVII and FX deficiencies. Also [146, 147]
siamensis Basel, used to detect prolonged clotting time in LA
Switzerland
ACC-C Serine Agkistrodon Protac® Pentapharm, Determination of Leiden mutation in FV [148]
contortrix Basel,
Switzerland
Noscarin Serine Notechis Pefakit® Pentapharm, Group D prothrombin activator, used in combination with [130]
scutatus APC-R Basel, RVV-X and ACC-C to detect Leiden mutation in FV
Factor V Switzerland
Leiden
Note: NA not applicable
505
506 K.N. Suvilesh et al.
polyvalent ASV raised against the BIG FOUR venoms in equine. Administration of
ASV poses some challenges and risks such as anaphylactic shock and allergic
symptoms as secondary complications which may also lead to death [141]. Hospital
records also evidence the administrations of prophylactic and antihistamine drugs to
majority of inpatients to combat the secondary complications mounted by ASV
[142]. These complications can be minimized by administering species-specific
monovalent ASV and for which identification of snake species responsible for bite
is very critical.
As of now, Australia and Papua New Guinea are the only two countries which are
using monovalent ASV through the means of snake venom detection kits. These kits
aid in the detection of the antigen(s)/protein(s) of the species-specific venom pres-
ent in the blood or plasma of the victims based on the ELISA method, and depend-
ing on the test outcome, a clinician may decide to administer species-specific
monovalent ASV. However, recent reports have shown the cross-reactivity existing
between these monovalent ASV against different species which may also reflect
with snake venom detection kits. These cross-reactivities are attributed to the anti-
genic similarity shared by snake venom proteins [38, 74, 79, 149–151]. Hence,
there is a need for an alternative detection method other than ELISA or perhaps a
non-antigen-based method to identify the snake species responsible for bite.
Evident from the aforementioned aspects, hemostatic system comprising plenty
of proteins is a preferred target for snake venom enzymes. Immediately after enven-
omation, hemostatic system gets affected and undergoes plenty of observable
changes, and these changes show marked differences depending on the family of
snake which has offended [152, 153]. On these grounds, our research group designed
a study in which different groups of rats were injected with BIG FOUR venoms and
the plasma obtained thereafter was used for the assessment of common routine
coagulation parameters such as recalcification time, prothrombin time, activated
partial thromboplastin time, and coagulation factor assays including fibrinogen levels.
In all the tests, striking differences existed between the plasma obtained from
viperid and elapid venom-injected groups, and results were promising enough to
differentiate between viperid and elapid species. If these result are consistent with
the human subjects, bivalent ASV specific against two elapids or viperids can be
administered rather than polyvalent ASV which in turn brings down the load of
ASV thereby reducing the secondary complications [154].
Among the diverse classes of toxins present in snake venom, proteinases being high
molecular weight components exhibit structural and functional diversity. In addi-
tion, it has given a wide platform for toxinologists not only to explore their toxic
properties but also to extrapolate the same to their pharmacological applications.
Although different domains contribute to structure and functions of SVMPs, cata-
lytic domain is vital in inducing pathological burden. Targeting the catalytic domain
will prove invaluable in SVMP-induced pathologies. Thus, chelation therapy might
23 Snake Venom Proteinases as Toxins and Tools 507
be a vital strategy in mitigating the extensive tissue damage induced by these pro-
teinases. Further, both SVSPs and SVMPs are capable of inducing systemic altera-
tion by specifically cleaving/inhibiting/activating the components of complement
system and blood coagulation pathway. Despite the observed toxicities, differential
selectivity and specificity of these toxins toward their substrates can be used as tools
to explore and expand the existing knowledge. In addition, the ability of SVMPs
and SVSPs to act similarly as blood coagulation factors has extended their applica-
tion to coagulation laboratories. Further, potential of these proteinases to differen-
tially alter the coagulation cascade upon bite by different snake species may be
harvested to identify the snake responsible for bite.
Acknowledgments The authors thank University Grants Commission (UGC) for providing
research fellowship to carry out the work and thank Dr. Yariswamy, M. and Mr. Vikram Joshi for
the critical reading of the manuscript.
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The World of Proteases Across Microbes,
Insects, and Medicinal Trees 24
Ratnakar Chitte and Sushma Chaphalkar
Abstract
Various types of proteases are present in all living organisms, and they play
important roles in physiology. There is interest in protease types of enzymes
because of their vital applications in industries. Proteases have specific inhibitors
are used for control of larval insect targets. Microbes produce proteolytic–fibri-
nolytic enzymes, which play important roles in pathological clot lysis. Proteases
from microbes, insects, medicinal plants, and endophytes have been studied.
Antimicrobial peptides (AMPs) produced by medicinal plants, as well endo-
phytic bacteria, play important roles in control of plant pathogens. Medicinal
trees in the Nakshatra Garden produce proteases and peptides, each of which has
its own physiological role and warrants investigation for various applications.
Keywords
Microbial • Insect & medicinal proteases • Antimicrobial peptides (AMPs) •
Endophytes • Medicinal trees
24.1 Introduction
understanding of the unique cellular processes of Archaea and mechanisms for their
adaptation to extreme environments. Over half of all archaeal genes encode unique
proteins with unknown functions [1].
Methanogens have been identified in the hindguts of many terrestrial arthropods,
including millipedes, cockroaches, termites, and scarab beetles [2]. The strict anaer-
obic conditions required for methanogen survival in the human colon and adapta-
tion to specific local nutrient environments must also apply to other members of the
microflora.
It is assumed that Eukaryotes originated from Archaea. This has been supported
by phylogenetic analyses in which Eukaryotes were found to be linked with Archaea.
Though Archaea and Eukaryotes share many basic features at the molecular level;
the archaeal mobilome resembles the bacterial mobilome more closely than the
eukaryotic mobilome.
Archaea and bacteria are also quite similar at the genomic level. Both have small
circular genomes. Otherwise, the gene organization of Archaea is not similar to that
of bacteria and exhibits a lot of “eukaryotic features” at the molecular level [3–6].
Many archaeal operational systems—such as ATP production, protein secretion,
cell division, and protein modification process—use proteins homologous to those
of Eukaryotes rather than the bacterial system [7–9].
Archaea and Eukaryotes share many features in all aspects of their cellular phys-
iology. Moreover, Archaea and Eukaryotes share a more complex evolutionary rela-
tionship than has previously been understood.
To date, several scenarios have been proposed to explain the origins of Archaea
and Eukaryotes [10–12].
These evolutionary domains also reflect changes in the nature of biomolecules
for adaptation to physiological conditions.
Thermophilic bacteria produce multiple proteases, such as keratinase, metallo-
proteases, and serine endopeptidases. Fibrinolytic proteases play important roles in
disease and thrombosis. Likewise, many peptidases play important roles in strate-
gies against disease targets (24.1).
Some medicinal plants produce antimicrobial peptides (AMPs) as part of their own
defensive mechanisms. These peptides also have potential as therapeutic peptides.
The Nakshatra Garden consists of medicinal trees. These trees produce varieties
of biologically active compounds such as alkaloids, flavonoids, terpenoids, pep-
tides, and enzymes. The production of these bioactive molecules by plants has phys-
iological significance. Processes to extract these bioactive molecules have been
attempted. Significant activities of proteases have been detected. These medicinal
trees are associated with endophytic consortia. These endophytes produce impor-
tant metabolites, such as plant growth hormones and promoters, which are symbi-
otically helpful to the plants and vice versa. Characterization of endophytic bacteria
and plasmids has been carried out [46].
The proteases produced by the Nakshatra Garden’s medicinal trees need to be
screened for various applications, and these plants may produce enzymes suitable
for food and pharmaceutical uses.
Proteases have been extracted from medicinal plants such as Azadirachta indica
and Terminalia arjuna to be studied for their potential as antiviral targets, and ana-
lytical studies of proteases extracted from Azadirachta indica have been carried out
[47, 48].
24 The World of Proteases Across Microbes, Insects, and Medicinal Trees 523
There are many bioactive compounds to be isolated and identified using mass
spectroscopy and NMR techniques, and further work is needed to identify as yet
unknown compounds. From our laboratory, various in vitro activities of com-
pounds—such as antidiabetic, anti-inflammatory, anticancer, and antimicrobial
activities—have been reported, and some data are still unpublished.
There is a lot of diversity and variation in the specificity of proteases from
microbes, insects, and medicinal trees, and they could be useful as effective thera-
peutic agents. Depending on the specificity of their hydrolytic actions, proteases
could be used for various applications in the food, detergent, leather, and pharma-
ceutical industries.
Gene cloning technology is being widely used to design molecules for better
understanding of structures and functional relationships.
Proteases are physiologically essential for all living organisms. They have wide
diversity, depending on the sources from which they are obtained, such as microbes,
insects, and medicinal plants [49]. Proteases such as papain, bromelain, and kerati-
nases are proteases of plant origin.
Proteases have a variety of functions according to their applications. Proteases at
cellular levels contribute greatly to metabolic pathways. They produce cascade sys-
tems for hemostasis and inflammation for normal physiological regulation of cells.
Proteases are involved in the life cycles of disease-causing organisms and thus are
potential targets for development of therapeutic agents for deadly diseases such as
cancer, AIDS, and cardiovascular diseases [17]. Microbial proteases are used in the
treatment of many disorders, such as cancer, inflammation, cardiovascular disor-
ders, necrotic wounds, etc. [50].
Proteases are used in the pharmaceutical industry for preparation of medicines,
such as fibrinolytic proteases for use as clot lysis agents.
Proteases also have wide applications in the detergent and food industries, where
they are prepared in bulk quantities and used as crude preparations. Keratinolytic
enzymes are used for dehairing and degradation of feathers to generate amino acids,
and they could be used for animal feed preparation.
Proteases used in medicines are produced in small amounts and are required in
absolutely pure forms. Proteases also have widespread applications in laundry
detergents [51, 52].
Alkaline proteases are used in different brands of detergents for day-to-day uses.
They are also used for removal of stains such as milk and blood. Protease enzymes
remove body secretions and foods such as milk, egg, fish, and meat. The stability of
the enzymes is most important, and they need to be stable and active in the detergent
solution, with temperature stability at different washing temperatures.
Proteases are used in the food industry, such as for cheese making, baking, prep-
aration of soya hydrolysates, and meat tenderization [53].
524 R. Chitte and S. Chaphalkar
24.7 Conclusion
Microbes are well known for production of therapeutic enzymes, and medicinal
plants have been in use from ancient times for treatment of diseases. These medici-
nal plants are well known to show a broad spectrum of activities due to the presence
of important metabolites. The active molecules are being specifically targeted
toward diseases. Microbes, insects, and medicinal plants produce proteases for their
own physiological significance, and their isolation and characterization have been
carried out and reported. Further study of the diversity of these proteases is needed
for understanding of their mechanisms of action for substrate catalysis to implement
applications in the food, pharmaceutical, and agricultural industries.
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A Review on the Mode
of the Interactions of Bacterial Proteases 25
with Their Substrates
Sanchari Bhattacharjee, Rakhi Dasgupta,
and Angshuman Bagchi
Abstract
Maintaining the quality of cellular proteome is a huge challenge to living cell as
the proper functioning of protein directly affects proper functioning as well as
survival of the cell. Due to different cellular stresses, the cellular proteins face
recurring threats which has a negative impact on cellular proteins. As a result of
these threats, proteins become damaged as well as misfolded, and these defective
proteins impose a load to the cellular machinery by elevating the level of cyto-
toxicity. Protease enzymes are a type of cellular machinery that is specifically
used to eliminate the damaged and short-lived regulatory proteins by their pro-
teolytic mechanisms. Bacteria evolves different types of bacterial proteases that
are highly diverse which correspond to their localization, sequence, structure,
active sites, proteolytic mechanism, substrate specificity as well as function.
Bacterial proteases not only eliminate the damaged protein but also act as chap-
erones in some special situation and thus act as charonin which makes a promis-
ing effect in cellular protein quality control. This study illustrates the detailed
comparison of bacterial FtsH, Lon and Clp protease and their modes of interac-
tion with substrates.
Keywords
FtsH protease • Lon protease • Clp protease
25.1 Introduction
Maintaining the integrity of proteome is vital for cell viability. Cells however face
with a continuous stream of misfolded proteins that hamper the survival of cells [1].
Proteins are highly susceptible to misfolding not only during their native folding
mechanism but also due to cellular stress. Protein misfolding results in loss of func-
tion of proteins and, if remain unchecked, can potentially form toxic protein inclu-
sions leading to cytotoxicity of the cells [2]. So, these consequences threaten the
protein homeostasis of the cell. To counteract these problems, cells emerge with a
mechanism, called stress response mechanism that protects a cell in unfavourable
environmental conditions, by employing mechanisms that reduce acute damage to
cellular machinery as well as provide the cell a recovery measure against adverse
conditions.
Cellular stress responses are primarily mediated through stress proteins. Stress
proteins are of two categories: those that operate only under stress and those that are
triggered on both in stress responses as well as in normal cellular functioning. These
stress proteins, because of their essential role in cell survival during normal condi-
tion as well as in stressed condition, are highly conserved throughout the phyla,
from simplest eukaryotic to the most complex prokaryotic cells. There are different
pathways to encounter stress. A particular anti-stress response is however depen-
dent on the type of stressor. Heat shock response, for example, is initiated by the
presence of denatured proteins [3]. The fate of the stressed cells is ultimately deter-
mined by whether they adopt a protective or a destructive pathway depending on the
nature and duration of the stress as well as the cell type.
With this cellular stress response, the cells also co-evolve the protein quality
control network, a network that maintains the quality of the cellular proteins by set-
ting different checkpoints at different levels and is mainly composed of molecular
chaperones and proteases. Due to cellular stresses, the proteins become misfolded,
aggregated as well as damaged. The molecular chaperones with their foldase activ-
ity not only refold the aggregated proteins to their native folding state but also pre-
vent further aggregation of misfolded proteins via their holdase activity, whereas the
protease eliminates the damaged proteins to reduce the cytotoxicity which will
compromise with the viability of the cell [4]. Thus this quality control network
functions in a concerted way to ensure the quality of the cellular proteins which is
essential for the proper functioning and survival of the cell.
Proteases play an important role in the elimination of stressed proteins which
accumulate during stress, thereby activating different types of stress response path-
ways [5]. In Escherichia coli there are several different proteases to maintain the
cellular protein quality [6].
25 A Review on the Mode of the Interactions of Bacterial Proteases with Their… 529
The misfolded proteins which are formed due to cellular stress impose a burden to
normal cellular functioning as well as cell survival. To combat these conditions,
post-translational cellular control is being carried out by degrading the short-lived
regulatory proteins and protein quality control by eliminating defective and poten-
tially damaged proteins from the cell. Proteases, a special kind of enzymes, facili-
tate the above phenomena. Usually these enzymes are ATP-dependent proteases,
having both ATPase and proteolytic activities in domains within a single polypep-
tide chain or in individual subunits forming a complex assembly.
The misfolded proteins which are formed due to cellular stress impose a burden
to normal cellular functioning as well as cell survival. Protease, a special kind of
enzymes, facilitates degradation and elimination of misfolded proteins. Rapid pro-
teolysis eliminates defective and potentially damaged proteins from the cell [7]
exhibiting a promising role in post-translational cellular control by degrading the
short-lived regulatory proteins and thus contributes in protein quality control. In all
cells, protein degradation is mediated by ATP-dependent proteases, which contains
both ATPase and protease as separate domains within a single polypeptide chain or
as individual subunits in complex assemblies.
Protease breaks the proteins into smaller polypeptide fragments by accelerating
the hydrolysis of peptide bonds. A unique feature of proteases is their preference to
cleave the peptide bond associated with a particular amino acid. This particular
amino acid belongs to the active site of the protease. The active site is actually in a
cavity of the protein, called catalytic pocket. The type of amino acid residues within
the pocket will determine the particular type of the proteases. Proteases hydrolyse
the peptide bonds by activating a nucleophile; this activated nucleophile in turn
attacks the carbon of the peptide bond [8]. As the nucleophile attaches itself, the
electrons in the carbon-oxygen double bond migrate onto the oxygen forming a
high-energy tetrahedral intermediate. So to stabilize this, the intermediate will then
decompose, usually releasing the two peptide fragments.
There are usually six main classes of protease such as serine protease, cysteine pro-
tease, aspartate protease, threonine protease, glutamic acid protease and metallopro-
tease. These different classes of protease utilize either a different nucleophile or a
different mechanism to activate it.
Serine protease is found in both prokaryotes and eukaryotes. In this protease, the
serine present in the active site of the protein acts as nucleophilic amino acid and
cleaves peptide bond in proteins. The active site of the enzyme is a catalytic triad
530 S. Bhattacharjee et al.
consisting of three amino acids: His 57, Ser 195 and Asp 102. This triad is preserved
in all serine protease enzymes [9].
These three catalytic triad residues make a charge relay system and serve a piv-
otal role in proteolysis. Though the amino acid members of the triad are located far
from one another in the primary sequence, due to folding these residues come very
close to one another in the core of the native enzyme forming the triad.
The catalytic mechanism generates several intermediates. The catalysis of the
peptide cleavage follows a ping-pong mechanism, in which a substrate binds, a
product is released, another substrate binds and another product is released. In the
case of serine protease, the –OH group of serine serves as nucleophile [10] attacking
the carbonyl carbon of the scissile peptide bond of the substrate. In this case, as a
result of substrate binding to the polypeptide, the N-terminus ‘half’ of the peptide is
being cleaved. Again water binds as another substrate and the C-terminus ‘half’ of
the peptide is released as product.
Threonine proteases involve a threonine (Thr) residue in their active site. The sec-
ondary alcohol of N-terminal threonine of threonine protease is used for nucleo-
philic attack to exhibit catalysis. The N-terminal amide of N-terminal threonine acts
as a general base which polarizes ordered water and deprotonates the alcohol to
increase its reactivity as a nucleophile [14].
Catalysis takes place in two steps:
Glutamic proteases contain a glutamic acid residue in the active. These residues
perform the nucleophilic attack, where the glutamic acid serves as a general acid
that donates a proton to the carbonyl oxygen of the peptide bond of the substrate. In
this reaction, one or two water molecules supply a hydroxyl group, and the glutamic
acid again donates a proton to the amide nitrogen, which results in the breakage of
the peptide bond. The glutamine and the glutamic acid restore to its initial state [15].
25.3.6 Metalloprotease
In E. coli, ftsH is the second gene of a bicistronic operon. The first gene, ftsJ, affects
cell division. These two genes have potential promoters, ftsJ is recognized by σ70
which is the vegetative sigma factor and ftsH is recognized by σ32 which is the heat
shock sigma factor. Though the function of ftsH gene is highly conserved in all
bacterial species, in different genera, the position of ftsh gene in their operon with
respect to the other neighbouring genes is not conserved. So, ftsH may belong to
either monocistronic, bicistronic or polycistronic operon [20].
A. B.
>FtsH
TISPKEKEKIAIHEAGHALMGLVSDD
DDKVHKISIIKHIYDKKDLYNKILVLL
GGRAAEEVFFGKDGITTGAENDLQRA
TDLAYRMVSMWGMSDKVGPIAIRRT
AVDTSPDLLREIDEEVKRIITEQYEKA
KAIVEEYKEPLKAVVKKLLEKETITCE
EFVEVFKLYGIELKDKCK
Fig. 25.1 (a) Crystal structure of protease domain of FtsH protease monomer (in flat ribbon
view). The region marked in blue indicates the location of the HEXXH motif. (b) FASTA sequence
of FtsH protease domain (retrieved from UniProt). The HEAGH motif required for proteolysis is
marked as bold in red colour
Fig. 25.2 (a) Different orientation of periplasmic N-terminal domain of FtsH protease crystal
structure (flat ribbon view) (PDB ID: 4VOB). (b) Crystal structure of AAA domain of FtsH prote-
ase (flat ribbon view) (PDB ID: 1LV7). (c) Crystal structure of cytoplasmic protease domain of
FtsH protease (flat ribbon view) (PDB ID: 2DI4)
Lon protease was the principle protease to be identified in Escherichia coli which is
actually an ATP-dependent protease. It is conserved in all living organisms and
catalyses the damaged as well as degraded proteins. Prokaryotic Lons are the prime
enzymes to execute proteolysis and eliminate mutant, abnormal, degraded proteins
as well as the catalysis of short-lived regulatory proteins which in turn maintains
protein quality control and cellular homeostasis [35]. Apart from the proteolytic
activity, AAA + proteins are contributed in many cellular functions such as mem-
brane fusion, protein and organelle translocation, DNA and RNA unwinding,
assembly and disassembly of multi-protein complexes and microtubule severing.
The cellular activity of AAA + protease is largely defined by the interacting func-
tional partners. In the case of Lon, the AAA + domain is interacted with protease
domain to execute protease activity [36].
It contains homohexamers having multidomains. The homohexamers of Lon
protease in E. coli form an encircled degradation cavity within which the misfolded
536 S. Bhattacharjee et al.
proteins are degraded, but in yeast, the Lon protease forms a seven-membered ring
[37].
The N Domain
The N domain is present in only Lon A subfamily members. The N-terminal
domain is composed of approximately 300 amino acid open reading frame but with
unknown biological functions. The N domain is divided into two sub-domains
which are generated by limited proteolysis. Limited proteolysis of E. coli Lon by
different proteases generates several transiently stable N-terminal fragments of
varying length, and the termination residue ranges from 223 to 240 [39]. Extended
incubation with protease results in the reduction of N-terminal domain to Lon
N-209 which is highly stable and exists as a monomer. Another possible suggested
boundary is residue 119. Lon N119 has a unique fold with three twisted β-sheets
which are folded to form a shallow U shape. The depression of the shallow U shape
occurred due to the presence of a single α-helix [40]. Most of the regions around
α-helix of the N domain are contributed by hydrophilic residues, whereas the
exposed surfaces have hydrophobic residues located across the β-sheet which are
opposite in position to the alpha helix. It was observed that the two sub-domains of
N domain of Lon protease are interacted to each other to form a single bimodal
structural unit [41] (Fig. 25.3).
The N domain of Lon protease has protein-binding ability, thus helping in sub-
strate recognition. In vitro experiments revealed that E. coli Lon protease which
lacks 107 N-terminal residues shows a drastic reduction in protein degradation
activity. In Mycobacterium smegmatis Lon protease deletion in 90, 225 or
277 N-terminal residues contributes in complete removal of proteolytic activity,
reduction in protein binding activity as well as an alteration in oligomeric state.
These deletions make some structural distortions that affect the different protein
activity as well as oligomerization state [42]. Experiments revealed that mutated
Lon N domain changes its substrate specificity. From the experimental evidences, it
can be assumed that N domain takes part during the interaction with specific sub-
strate protein which involves the disordered regions in substrates with some recog-
nizable specific motifs, domains or sequences that are being identified by Lon
protease.
25 A Review on the Mode of the Interactions of Bacterial Proteases with Their… 537
Fig. 25.4 Crystal structure of AAA domain of Lon protease (flat ribbon view) (PDB ID: 4GIT)
allosteric changes that affect the configuration of the domain involving the catalytic
residues that make an alteration in the interactions, and thus a decision is being
made whether or not the bound substrate protein within AAA module will transfer
to protease domain. In oligomeric structure of Lon protease, this effect arises in dif-
ferent subunits with multiple active sites in a divergent way but in ordered or
sequential manner [47].
It is observed that, within the proteolytic domain, Lon exhibits Ser679-Lys722 dyad
in active site to promote catalysis [50]. The classical catalytic triad residues take
part in intra- and intermolecular interactions. In the case of proteases with catalytic
triad mechanism, substrates are attacked from the re-face of the amide bond,
whereas the protease with the catalytic dyad mechanism performs the attack from
si-face in the active site. The side chain of Asp676 forms hydrogen bonds with
Val633 and Met634 amino acid residues. His665 and His667 are placed on the sur-
face of the molecule and interact with Leu709 and Thr643; His667 also produces an
ion pair with Glu614 [51]. If the catalytic triad residues are mutated, these do not
affect the oligomerization of Lon protease which is a prerequisite for proteolytic
activity. It illustrates that these triad residues are not utilized to form an active site.
These residues all belong to the 15 amino acid fragment HVHVPEGATPKDGPS
(665–679), preceded and included the catalytic Ser679. Mutation in Ser679 with
alanine causes the loss of proteolytic activity, but there is no change in ATPase
activity (Fig. 25.6).
Proteolytic domain of Lon protease contains a single conserved lysine at amino
acid position 722 which is located 43 residues beyond the catalytic serine 679 and
if this lysine722 is mutated to glutamine by site directed mutagenesis, the Lon
K722Q mutant loses its hydrolytic activity and generates the similar properties of
S679A mutant, i.e. to be proteolytically inactive, but it does not affect the property
of solubility [52].
Experimental data explains that Lys722 forms a hydrogen bond with Gly717,
and this hydrogen bond remains strictly conserved in Lon superfamilies. ε-amino
group of Lys722 is in its position to form the hydrogen bond with the catalytic
Ser679 residue. In addition to maintain the position of the general base, the con-
served Thr704 residue is involved in hydrogen bonding with the Lys722 side chain.
So, the experimental analysis clearly portrays that P domain of Lon protease utilizes
a unique Ser679-Lys722 catalytic dyad [53].
Lon-mediated degradation is mediated by peptide bond hydrolysis but without
the dissociation of substrate. Both the prokaryotic and eukaryotic Lon proteases
exhibit proteolysis of substrates by producing short peptide products consisting of
~5–30 amino acids. Hydrolysis of peptide bond proceeds via sequential linear man-
ner from the amino to the carboxyl-termini or vice versa.
It is possible that protein substrates are proteolysed from the bulk solvent by the
Lon complex and degraded by repetitive rounds of substrate binding, cleavage and
release and rebinding to the proteolytic site in a sequential way, thereby resulting in
small hydrolysed peptide products [54]. Results also show that Lon does not neces-
sarily cleave substrates at a specific peptide consensus sequence; rather it shows a
preference for hydrophobic residues. In addition to have cleavage specificity, pep-
tide sequences within an exposed or unstructured region of a substrate also may
serve to trigger substrate recognition and interaction, which facilitates the initiation
of degradation mechanism [55].
540
A. B.
>Lon protease
RVGQVTGLAWTEVGG
DLLTIETACVPGKGKL
TYTGSLGEVQESIQAA
LTVVRARAEKLGINPD
FYEKRDIHVHVPEGAT
PKDGPSAGIACTALVS
CLTGNPVRADVATGEI
TLRGQVLPIGGLKEKL
LAAHRGGIKTVLIPFE
NKRDLEEIPDNVIADL
DIHPVKRIEEVLTLALQ
NEPS
Fig. 25.5 (a) Crystal structure of homohexamers of protease domain of Lon protease (flat ribbon view) (PDB ID: 1RRE). (b) FASTA sequence of Lon protease
proteolytic domain (retrieved from UniProt). Catalytic dyad residue serine 679 and lysine 722 are marked as bold in red colour
S. Bhattacharjee et al.
25 A Review on the Mode of the Interactions of Bacterial Proteases with Their… 541
Fig. 25.6 (a) Crystal structure of protease domain of Lon protease (flat ribbon view). The region
marked in yellow indicates the location of HVHVPEGATPKDGPS fragment. (b) Catalytic Ser679-
Lys722 dyad of Lon protease
A. B.
C.
>Clp protease
SRGERSFDIYSRLLKERVIFLTGQVEDHMANLIVAQMLFLEAENPEKDIYLYINSPGGVITAGMSIYDTMQ
FIKPDVSTICMGQAASMGAFLLTAGAKGKRFCLPNSRVMIHQPLGGYQGQATDIEIHAREILKVKGRMN
ELMALHTGQSLEQIERDTERDRFLSAPEAVEYGLVDSILTHRN
Fig. 25.7 Crystal structure of Clp protease in (a). Front view and (b) back view (flat ribbon view)
(PDB ID 1TYF) (c) FASTA sequence of protease domain of Clp protease (retrieved from UniProt).
Catalytic triad residues (serine 97, histidine 122, aspartate 171) are marked as bold in red colour
subunits each. When the peptides are fewer than 6 residues, it exhibits limited
serine-peptidase activity and preferentially cleaves them after hydrophobic resi-
dues. Degradation of peptides longer than approximately 6 residues needs the
involvement of an ATPase subunit such as ClpA to form the active protease (ClpAP),
but not ATP hydrolysis; by contrast, proteolysis of protein substrates demands ATP
hydrolysis as well as ClpAP. Protein substrates are catalysed in a highly sequential
manner, producing 7- to 10-residue peptides; the pattern of cleavage does not show
any clear sequence specificity [58].
Each ClpP subunit composes an α-/β-fold buildup of six repeats of the α-/β-unit
along with an additional protruding α-/β-unit. The ten β-strands form two layers of
β-sheets that are packed against a layer of α-helices. The layers of sheets remain
perpendicular to each other to create one side of the substrate-binding cleft. In pro-
jection, the ClpP monomer shows a similarity with a hatchet having wedge-shaped
head (head domain) and a short handle. Residues 28–120 and160–188 comprises
the head domain, and the ‘handle’ is formed by residues 125–130 and 132–157.
Residues 11–27 belong to the amino termini, and residues 189–193 belong to the
carboxyl-termini which lie at opposite ends of the head domain, extending away
from its central core [59]. The catalytic triad, Ser-97, His-122 and Asp-171, is situ-
ated in a cleft at the connection point of the head domain and handle. The structure
of ClpP defines a fifth family of serine proteases which is different from the other
25 A Review on the Mode of the Interactions of Bacterial Proteases with Their… 543
25.4 Discussion
Even though the bacterial proteases perform the same proteolytic activity, there are
many differences regarding different aspects. These proteases vary in their subcel-
lular localization. Both Lon and Clp are cytoplasmic protease, whereas Ftsh has a
periplasmic domain, membrane-bound domain as well as cytoplasmic domain.
Sequence analysis of these proteins shows huge diversities in respect of sequence
length, amino acid composition and even the structures of catalytic pockets. FtsH
and Clp both utilize conventional catalytic triad consisting of serine, histidine and
aspartate, whereas Lon employs a unique catalytic dyad consisting of serine and
lysine to promote catalysis. Hydrophobicity analysis of catalytic pockets (Fig. 25.8)
of FtsH protease, Lon protease and Clp protease reveals that even if they perform
the same proteolytic activity, there are some differences regarding their hydropho-
bicity. The catalytic pocket of FtsH indicates less hydrophobicity, Lon protease has
neutral hydrophobicity, whereas Clp protease reveals increased hydrophobicity.
Though these three proteases belong to Escherichia coli, they also possess a
substrate diversity as well as functional diversity. Lon and Clp both promote the
proteolysis and degradation of misfolded proteins, but FtsH, being a protease, not
only engaged in the selective degradation of damaged proteins but also performs as
a molecular chaperone. FtsH can bind to denatured proteins, and this binding does
not necessarily ensure the proteolysis of the bound protein which is a unique char-
acteristic feature of molecular chaperones. FtsH is actively participated in folding
activity of non-native proteins, i.e. acts as foldase and then sends them either to its
own protease pathway or to the folding pathway. By satisfying both the protease and
the chaperone activity, FtsH can be considered as a charonin.
25 A Review on the Mode of the Interactions of Bacterial Proteases with Their… 545
Fig. 25.8 (a) Catalytic pocket of FtsH protease. (b) Catalytic pocket of Lon protease. (c) Catalytic
pocket of Clp protease. (d) Hydrophobicity scale where the blue region denotes less hydrophobic-
ity, white region denotes neutral hydrophobicity and brown region denotes increased hydrophobic-
ity. Yellow arrow denotes the exact location of catalytic pocket
Acknowledgement The authors are thankful to the Department of Biochemistry and Biophysics
and Bioinformatics Infrastructure Facility Centre, University of Kalyani, for their continuous sup-
port and for providing the necessary instruments to carry out the experiments. The authors would
like to acknowledge the DBT (project no.BT/PR6869/BID/7/417/2012) and the ongoing DST-
PURSE programme (2012-2015) for their support.
546 S. Bhattacharjee et al.
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The Ubiquitin Proteasome System
with Its Checks and Balances 26
Prranshu Yadav, Ankita Doshi, Yong Joon Yoo,
and C. Ratna Prabha
Abstract
Cells need to quickly change according to changing environment to survive, and
for that, they must not just make new proteins but also degrade others equally
promptly. For this purpose, cells have evolved the ubiquitin system, which con-
sists of ubiquitin molecules which are used to tag proteins in a process called
ubiquitination; E1, E2 and E3 enzymes which carry out the process of ubiquiti-
nation; and deubiquitinating enzymes (DUBs) that remove the ubiquitin from the
substrate proteins in a process called deubiquitination. Ubiquitination involves
various lysine residues on ubiquitin; among them K48 and K63 are the most
significant and well understood. Ubiquitination with K48 linkage leads to degra-
dation of substrate proteins by a multi-protein complex called proteasome.
Proteasome-mediated degradation is involved in numerous different processes in
cells, due to which defects in it are responsible for several diseases. But due to
the high diversity of E3 enzymes and ubiquitin target proteins, there are many
drug targets that can be utilized to treat diseases. This makes it vital to under-
stand ubiquitin system for advancement of health care.
Keywords
Ubiquitin • Ubiquitin structure • Proteasomes • Ubiquitination • Deubiquitination
26.1 Introduction
26.2.1 Ubiquitin
turns and a 310 helix. Additionally, ubiquitin has two β-bulges in its structure.
β-bulges are regions in a β-sheet, where two residues in a β-strand are present oppo-
site a single residue in the neighbouring strand. The space required by the extra resi-
due causes this region to bulge out from the β-strand, hence the name. β-bulge
affects β-sheet’s structure firstly by accentuating the sheet’s inherent twist and sec-
ondly by interfering with the alternating arrangement of side chains on the two sides
of the backbone. The first β-bulge of ubiquitin is located in the N-terminal region
between the two antiparallel β-strands forming a hairpin, while the second β-bulge
is present in the β-sheet, adjacent to K63. The second β-bulge is a parallel G1β-
bulge formed by residues E64 and S65 at positions 1 and 2 and Q2 at position X on
the other strand.
Ubiquitin sequence shows a very high degree of conservation among all eukary-
otes, with only three residues being replaced from yeast to humans.
In the 1990s, Makhatadze et al. studied the effects of different salts on ubiquitin’s
stability at pH 2.0, using differential scanning calorimetry, circular dichroism and
fluorescence spectroscopy [20, 21]. They found that all salts tested increased the
thermostability of ubiquitin through anion binding. They then studied the effect of
surface charge on ubiquitin’s stability using site-directed mutagenesis and specific
chemical modifications. Robertson et al. too studied significance of charges and ion
pairs on ubiquitin’s surface using site-directed mutagenesis of specific surface resi-
dues and determining pKa of neighbouring charged residues by 2D NMR [22, 23].
Makhatadze et al. have produced an ubiquitin mutant more stable than its wild-type
counterpart [24]. They first converted all arginine residues on ubiquitin surface to
lysines and then carbamoylated their amino groups. They observed that ubiquitin
was most stable when all these carboxyl groups were protonated and hence all sur-
face charges were neutralized. Surface charges therefore do not appear to contribute
to ubiquitin’s stability. The outcome of this study helped in framing guidelines for
engineering of surface charges to increase protein stability [25]. The importance of
hydrophobic residues in ubiquitin’s core was studied using site-directed mutagene-
sis and measurement of consequent heat capacity changes by differential scanning
calorimetry [26, 27]. Replacing nonpolar residues with polar ones decreased the
stability of ubiquitin, while replacing naturally occurring polar residues in core with
nonpolar ones increased its stability. Replacing nonpolar residues with other nonpo-
lar residues had no significant effect. There have been other studies too on the
importance of hydrophobic residues which are present in the core of ubiquitin [28–
30]. Significance of the interaction between I30 and I36 at the C-terminus of the
α-helix has also been studied. Out of 16 variants produced in the study, none were
found as stable as the wild type [31].
Ubiquitin residues essential for vegetative growth of yeast are clustered in three
regions on ubiquitin surface. They are hydrophobic patches formed by L8, I44 and
V70 and their surrounding residues, F4 and its surrounding residues and the
C-terminal tail [32]. I44 patch is essential for proteasomal degradation and endocy-
tosis [33], F4 patch is essential for endocytosis and for proteasomal degradation as
well [32–35], and the C-terminal tail is essential for most ubiquitin functions, owing
to its fundamental role in ubiquitination. The L8, I44 and V70 patch interacts with
552 P. Yadav et al.
regulatory subunit(s) of proteasome, while this patch together with F4 patch may
either form a common binding site for proteins involved in endocytosis, or both
patches may bind separate sets of proteins is yet to be known.
Functional studies with ubiquitin carrying E64G, S65D and Q2N substitutions and
their combinations showed no effect on growth under normal conditions, survival
under heat stress and adherence to N-end rule [59–62]. However, the mutations led
to increased cycloheximide sensitivity. Absence of any significant structural changes
in ubiquitin due to these substitutions establishes that increased cycloheximide sen-
sitivity results from functional rather than structural defect. Ribosomal protein L28
is modified by K63-linked polyubiquitin chain, which is important for making the
organism resistant to translational antibiotics. Here the mutations E64G, S65D and
Q2N hamper the formation of K63 polyubiquitin chain, because of their spatial
proximity to K63 [59–62]. PTEN-induced kinase 1 (PINK1) is a Ser/Thr kinase that
phosphorylates S65 on ubiquitin. S65-phosphorylated ubiquitin in turn activates
Parkin, which is an E3 implicated in Parkinson’s disease, and ubiquitinates mito-
chondrial proteins. Phosphorylation of ubiquitin by PINK1 occurs in response to
mitochondrial membrane depolarization. Parkin itself is also known to be phos-
phorylated by PINK1. Together, PINK1 and parkin are involved in mitochondrial
membrane quality control [63]. Missense mutations in PINK1 gene are also impli-
cated in autosomal recessive inherited Parkinson’s disease.
Hence, the functional importance and in some cases the medical relevance of the
conserved residues can be understood by generating mutations of ubiquitin and
studying their differential effects. Ubiquitin gene was evolved in vitro, and a dosage-
dependent lethal mutation UbEP42 was isolated in our laboratory [64]. UbEP42
carried amino acid substitutions in four positions, namely S20F, A46S, L50P and
I61T. In spite of being incorporated into polyubiquitin chains, the mutant caused G1
phase arrest of the cell cycle by changing the Cdc28 protein kinase levels. Further,
it displayed increased sensitivity towards heat stress and exposure to cycloheximide
[65]. Ubiquitin’s core is significantly hydrophobic, with 16 of its 21 leucine, isoleu-
cine, methionine and valine residues buried in the interior. This may explain ubiqui-
tin’s high thermostability, which is necessary since the molecule’s role in stress
management often requires it to operate at high temperature.
[74], which probably allows easy transfer of ubiquitin from the first site to the sec-
ond. Only a single E1 transfers primed ubiquitin to all the different E2 enzymes in
most organisms. Catalysing the first step of the cascade, therefore, E1 is responsible
for ensuring unlimited supply of primed ubiquitin to all the downstream conjuga-
tion reactions, and so it would be expected to be present in high concentrations.
However, E1 concentration is less than total E2 concentration [4]. This requires E1
to be a highly efficient enzyme. Indeed, catalytic rate values for substrate ubiquiti-
nation are 10–100-fold slower than that of all E1-catalysed steps from ATP binding
to thioester formation [75]. However, E1 affinity for ubiquitin increases tremen-
dously after ATP binding [4, 66]. ATP binding causes a conformational change in
E1, which makes the binding site more accessible to ubiquitin. The interactions
between AMP-ubiquitin and E1 are extensive [76]. The only known residue in E1
active site is cysteine, but as arginine residues in ubiquitin are essential for its bind-
ing with E1, certain acidic residues may also be involved [77, 78]. E1s for ubiquitin
and ubiquitin-like proteins (UBLs) are related. E1 for ubiquitin is a monomer of
110 K Da. Initially, the role of ubiquitin proteasome system in the degradation of a
diverse array of short-lived proteins was demonstrated by using temperature-
sensitive E1 mutants expressed in mammalian cell lines [79]. These mutants also
help determine if a process is ubiquitin dependent [80, 81]. Hypomorphic allele of
E1 was isolated, which produces mutant form of E1 that is less efficient than wild-
type E1 in performing its function. It was demonstrated that the hypomorphic allele
of E1 of yeast can successfully replace the temperature-sensitive variant in the
experiments where ubiquitin dependence of degradation is investigated [35, 82].
E2 accepts ubiquitin from E1. A thioester bond is formed between C-terminal gly-
cine of the ubiquitin and active site cysteine in E2, as the Ub-E1 thioester bond
breaks [4]. This step does not involve any change in energy level since energy stored
in the thioester bond between ubiquitin and E1 cysteine is conserved in the thioester
bond between ubiquitin and E2 cysteine. E2s are much more diverse than E1s. S.
cerevisiae has 14 E2s, and higher organisms have even more. Some of these may be
different isoforms of same E2 [83, 84], while others may have evolved indepen-
dently [85–87]. This diversity confers first level of substrate specificity to ubiquitin
system, the other being at the level of E3. Structural studies on E2 enzymes show
that a core domain of 150 amino acids is conserved across all E2s and may account
for their chief function of transferring ubiquitin to E3 enzymes. The core domain
contains a four-stranded antiparallel β-sheet, four α-helices and a 310 helix. Helix 2
and the β-sheet make up a central region bound by helices 3 and 4 on one side and
helix 1 on the other. The loop connecting β-strand S4 to helix 2 contains the active
site cysteine. It is located in a shallow groove formed by upstream residues of the
same loop on one side and those of loop connecting helix 2–3 on the other. The
region surrounding active site cysteine contains many of the most conserved resi-
dues [88–94], some of which interact with ubiquitin and others probably with E1.
26 The Ubiquitin Proteasome System with Its Checks and Balances 555
The side opposite to the active site, on the other hand, contains most of the poorly
conserved residues. Presently, it is not clear if this variation in sequence in different
E2s is due to low selective pressure or because this region may be responsible for
selecting specific E3s [95]. Some E2s also have N- or C-terminal extensions, which
may be responsible for conferring either substrate or E3 specificity in these cases
[96–98]. Recent structural studies on complexes of E3 with Ub-E2 conjugates are
beginning to unveil the mechanism of ubiquitin transfer from E2 to E3 [99–104].
E3s catalyse the final step of ubiquitination, i.e. forming the isopeptide bond
between the C-terminal glycine of ubiquitin accepted from an E2 and the lysine on
the substrate protein. In some cases, substrate protein is ubiquitinated on the
α-amino group of its N-terminal residue, which may not be lysine [105]. E3s are
extremely diverse in their substrate specificity. This diversity enables selective ubiq-
uitination of proteins and hence makes ubiquitination a powerful tool for regulating
cellular activities. E3s are of two types, namely, RING E3s and HECT E3s. HECT
E3s simply mimic the step of ubiquitin conjugation to E2, by breaking the thioester
bond of Ub-E2 and forging another thioester bond between the C-terminus of ubiq-
uitin and -SH group of the cysteine present in the active site of E3, leading to forma-
tion of Ub-E3. In this process, energy from E2-Ub thioester bond is conserved in
E3-Ub thioester bond. Subsequently, Ub is transferred from E3 to a substrate pro-
tein, by forming an isopeptide bond between the carboxyl group of C-terminal gly-
cine of ubiquitin and ε-amino group of lysine on the substrate protein. RING E3s,
unlike HECT E3s, do not form a covalent bond with ubiquitin; instead, they act as
adapters by binding to the substrate protein and E2 loaded with ubiquitin simultane-
ously. They facilitate transfer of ubiquitin from E2 to substrate protein directly.
Majority of E3s belong to RING domain type. RING domain was discovered and
characterized in the early 1990s. The canonical sequence found in RING E3s is
Cys-X2-Cys-X(9–39)-Cys-X(1–3)-His-X(2–3)-Cys-X2-Cys-X(4–48)-Cys-X2-Cys
(where X is any amino acid). Cysteines 1, 2, 4 and 5 coordinate one Zn+2 ion, and
cysteines 3, 6 and 7 along with the histidine coordinate a second Zn+2 ion. RING
domain is thus structurally related to zinc finger domain. Unlike zinc finger, how-
ever, RING domain assumes a rigid and compact shape owing to the presence of the
two zinc ions in the coordination sites. Sequence conservation in RING domain is
not absolute. There are variants in which cysteines and histidines are swapped, as
well as those in which another residue capable of coordinating zinc replaces a cys-
teine, e.g. Asp in Rbx1/Roc1. There is a third group of E3s known as U-box E3s,
which also recruits E2s. They are often clubbed along with RING group of E3s, as
the two are closely related. In U-box domain, which is structurally similar to RING
domain, zinc ions are replaced by a network of hydrogen bonds [106].
HECT E3s have a modular architecture. HECT domain has a bilobal shape, with
E2-binding site in the N-terminal lobe and the active site cysteine in the C-terminal
lobe [86]. As the distance between the two lobes is large, both lobes must come
556 P. Yadav et al.
close together for catalysis, perhaps covering a distance of around 50 Å [107].
Further, significant decrease in catalytic activity observed due to the mutations of
the hinge between the lobes as a consequence of restricted movement supports the
above model [108]. N-terminal region extending from N-terminal lobe of HECT
domain differs from enzyme to enzyme and confers substrate specificity adding one
more functional domain. Interestingly, substrate binding to E3 is not affected by the
deletion of HECT domain [109–111]. Besides, isolated HECT domains retain bind-
ing to E2 enzymes. HECT domains do not show functional redundancy as they
cannot substitute for each other [112], because they contribute to substrate specific-
ity of their respective E3s [113] (Fig. 26.1).
individual ubiquitins [116, 117]. They also cleave free polyubiquitin chains pro-
duced by ubiquitin conjugation machinery in the absence of any target protein.
Thirdly, three of the four ubiquitin genes (UBI1, UBI2 and UBI3 in yeast) produce
ubiquitin fused to ribosomal proteins, and the fourth (UBI4) produces a fusion of 4
or 5 ubiquitins in yeast [118–120]. Hence, DUBs are needed for producing free and
functional ubiquitin from the fusions. Lastly, unwanted reactions involving ubiqui-
tin in cell create species, which do not seem to serve any function. Such species
include thiol esters between ubiquitin C-terminus and cellular thiols like glutathi-
one, amide derivatives of ubiquitin formed with lysine and spermidine and free
ubiquitin adenylate. DUBs release free ubiquitin from these species [121, 122].
There are five different families of DUBs in humans, which together constitute
about 100 known DUBs. Four of these families are cysteine-like proteases. These
are Josephin domain or MJD proteases, ubiquitin-specific proteases (USP/UBP),
ubiquitin C-terminal hydrolases (UCH) and ovarian tumour (OTU) families. The
fifth one is of JAB1/MPN/Mov34 metalloenzyme (JAMM) domain zinc-dependent
metalloprotease family. The active site in DUBs of Cys protease families consists of
the catalytic cysteine and a histidine. Histidine increases polarity of cysteine -SH
group, making S more negative, enabling it to make a nucleophilic attack on the
isopeptide bond linking ubiquitin to substrate protein. Cleavage of isopeptide bond
is followed by formation of a transient acyl intermediate-linking carboxyl group of
ubiquitin to catalytic Cys of DUB. This acyl intermediate is then cleaved by a water
molecule, releasing ubiquitin. In many, though not all cases, a third residue, which
usually is Asn or Asp, polarizes and aligns histidine. The catalytic Cys, His and Asn/
Asp are called the catalytic triad. All the four Cys protease families have the same
basic catalytic site structure and mechanism. What distinguishes them from one
another is their tertiary structure.
Besides the DUBs, which cleave SUMO and Nedd8, ubiquitin-like proteases
(ULPs) include Adenain family of cysteine proteases [123–127]. The members of
Adenain family resemble adenovirus protease. The diversity seen with DUBs is
mainly responsible for their immense substrate specificity, which enables selective
deubiquitination of proteins. Similar to DUBs, the feature of ‘structural diversity
resulting in functional selectivity’ is observed with E3 enzymes as well, while cata-
lysing ubiquitination. This makes deubiquitination as useful as ubiquitination in the
regulation of cellular activities such as regulation of DNA repair [128], gene expres-
sion [129], cell cycle regulation [130], kinase activation [131, 132], proteasome-
mediated degradation [133, 134], lysosome-mediated degradation [134], microbial
pathogenesis [135, 136] and myriad other activities. Many pathogenic bacteria
[136, 137] and viruses [135, 138–145] have acquired DUB genes through parallel
transfer from eukaryotic genomes. These microbes may use DUBs to shield their
proteins against host’s ubiquitination machinery. For example, DUBs in Adenain
family have been acquired by bacteria and viruses to cleave ubiquitin and interferon-
stimulated gene 15 (ISG15) conjugates [123–127]. Mutant DUBs have also been
implicated in numerous diseases [128, 146–148]. To date, very few DUBs have
been characterized in terms of their substrates and physiological roles, and much of
this area remains to be explored.
558 P. Yadav et al.
In UCH domain family UCH catalytic core is formed by a domain of 230 amino
acid residues. The salient feature of this family is that the active site is covered by a
loop. The loop restricts access to ubiquitinated proteins and polyubiquitin chains as
they are too big and ensures that ubiquitin attached through its C-terminal to small
peptides, and small chemical groups only can access the active site of the DUB for
processing. The space afforded by this arrangement is too small for large, folded
ubiquitin conjugates or even polyubiquitin chains to fit in. In a study conducted on
UCHL3, the loop was systematically extended until UCHL3 gained ability to
hydrolyse polyubiquitin. The amount of extension needed was significant. Hence,
UCHs act not on ubiquitin linkage with whole proteins but with small peptides pro-
duced as by-products of proteasomal or lysosomal degradation, molecules like alde-
hydes and C-terminal extensions of polymeric proubiquitin. However, unfolded
whole proteins may also be targeted, if they can manage to thread through confined
space provided by the loop [149]. In some UCHs, active site exists in an unproduc-
tive conformation in the absence of activation signals [150].
Members of USP domain family consist of three subdomains in their structure,
namely, finger, palm and thumb [151]. CYLD is the only USP that lacks finger sub-
domain [152]. Though sequence similarity of USP domain across the family is low,
it is structurally well conserved [151–155]. The active site lies in a cleft between
palm and thumb, and the C-terminus of ubiquitin binds the active site, while the
globular portion of ubiquitin binds the fingers [151, 153, 155]. Crystal structures of
both ubiquitin-bound and ubiquitin-unbound forms of many USPs have been solved,
and their comparison shows that some USPs exist in inactive conformation in the
absence of ubiquitin, while others maintain active conformation irrespective of
ubiquitin’s presence. In the former case, inactive conformation of the USP may be
a result of either improper positioning of catalytic triad or blocking of correctly
positioned triad by ubiquitin-binding surface loops. In many cases, USP domain has
insertions or terminal extensions capable of folding into independent domains with
some functional relevance. In USP5, these domains have additional ubiquitin-
binding sites, whereas in CYLD, they determine subcellular localization [152].
In the OTU domain family, the structure of Otu1 covalently bound to ubiquitin
shows that most of its interactions with ubiquitin are mediated by a large surface
loop. In the absence of ubiquitin, this surface loop has been found to be disordered
in OTUB1 and the other members of the family OTUB2 and A20. Superposition of
ubiquitin-bound Otu1 structure on the structure of A20 shows that a helical domain
blocks binding site for ubiquitin moiety, suggesting architectural variation of this
site in A20. The structure of OTUB1 apoprotein shows nonproductive alignment of
His residue with catalytic Cys in the catalytic triad, suggesting that OTUs, like
USPs and UCHs, may exist in catalytically inactive conformation in the absence of
ubiquitin. In OTU core domain, five β-strands are sandwiched between helical
domains. However, the lengths of the β-strands are variable within the OTU family
[152].
MJD family has four members in humans, of which the best studied is Ataxin 3
[156–160]. Ataxin 3 probably acts specifically on K63 polyubiquitin. It has an
extended helical arm, which may regulate access of polyubiquitin chain to the active
26 The Ubiquitin Proteasome System with Its Checks and Balances 559
site [157–159]. A second ubiquitin-binding site, which is distinct from the active
site, lies at the back of this arm, suggesting that ataxin 3 may interact with two distal
ubiquitins simultaneously in a polymer. Ataxin 3 also contains a polyglutamine
stretch whose extension causes Machado-Joseph disease [161].
JAMM domain DUBs are commonly associated with large complexes [162–
169]. Solving the crystal structure of one of the JAMM domain DUB, AMSH-like
protease bound to a K63 diubiquitin has helped unravel the catalytic mechanism of
JAMM domain DUBs [169]. JAMM domain coordinates two Zn ions, one of which
primes an H2O for hydrolysis of isopeptide bond, while the other is included in an
AMSH-specific insert that forms a motif recognizing proximal ubiquitin of K63
diubiquitin. Another AMSH-specific insert recognizes distal ubiquitin. Hence,
JAMM domain recognizes distal ubiquitin and sequence Gln62-Lys63-Glu64 in
proximal ubiquitin, which makes AMSH-like protease K63 linkage specific.
AMSH-specific inserts are absent in JAMM domain proteases that are not specific
for polyubiquitin.
UBDs interact with the L8-I44-V70 patch of ubiquitin, making their footprints on
ubiquitin overlapping, although some recognize other surfaces such as Asp58 and
Gln62 [178] or the C-terminal residues [179]. Moreover, the footprints on I44 patch
show marked variation in spite of some overlap. Although most UBDs characterized
so far contact the I44 face of ubiquitin, high degree of conservation seen with other
surfaces of ubiquitin suggests that many more UBDs remain to be discovered.
Besides, many proteins involved in ubiquitin-dependent processes have been shown
to have unidentified ubiquitin-binding sites.
Mechanisms of action of UBDs are unclear, but their properties give key insights
into their functioning in vivo. Affinity of UBDs for ubiquitin is typically low with
their Kd values in the range of 10–500 μM. This may make complexes based on
UBD-Ub interactions capable of rapid assembly and disassembly, making them
more dynamic [180]. Low affinity may also make regulation of these complexes
easier, as disruption of even a single interaction may destabilize the complex.
Besides, as ubiquitin concentration in cell is very high [181], low affinity may be a
strategy to ensure availability of free UBDs. Proteins needed to bind strongly to
ubiquitin may do so by having multiple UBDs. The overlapping footprints of differ-
ent UBDs on ubiquitin may prevent simultaneous binding of more than one ubiqui-
tin receptor to ubiquitinated protein. This may be desirable in pathways that need
sequential handing over of ubiquitinated protein from one ubiquitin receptor to
another. Different footprints may also help UBDs in distinguishing between monou-
biquitination and polyubiquitination and also between different linkages of polyu-
biquitination [172, 182, 183]. Structural studies show that ubiquitin structure
slightly changes on binding to different UBDs, increasing their affinity for ubiquitin
[184]. Some UBDs appear to be linkage specific, while others are not [185, 186].
Linkage specificity can be conferred either by interaction of UBD with linker region
of polyubiquitin [187] or by fixed length of the linker sequence between tandem
UBDs that favourably positions them to recognize a particular polyubiquitin [158,
188, 189].
heterohexameric ring [211], though the order in which they are arranged in the ring
is debated [212–214]. The two non-ATPase subunits are Rpn1 and Rpn2. The lid
also has eight subunits, Rpn3 and from Rpn5 to Rpn11. Out of these, Rpn10 attaches
lid to base. The 19S complex imparts substrate specificity to proteasome by favour-
ing K48 polyubiquitinated proteins over others [215]. The K48 polyubiquitin must
contain at least four to five ubiquitins for recognition by proteasome [215], which
suggests that both ubiquitin structure and polyubiquitin topology determine recog-
nition. Only a few proteins are known to be recognized by 19S in nonubiquitinated
state [216–220]. Rpn10 [221] and Rpn13 [222] are the subunits, which are known
to bind polyubiquitinated proteins. Rpn10 binds ubiquitin through its ubiquitin-
interacting motif (UIM) consisting of three helices connected by flexible linkers
[223]. The UIM does not display any definite tertiary structure due to the flexible
linkers. Rpn13 binds ubiquitin through a pleckstrin-like receptor of ubiquitin (PRU)
domain [224]. Ubiquitin binds to loops in PRU domain, unlike other UBDs in which
it binds to secondary structures. Studies suggest that Rpn1, Rpn2 and Rpt5 are ubiq-
uitin receptors as well [225–227]. Rpt5 is also known to bind ornithine decarboxyl-
ase, the nonubiquitinated substrate of proteasome [218]. Before the unfolded
proteins are inserted into 20S complex, the polyubiquitin tail is cleaved off by
Rpn11 using its zinc finger containing JAMM motif [162, 228]. Rpn11 functions
only in association with the rest of the 19S, as its deubiquitinating activity is ATP
dependent. The 19S particle also opens the gates formed by N-terminal regions of
α-subunits at the mouth of 20S complex. The 20S and 19S complexes together make
the 26S proteasome. Proteasomes can exist in the nucleus and cytoplasm and
attached to the outer surface of the endoplasmic reticulum.
The tertiary structures of proteasomal subunits exhibit properties typical of and
necessary for subunits of large protein assemblies. Proteasomal-activating nucleo-
tidase (PAN), an orthologue of proteasomal AAA ATPases, has coiled coils protrud-
ing from an oligonucleotide binding (OB) fold called PAN-N and an AAA fold
[213, 229]. The long and slender topology enables coiled coils of PAN to establish
extensive contact with their binding proteins. Rpn1 and Rpn2 have proteasome
cyclosome (PC) repeats [230], which may form two helix ARM/HEAT units, which
in turn form α-solenoids [231]. The α-solenoids are superhelical quaternary struc-
tures having extensive surface area for establishing contact with binding partners.
Rpn3, Rpn5, Rpn6, Rpn7, Rpn9, and Rpn12 have PCI domain [232], which consists
of an N-terminal helical bundle fold and a C-terminal-winged helix fold. Repetitive
bihelical blocks preceding PCI domain in these subunits may form α-solenoids
together with helical bundle fold in PCI domain [232].
26 The Ubiquitin Proteasome System with Its Checks and Balances 563
26.5 R
ole of Proteasomal Degradation in Cell Physiology,
Disease and Therapy
There are various aspects of cell physiology that are regulated by proteasomal deg-
radation, including DNA repair, transcription, protein synthesis, cell signalling, cell
cycle and autophagy. During the progression of cell cycle, ubiquitin system ensures
timely degradation of cell cyclins, inhibitors of cyclin-dependent kinase and other
important proteins. Often defective regulation of UPS in relation to degradation of
these key proteins is a major cause of tumorigenesis. There are two main families of
E3s involved in cell cycle: the SCF (Skp1/Cul1/F-box protein) complex and the
anaphase promoting complex or cyclosome (APC/C). APC/C is responsible for exit
from mitosis and establishing a stable G0/G1 phase, while SCF is involved in all
stages of cell cycle. In transcription-coupled DNA repair, proteasomal degradation
of the largest subunit of RNA polymerase II occurs after its ubiquitination by Rsp5,
which is a HECT domain E3 [110]. Besides, transcriptional activators are also
degraded by proteasomes [233, 234]. Transcriptional activation domains (TADs)
are responsible for signalling proteasomal degradation. Transcriptional factors con-
taining TADs may be both activated and degraded through the same pathway.
Proteasomal ATPases unfold the yeast Gal4 activator irreversibly, leading to its pro-
teasomal degradation [7]. Interestingly, monoubiquitination is sufficient to serve as
a marker for degradation in the case of Gal4, instead of polyubiquitination with K48
linkage. Genome-wide ChIP-chip studies show that proteasomal ATPases are resi-
dent on hundreds of yeast genes, as several trans- and co-activators of transcription
undergo monoubiquitination. Hence, ubiquitination is used by cells as a mechanism
in transcriptional regulation. Proteasomal degradation is also involved in protein
synthesis. In order to prevent formation of defective ribosomes, cell must produce
all ribosomal proteins in equimolar amounts. This requires an unattainably high
level of coordination between the large numbers of ribosomal protein genes. Cells
solve this problem by producing excess of all ribosomal proteins and subjecting
those subunits which fail to get incorporated into ribosomes to proteasome-mediated
degradation [11]. Proteasomal degradation therefore sculpts the ribosomal protein
set into equimolarity. Interestingly, ribosomal proteins might be protected from pro-
teasomal degradation before being incorporated into ribosomes. For example, Ubi1
and Ubi2 genes are expressed as ubiquitin fusions with small ribosomal subunit
protein S27a, while Ubi3 gene is expressed as ubiquitin fused to large ribosomal
subunit protein L40 [120, 121]. Ubiquitin fused to these proteins may protect them
not only from proteasomal degradation but also from N-terminal-specific proteases,
as the ubiquitin is fused to their N-terminal. Proteasomal degradation is also
involved in regulating the action of two prosurvival switches in TNFR1-mediated
cell signalling. NF-κB pathway is driven towards cell survival by proteasome-
mediated degradation of Ik-Bα, the inhibitor of NF-κB. Degradation of Ik-Bα
enables NF-κB to migrate to nucleus and upregulate its target genes [235]. The
second switch in the same pathway is receptor-interacting serine/ threonine protein
564 P. Yadav et al.
kinase 1 or RIP1. RIP1 serves as a dual switch. When RIP1 is conjugated with a
K63-linked polyubiquitin chain, it functions as an inhibitor to proapoptotic pathway
by NF-κB-independent mechanism initially and later by NF-κB-dependent mecha-
nism [236]. Alternatively, a deubiquitinated RIP1 may also interact with FADD and
caspase 8, causing apoptosis [237]. The enzyme A20 has both DUB and E3 activi-
ties and replaces the K63 chain on RIP1 with a K48 chain, causing its proteasomal
degradation [238]. It is interesting to study the consequences when A20 fails to
polyubiquitinate RIP1 with K48 chain, after removal of its K63 chain. Hence, the
ubiquitination status of RIP1 acts as a checkpoint, as polyubiquitin chains with K48
linkage and K63 linkage serve as negative and positive regulators of NF-κB signal-
ling, respectively, and in turn act as switches for apoptosis and cell survival.
Ubiquitin system also plays a vital role in stress response. During stress, heat-shock
proteins (HSPs) act as chaperones, folding unfolded proteins back to their native
state. However, when the proteins are truncated or damaged beyond repair, these
same HSPs associate with E3s like CHIP and Parkin and facilitate their degradation
by proteasomes. If the rate of denaturation of proteins is so high that neither HSPs
nor proteasomes can clear them quickly, the unfolded proteins form intracellular
aggregates. Such potentially toxic aggregates may be responsible for neurodegen-
erative diseases like Parkinson’s and Alzheimer’s. Indeed, several protein aggre-
gates have been shown to be ubiquitin positive. Interestingly, ubiquitin system plays
a role not just in proteasomal degradation but also in lysosomal degradation [239–
241]. Ubiquitin has been shown to be responsible for marking membrane proteins
for selective degradation by lysosomes.
a RING finger E3. Parkin protein contains an N-terminal Ubl domain and two
C-terminal RING finger domains flanking an in-between RING (IBR) domain.
Parkin binds to proteasome through its S5a subunit, and this binding is reduced by
mutations at R42 residue. Such mutations may compromise substrate degradation
through parkin, and resultant parkin substrate accumulation may result in Parkinson’s
disease pathogenesis. Possible parkin substrates include Parkin-associated endothe-
lin receptor-like receptor (PAEL-R), α-synuclein, synaptotagmin XI, cyclin E, tubu-
lin, misfolded dopamine transporters and polyglutamine-repeat proteins. None of
them, however, showed increased abundance in neurons of parkin-lacking mice
[15].
Huntington’s disease is a CAG repeat disorder. The protein Huntingtin is crucial
to the function of neuronal cells. Polyglutamine (polyQ) tracks are expanded in
Huntingtin protein giving rise to a mutant form. Role of ubiquitin system is sus-
pected in Huntington’s disease as the inclusion bodies formed contain ubiquitin, E2/
E3s and proteasomal subunits. Moreover, Huntingtin undergoes ubiquitination, and
overexpression of its mutant form inhibits proteasomal activity, leading to cell cycle
arrest [15].
Defects in ubiquitin system have also been implicated in several types of cancers.
Products of numerous oncogenes and suppressor genes undergo ubiquitination [81,
242, 243].
The protein p53 is a tumour suppressor. It is involved in numerous cell prolifera-
tion and apoptosis pathways, which makes it a good drug target. About 50% of all
human tumours contain p53 mutations and many of those that do not have defects
in other components of p53 network. MDM2 is a RING finger E3 that ubiquitinates
p53 causing its proteasomal degradation. It also inhibits p53 activity by physically
blocking its N-terminal transactivation domain and preventing its nuclear export.
p53 transcriptionally induces MDM2, creating a negative feedback loop. Hence,
p53 activity could be boosted in cancer cells by inhibiting MDM2, helping tumour
suppression. Moreover, MDM2 may also be responsible for degradation of other
antioncogenic proteins.
SCF E3s are multisubunit E3s composed of four components. The subunit of
SCF, CUL1 serves as the scaffold for assembly of the rest of ubiquitin-conjugating
machinery. RBX1 is a RING finger protein interacting with C-terminus of CUL1,
while N-terminus of CUL1 binds to SKP1, and in turn SKP1 interacts with F-box
proteins. At least 68 F-box proteins are found in human genome, each of which
recognizes multiple substrates. SCF E3s interact with different F-box proteins to
recognize different substrates. F-box proteins therefore modulate specificity of SCF
E3s. Mutations in many F-box protein-substrate pairs are involved in cancers. SCF-
FBW7 is an E3 promoting degradation of proteins involved in cell proliferation, like
cyclin E, c-Myc, c-Jun, Notch and sterol regulatory element-binding proteins
(SREBPs). Arginine residues in WD40 repeats of FBW7 interact with phosphode-
grons in these substrate proteins. Mutations targeting these arginines cause many
types of cancers. Mutations in substrate phosphodegron, preventing their recogni-
tion by FBW7, may also cause malignant transformations. SCF-SKP2 is involved in
the degradation of several negative cell cycle regulators like p130 (a protein
566 P. Yadav et al.
26.6 Conclusion
cell’s survivability is evident from lethality of ubiquitin mutations like EP42. And
the high degree of ubiquitin’s sequence and structure conservation across species
underlines its functional relevance. Numerous proteins have evolved to interact with
different surfaces on ubiquitin through various ubiquitin-binding domains. E1, E2
and E3 are the enzymes involved in ubiquitination, and high diversity of E3s is
responsible for the specificity of ubiquitination. Deubiquitinating enzymes do not
just recycle of ubiquitin but confer additional control and specificity to the system.
The simple structure of ubiquitin makes it an ideal system to study protein folding,
and many structural studies have been carried out on it. Proteasomal degradation is
involved in numerous cellular processes including DNA repair, transcription, pro-
tein synthesis and cell signalling. Besides, ubiquitination is involved not just in
proteasomal degradation but lysosomal degradation as well. Owing to its diverse
functions, defects in ubiquitin system have been implicated in several diseases like
Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, various cancers and
microbial infections. Nevertheless ubiquitin proteasome system along with deubiq-
uitinating enzymes offers several prime drug targets which could be exploited in
future to treat these diseases. This has triggered intense research on ubiquitin sys-
tem and makes it an important field of study in our quest to improve human health.
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A Brief Account of Structure-Function
Relationship of the Traditional Cysteine 27
Protease Inhibitor - Cystatin
with a Special Focus on Human Family 1
and 2 Cystatins
Suman K. Nandy
Abstract
Cystatins are well-documented cysteine protease inhibitors with highly con-
served structural folds, distributed in a variety of species. Involvement of cystatin
in various biochemical pathways through regulation of protein degradation
makes it an element of amazing therapeutic possibilities for treatment of a broad
range of diseases. Cystatin superfamily has been divided into four groups: ste-
fins or family 1; cystatins or family 2; kininogens or family 3; and family 4 cys-
tatin. The cystatin superfamily shares a common cystatin fold constituting five
antiparallel β-sheets enfolded around a five-turn α-helix forming a cuneus-
shaped structure that blocks the access of the active site of papain-like cysteine
proteases (CPs). Crystallographic and mutagenesis studies identify three con-
served regions mainly involved in the interaction with papain (C1) family of
CPs, namely, (a) N-terminal region, (b) L1 loop, and (c) L2 loop. Despite sharing
the same structural fold and inhibiting through the same mechanism, cystatin
demonstrates huge variation in inhibitory affinity toward C1 family of CPs.
Relative contribution and sequential dissimilarity of three conserved sites con-
trolled the diverse interaction patterns of cystatins, which in turn determined the
wide-ranging affinity of cystatins toward papain family of CPs. Some of the
members of family 2 cystatins show additional affinity toward legumain family
of CPs through an alternate binding site compared to papains.
Keywords
Cystatin • Cysteine protease inhibitor • Papain • C1 family • Cysteine protease
27.1 Introduction
Cystatin superfamily was first classified into three protein families based on evolu-
tionary relationship, at least a 50% sequence identity [30] with cc [25] and presence
or absence of disulfide bonds [1]. Later with the discovery of new members, the
superfamily was classified into four types, on the basis of number of cystatin-like
domains, disulfide bonds, and physiological localization [31]. Whereas the first
27 A Brief Account of Structure-Function Relationship of the Traditional Cysteine… 581
three types, viz., stefins, cystatins, and kininogens, are cysteine protease inhibitors
(CPIs), the fourth family includes members inactive to CP and with functions uncor-
related to CP inhibition [3, 4, 19, 31, 32]. Type 4 cystatins might serve as a classic
example of loss of certain protein function due to sequential change, while the struc-
tural fold still remains conserved in the course of evolution, owing to much lower
structural mutation sensitivity [33].
Cystatins (also cited as family 2 or class 2 cystatins) are generally 120–125 amino
acid protein (MW 13–14 kDa) with two intra-chain disulfide bonds [37]. In all
mammalian type 2 cystatins, two disulfide bridges are located near C-terminal.
Although cystatins are translated with a signal peptide and meant to found extracel-
lularly [5, 38], they are also detected intracellularly [32, 34]. For example, signifi-
cant uptake of extracellular cystatin C has been reported in human epithelial or
neuroblastoma cells [39, 40]. Cystatins are broadly distributed compared to stefins
and can be found in most of the body fluids [4, 35]. Presence of multigene families
and the effect of polymorphism in coding sequences induce substantial diversity in
sequence and function of the cystatins compared to stefins.
In addition, several posttranslational modifications are found in the members of
this family. Asn-108 of cystatin M/E is glycosylated [41]; in cystatin S phosphoryla-
tion is detected at Ser1 and Ser3 [42, 43]. Cystatin S with variable states of phos-
phorylation is isolated from bronchoalveolar (BAL) and nasal fluids, but the
implication of the variance is yet to be established [44].
and cathepsins are the two key routes of kinin release [45]. Kininogens are of three
types: high-molecular-weight kininogen (HK), low-molecular-weight kininogen
(LK), and an acute phase T-kininogen (TK) isolated only in rats, originated from TK
gene [46, 47]. Both human HK and LK are released from the same gene through
alternative mRNA splicing [48]. Kinin segment is released by kallikreins, and the
rest of the protein has two parts: an N-terminal heavy chain and a C-terminal light
chain linked through a disulfide bond [49]. HK (MW ≈ 90–120 kDa) and LK
(MW ≈ 50–70 kDa) share an identical heavy chain and differ in light chain, which
is much longer in HK [5, 32, 50, 51]. These proteins are equipped with three cys-
tatin domains (D1, D2, and D3) [52] and eight disulfide bonds, six characteristic to
cystatins and two additional between D2 and D3 in the heavy chains of HK and
LK. The D2 and D3 domains possess the conserved QXVXG domain and in turn
exhibit CP inhibitory activity [52]. Although kininogens are glycoproteins, no gly-
cosylation sites are reported within cystatin domains. Both HK and LK bind two
CPs per protein with high affinity including cathepsins and cruzipain [50, 51].
Kininogens apart from being CPI also act as substrates to a variety of serine protein-
ases, thus providing the nickname kininogenases.
The type 4 cystatins (also quoted as family 4 or class 4 cystatins) represent cystatin
homologs devoid of CP inhibitory activity, to wit fetuins [53], histidine-rich glyco-
protein (HRG) [54], and alpha-2-Heremans-Schmid glycoprotein (α-2-HS glyco-
protein), each with two cystatin-like domains [55]. Both glycosylation (N and O)
and phosphorylation are observed in fetuins. Two cystatin-like domains built the
N-terminal region linked through a disulfide bridge to the C-terminal region consist-
ing of a histidine-rich domain surrounded by two proline-rich domains [44]. HRG
exhibits substantial structural similarity with α-2-HS glycoprotein except the latter
lacks the histidine-rich tandem repeat. In the course of evolution, these cystatins
have lost their traditional inhibitory activity toward CPs due to mutations in the
structurally conserved regions and gained new functions [35, 55], namely, inhibi-
tory activity against subtilisin (S8) [56] and astacin/adamalysin (M12) [57] family
of proteases.
The differences in expression profiles and genetic organization of stefins and cys-
tatins indicated early occurrence of neofunctionalization in the cystatin superfamily
[35]. Considering the most of the eukaryotes are unicellular, the specialization of
subcellular localization of cystatins and stefins might be of enormous importance
for their hosts in the early days. It has been evidenced in several taxonomic groups
that loss of any one of the ancestral lineages is compensated by the remaining one
by gaining an additional function, i.e., inhibition of endogenous or exogenous CPs.
For example, loss of stefins in plants is counterbalanced by cystatins, which addi-
tionally inhibits the endogenous CPs. Signal peptides have been found in many
unicellular eukaryotic stefins (e.g., in Hyperamoeba, Capsaspora, and Karlodinium)
which might be useful in novel host defense-related function. In major vertebrate
orthologous families, cystatins are mostly subjected to loss of their traditional inhib-
itory activity and gaining new function in innate immunity. Stefins, kininogens, and
cystatins C, M/E, and F conserved their inhibitory activity toward CPs, while some
of them became more specialized in terms of tissue, cell type, and pathogen [3, 44,
62]. On the other hand, other members of the superfamily inhibit matrix metallopro-
teases (e.g. latexin) [63], gained new role in angiogenesis (e.g. HRG) [64], showed
antimicrobial activity (e.g., HRG, latexin, and cathelicidin) [63–65], and partici-
pated in bone regulation and calcification [63, 66, 67]. The number of modified
27 A Brief Account of Structure-Function Relationship of the Traditional Cysteine… 585
functions of cystatins in the vertebrate families points toward the very diverse pro-
tein-protein interaction module of cystatin fold and its ability to interact with novel
targets which might also be useful in protein designing [3, 44, 62].
27.5.1 Cystatin A
27.5.2 Cystatin B
27.5.3 Cystatin C
27.5.4 Cystatin D
Function:
• Acts as CPI; mainly its activity remains limited to oral cavity.
• Exhibits potential inhibition against cathepsins B, L, H, and S in ascending
order [83].
27.5.5 Cystatin F
27.5.7 Cystatin S
27.5.8 Cystatin SA
Function:
• Cystatin SA acts against the harmful dietary CPs and protects salivary pro-
teins from degradation [44].
• Cystatin SA is a physiologic inhibitor of acid ceramidase that contributes to
regulation of cellular ceramide content [98].
27.5.9 Cystatin SN
Fig. 27.1 Cystatin fold of human cystatin C [155]. Papain (N-terminal, L1 and L2 loop) and
legumain (Asn-39 containing post-α1-helix region and part of AS loop) binding sites are labeled,
and disulfide bonds are shown in stick
Mutational and X-ray crystallographic studies of three CPIs, cc [26, 102, 103], ste-
fin B in complex with papain [29], and stefin A [104], marked three conserved
regions of cystatins distributed at the edges of the cuneal-shaped structure that pre-
vent the access of the active site of C1 family of CPs [32]. The exposed L1 loop
consists of a highly conserved Glu_Val_Gly (QXVXG) region flanked between the
protruding N-terminal, and a second C-terminal β-hairpin loop (L2) with highly
conserved Pro-Trp (PW) residues (Figs. 27.1 and 27.2) constitutes the tripartite,
hydrophobic, cuneal-shaped structure – highly complementary to the active site of
C1 CPs. It should, however, be noted that in human stefins, the PW motif is replaced
by PG in stefin A and PH in stefin B [26, 29] (Fig. 27.2a). The cystatin-CP com-
plexes are largely stabilized by the hydrophobic side-chain interactions. The inter-
actions in the S2 subsite, second binding site at the catalytic site of papain toward
N-terminal starting from scissile bond of the substrate [105], further fortify the
complexes significantly [106]. Cystatins are exosite inhibitors, bind around the cat-
alytic cleft without interacting directly with the active site residues, and block the
entry of substrate [107]. This marks a fundamental difference in mechanism of inhi-
bition of cystatins in contrast to serine proteases and their inhibitors [20].
27 A Brief Account of Structure-Function Relationship of the Traditional Cysteine… 591
Fig. 27.2 Alignment of human family 1 and 2 cystatins. Disulfide bonds, conserved regions, and
reactive sites for papain (G, QXVXG, and PX/PW) and legumain (N) binding are marked
Despite being the exosite inhibitors, cystatins are able to distinguish between endo-
peptidases and exopeptidases, by recognizing the differences in the active site sce-
nario of these enzymes. The easily accessible catalytic site clefts of endopeptidases,
like cathepsins K, L, and S and papain, readily accommodate cystatins. The more
rigid β-hairpin loops (L1 and L2) participate in initial binding, and the flexible
N-terminal region stabilizes the cystatin-endopeptidase complex in the second step
by reducing the dissociation rate (Fig. 27.3a) [108]. This two-step binding is further
demonstrated in stefin A-papain complex, where N-terminal interaction is preceded
by the two β-hairpin loops binding in the catalytic core of papain [109].
In contrast, exopeptidase cathepsin B also illustrates a two-step binding, but in
reverse order, initial weak interactions through N-terminal segment of cystatin A or
C trigger a conformational change by displacing the occluding loop of cathepsin B
and in turn disrupting a major salt bridge between His110 and Asp22 to facilitate
β-hairpin loops binding in the catalytic site of the endopeptidase [108, 110–112].
Cystatin C displaces the loop more efficiently than stefin A [111]. Similar confor-
mational change is also observed in cathepsin H-stefin A complex, where the
592 S.K. Nandy
Fig. 27.3 Mechanism of inhibition – two-step binding. (a) Endopeptidase (papain-stefin B com-
plex, PDB ID-1STF) and (b) exopeptidase (cathepsin H-stefin A complex, PDB ID-1NB3) com-
plex. Papain and cathepsin H are shown in green, stefins A and B are shown in purple, and cathepsin
H mini-chain is red in color
N-terminal residue of stefin A adopted a hooklike form and marginally displaced the
cathepsin H mini-chain causing conformational distortion, which becomes charac-
teristic for exopeptidases (Fig. 27.3b) [113].
Thus the comparison of the kinetics of binding shows that N-terminal segment
interacts differently with endo- and exopeptidases. For endopeptidases the
N-terminal stabilizes the complex by reducing the dissociation rate of the complex.
In exopeptidases, N-terminal region of cystatins contributes to the association rate
of the inhibitors, whereas the conformational change determines the rate-limiting
step (Fig. 27.3). Although the rate of interaction of β-hairpin loops of cystatins
remains almost the same with exo- and endopeptidases, the steric hindrance caused
by occluding loops in carboxypeptidases (e.g., cathepsins B, X) and propeptide
regions in aminopeptidases (e.g., cathepsins H, C) decreases the overall affinity of
cystatins toward exopeptidases [20].
The inhibitory interactions of cystatins of the first three families toward few selected
members of C1 family of CPs are summarized in Table 27.2. Human cystatin C
(hcc) and cc are the most potent inhibitors, whereas SD-type cystatins are the back
benchers. Exopeptidases report lower binding affinity for cystatins in comparison to
endopeptidases [116] (Table 27.2). Moreover, the affinities of the cystatins differ
remarkably, from μM (10−6) to pM (10−12) range, even toward a particular CP despite
sharing the same cystatin fold and mechanism of inhibition (Table 27.2). These
huge affinity differences between CPs cannot only be attributed to the differences in
the two-step binding between endo- and exopeptidases [7, 117, 118]. Further, S-type
cystatins illustrate ≈90% sequence similarity, and still cystatin S is recognized as
considerably poorer inhibitor than the other two (Table 27.2). It may be viewed as a
gradual specialization of SD-type cystatins in order to protect mucosa from foreign
27 A Brief Account of Structure-Function Relationship of the Traditional Cysteine… 593
Table 27.2 Binding affinity of cystatins toward papain (C1) family of cysteine proteases
Ki or Ki, app* (nM)
Inhibitor Papain Cathepsin B Cathepsin H Cathepsin L
Stefin A 0.019 8.2 0.31 1.3
Stefin B 0.012 73 0.58 0.23
Cystatin C 0.00001 0.27 0.28 <0.005
Cystatin D 1.2 >1000 7.5 18
Cystatin E/M 0.39 32 n.d. 1.78#
Cystatin F 1.1 >1000 n.d. 0.31
Cystatin S 108 >6000* >6000* >6000*
Cystatin SA 0.32 >6000* >6000* 7300*
Cystatin SN 0.016 19 900* 400*
Chicken cystatin 0.005 1.7 0.06 0.019
L-kininogen 0.015 600 0.72 0.017
Inhibitor constants [Ki] for human cystatins [3, 5], chicken cystatins [5], *apparent inhibitor con-
stant (Ki, app.*) [114]; # [95]; n.d. not determined
Note: Determination of the equilibrium dissociation constant provides a measure of the affinity of
an inhibitor for an enzyme. In most cases, cystatins bind so tightly that Ki cannot be measured
directly, so it is determined as the ratio of the separately measured rate constants or as the inhibi-
tion constant Ki, obtained by measurement of the substrate-dependent apparent inhibition constant
Ki, app, followed by a mathematical correction for the presence of the inhibitor [115]
CPs without interfering with the endogenous CPs in a great extent. Collectively, the
diverse role of the three conserved regions to the particular CP-cystatin binding and
sequential variation in the binding interface may be the major determinant of the
dissimilarities in inhibition constant (Ki) values, i.e., for the free energy change in
complex formation between different cystatins and CPs [119].
Molecular docking studies of cc and papain point toward the conserved Gly9 resi-
due that might provide the additional flexibility to the N-terminal region to maxi-
mize the binding contribution [26]. N-terminally truncated forms of cc showed
5000- to 10,000-fold lower affinities for papain [120] and further confirm the vital
role of the region in binding [121].
Consistent with this model, mutation in conserved Gly11 residue of cystatin C
(Fig. 27.2b) decreases binding affinity, and the size of the side chain of the substi-
tuted amino acid determines the magnitude of the effect [122]. Truncated forms of
these mutated cystatin C show slightly lower affinities, in contrast to truncated wild-
type cystatin C forms where binding affinity decreases significantly and further
affirms the importance of conserved Gly residue. Inconsistent reduction of binding
affinity of modified cystatin C is observed toward cathepsins, dubious reports are
obtained about the extent of decrease [123, 124]. Apart from the conserved Gly11,
Val10 is found to contribute largely to the binding of cathepsins B, H, L, and S;
Arg8 and Leu9 are also known to do minor, enzyme-specific contributions to
594 S.K. Nandy
binding affinity (Fig. 27.2b). Effect of some mutations are found to be highly
enzyme dependent, for example, Val10Trp substitution causes about twofold
decrease in cathepsin S affinity but triggers a tenfold increase in cathepsin L affinity
[124, 125]. Thus, N-terminal Arg8, Leu9, and Val10 determine the specificity of
cystatin C binding, and by substituting these residues suitably, cathepsin-specific
cystatins might be designed. In fact, N-terminal cystatin C decapeptide substitution
in place of the first three residues of stefin A results in about 15-fold increase in
binding affinity in stefin A-cathepsin B complex. The higher binding affinity is pre-
dominantly due to an increase in overall association rate constant resulting from
better displacement of cathepsin B occluding loop by the elongated N-terminal.
This substitution neither affect the dissociation rate in cathepsin B-stefin A interac-
tion nor the modified stefin A’s binding capability toward endopeptidases [126].
Truncated forms of human salivary cystatins show the N-terminal region is not
of that importance for inhibition [127–130]. Three rat cystatin S forms of varying
N-terminal length differ only moderately (<50-fold) in their inhibitory activity
toward papain and ficin [131]. Two conserved consecutive Gly to Ala mutation
(Fig. 27.2b) in human cystatin SN also illustrate only minute change in binding
affinity toward papain [132]. In contrast a six-residue truncated form of cystatin SA
shows 1000-fold poorer inhibitory activity to cathepsin L [127]. A cystatin D form
lacking all residues including conserved Gly reported to be virtually inactive against
cathepsins H, L, or S (Ki > 1 mM) pointed two to four orders of contribution of
N-terminal in binding affinity in an enzyme-specific manner. Exchange of
N-terminal regions between S-type cystatins and human cystatin C/D only moder-
ately alters binding affinities for cathepsins [133]. Thus we can say N-terminal
region of SD-type cystatin is more important for cathepsin binding than papain.
The requirement of N-terminal region of human stefin A, not only for specificity
of binding toward CPs but also for maintenance of conformational integrity of tri-
partite reactive wedge, is well documented [134]. Even the smallest replacement of
evolutionarily conserved Gly-4 residue, by Ala (Fig. 27.2a), causes ≈103-fold
decrease in binding affinity for papain and cathepsin B, while cathepsin L stays
least affected [135]. N-terminally truncated variants, sequential deletion mutant of
the first three amino acids, show progressive decrease of cystatin A affinity and
estimate about 40% contribution in total binding free energy of cystatin A to papain
and cathepsin B. Pro3 and Ile2 are the most prominent contributor in the interaction.
For stefin B, in contrast to the previous observations [120], Pol et al. have described
that N-terminal region is accountable for 12–40% of the total binding affinity in
stefin B-papain and stefin B-cathepsin B and L complexes; cathepsin H remains
least affected [136]. Cys3 of stefin B contributes most in papain, cathepsin L and H
inhibition; the contribution of the rest of the residues of N-terminal varies with the
concerned CP [136, 137].
An earlier study of oryzacystatin-papain interaction indicates that N-terminal or
C-terminal truncation of oryzacystatin fails to affect its inhibitory activity [138]. In
the contrary to this report, the latter studies notify the necessity of N-terminal
region, in particular the highly conserved Gly10 residue of oryzacystatin in papain
inhibition [139]. A mature form of sunflower cystatin (SCA), lacking 15 amino
27 A Brief Account of Structure-Function Relationship of the Traditional Cysteine… 595
acids at the N-terminus, illustrates a higher dissociation rate constant and lower
affinity toward papain in comparison to the full-length recombinant SCA (rSCA)
due to the lack of additional stabilization of the complex by the elongated N-terminal
region [140]. Another sunflower cystatin SCB also evidences the instrumental role
of the first four N-terminal amino acids (IPGG) manifesting the inhibitory activity
toward papain [141].
The highly conserved Pro-Trp motif of cystatin L2 loop contributes to the free
energy of binding by lowering the dissociation rate in an enzyme-specific manner.
For cc-cathepsin B and cc-papain complexes, L2 loop contributes moderately but
rather lowly in case of cathepsin L, where the N-terminal and L2 loop are the major
determinant of the interaction [119]. Modification in conserved Trp residue of cc
lowers its affinity toward papain by 105-fold [147].
Substitution of Trp106 with Gly in hcc results decrease in binding affinity for
actinidin, papain, and cathepsins B and H by 300- to 900-fold; a two-order increase
in inhibition constant is also reported in case of cathepsin B inhibition for Trp106Ser
mutation though cathepsins H and L remain less affected [143, 148, 149]. These
studies estimate L2 loop is responsible for 20–30% of the total binding affinity
[143, 148]. PW motif substitutions by Gly in cystatin SN show a 100-fold decrease
in binding free energy in case of cathepsin C inhibition but of negligible interest for
papain binding [132].
596 S.K. Nandy
Cystatins can also act as inhibitor of mammalian legumain – a family C13 CP with
dual protease-ligase activity, evolutionarily unrelated to C1 family of CP and
equipped with distinctly different catalytic site and structural fold [79]. Dall et al.
[151] recently crystallographically evidenced the much speculated inhibition of
legumain by cystatin M/E through dual interaction of legumain reactive center loop
(RCL – residues 38–43 in post-α1-helix region, hcc numbering) and legumain
exosite loop (LEL – residues 74–94 in AS loop region, hcc numbering) (Figs. 27.2
and 27.4). RCL exhibits a canonical, substrate-like binding mode, typical within the
type 2 cystatin family, while overall hydrophobic LEL serves as exosite binder, and
Asn39 residue (hcc numbering) plays a very important role in ligase activity of
legumain as hypothesized previously [151, 152]. Cystatin M/E emerges as the most
potent inhibitor of legumain (Ki = 0.0016 nM), and cystatins C and F come next
(Ki = 0.2 nM and 10 nM with pig legumain, respectively) [152]. Cystatin D does not
show any inhibitory activity toward legumain despite the presence of Asn around
this region, possibly because of the positively charged Lys residue in the very next
position, instead of the highly conserved negatively charged residue in cystatins
(Fig. 27.2b). The S-type cystatins are devoid of Asn in the post-helix region and
appear to be non-inhibitor of legumain (Fig. 27.2b).
Cystatin SN is reported to bind stably in a second site of papain, other than its
active site, without affecting the proteolytic activity of the enzyme. Even when the
catalytic site is preoccupied with an irreversible inhibitor like E-64, binding of cys-
tatin SN at the second site is not hampered. Unfortunately the details of these inter-
actions are still unknown [153].
27 A Brief Account of Structure-Function Relationship of the Traditional Cysteine… 597
Fig. 27.4 Binding conformation of legumain-cystatin M/E complex (PDB ID: 4N6N). (a)
Legumain (left)-cystatin M/E (right) interaction is mediated by the reactive center loop (RCL in
yellow) containing Asn39 and the legumain exosite loop (LEL in purple). (b) Binding site sce-
nario. Amino acid residues of legumain and cystatin M/E are labeled in black and blue,
respectively
27.9 Conclusion
To summarize, the mutation, substitution, deletion, and grafting studies point toward
the differential inhibitory approaches of cystatins toward CPs. The N-terminal seg-
ment and QXVXG loop make the most significant contributions in cc-papain (≈87%
of binding energy) and cc-cathepsin B complexes; in case of cathepsin L complexes,
the N-terminal holds the key [119, 120]. Two β-hairpin loops are jointly recognized
by Björk et al. [143] for the major part (60–90%) of human cystatin C affinity
toward endopeptidase papain and exopeptidase cathepsin B; in contrast to Hall
et al.’s report [124], the side chains of N-terminal segment and the Trp residue of L2
loop are mainly responsible for cystatin C affinity toward endopeptidase cathepsin
L and exopeptidase cathepsins B and H. Arg8, Leu9, and Val10 of hcc are recog-
nized for their contribution in selectivity and affinity [124, 125]. In case of SD-type
cystatins, the first β-hairpin loop plays the major role in papain inhibition, whereas
all three conserved regions chip in for cathepsin CPs [44, 2002]. In human family 1
cystatins, the flexible N-terminal and the PW motif of L2 loop together contribute
most of the binding energy to papain and cathepsin B. The highly conserved
Leu73 in type 1 cystatins, Cys3, Tyr97 for stefin B and Ile2, and Pro3 for stefin A
are the key residues for inhibition [29, 108, 135–137, 148, 150]. All three conserved
regions of plant cystatins participate in papain inhibition, although N-terminal and
first hairpin loop plays the central role [154].
598 S.K. Nandy
Acknowledgment I would like to thank Dr. Alpana Seal, Retired Professor in Biophysics,
Department of Biochemistry and Biophysics, University of Kalyani, for her help and encourage-
ment. Thanks are also due to Bioinformatics Centre, University of Kalyani and Bioinformatics
Centre, NEHU, Tura campus.
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Solid Support Synthesis
of a Dnp-Labeled Peptide for Assay 28
of Matrix Metalloproteinase-2
Abstract
Herein, synthesis of a Dnp-labeled peptide, Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-
D-Arg-CONH2, is described by the Fmoc solid-phase method. Post-synthesis of
the peptide was purified by reversed-phase HPLC. The purity of the peptide was
determined by nuclear magnetic resonance total correlation spectroscopy (NMR
TOCSY), and the validity of the peptide as a specific synthetic substrate for
matrix metalloproteinase-2 (MMP-2) was also assessed by measuring the spe-
cific activities of the MMP-2 using the peptide as a substrate. It was found to be
a suitable substrate with respect to MMP-2 and correlated well with the
[14C]-gelatin degradation assay of MMP-2. Pretreatment of the pure MMP-2
with tissue inhibitor of metalloproteinase-2 (TIMP-2), a specific endogenous
inhibitor of MMP-2, prevented both the Dnp-labeled peptide substrate degrada-
tion and the [14C]-gelatin degradation. Thus, the Dnp-labeled peptide can be used
as a synthetic substrate for in vitro assay of MMP-2.
A. Mandal
Department of Biochemistry and Biophysics,
University of Kalyani, Kalyani 741235, West Bengal, India
Department of Physiology, University of Arizona,
1501 N Campbell Ave, Tucson AZ85724, USA
A. Maiti
Department of Biophysics, Bose Institute, P-1/12 C.I.T. Scheme VIIM, Kolkata 700054, India
Department of Biochemistry and Molecular Biology and Greenebaum Cancer Center,
University of Maryland School of Medicine, Baltimore, MD 21201, USA
T. Chakraborti • S. Chakraborti (*)
Department of Biochemistry and Biophysics,
University of Kalyani, Kalyani 741235, West Bengal, India
e-mail: saj_chakra@rediffmail.com
Keywords
Dnp-labeled peptide • Solid-phase peptide synthesis • MMP-2 • TIMP-2 • HPLC
• TOCSY
Abbreviations
28.1 Introduction
28.2.1 General
MBHA resin, HOBt, PyBOP, and Fmoc-protected amino acids except Fmoc-
protected proline were obtained from Novabiochem (Darmstadt, Germany);
610 A. Mandal et al.
Dnp-labeled Fmoc-protected proline was obtained from Sigma Chemical Co. (St.
Louis, MO, USA); and DIPEA, TFA (spectrofluorometric grade), water (HPLC
grade), acetonitrile, DMF, piperidine, TNBS, ethane dithiol, anisole, phenol, diethyl
ether, t-amyl alcohol, and glacial acetic acid were obtained from E. Merck
(Darmstadt, Germany). Molecular sieve 4a was obtained from Delta Adsorbents
(Roselle, IL, USA). Pure MMP-2 and TIMP-2 were obtained from Chemicon
International Inc. (Temecula, CA, USA). All other chemicals used were of analyti-
cal grade and purchased from E. Merck (Darmstadt, Germany).
The solid-phase peptide synthesis was done by following the method described by
Merrifield et al.[3] with some modifications [6]. The Fmoc chemistry for the solid-
phase peptide synthesis was adopted from Sheppard et al. [20–22]. For the synthesis
of the peptide of interest, amide MBHA resin was chosen. To achieve the targeted
quantity of the final synthesis, each constituent amino acids used were in excess (3
times of the final concentration). To synthesize 0.05 mmole peptide, 0.15mmole of
individual amino acids were used.
After equilibration at 4 °C, the column was allowed to attain room temperature
(23–25 °C) to match with the operational temperature of the peptide synthesizer
(Biolynx 4175 (LKB Biochrom, Cambridge, UK). Unless stated otherwise, the
entire procedure of the solid support peptide synthesis was performed in an inert
nitrogen atmosphere. Near-zero moisture content of the solvents used in the synthe-
sis process has been ascertained by passing the solvents through molecular sieve 4a.
The column was washed with DMF for 10 min (3.5mlmin−1) to prime the entire
system. Following this priming step, 20% piperidine in DMF was run through the
column with the same flow rate for 5 min, and finally the column was again washed
with DMF for 15 min.
28 MMP-2 Assay by Dnp-Labeled Peptide 611
Since the peptide is synthesized from C-terminus to N-terminus, the first amino
acid to be coupled was D-Arg-OH following the target sequence. The complete
availability of the N-terminus of the resin was confirmed by trinitrobenzenesulfonic
acid (TNBS) test before loading the column with the first Fmoc-D-Arg-OH.
Freshly prepared TNBS reagent was used. DMF (450 μl) was taken in a micro-
centrifuge tube protected from light, and to it DIPEA (50 μl) was added. In the
resulting solution, TNBS (50 μl) was added. The resulting reddish orange solution
was used as TNBS reagent to check the availability of N-terminus of the resin to
couple with the first amino acid. A small quantity of the pre-swollen and primed
resin from the peptide synthesizer column was carefully withdrawn and placed in a
small wide-mouth glass vial. The resin was washed with a sequential addition (2 ml
each) of DMF, t-amyl alcohol, glacial acetic acid, t-amyl alcohol, DMF, and diethyl
ether. Freshly prepared TNBS reagent (500 μl) was added to the resin and allowed
to react for 1 min at room temperature. The resin was washed repetitively with
DMF and finally washed twice with diethyl ether. A bright orange color of the
bead indicates the presence of free amino group available for coupling of the first
Fmoc-D-Arg-OH.
Fmoc-D-Arg-OH (98 mg, MW 648.8), PyBOP (80 mg, MW 520.3), and HOBt
(20 mg, MW 135.13) were taken in a clean and dry glass vial. To it DMF (500 μl)
was added and the entire material was carefully dissolved. The final volume of this
amino acid reagent was made up to 2 ml with the addition of requisite amount of
DMF. Before loading the column with Fmoc-amino acid reagent, the eluate of the
column was carefully monitored by a Nova spectrophotometer (Biochrom,
Holliston, MA, USA) at 360 nm. Before loading the column, the absorbance at
360 nm read negative. The Fmoc-D-Arg-OH reagent was then loaded into the col-
umn slowly to minimize the possibility to trap any air bubble. DIPEA (50 μl) was
directly applied over the column. The loading flow rate of the column was 2 ml/min.
The absorbance at 360 nm increased gradually from negative to positive values,
indicating amino acid coupling reaction with the resin. After the completion of the
loading, the column was recirculated (3.5 ml/ min) for another 5 h for complete
coupling reaction to maximize the yield. Next, TNBS test was performed to ascer-
tain completion of the coupling as indicated by a negative result. The column was
again washed (3.5 ml/ min) with DMF (10 min), 20% piperidine (10 min), and
finally DMF (15 min). TNBS test was again performed as described. A positive test
indicated the presence of free N-terminus of the first coupled amino acid, an indica-
tion for the readiness of the resin for next amino acid coupling.
612 A. Mandal et al.
The second and subsequent amino acids (0.05mmole each) were loaded following
the method described for the first amino acid. In case of glycine, Pentaflurophenyl
ester (OPfp) ester form was used keeping other conditions unaltered. After completion
of the synthesis of the peptide, it was cleaved from the solid resin by the following
method.
The synthesized peptide from the solid resin support was cleaved following the
method of Guy et al. [23]. Briefly, the resin-bound peptide was withdrawn and taken
in a sintered glass funnel connected to a low vacuum pump via Buchner flask. The
resin was washed in the sequence as follows, DMF, t-amyl alcohol, acetic acid,
t-amyl alcohol, DMF, and diethyl ether followed by drying under vacuum and sub-
sequently taken for the peptide cleavage. The peptide cleavage reagent consists of
TFA, ethane dithiol, anisole, and phenol (94:2:2:2 v/v) and is prepared fresh. The
peptide cleavage reagent was added slowly to the dried peptide attached to resin
over the Buchner funnel, and the filtration was continued. This step was repeated
three times to ascertain complete recovery of the cleaved peptide. The pooled com-
bined filtrate was transferred to a round-bottomed flask and evaporated to dryness
using a refrigerated rotary evaporator (Savant, Holbrook, NY, USA). The peptide
was purified further by adding diethyl ether which helps removal of organic con-
taminants. Diethyl ether was aspirated carefully to get rid of contaminants. The
residual diethyl ether was evaporated with rotary evaporator. The entire procedure
for contaminant removal using diethyl ether was repeated 7–8 times, and finally the
dried peptide was stored at 70 °C for purification.
The peptide was purified by HPLC following the method of Miranda et al. [24] with
some modifications. Preparative reversed-phase HPLC was performed with Waters
510 solvent delivery system (Waters Corporation, Milford, MA, USA) using Waters
Baseline 810 software at an operating pressure of 4 × 1000PSI (sample injection
volume, 200 μl). The peptide solution was thoroughly filtered through Whatman
filter paper (0.45 μm) by pressure filtration prior to loading to avoid particulate
contamination. The chromatographic separation was monitored using a Waters 484
Lambda Max HPLC UV-Visible Detector (Waters Corporation, Milford, MA, USA)
at 210 nm. Chromatographic separations were achieved with a 1% min−1 linear
gradient of buffer B in A (A = 0.1% TFA in HPLC grade water; B = 60% CH3CN in
A) over 55 min at a flow rate of 8 ml min−1 using Waters C18 preparative reversed-
phase HPLC (RP-HPLC) column (10 μm, 2.2 cm × 25 cm) (Waters Corporation,
Milford, MA, USA).
28 MMP-2 Assay by Dnp-Labeled Peptide 613
The 210 nm peak fraction (resolved at 35.35 min) was collected in a thoroughly
cleaned and dried round-bottomed flask, evaporated to dryness by refrigerated
rotary evaporator (Savant, Holbrook, NY, USA) for 3 h, solubilized with HPLC
grade water, and lyophilized for future use.
gelatin was precipitated by the addition of 10% TCA (60 μl). After cooling down on
ice for 10 min, samples were centrifuged at 10,000 × g for 10 min, and the radioac-
tivity in the supernatant was determined [29].
To study the effect of TIMP-2 on MMP-2, the enzyme was preincubated with
TIMP-2 (5 μg/ml) for 1 h at 37 °C after APMA activation prior to [14C]-gelatin
degradation.
The general scheme of the solid support peptide synthesis was shown in Fig. 28.1.
The Dnp-labeled peptide was purified by RP-HPLC. The peak fractions of the
HPLC run were collected by measuring the absorbance at 210 nm (Fig. 28.2). The
HPLC purified and lyophilized peptide was analyzed by 1D 1H NMR and TOCSY
(Fig. 28.3). The outcome of the 1H 1D NMR and the TOCSY was shown in
Fig. 28.3. The presence of the purified peptide in the 35.35 min peak (Fig. 28.2)
was also confirmed by the TOCSY spectra (Fig. 28.3). The 1D spectra were
assigned using the backbone amide 1H chemical shift (ppm) for the random coil
[30]. The 1D 1H NMR spectra confirm the presence of the following amino acids
Pro, Gln, Gly, Ile, Ala, Gln, and D-Arg (Fig. 28.3). The peak at the 8.73 ppm cor-
responds to Dnp. From the data it was found that all amino acids were incorpo-
rated in the synthesized peptide and since the peptide was synthesized in the solid
support method so the chance of misincorporation of any amino acid is ruled out
and hence all of the amino acids were added in the sequence Dnp-Pro-Gln-Gly-
Ile-Ala-Gly-Gln-D-Arg-CONH2 (Fig. 28.3). The presence of proline was not vis-
ible due to the absence of any αH atom (Fig. 28.3). The 35.35 min peak was
intense yellow colored and confirmed as purified peptide (Figs. 28.2 and 28.3) and
validated as an MMP-2 substrate by degradation of the peptide by MMP-2 which
was also compared with [14C]-gelatin degradation studies (Fig. 28.4a, b).
Pretreatment of the pure MMP-2 with TIMP-2 prevents both the Dnp-labeled pep-
tide substrate degradation and [14C]-gelatin degradation (Fig. 28.4c, d). Thus, it
can be concluded that the Dnp-labeled peptide synthesized in the solid-phase pep-
tide synthesis method can be effectively used as a synthetic substrate for MMP-2
(Fig. 28.4).
28 MMP-2 Assay by Dnp-Labeled Peptide 615
R1
Piperidine
R1
Fmoc-NH C COOBt
H
R2 R1
TFA treatment
Rn Rn-1 R2 R1
H2N C CO N C CO N C CO N C COOH
H H H H H H H
AAn: N CHRn CO
H
Fig. 28.1 Schematic representation of the individual reaction steps associated with solid-phase
peptide synthesis employing the use of Fmoc-protected amino acids
Fig. 28.3 The TOCSY spectra of the HPLC-purified peptide at 27 °C. The 1D 1H NMR spectra
confirm the presence of the amino acids Pro, Gln, Gly, Ile, Ala, Gln, and D-Arg. The peak at the
8.73 ppm corresponds to Dnp. Proline was not detected in this spectra due to the absence of αH atom
28 MMP-2 Assay by Dnp-Labeled Peptide 617
A 2500 B
1800 * *
2000
1400 * *
MMP-2 activity
1200 * 1500 *
1000
800 * 1000 *
600
400 500
200
0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time of incubation (min) with APMA (2 mM) Time of incubation (min) with APMA (2 mM)
1.4 C 30 D
MMP-2 activity (mU/mg)
1.2
0.2 5
0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time of incubation (min) with APMA (2 mM) Time of incubation (min) with APMA (2 mM)
Fig. 28.4 Relative degradation study of Dnp-labeled peptide with [14C]-gelatin by APMA
(2 mM)-activated pure MMP-2 (0.1 μg) at different times in the absence and presence of pure
TIMP-2 (5 μg/ml). Pure MMP-2 was activated with APMA at 37 °C for indicated times. Then, the
APMA-activated MMP-2 was (a) incubated with the Dnp-labeled synthetic peptide ( ) for 1 h
at 37 °C, (b) incubated with the [14C]-gelatin ( ) for 1 h at 37 °C, (c) preincubated with TIMP-2
(5 μg/ml) for 1 h at 37 °C prior to synthetic substrate degradation ( ), and (d) preincubated
with TIMP-2 (5 μg/ml) for 1 h at 37 °C prior to [14C]-gelatin degradation ( ). The degree of
hydrolysis of the synthetic peptide substrate was determined by measuring the absorbance of the
organic layer at 365 nm. 1 U gelatinase catalyzes the hydrolysis of 1 μmol Dnp-labeled pep-
tide/30 min at 37 °C. The [14C]-gelatinolytic activity was determined by taking the radioactive
count and is expressed as cpm/mg protein/30 min. Results are mean ± SE (n = 4). *p < 0.001
compared with basal value
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