The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has... more
The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has been investigated as a biomarker for pancreatic ductal adenocarcinoma (PDAC). Here we show that cathepsin E expression in mouse PDAC tumors is increased by more than 400-fold when compared to healthy pancreatic tissue. Cathepsin E accumulates over the course of disease progression and accounts for more than 3% of the tumor protein in mice with end-stage disease. Through immunoblot analysis we determined that only procathepsin E exists in mouse PDAC tumors and cell lines derived from these tumors. By decreasing the pH, this procathepsion E is converted to the mature form, resulting in an increase in proteolytic activity. Although active site inhibitors can bind procathepsin E, treatment of PDAC mice with the aspartyl protease inhibitor ritonavir did ...
Research Interests: Cancer, Biology, Biological Chemistry, Cancer Research, Rare diseases, and 15 moreMedicine, Pancreatic Cancer, Cell line, cathepsin D, Mice, Animals, Cathepsin, Aspartyl protease, Protease Inhibitors, Disease Progression, Amino Acid Sequence, Proteases, Biochemistry and cell biology, Gene Expression Regulation, and Pancreatic neoplasms
Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quanti-tative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular... more
Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quanti-tative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibod-ies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrys-tal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human can-cers. Confocal imaging...
Viral infection and pathogenesis is mediated by host protein—viral protein complexes that are important targets for therapeutic intervention as they are potentially less prone to development of drug resistance. We have identified human,... more
Viral infection and pathogenesis is mediated by host protein—viral protein complexes that are important targets for therapeutic intervention as they are potentially less prone to development of drug resistance. We have identified human, recombinant antibodies (Fabs) from a phage display library that bind to three HIV-host complexes. We used these Fabs to 1) stabilize the complexes for structural studies; and 2) facilitate characterization of the function of these complexes. Specifically, we generated recombinant Fabs to Vif-CBF-β-ELOB-ELOC (VCBC); ESCRT-I complex and AP2-complex. For each complex we measured binding affinities with KD values of Fabs ranging from 12–419 nM and performed negative stain electron microscopy (nsEM) to obtain low-resolution structures of the HIV-Fab complexes. Select Fabs were converted to scFvs to allow them to fold intracellularly and perturb HIV-host protein complex assembly without affecting other pathways. To identify these recombinant Fabs, we devel...
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Here we present a platform for discovery of protease-activated prodrugs and apply it to antibiotics that target Gram-negative bacteria. Because cleavable linkersfor prodrugs had not been developed for bacterial proteases, we used... more
Here we present a platform for discovery of protease-activated prodrugs and apply it to antibiotics that target Gram-negative bacteria. Because cleavable linkersfor prodrugs had not been developed for bacterial proteases, we used substrate phage to discover substrates for proteases found in the bacterial periplasm. Rather than focusing on a single protease, we used a periplasmic extract to find sequences with the greatest susceptibility to the endogenous mixture of periplasmic proteases. Using a fluorescence assay, candidate sequences were evaluated to identify substrates that release native amine-containing payloads without an attached peptide “scar”. We next designed conjugates consisting of: 1) an N-terminal siderophore to facilitate uptake; 2) a protease-cleavable linker; 3) an amine-containing antibiotic. Using this strategy, we converted daptomycin – which by itself is active only against Gram-positive bacteria – into an antibiotic capable of targeting Gram-negative Acinetobac...
OBJECTIVES/GOALS: Pancreatic cysts are comprised of both precancerous mucinous lesions and non-mucinous lesions with minimal malignant potential. Our goal is to improve our ability to classify the type of cyst using a combination of novel... more
OBJECTIVES/GOALS: Pancreatic cysts are comprised of both precancerous mucinous lesions and non-mucinous lesions with minimal malignant potential. Our goal is to improve our ability to classify the type of cyst using a combination of novel radiomic features and cyst fluid proteolytic activity. METHODS/STUDY POPULATION: Preoperative pancreatic protocol CT images from 30 patients with proteolytic assay characterization, followed by surgical resection with a pathologically confirmed pancreatic cyst diagnosis between 2016-2019 will be used in this study. We will contour images using the widely available software 3D Slicer, and extract radiomic features using IBEX software. We will analyze area under the ROC curves to identify the radiomic features that best differentiate mucinous from non-mucinous cysts, and identify features to be cross validated. The predictive ability of identified radiomic features combined with proteolytic assay will be determined by performing multiple logistic reg...
Background and Aims The role of suPAR as a biomarker and/or causative factor in the pathogenesis of recurrent FSGS remains unclear (Harel E., et al. Transplantation 2020; 104:54-60). While anti-suPAR antibodies have been shown to block... more
Background and Aims The role of suPAR as a biomarker and/or causative factor in the pathogenesis of recurrent FSGS remains unclear (Harel E., et al. Transplantation 2020; 104:54-60). While anti-suPAR antibodies have been shown to block suPAR induced podocyte injury in mouse models of FSGS, this beneficial effect has not yet been demonstrated in FSGS patients. We report here the inhibitor effects of 2G10, a fully human anti-suPAR antibody that blocks the interaction of suPAR with the B3 integrin on podocytes. Method The immortalized podocyte cell line was developed by transfection with the temperature sensitive SV40 T gene. These cells proliferate at the “permissive” temperature (33°C) and are considered undifferentiated. After transferring to the “nonpermissive” temperature (37°C), they enter growth arrest and by day 10-14 express markers of differentiated podocytes in vivo, such as nephrin, podocin, CD2 associated protein (CD2AP), synaptopodin, and known molecules of the slit diaph...
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A promising molecular target for aggressive cancers is the urokinase receptor (uPAR). A fully human, recombinant antibody that binds uPAR to form a stable complex that blocks uPA-uPAR interactions (2G10) and is internalized primarily... more
A promising molecular target for aggressive cancers is the urokinase receptor (uPAR). A fully human, recombinant antibody that binds uPAR to form a stable complex that blocks uPA-uPAR interactions (2G10) and is internalized primarily through endocytosis showed efficacy in a mouse xenograft model of highly aggressive, triple negative breast cancer (TNBC). Antibody-drug conjugates (ADCs) of 2G10 were designed and produced bearing tubulin inhibitor payloads ligated through seven different linkers. Aldehyde tag technology was employed for linking, and either one or two tags were inserted into the antibody heavy chain, to produce site-specifically conjugated ADCs with drug-to-antibody ratios of either two or four. Both cleavable and non-cleavable linkers were combined with two different antimitotic toxins—MMAE (monomethylauristatin E) and maytansine. Nine different 2G10 ADCs were produced and tested for their ability to target uPAR in cell-based assays and a mouse model. The anti-uPAR AD...
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Incidental detection of pancreatic cysts has increased dramatically over the last decade, but risk stratification and clinical management remain a challenge. Mucinous cysts are precursor lesions to pancreatic cancer, however, the majority... more
Incidental detection of pancreatic cysts has increased dramatically over the last decade, but risk stratification and clinical management remain a challenge. Mucinous cysts are precursor lesions to pancreatic cancer, however, the majority are indolent. Current diagnostics cannot identify mucinous cysts that harbor cancer or reliably differentiate these lesions from nonmucinous cysts, which present minimal risk of malignant progression. We previously determined that activity of two aspartyl proteases was increased in mucinous cysts. Using a global protease activity profiling technology, termed multiplex substrate profiling by mass spectrometry (MSP-MS), we now show that aminopeptidase activity is also elevated in mucinous cysts. The serine aminopeptidase, tripeptidyl peptidase 1 (TPP1), was detected by proteomic analysis of cyst fluid samples and quantitation using targeted mass spectrometry demonstrated that this protease was significantly more abundant in mucinous cysts. In a cohor...
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Human KLK8/neuropsin, a kallikrein-related serine peptidase, is mostly expressed in skin and the hippocampus regions of the brain, where it regulates memory formation by synaptic remodeling. Substrate profiles of recombinant KLK8 were... more
Human KLK8/neuropsin, a kallikrein-related serine peptidase, is mostly expressed in skin and the hippocampus regions of the brain, where it regulates memory formation by synaptic remodeling. Substrate profiles of recombinant KLK8 were analyzed with positional scanning using fluorogenic tetrapeptides and the proteomic PICS approach, which revealed the prime side specificity. Enzyme kinetics with optimized substrates showed stimulation by Ca and inhibition by Zn, which are physiological regulators. Crystal structures of KLK8 with a ligand-free active site and with the inhibitor leupeptin explain the subsite specificity and display Ca bound to the 75-loop. The variants D70K and H99A confirmed the antagonistic role of the cation binding sites. Molecular docking and dynamics calculations provided insights in substrate binding and the dual regulation of activity by Ca and Zn, which are important in neuron and skin physiology. Both cations participate in the allosteric surface loop network...
Serine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat... more
Serine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis. SmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restric...
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Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs... more
Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores. Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase, as well as subnanometre-resolution electron cryo-microscopy. Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The struc...
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Enzymes that modify the proteome, referred to as post-translational modifying (PTM) enzymes, are central regulators of cellular signaling. Determining the substrate specificity of PTM enzymes is a critical step in unraveling their... more
Enzymes that modify the proteome, referred to as post-translational modifying (PTM) enzymes, are central regulators of cellular signaling. Determining the substrate specificity of PTM enzymes is a critical step in unraveling their biological functions both in normal physiological processes and in disease states. Advances in peptide chemistry over the last century have enabled the rapid generation of peptide libraries for querying substrate recognition by PTM enzymes. In this article, we highlight various peptide-based approaches for analysis of PTM enzyme substrate specificity. We focus on the application of these technologies to proteases and also discuss specific examples in which they have been used to uncover the substrate specificity of other types of PTM enzymes, such as kinases. In particular, we highlight our multiplex substrate profiling by mass spectrometry (MSP-MS) assay, which uses a rationally designed, physicochemically diverse library of tetradecapeptides. We show how...
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The immunoproteasome (iP) has been proposed to perform specialized roles in MHC class I antigen presentation, cytokine modulation, and T cell differentiation and has emerged as a promising therapeutic target for autoimmune disorders and... more
The immunoproteasome (iP) has been proposed to perform specialized roles in MHC class I antigen presentation, cytokine modulation, and T cell differentiation and has emerged as a promising therapeutic target for autoimmune disorders and cancer. However, divergence in function between the iP and the constitutive proteasome (cP) has been unclear. A global peptide library-based screening strategy revealed that the proteasomes have overlapping but distinct substrate specificities. Differing iP specificity alters the quantity of production of certain MHC I epitopes but does not appear to be preferentially suited for antigen presentation. Furthermore, iP specificity was found to have likely arisen through genetic drift from the ancestral cP. Specificity differences were exploited to develop isoform-selective substrates. Cellular profiling using these substrates revealed that divergence in regulation of the iP balances its relative contribution to proteasome capacity in immune cells, resul...
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120 Background: Identification, enumeration, and genomic analysis of circulating tumor cells (CTCs) may allow for a better understanding of the mechanisms of resistance to therapies in metastatic castration-resistant prostate cancer... more
120 Background: Identification, enumeration, and genomic analysis of circulating tumor cells (CTCs) may allow for a better understanding of the mechanisms of resistance to therapies in metastatic castration-resistant prostate cancer (mCRPC). The Vitatex VitaAssay platform captures invasive CTCs (iCTCs) in a cell surface marker-independent fashion based on their ability to invade a fluorescently-labeled cell-adhesion matrix (CAM), allowing for the analysis of multiple CTC subpopulations. Here we sought to estimate epithelial, mesenchymal, and stem-like iCTC subpopulation diversity in men with CRPC starting abiraterone acetate therapy, to compare the genomic profiles of iCTCs to matched metastatic biopsies, and to explore the potential for 2D and 3D CTC culture. Methods: iCTCs were isolated from men with mCRPC using the CAM platform, and paired metastatic biopsies were performed. iCTCs were defined as CAM+/CD45-/CD14-/DAPI+, mesenchymal iCTCs as vimentin+/CAM+/CD45-/CD14-/DAPI+, and s...
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Heterochromatin protein 1 (HP1) family proteins are conserved chromatin binding proteins involved in gene silencing, chromosome packaging, and chromosome segregation. These proteins recognize histone H3 lysine 9 methylated tails via their... more
Heterochromatin protein 1 (HP1) family proteins are conserved chromatin binding proteins involved in gene silencing, chromosome packaging, and chromosome segregation. These proteins recognize histone H3 lysine 9 methylated tails via their chromodomain and recruit additional ligand proteins with diverse activities through their dimerization domain, the chromoshadow domain. Species that have HP1 proteins possess multiple paralogs that perform non-overlapping roles in vivo. How different HP1 proteins, which are highly conserved, perform different functions is not well understood. Here, we use the two Schizosaccharomyces pombe HP1 paralogs, Swi6 and Chp2, as model systems to compare and contrast their biophysical properties. We find that Swi6 and Chp2 have similar dimerization and oligomerization equilibria, and that Swi6 binds slightly (~3-fold) more strongly to nucleosomes than Chp2. Furthermore, while Swi6 binding to the H3K9me3 mark is regulated by a previously described auto-inhibition mechanism, the binding of Chp2 to the H3K9me3 mark is not analogously regulated. In the context of chromoshadow domain interactions, we show using a newly identified peptide sequence from the Clr3 histone deacetylase and a previously identified sequence from the protein Shugoshin that the Swi6 chromoshadow domain binds both ligands more strongly than the Chp2. Overall, our findings uncover quantitative differences in how Swi6 and Chp2 interact with nucleosomal and non-nucleosomal ligands and qualitative differences in how their assembly on nucleosomes is regulated. These findings provide a biochemical framework to explain the varied functions of Chp2 and Swi6 in vivo.
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Targeting of cryptic binding sites represents an attractive but underexplored approach to modulating protein function with small molecules. Using the dimeric protease (Pr) from Kaposi's sarcoma-associated herpesvirus (KSHV) as a model... more
Targeting of cryptic binding sites represents an attractive but underexplored approach to modulating protein function with small molecules. Using the dimeric protease (Pr) from Kaposi's sarcoma-associated herpesvirus (KSHV) as a model system, we sought to dissect a putative allosteric network linking a cryptic site at the dimerization interface to enzyme function. Five cryogenic x-ray structures were solved of the monomeric protease with allosteric inhibitors bound to the dimer interface site. Distinct coordinated movements captured by the allosteric inhibitors were also revealed as alternative states in room temperature X-ray data and comparative analyses of other dimeric herpesvirus proteases. A two-step mechanism was elucidated through detailed kinetic analyses and suggests an enzyme isomerization model of inhibition. Finally, a representative allosteric inhibitor from this class was shown to be efficacious in a cellular model of viral infectivity. These studies reveal a coor...
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Objectives To assess the ability of the type II transmembrane serine proteinase matriptase to promote aggrecan loss from cartilage of osteoarthritis (OA) patients, and whether matriptase inhibition could prevent aggrecan loss and... more
Objectives To assess the ability of the type II transmembrane serine proteinase matriptase to promote aggrecan loss from cartilage of osteoarthritis (OA) patients, and whether matriptase inhibition could prevent aggrecan loss and cartilage damage in experimental OA. Methods Aggrecan release was assessed from human OA cartilage explants and human stem cell-derived cartilage discs, whilst cartilage conditioned media were used for western blotting. Gene expression was analysed by real-time PCR. Murine OA was induced by surgical destabilisation of the medial meniscus, and matriptase inhibitors delivered via osmotic mini-pump or intra-articular injection. Cartilage damage was scored histologically and aggrecan cleavage visualised immuno-histochemically using specific neo-epitope antibodies. Results Addition of soluble recombinant matriptase promoted a time-dependent release of aggrecan (and collagen) from OA cartilage, which was sensitive to metalloproteinase inhibition and protease-acti...
Research Interests: Membrane Proteins, Immunohistochemistry, Western blotting, Humans, Mice, and 12 moreFemale, Animals, Male, Aged, Middle Aged, Knee Osteoarthritis, Articular Cartilage Wear, Matrix Metalloproteinases, Recombinant Proteins, Gene expression profiling, real time polymerase chain reaction, and In Vitro Techniques
The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut... more
The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis, we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the National Institutes of Health Human Microbiome Project (NIH HMP) stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics, synthetic biology, and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic pote...
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Cardiovascular diseases (CVDs) account for over 17 million deaths globally each year, with atherosclerosis as the underlying cause of most CVDs. Herein we describe the synthesis and in vitro mechanistic evaluation of novel... more
Cardiovascular diseases (CVDs) account for over 17 million deaths globally each year, with atherosclerosis as the underlying cause of most CVDs. Herein we describe the synthesis and in vitro mechanistic evaluation of novel N'-benzylidene-carbohydrazide-1H-pyrazolo[3,4-b]pyridines (3-22) designed as non-anionic antiplatelet agents and presenting a 30-fold increase in potency compared to aspirin. The mechanism underlying their antiplatelet activity was elucidated by eliminating potential targets through a series of in vitro assays including light transmission aggregometry, clot retraction, and quantitative ELISA, further identifying the reduction in biosynthesis of thromboxane B2 as their main mechanism of action. The intrinsic fluorescence of the compounds permits their binding to platelet membranes to be readily monitored. In silico structure-activity relationship, molecular docking and dynamics studies support the biological profile of the series revealing the molecular basis o...
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The opportunistic fungal pathogen Cryptococcus neoformans is a major cause of mortality in immunocompromised individuals, resulting in more than 600,000 deaths per year. Many human fungal pathogens secrete peptidases that influence... more
The opportunistic fungal pathogen Cryptococcus neoformans is a major cause of mortality in immunocompromised individuals, resulting in more than 600,000 deaths per year. Many human fungal pathogens secrete peptidases that influence virulence, but in most cases the substrate specificity and regulation of these enzymes remains poorly understood. The paucity of such information is a roadblock to our understanding of the biological functions of peptidases and whether or not these enzymes are viable therapeutic targets. We report here an unbiased analysis of secreted peptidase activity and specificity in C. neoformans using a mass spectrometry-based substrate profiling strategy and subsequent functional investigations. Our initial studies revealed that global peptidase activity and specificity are dramatically altered by environmental conditions. To uncover the substrate preferences of individual enzymes and interrogate their biological functions, we constructed and profiled a ten-member...
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Disulfide-rich peptides (DRPs) are found throughout nature. They are suitable scaffolds for drug development due to their small cores, whose disulfide bonds impart extraordinary chemical and biological stability. A challenge in developing... more
Disulfide-rich peptides (DRPs) are found throughout nature. They are suitable scaffolds for drug development due to their small cores, whose disulfide bonds impart extraordinary chemical and biological stability. A challenge in developing a DRP therapeutic is to engineer binding to a specific target. This challenge can be overcome by (i) sampling the large sequence space of a given scaffold through a phage display library and by (ii) panning multiple libraries encoding structurally distinct scaffolds. Here, we implement a protocol for defining these diverse scaffolds, based on clustering structurally defined DRPs according to their conformational similarity. We developed and applied a hierarchical clustering protocol based on DRP structural similarity, followed by two post-processing steps, to classify 806 unique DRP structures into 81 clusters. The 20 most populated clusters comprised 85% of all DRPs. Representative scaffolds were selected from each of these clusters; the represent...
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ABSTRACTCandida albicansis a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections.C. albicanscells proliferate in a planktonic (suspension) state,... more
ABSTRACTCandida albicansis a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections.C. albicanscells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of newC. albicansinfections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically byC. albicansbiofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted byC. albicans. Fluorogenic peptide su...
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We report label-free electrical detection of enzymatic reactions using 2-D nanofluidic channels and investigate reaction kinetics of enzymatic reactions on immobilized substrates in nanoscale confined spaces. Trypsin proteolysis is chosen... more
We report label-free electrical detection of enzymatic reactions using 2-D nanofluidic channels and investigate reaction kinetics of enzymatic reactions on immobilized substrates in nanoscale confined spaces. Trypsin proteolysis is chosen for demonstration of the detection scheme. When trypsin cleaves Poly-L-Lysine (PLL) coated on the surface of silica nanochannels, the resulting change of surface charge density can be detected by monitoring the ionic conductance of the nanochannels. Our results show that detection of such surface enzymatic reactions is faster than detection of surface binding reactions in nanochannels for low-concentration analytes. Furthermore, the nanochannel sensor has a sensitivity down to 5 ng/ml, which statistically corresponds to single enzyme per nanochannel. Our results also suggest that enzyme kinetics in nanochannels is fundamentally different from that in bulk solutions or plain surfaces. Such enzymatic reactions form two clear self-propagating reaction...
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Research Interests: Biochemistry, Computer Graphics, Kinetics, Escherichia coli, Animals, and 15 morePlasmids, Enzyme, Cattle, Computer Graphic, Lysine, Rats, Substrate Specificity, Protein Conformation, Amino Acid Sequence, Base Sequence, Recombinant Proteins, Catalytic Activity, Biochemistry and cell biology, binding sites, and Medical biochemistry and metabolomics
Fasciola parasites (liver flukes) express numerous cathepsin L proteases that are believed to be involved in important functions related to host invasion and parasite survival. These proteases are evolutionarily divided into clades that... more
Fasciola parasites (liver flukes) express numerous cathepsin L proteases that are believed to be involved in important functions related to host invasion and parasite survival. These proteases are evolutionarily divided into clades that are proposed to reflect their substrate specificity, most noticeably through the S(2) subsite. Single amino acid substitutions to residues lining this site, including amino acid residue 69 (aa69; mature cathepsin L5 numbering) can have profound influences on subsite architecture and influence enzyme specificity. Variations at aa69 among known Fasciola cathepsin L proteases include leucine, tyrosine, tryptophan, phenylalanine and glycine. Other amino acids (cysteine, serine) might have been expected at this site due to codon usage as cathepsin L isoenzymes evolved, but C69 and S69 have not been observed. The introduction of L69C and L69S substitutions into FhCatL5 resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants in Fasciola. An FhCatL5 L69F variant showed an increase in the ability to cleave substrates with P(2) proline, indicating F69 variants expressed by the fluke would likely have this ability. An FhCatL2 Y69L variant showed a decreased acceptance of P(2) proline, further highlighting the importance of Y69 for FhCatL2 P(2) proline acceptance. Finally, the P(1)-P(4) specificity of Fasciola cathepsin L5 was determined and, unexpectedly, aspartic acid was shown to be well accepted at P(2,) which is unique amongst Fasciola cathepsins examined to date.
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The crystal structure of trypsin-G226A has been determined, in the presence of benzamidine, to a resolution of 1.75 A with an R-factor of 14.6%. The mutation was designed to alter substrate specificity by disrupting arginine binding, but... more
The crystal structure of trypsin-G226A has been determined, in the presence of benzamidine, to a resolution of 1.75 A with an R-factor of 14.6%. The mutation was designed to alter substrate specificity by disrupting arginine binding, but was previously found to disrupt catalysis to a greater extent than binding. The arginine analog, benzamidine, has rotated 40 degrees and 49 degrees and translated 1.1 A in the specificity pocket, relative to the position in wild-type trypsin. The salt-bridge between the amidinium group of benzamidine and the carboxylate of D189 as well as four other hydrogen bonds have been replaced by a set of six new hydrogen bonds. Based on these interactions, computer modeling of an arginine substrate demonstrates that arginine terminal nitrogen atoms can occupy the new benzamidine nitrogen positions with torsion angle adjustments and without short contacts. In the secondary orientation, arginine substrates appear to be forced out of alignment with the active site. This may account for the larger drop in kcat with arginine relative to lysine substrates. A second possible cause of the altered activity is a change of the enzyme structure with concomitant loss of activity. No evidence of such a change is seen in the co-ordinates or temperature factors of the trypsin-G226A-benzamidine complex. A226 disrupts mainly the co-ordinates of amino acids with which it has direct contacts such that the effects of the mutation are absorbed locally.
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Research Interests: Protein Folding, Prostate Cancer, Protein Engineering, Biological Sciences, Cell line, and 15 moreHumans, Escherichia coli, Mice, Female, Animals, Male, Affinity chromatography, Enzyme, Snake Venom, Mammary gland, Biological Process, Amino Acid Sequence, Serine Protease, Plasminogen Activator, and Molecular Sequence Data
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ABSTRACT The aim of this study is to examine the capability of the Restricted Interaction Peptide (RIP), Cy3-Protonectin-PAR1, to image and quantify in-vivo thrombin activity. Upon thrombin activation at the cleavage site, the RIP’s... more
ABSTRACT The aim of this study is to examine the capability of the Restricted Interaction Peptide (RIP), Cy3-Protonectin-PAR1, to image and quantify in-vivo thrombin activity. Upon thrombin activation at the cleavage site, the RIP’s membrane interacting region linked to an imaging agent will bind to a phospholipid bilayer producing a high-resolution, non-invasive, real-time image of blood clot formation. Using a bottom-up approach, we observed the properties of the RIP probe through three stages: testing fluorescence at the molecular level, in-vitro staining of murine blood, and in vivo imaging of blood clot in mouse model. We concluded that Cy3-Protonectin-PAR1 has a high selectivity for thrombin with a catalytic efficiency (Kcat/K m) of 5.3 uM/s and a turnover number (Kcat) of 1.3 s-1. In addition, a minute sample (~50 nM) of the peptide is capable of producing high resolution blood clot images. Further investigation of the design of RIP is necessary to produce probes that are capable of detecting blood clot in stroke, coronary thrombosis, and pulmonary embolism.
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Research Interests: Biochemistry, Kinetics, Mutation, Escherichia coli, Animals, and 15 moreEnzyme, Genetically Modified, Active site, Cysteine endopeptidases, Cysteine, Amino Acid Sequence, Base Sequence, Fusion Protein, Catalytic Activity, Biochemistry and cell biology, Molecular Sequence Data, binding sites, Cysteine Protease, Medical biochemistry and metabolomics, and HPLC Chromatography
The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and... more
The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2)...