Research Interests: Biophysics, Chemistry, Crystallography, Structure, Magnetic Resonance Spectroscopy, and 15 moreAtomic Force Microscopy, Medicine, Biological Sciences, Humans, Collagen, Triple Helix, Metalloproteinase, CHEMICAL SCIENCES, Neoplasm Invasiveness, Protein Conformation, Amino Acid Sequence, Protein Binding, Matrix Metalloproteinase, Molecular Sequence Data, and proteolysis
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Research Interests: Biochemistry, Chemistry, Biological Chemistry, Medicine, Biological Sciences, and 13 moreHumans, Collagen, Triple Helix, Animals, Biological, CHEMICAL SCIENCES, Protein Secondary Structure Prediction, Substrate Specificity, Hydrogen Deuterium Exchange, Proteases, Catabolism, Matrix Metalloproteinase, and Medical and Health Sciences
Research Interests: Biochemistry, Chemistry, Organic Chemistry, Fluorescent Dyes and Reagents, Medicine, and 15 moreHumans, Triple Helix, Temperature, Enzyme, Protein Secondary Structure Prediction, Substrate Specificity, Amino Acid Sequence, Amino Acid Substitution Rates, Hydrolysis, Biochemistry and cell biology, Matrix Metalloproteinase, Molecular Sequence Data, fluorescent dyes, Medical biochemistry and metabolomics, and Collagen type I
ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive breast cancer in response to trastuzumab. The majority of ADAM10 and ADAM17 inhibitor development has been focused on the discovery of... more
ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive breast cancer in response to trastuzumab. The majority of ADAM10 and ADAM17 inhibitor development has been focused on the discovery of compounds that bind the active site zinc, however, in recent years, there has been a shift from active site to secondary substrate binding site (exosite) inhibitor discovery in order to identify non-zinc-binding molecules. In the present work a glycosylated, exosite-binding substrate of ADAM10 and ADAM17 was utilized to screen 370,276 compounds from the MLPCN collection. As a result of this uHTS effort, a selective, time-dependent, non-zinc-binding inhibitor of ADAM10 with Ki = 883 nM was discovered. This compound exhibited low cell toxicity and was able to selectively inhibit shedding of known ADAM10 substrates in several cell-based models. We hypothesize that differential glycosylation of these cognate substrates is the source of selectivity of our nove...
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Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiquitous cell adhesion mechanism. However, the HSPG binding sites on type I collagen are unknown. Previously we mapped heparin binding to the... more
Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiquitous cell adhesion mechanism. However, the HSPG binding sites on type I collagen are unknown. Previously we mapped heparin binding to the vicinity of the type I collagen N terminus by electron microscopy. The present study has identified type I collagen sequences used for heparin binding and endothelial cell-collagen interactions. Using affinity coelectrophoresis, we found heparin to bind as follows: to type I collagen with high affinity (Kd≈ 150 nM); triple-helical peptides (THPs) including the basic N-terminal sequence α 1(I)87-92, KGH-RGF, with intermediate affinities (Kd≈ and THPs including other collagenous sequences, or single-stranded sequences, negligibly (Kd >> 10 μ M). Thus, heparin-type I collagen binding likely relies on an N-terminal basic triple-helical domain represented once within each monomer, and at multiple sites within fibrils. We next defined the features of type I collagen necessary for angiogenesis in a system in which type I collagen and heparin rapidly induce endothelial tube formation in vitro. When peptides, denatured or monomeric type I collagen, or type V collagen was substituted for type I collagen, no tubes formed. However, when peptides and type I collagen were tested together, only the most heparin-avid THPs inhibited tube formation, likely by influencing cell interactions with collagen-heparin complexes. Thus, induction of endothelial tube morphogenesis by type I collagen may depend upon its triple-helical and fibrillar conformations and on the N-terminal heparin-binding site identified here.
Research Interests: Electron Microscopy, Cell Adhesion, Multidisciplinary, Humans, Heparin, and 12 moreCollagen, Vascular endothelium, Molecular Mimicry, Endothelial cell, Surface Properties, Protein Conformation, Amino Acid Sequence, Protein Binding, Cell Surface Markers, Binding Site, single stranded DNA, and Type I Collagen
Biomimetic membrane surfaces functionalized with fragments of the extracellular matrix protein, fibronectin, are constructed from mixtures of peptide and polyethylene glycol (PEG) amphiphiles. Peptides from the primary binding loop,... more
Biomimetic membrane surfaces functionalized with fragments of the extracellular matrix protein, fibronectin, are constructed from mixtures of peptide and polyethylene glycol (PEG) amphiphiles. Peptides from the primary binding loop, GRGDSP, were used in conjunction with the synergy site peptide, PHSRN, in the III9–10 sites of human fibronectin. These peptides were attached to dialkyl lipid tails to form peptide amphiphiles. PEG amphiphiles were mixed in the layer to minimize non-specific adhesion in the background. GRGDSP and PEG amphiphiles or GRGDSP, PHSRN, and PEG amphiphiles were mixed in various ratios and deposited on solid substrates from the air–water interface using Langmuir–Blodgett techniques. In this method, peptide composition, density, and presentation could be controlled accurately. The effectiveness of these substrates to mimic native fibronectin is evaluated by their ability to generate adhesive forces when they are in contact with purified activated α5β1 integrin receptors that are immobilized on an opposing surface. Adhesion is measured using a contact mechanical approach (JKR experiment). The effects of membrane composition, density, temperature, and peptide conformation on adhesion to activated integrins in this simulated cell adhesion setup were determined. Addition of the synergy site, PHSRN, was found to increase adhesion of α5β1 to biomimetic substrates markedly. Increased peptide mobility (due to increased experimental temperature) increased integrin adhesion markedly at low peptide concentrations. A balance between peptide density and steric accessibility of the receptor binding face to α5β1 integrin was required for highest adhesion.
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Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically active and play important roles in wound healing. Following myocardial infarction (MI), collagen I, a major component of cardiac ECM, is... more
Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically active and play important roles in wound healing. Following myocardial infarction (MI), collagen I, a major component of cardiac ECM, is cleaved by matrix metalloproteinases (MMPs). This study identified novel collagen-derived matricryptins generated post-MI that mediate remodeling of the left ventricle (LV). Recombinant collagen Ia1 was used in MMPs cleavage assays, the products were analyzed by mass spectrometry for identification of cleavage sites. C57BL6/J mice were given MI and animals were treated either with vehicle control or p1158/59 matricryptin. Seven days post-MI, LV function and parameters of LV remodeling were measured. Levels of p1158/59 were also measured in plasma of MI patients and healthy controls. In situ, MMP-2 and -9 generate a collagen Iα1 C-1158/59 fragment, and MMP-9 can further degrade it. The C-1158/59 fragment was identified post-MI, both in human plasma and mou...
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A great variety of cells, such as melanoma cells, fibroblasts, platelets, keratinocytes, and epithelial cells, adhere to and migrate on specific regions within the triple-helical domains of types I, III, and IV collagen. The relative... more
A great variety of cells, such as melanoma cells, fibroblasts, platelets, keratinocytes, and epithelial cells, adhere to and migrate on specific regions within the triple-helical domains of types I, III, and IV collagen. The relative importance of collagen primary, secondary, and tertiary structures on these cellular activities has not been ascertained, as no general synthetic methodology exists to allow for the study of peptides incorporating biologically active sequences in triple-helical conformation. We have thus developed a novel, generally applicable solid-phase branching methodology for the synthesis of aligned, triple-helical collagen-model polypeptides (i.e. "mini-collagens"). Three nascent peptide chains are carboxyl-terminally linked through one N alpha-amino and two N epsilon-amino groups of Lys, while repeating Gly-Pro-Hyp triplets induce triple helicity. A homotrimeric triple-helical polypeptide (THP) of 124 amino acids, incorporating residues 1263-1277 of al...
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... 23-55 appears to exist in aqueous solution in a "random coil" distribution of highly ... Hexamer formation resulted in a hydrophobic core, not the expected hydrophobic packing, whiie in the ... Acetyl-a4 is believed to exist... more
... 23-55 appears to exist in aqueous solution in a "random coil" distribution of highly ... Hexamer formation resulted in a hydrophobic core, not the expected hydrophobic packing, whiie in the ... Acetyl-a4 is believed to exist in two folding topologies, right-handed and lefthanded bundles ...
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Analysis of matrix metalloproteinases (MMPs) expression profiles in various pathologies correlated with their presence in promoting disease progression. Drugs were designed to inhibit MMPs in an extreme manner by chelating the active site... more
Analysis of matrix metalloproteinases (MMPs) expression profiles in various pathologies correlated with their presence in promoting disease progression. Drugs were designed to inhibit MMPs in an extreme manner by chelating the active site zinc ion. This approach did not distinguish between the 24 members of the MMP family and had devastating consequences during clinical trials. Subsequent knockout mouse studies showed that some MMPs are beneficial in regulating tumor growth, metastasis and indirectly stimulating the immune system. The broad-spectrum inhibitor approach was rethought and modified in order to increase specificity by taking into account the non-conserved secondary binding sites or differences in structures within MMPs and also generating antibodies. These showed interesting results in vitro and in vivo. The recent technological advances that allow us to better understand the function and structure of MMPs are aiding in the development of selective inhibitors.
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Numerous studies have shown that the physical characteristics of a surface, such as topography and chemical composition [1-5], can greatly affect how cells respond to the surface. Our initial studies have focused on an amphiphile that has... more
Numerous studies have shown that the physical characteristics of a surface, such as topography and chemical composition [1-5], can greatly affect how cells respond to the surface. Our initial studies have focused on an amphiphile that has a peptide sequence in ...
Matrix metalloproteinases (MMPs) have been implicated in numerous pathologies. An overall lack of selectivity has rendered active-site-targeted MMP inhibitors problematic. The present study describes MMP inhibitors that function by... more
Matrix metalloproteinases (MMPs) have been implicated in numerous pathologies. An overall lack of selectivity has rendered active-site-targeted MMP inhibitors problematic. The present study describes MMP inhibitors that function by binding both secondary binding sites (exosites) and the active site. Heterotrimeric triple-helical peptide transition-state analogue inhibitors (THPIs) were assembled utilizing click chemistry. Three different heterotrimers were constructed, allowing for the inhibitory phosphinate moiety to be present uniquely in the leading, middle, or trailing strand of the triple helix. All heterotrimeric constructs had sufficient thermally stability to warrant analysis as inhibitors. The heterotrimeric THPIs were effective against MMP-13 and MT1-MMP, with Ki values spanning 100-400 nM. Unlike homotrimeric THPIs, the heterotrimeric THPIs offered complete selectivity between MT1-MMP and MMP-1. Exosite-based approaches such as this provide inhibitors with desired MMP sel...
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The main focus of enzymology is on the enzyme rates, substrate structures, and reactivity, whereas the role of solvent dynamics in mediating the biological reaction is often left aside owing to its complex molecular behavior. We used... more
The main focus of enzymology is on the enzyme rates, substrate structures, and reactivity, whereas the role of solvent dynamics in mediating the biological reaction is often left aside owing to its complex molecular behavior. We used integrated X-ray- and terahertz- based time-resolved spectroscopic tools to study protein-water dynamics during proteolysis of collagen-like substrates by a matrix metalloproteinase. We show equilibration of structural kinetic transitions in the millisecond timescale during degradation of the two model substrates collagen and gelatin, which have different supersecondary structure and flexibility. Unexpectedly, the detected changes in collective enzyme-substrate-water-coupled motions persisted well beyond steady state for both substrates while displaying substrate-specific behaviors. Molecular dynamics simulations further showed that a hydration funnel (i.e., a gradient in retardation of hydrogen bond (HB) dynamics toward the active site) is substrate-dependent, exhibiting a steeper gradient for the more complex enzyme-collagen system. The long-lasting changes in protein-water dynamics reflect a collection of local energetic equilibrium states specifically formed during substrate conversion. Thus, the observed long-lasting water dynamics contribute to the net enzyme reactivity, impacting substrate binding, positional catalysis, and product release.
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Proliferating cell nuclear antigen (PCNA) is a highly conserved protein necessary for proper component loading during the DNA replication and repair process. Proteins make a connection within the interdomain connector loop of PCNA, and... more
Proliferating cell nuclear antigen (PCNA) is a highly conserved protein necessary for proper component loading during the DNA replication and repair process. Proteins make a connection within the interdomain connector loop of PCNA, and much of the regulation is a result of the inherent competition for this docking site. If this target region of PCNA is modified, the DNA replication and repair process in cancer cells is potentially altered. Exploitation of this cancer-associated region has implications for targeted breast cancer therapy. In the present communication, we characterize a novel peptide (caPeptide) that has been synthesized to mimic the sequence identified as critical to the cancer-associated isoform of PCNA. This peptide is delivered into cells using a nine-arginine linking mechanism, and the resulting peptide (R9-cc-caPeptide) exhibits cytotoxicity in a triple-negative breast cancer cell line, MDA-MB-436, while having less of an effect on the normal counterparts (MCF10A...
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dependent platelet adhesion, without appreciably activating or aggregating platelets. Notably, endorepellin enhanced collagen-evoked responses in platelets, in a src kinase-dependent fashion, and enhanced the collagen-inhibitory effect of... more
dependent platelet adhesion, without appreciably activating or aggregating platelets. Notably, endorepellin enhanced collagen-evoked responses in platelets, in a src kinase-dependent fashion, and enhanced the collagen-inhibitory effect of an 21-integrin function-blocking an- tibody. Collectively, these results suggest that endorepellin/21-integrin interac- tion and effects are specific and depen- dent on cell type, differ from those ema- nated by exposure to collagen, and may be due to cellular differences in 21- integrin activation/ligand affinity state. These studies also suggest a heretofore unrecognized role for angiostatic base- ment membrane fragments in platelet bi- ology. (Blood. 2007;109:3745-3748)
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Venomous marine molluscs belonging to the genus Conus (cone snails) utilize a unique neurochemical strategy to capture their prey. Their venom is composed of a complex mixture of highly modified peptides (conopeptides) that interact with... more
Venomous marine molluscs belonging to the genus Conus (cone snails) utilize a unique neurochemical strategy to capture their prey. Their venom is composed of a complex mixture of highly modified peptides (conopeptides) that interact with a wide range of neuronal targets. In this chapter, we describe a set of modifications based upon the hydroxylation of polypeptidic chains that are defining within the neurochemical strategy used by cone snails to capture their prey. In particular, we present a differential hydroxylation strategy that affects the neuronal targeting of a new set of a-conotoxins, mini-M conotoxins, conophans, and y-hydroxyconophans. Differential hydroxylation, preferential hydroxylation and hyperhydroxylation have been observed in these conopeptide families as a means of augmenting the venom arsenal used by cone snails for neuronal targeting and prey capture.
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The prevalence of the gelatinases, MMP-2 and MMP-9, in many human tumors, including breast, colorectal, prostate and gastric cancer, make them an attractive target for molecular imaging. A self assembling homotrimeric triple helical... more
The prevalence of the gelatinases, MMP-2 and MMP-9, in many human tumors, including breast, colorectal, prostate and gastric cancer, make them an attractive target for molecular imaging. A self assembling homotrimeric triple helical peptide (THP), incorporating ...
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The peptide-amphiphiles described here provide a simple approach for building stable protein structural motifs using peptide head groups. One of the most intriguing features of this system is the possible formation of stable lipid films... more
The peptide-amphiphiles described here provide a simple approach for building stable protein structural motifs using peptide head groups. One of the most intriguing features of this system is the possible formation of stable lipid films on solid substrates, or the use of the novel amphiphiles in bilayer membrane systems, where the lipid tail serves not only as a peptide structure-inducing
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The gelatinase members of the MMP family have consistently been associated with tumor invasiveness, which make them an attractive target for molecular imaging. We report new activatable proteolytic optical imaging agents that consist of... more
The gelatinase members of the MMP family have consistently been associated with tumor invasiveness, which make them an attractive target for molecular imaging. We report new activatable proteolytic optical imaging agents that consist of triple-helical peptide (THP) conjugates, with high specificity to the gelatinases, bearing quenched cypate dyes. With quenching efficiencies up to 51%, the amplified fluorescence signal upon cypate3-THP hydrolysis by the gelatinases (kcat/KM values of 6.4×10(3) M(-1) s(-1) to 9.1×10(3) M(-1) s(-1) for MMP-2 and MMP-9, respectively) in mice bearing human fibrosarcoma xenografted tumors was monitored with fluorescence molecular tomography. There was significant fluorescence enhancement within the tumor and this enhancement was reduced by treatment with pan-MMP inhibitor, Ilomastat. These data, combined with the gelatinase substrate specificity observed in vitro, indicated the observed fluorescence at the site of the tumor was due to gelatinase mediated hydrolysis of cypate3-THP.
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Matrix metalloproteinases (MMP) 2 and 9, the gelatinases, have consistently been associated with tumor progression. The development of gelatinase-specific probes will be critical for identifying in vivo gelatinoic activity to understand... more
Matrix metalloproteinases (MMP) 2 and 9, the gelatinases, have consistently been associated with tumor progression. The development of gelatinase-specific probes will be critical for identifying in vivo gelatinoic activity to understand the molecular role of the gelatinases in tumor development. Recently, a self-assembling homotrimeric triple-helical peptide (THP), incorporating a sequence from type V collagen, with high substrate specificity to the gelatinases has been developed. To determine whether this THP would be suitable for imaging protease activity, 5-carboxyfluorescein (5FAM) was conjugated, resulting in 5FAM3-THP and 5FAM6-THP, which were quenched up to 50%. 5FAM6-THP hydrolysis by MMP-2 and MMP-9 displayed kcat/KM values of 1.5 × 104 and 5.4 × 103 M-1 s-1, respectively. Additionally 5FAM6-THP visualized gelatinase activity in gelatinase positive HT-1080 cells, but not in gelatinase negative MCF-7 cells. Furthermore, the fluorescence in the HT-1080 cells was greatly attenuated by the addition of a MMP-2 and MMP-9 inhibitor, SB-3CT, indicating that the observed fluorescence release was mediated by gelatinase proteolysis and not non-specific proteolysis of the THPs. These results demonstrate that THPs fully substituted with fluorophores maintain their substrate specificity to the gelatinases in human cancer cells and may be useful in in vivo molecular imaging of gelatinase activity.
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Cancer treatment by chemotherapy is typically accompanied by deleterious side effects, attributed to the toxic action of chemotherapeutics on proliferating cells from nontumor tissues. The cell surface proteoglycan CD44 has been... more
Cancer treatment by chemotherapy is typically accompanied by deleterious side effects, attributed to the toxic action of chemotherapeutics on proliferating cells from nontumor tissues. The cell surface proteoglycan CD44 has been recognized as a cancer stem cell marker. The present study has examined CD44 targeting as a way to selectively deliver therapeutic agents encapsulated inside colloidal delivery systems. CD44/chondroitin sulfate proteoglycan binds to a triple-helical sequence derived from type IV collagen, α1(IV)1263-1277. We have assembled a peptide-amphiphile (PA) in which α1(IV)1263-1277 was sandwiched between 4 repeats of Gly-Pro-4-hydroxyproline and conjugated to palmitic acid. The PA was incorporated into liposomes composed of DSPG, DSPC, cholesterol, and DSPE-PEG-2000 (1 : 4 : 5 : 0.5). Doxorubicin-(DOX-)loaded liposomes with and without 10% α1(IV)1263-1277 PA were found to exhibit similar stability profiles. Incubation of DOX-loaded targeted liposomes with metastatic ...