Posttranslational Modifications

Protein-damaging stresses, such as exposure to elevated temperature, alcohol, and heavy metals and microbial infections, increase the amount of misfolded proteins in cells, which may lead to formation of toxic protein aggregates. In order to counteract detrimental effects of proteotoxic stresses, all organisms have developed a specific response, called the heat shock response (HSR). Upon the HSR activation, synthesis of heat shock proteins (HSPs) increases. HSPs function as molecular chaperones to aid in the refolding of misfolded proteins and prevent formation of protein aggregates. The inducible expression of HSP genes is mediated by heat shock factors (HSFs). Mounting the HSR involves trimerization, nuclear accumulation, gain of DNA-binding and transactivation capacity of HSF1 and coincides with extensive post-translational modifications of HSF1. However, the importance of these modifications on HSF1 activity has remained elusive. This PhD thesis describes the functional impact of stress-inducible phosphorylation of HSF1 on the HSR. While HSF1 phosphorylation upon stress is not required for the HSR activation, it defines the onset of the HSR and fine-tunes the magnitude of the response. Moreover, a chaperone co-inducer BGP-15 was found to augment the HSR, due to its ability to inhibit histone deacetylases (HDACs). BGP-15 as an HDAC inhibitor increases chromatin accessibility at multiple genomic loci including HSP genes and accelerates HSF1 activation in the event of proteotoxic stress. Furthermore, HDAC inhibitors can function as chaperone co-inducers and have cytoprotective effect for cells exposed to stress. Taken together, this study provides valuable information on molecular mechanisms regulating the HSR, which can serve as a basis for further development of pharmaceuticals aimed to activate, enhance or repress the HSR.

Chromosome 21, triplicated in Down Syndrome, contains several genes that are thought to play a critical role in the development of AD neuropathology. The overexpression of the gene for the amyloid precursor protein (APP), on chromosome 21, leads to early onset beta-amyloid (Aβ) plaques in DS. In addition to Aβ accumulation, middle-aged people with DS develop neurofibrillary tangles, cerebrovascular pathology, white matter pathology, oxidative damage, neuroinflammation and neuron loss. There is also evidence of potential compensatory responses in DS that benefit the brain and delay the onset of dementia after there is sufficient neuropathology for a diagnosis of AD. This review describes some of the existing literature and also highlights gaps in our knowledge regarding AD neuropathology in DS. It will be critical in the future to develop networked brain banks with standardized collection procedures to fully characterize the regional and temporal pathological events associated with aging in DS. As more information is acquired regarding AD evolution in DS, there will be opportunities to develop interventions that are age-appropriate to delay AD in DS.

In biological applications, the tandem mass spectrometry is a widely used method for determining protein and peptide sequences from an “in vitro” sample. The sequences are not determined directly, but they must be interpreted from the mass spectra, which is the output of the mass spectrometer. This work is focused on a similarity-search approach to mass spectra interpretation, where the parameterized Hausdorff distance (dHP) is used as the similarity. In order to provide an efficient similarity search under dHP, the metric access methods and the TriGen algorithm (controlling the metricity of dHP) are employed. Moreover, the search model based on the dHP supports posttranslational modifications (PTMs) in the query mass spectra, what is typically a problem when an indexing approach is used. Our approach can be utilized as a coarse filter by any other database approach for mass spectra interpretation.

In eukaryotes, heme attachment through two thioether bonds to mitochondrial cytochromes c and c1 is catalyzed by either multisubunit cytochrome c maturation system I or holocytochrome c synthetase (HCCS). The former was inherited from the alphaproteobacterial progenitor of mitochondria; the latter is a eukaryotic innovation for which prokaryotic ancestry is not evident. HCCS provides one of a few exemplars of de novo protein innovation in eukaryotes, but structure-function insight of HCCS is limited. Uniquely, euglenozoan protists, which include medically relevant kinetoplastids Trypanosoma and Leishmania parasites, attach heme to mitochondrial c-type cytochromes by a single thioether linkage. Yet the mechanism is unknown, as genes encoding proteins with detectable similarity to any proteins involved in cytochrome c maturation in other taxa are absent. Here, a bioinformatics search for proteins conserved in all hemoprotein-containing kinetoplastids identified kinetoplastid cytochrome c synthetase (KCCS), which we reveal as essential and mitochondrial and catalyzes heme attachment to trypanosome cytochrome c. KCCS has no sequence identity to other proteins, apart from a slight resemblance within four short motifs suggesting relatedness to HCCS. Thus, KCCS provides a novel resource for studying eukaryotic cytochrome c maturation, possibly with wider relevance, since mutations in human HCCS leads to disease. Moreover, many examples of mitochondrial biochemistry are different in euglenozoans compared to many other eukaryotes; identification of KCCS thus provides another exemplar of extreme, unusual mitochondrial biochemistry in an evolutionarily divergent group of protists.

Two-dimensional remains one of the main experimental approaches in proteome analysis. However, comigration of protein leads to several limitations: lack of accuracy in protein identification , impaired comparative quantification, and PTM detection. We have optimized a third additional step of in-gel separation to alleviate comigration associated drawbacks. Spot resolution is strikingly improved following this simple and rapid method and the positive impact on protein and peptide identification from MS/MS data, on the analysis of relative changes in protein abundance, and on the detection of PTM is described.

Ribonucleoprotein complexes involved in pre-mRNA splicing and mRNA decay are often regulated by phosphorylation of RNA-binding proteins.  Cells use phosphorylation-dependent signaling pathways to turn on and off gene expression.  Not much is known about how phosphorylation-dependent signals transmitted by exogenous factors or cell cycle checkpoints regulate RNA-mediated gene expression at the atomic level.  Several human diseases are linked to an altered phosphorylation state of an RNA binding protein.  Understanding the structural response to the phosphorylation “signal” and its effect on ribonucleoprotein assembly provides mechanistic understanding, as well as new information for the design of novel drugs.  In this review, I highlight recent structural studies that reveal the mechanisms by which phosphorylation can regulate protein-protein and protein-RNA interactions in ribonucleoprotein complexes.

Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.

Temperature miRNA-21 miRNA-155 miRNA-206 Atlantic cod (Gadus morhuaL A B S T R A C T Extracellular vesicles (EVs) are released from cells and carry protein and genetic cargo involved in cell communication. EVs isolated from bodyfluids, including plasma and serum, can also be used as markers of patho-physiological changes. Peptidylarginine deiminases (PADs) are phylogenetically conserved enzymes with physiological and pathophysiological roles and cause post-translational protein deimination. This can affect function of target proteins and deimination is also linked to EV release. Atlantic cod (Gadus morhua L.) reared at 4 °C and 9 °C respectively for 18 months, were here assessed for changes in serum derived EVs, including analysis of deiminated protein and micro-RNA cargo markers related to stress and growth. We found that cod reared at 9 °C showed significantly reduced numbers of EVs in serum, compared to cod reared at 4 °C. Some deiminated protein targets, including complement component C3, were found to be considerably higher in EVs of cod reared at 4 °C. Proteomic analysis revealed further differences in deiminated protein targets in EVs isolated from sera of the two temperature groups. Whole cod sera from the two temperature groups furthermore showed differences in dei-minated protein targets, including C3, CRP and histone H3, which is a marker of neutrophil extracellular trap formation. MicroRNAs related to inflammation (miRNA-21) and stress (miRNA-155) were elevated in both total cod serum and serum-derived EVs from the 9 °C group, while the growth-related miRNA-206 was higher in the 4 °C group. Our findings highlight EVs as novel biomarkers to assess fish health in response to environmental rearing temperature.

Oxygen deprivation and infection are major causes of perinatal brain injury leading to cerebral palsy and other neurological disabilities. The identification of novel key factors mediating white and grey matter damage are crucial to allow better understanding of the specific contribution of different cell types to the injury processes and pathways for clinical intervention. Recent studies in the Rice-Vannucci mouse model of neonatal hypoxic ischaemia (HI) have highlighted novel roles for calcium-regulated peptidylarginine deiminases (PADs) and demonstrated neuro-protective effects of pharmacological PAD inhibition following HI and synergistic infection mimicked by LPS stimulation.

Abstract
INTRODUCTION:
Protein deimination, defined as the post-translational conversion of protein-bound arginine to citrulline, is carried out by a family of 5 calcium-dependent enzymes, the peptidylarginine deiminases (PADs) and has been linked to various cancers. Cellular microvesicle (MV) release, which is involved in cancer progression, and deimination have not been associated before. We hypothesize that elevated PAD expression, observed in cancers, causes increased MV release in cancer cells and contributes to cancer progression.
BACKGROUND:
We have previously reported that inhibition of MV release sensitizes cancer cells to chemotherapeutic drugs. PAD2 and PAD4, the isozymes expressed in patients with malignant tumours, can be inhibited with the pan-PAD-inhibitor chloramidine (Cl-am). We sought to investigate whether Cl-am can inhibit MV release and whether this pathway could be utilized to further increase the sensitivity of cancer cells to drug-directed treatment.
METHODS:
Prostate cancer cells (PC3) were induced to release high levels of MVs upon BzATP stimulation of P2X7 receptors. Western blotting with the pan-protein deimination antibody F95 was used to detect a range of deiminated proteins in cells stimulated to microvesiculate. Changes in deiminated proteins during microvesiculation were revealed by immunoprecipitation and immunoblotting, and mass spectrometry identified deiminated target proteins with putative roles in microvesiculation.
CONCLUSION:
We report for the first time a novel function of PADs in the biogenesis of MVs in cancer cells. Our results reveal that during the stimulation of prostate cancer cells (PC3) to microvesiculate, PAD2 and PAD4 expression levels and the deimination of cytoskeletal actin are increased. Pharmacological inhibition of PAD enzyme activity using Cl-am significantly reduced MV release and abrogated the deimination of cytoskeletal actin. We demonstrated that combined Cl-am and methotrexate (MTX) treatment of prostate cancer cells increased the cytotoxic effect of MTX synergistically. Refined PAD inhibitors may form part of a novel combination therapy in cancer treatment.
KEYWORDS:
chloramidine; microvesicles; microvesiculation; peptidylarginine deiminases; prostate cancer cells (PC3)

Chromosome 21, triplicated in Down Syndrome, contains several genes that are thought to play a critical role in the development of AD neuropathology. The overexpression of the gene for the amyloid precursor protein (APP), on chromosome 21, leads to early onset beta-amyloid (Aβ) plaques in DS. In addition to Aβ accumulation, middle-aged people with DS develop neurofibrillary tangles, cerebrovascular pathology , white matter pathology, oxidative damage, neuroinflammation and neuron loss. There is also evidence of potential compensatory responses in DS that benefit the brain and delay the onset of dementia after there is sufficient neuropa-thology for a diagnosis of AD. This review describes some of the existing literature and also highlights gaps in our knowledge regarding AD neuropathology in DS. It will be critical in the future to develop networked brain banks with standardized collection procedures to fully characterize the regional and temporal pathological events associated with aging in DS. As more information is acquired regarding AD evolution in DS, there will be opportunities to develop interventions that are age-appropriate to delay AD in DS.

Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by 5 sugars via the action of three glycosyltransferases, Gnt1, PgtA and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar ...

Peptidylarginine deiminases (PADs) are calcium dependent enzymes with physiological and pathophysiological roles conserved throughout phylogeny. PADs promote post-translational deimination of protein arginine to citrulline, altering the structure and function of target proteins. Deiminated proteins were detected in the early developmental stages of cod from 11 days post fertilisation to 70 days post hatching. Deiminated proteins were present in mucosal surfaces and in liver, pancreas, spleen, gut, muscle, brain and eye during early cod larval development. Deiminated protein targets identified in skin mucosa included nuclear histones; cytoskeletal proteins such as tubulin and beta-actin; metabolic and immune related proteins such as galectin, mannan-binding lectin, toll-like receptor, kininogen, Beta2-microglobulin, aldehyde dehydrogenase, bloodthirsty and preproapolipoprotein A-I. Deiminated histone H3, a marker for anti-pathogenic neutrophil extracellular traps, was particularly el...

Autosomal-recessive optic neuropathies are rare blinding conditions related to retinal ganglion cell (RGC) and optic-nerve degeneration, for which only mutations in TMEM126A and ACO2 are known. In four families with early-onset recessive optic neuropathy, we identified mutations in RTN4IP1, which encodes a mitochondrial ubiquinol oxydo-reductase. RTN4IP1 is a partner of RTN4 (also known as NOGO), and its ortholog Rad8 in C. elegans is involved in UV light response. Analysis of fibroblasts from affected individuals with a RTN4IP1 mutation showed loss of the altered protein, a deficit of mitochondrial respiratory complex I and IV activities, and increased susceptibility to UV light. Silencing of RTN4IP1 altered the number and morphogenesis of mouse RGC dendrites in vitro and the eye size, neuro-retinal development, and swimming behavior in zebrafish in vivo. Altogether, these data point to a pathophysiological mechanism responsible for RGC early degeneration and optic neuropathy and linking RTN4IP1 functions to mitochondrial physiology, response to UV light, and dendrite growth during eye maturation.

There are three prolyl hydroxylases (PHD1, 2 and 3) that regulate the hypoxia-inducible factors (HIFs), the master transcriptional regulators that respond to changes in intracellular O(2) tension. In high O(2) tension (normoxia) the PHDs hydroxylate two conserved proline residues on HIF-1α, which leads to binding of the von Hippel-Lindau (VHL) tumour suppressor, the recognition component of a ubiquitin-ligase complex, initiating HIF-1α ubiquitylation and degradation. However, it is not known whether PHDs and VHL act separately to exert their enzymatic activities on HIF-1α or as a multiprotein complex. Here we show that the tumour suppressor protein LIMD1 (LIM domain-containing protein) acts as a molecular scaffold, simultaneously binding the PHDs and VHL, thereby assembling a PHD-LIMD1-VHL protein complex and creating an enzymatic niche that enables efficient degradation of HIF-1α. Depletion of endogenous LIMD1 increases HIF-1α levels and transcriptional activity in both normoxia and hypoxia. Conversely, LIMD1 expression downregulates HIF-1 transcriptional activity in a manner depending on PHD and 26S proteasome activities. LIMD1 family member proteins Ajuba and WTIP also bind to VHL and PHDs 1 and 3, indicating that these LIM domain-containing proteins represent a previously unrecognized group of hypoxic regulators.

Glutathione transferase P1-1 (GSTP1-1) plays crucial roles in
cancer chemoprevention and chemoresistance and is a key
target for anticancer drug development. Oxidative stress or
inhibitor-induced GSTP1-1 oligomerization leads to the activation
of stress cascades and apoptosis in various tumor cells.
Therefore, bivalent glutathione transferase (GST) inhibitors with
the potential to interact with GST dimers are been sought as
pharmacological and/or therapeutic agents. Here we have
characterized GSTP1-1 oligomerization in response to various
endogenous and exogenous agents. Ethacrynic acid, a classic
GSTP1-1 inhibitor, 4-hydroxy-nonenal, hydrogen peroxide, and
diamide all induced reversible GSTP1-1 oligomerization in Jurkat
leukemia cells through the formation of disulphide bonds
involving Cys47 and/or Cys101, as suggested by reducing and
nonreducing SDS-polyacrylamide gel electrophoresis analysis
of cysteine to serine mutants. Remarkably, the electrophilic
prostanoid 15-deoxy-
12,14-prostaglandin J2 (15d-PGJ2) induced
irreversible GSTP1-1 oligomerization, specifically involving
Cys101, a residue present in the human but not in the
murine enzyme. 15d-PGJ2-induced GSTP1-1 cross-linking required
the prostaglandin (PG) dienone structure and was associated
with sustained c-Jun NH2-terminal kinase activation and
induction of apoptosis. It is noteworthy that 15d-PGJ2 elicited
GSTP1-1 cross-linking in vitro, a process that could be mimicked
by other dienone cyclopentenone PG, such as
12-PGJ2,
and by the bifunctional thiol reagent dibromobimane, suggesting
that cyclopentenone PG may be directly involved in oligomer
formation. Remarkably,
12-PGJ2-induced oligomeric
species were clearly observed by electron microscopy showing
dimensions compatible with GSTP1-1 tetramers. These results
provide the first direct visualization of GSTP1-1 oligomeric
species. Moreover, they offer novel strategies for the modulation
of GSTP1-1 cellular functions, which could be exploited to
overcome its role in cancer chemoresistance.

Protein palmitoylation, a common post-translational lipid modification, plays an important role in protein trafficking and functions. Recently developed palmitoyl-proteomic methods identified many novel substrates. However, the whole picture of palmitoyl substrates has not been clarified. Here, we performed global in silico screening using the CSS-Palm 2.0 program, free software for prediction of palmitoylation sites, and selected 17 candidates as novel palmitoyl substrates. Of the 17 candidates, 10 proteins, including six synaptic proteins (Syd-1, TARP γ-2, TARP γ-8, cornichon-2, CaMKIIα and neurochondrin [Ncdn]/norbin), one focal adhesion protein (zyxin), two ion channels (TRPM8 and TRPC1), and one G-protein coupled receptor (orexin 2 receptor), were palmitoylated. Using the DHHC palmitoylating enzyme library, we found that all tested substrates were palmitoylated by the Golgi-localized DHHC3/7 subfamily. Ncdn, a regulator for neurite outgrowth and synaptic plasticity, was robustly palmitoylated by the DHHC1/10 (zDHHC1/11; z1/11) subfamily, whose substrate has not yet been reported. As predicted by CSS-Palm 2.0, Cys-3 and Cys-4 are the palmitoylation sites for Ncdn. Ncdn was specifically localized in somato-dendritic regions, not in the axon of rat cultured neurons. Stimulated emission depletion (STED) microscopy revealed that Ncdn was localized to Rab5-positive early endosomes in a palmitoylation-dependent manner, where DHHC1/10 (z1/11) were also distributed. Knockdown of DHHC1, 3, or 10 (z11) resulted in the loss of Ncdn from Rab5-positive endosomes. Thus, through in silico screening, we demonstrate that Ncdn and the DHHC1/10 (z1/11) and DHHC3/7 subfamilies are novel palmitoyl substrate-enzyme pairs and that Ncdn palmitoylation plays an essential role in its specific endosomal targeting.