Escherichia coli Proteome Microarrays Identified the Substrates of ClpYQ Protease

Proteolysis is a vital mechanism to regulate the cellular proteome in all kingdoms of life, and ATP-dependent proteases play a crucial role within this process. In Escherichia coli, ClpYQ is one of the primary ATP-dependent proteases. In addition to function with removals of abnormal peptides in the cells, ClpYQ degrades regulatory proteins if necessary and thus let cells adjust to various environmental conditions. In E. coli, SulA, RcsA, RpoH and TraJ as well as RNase R, have been identified as natural protein substrates of ClpYQ. ClpYQ contains ClpY and ClpQ. The ATPase ClpY is responsible for protein recognition, unfolding, and translocation into the catalytic core of ClpQ. In this study, we use an indirect identification strategy to screen possible ClpY targets with E. coli K12 proteome chips. The chip assay results showed that YbaB strongly bound to ClpY. We used yeast two-hybrid assay to confirm the interactions between ClpY and YbaB protein and determined the K_d between ClpY and YbaB by quartz crystal microbalance. Furthermore, we validated that YbaB was successfully degraded by ClpYQ protease activity using ClpYQ in vitro and in vivo degradation assay. These findings demonstrated the YbaB is a novel substrate of ClpYQ protease. This work also successfully demonstrated that with the use of recognition element of a protease can successfully screen its substrates by indirect proteome chip screening assay.     

Proteomic techniques and activity-based probes for the system-wide study of proteolysis

Proteolysis constitutes a major post-translational modification but specificity and substrate selectivity of numerous proteases have remained elusive. In this review, we highlight how advanced techniques in the areas of proteomics and activity-based probes can be used to investigate i) protease active site specificity; ii) protease in vivo substrates; iii) protease contribution to proteome homeostasis and composition; and iv) detection and localization of active proteases. Peptide libraries together with genetical or biochemical selection have traditionally been used for active site profiling of proteases. These are now complemented by proteome-derived peptide libraries that simultaneously determine prime and non-prime specificity and characterize subsite cooperativity. Cell-contextual discovery of protease substrates is rendered possible by techniques that isolate and quantitate protein termini. Here, a novel approach termed Terminal Amine Isotopic Labeling of Substrates (TAILS) provides an integrated platform for substrate discovery and appropriate statistical evaluation of terminal peptide identification and quantification. Proteolytically generated carboxy-termini can now also be analyzed on a proteome-wide level. Proteolytic regulation of proteome composition is monitored by quantitative proteomic approaches employing stable isotope coding or label free quantification. Activity-based probes specifically recognize active proteases. In proteomic screens, they can be used to detect and quantitate proteolytic activity while their application in cellular histology allows to locate proteolytic activity in situ. Activity-based probes – especially in conjunction with positron emission tomography – are also promising tools to monitor proteolytic activities on an organism-wide basis with a focus on in vivo tumor imaging. Together, this array of methodological possibilities enables unveiling physiological protease substrate repertoires and defining protease function in the cellular- and organism-wide context.     

Time-resolved Analysis of the Matrix Metalloproteinase 10 Substrate Degradome

Proteolysis is an irreversible post-translational modification that affects intra- and intercellular communication by modulating the activity of bioactive mediators. Key to understanding protease function is the system-wide identification of cleavage events and their dynamics in physiological contexts. Despite recent advances in mass spectrometry-based proteomics for high-throughput substrate screening, current approaches suffer from high false positive rates and only capture single states of protease activity. Here, we present a workflow based on multiplexed terminal amine isotopic labeling of substrates for time-resolved substrate degradomics in complex proteomes. This approach significantly enhances confidence in substrate identification and categorizes cleavage events by specificity and structural accessibility of the cleavage site. We demonstrate concomitant quantification of cleavage site spanning peptides and neo-N and/or neo-C termini to estimate relative ratios of noncleaved and cleaved forms of substrate proteins. By applying this strategy to dissect the matrix metalloproteinase 10 (MMP10) substrate degradome in fibroblast secretomes, we identified the extracellular matrix protein ADAMTS-like protein 1 (ADAMTSL1) as a direct MMP10 substrate and revealed MMP10-dependent ectodomain shedding of platelet-derived growth factor receptor alpha (PDGFRα) as well as sequential processing of type I collagen. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000503.Historically regarded as a mechanism for unspecific degradation of proteins, proteolysis is now recognized as a specific irreversible post-translational modification that affects major intra- and intercellular signaling processes (1, 2). Proteases specifically process bioactive proteins, their receptors, and associated proteins in an interconnected interaction network termed the protease web (3). Dysregulation of the protease web might cause or result from pathologies, such as impaired tissue repair, cancer and neurodegenerative diseases. Therefore, a better understanding of the functions of individual proteases and their interconnections within proteolytic networks is a prerequisite for exploiting proteases as targets for therapeutic intervention (4).To address this issue, several powerful technologies have been developed for the system-wide discovery of protease substrates, i.e. substrate degradomes, in complex and active proteomes (5, 6). A common principle of these mass spectrometry-based methods is the enrichment and monitoring of N-terminal peptides (protein neo-N termini) that are newly generated by a test protease (7). Protein N termini are enriched from complex proteomes either by chemical tagging and affinity resins (positive selection) or by depletion of internal peptides (negative selection) (7). Both principles have been successfully applied in various studies to characterize N-terminomes and to identify protease substrates using in vitro or cell-based systems and more recently also in vivo (8, 9). Negative enrichment approaches were further extended to the analysis of protein C termini (10, 11) and have the general advantage of recording data on naturally blocked (e.g. acetylated) N termini and internal peptides in the same experiment (8).Even if successful in identifying novel proteolytic cleavage events, which could also be validated by orthogonal methods, high-throughput substrate discovery approaches potentially suffer from high numbers of false positive identifications, particularly when employing in vitro systems (12). These have been reduced by monitoring abundances of N-terminal peptides at multiple time points after incubation of a proteome with a test protease (12). In this SILAC-based approach the authors efficiently distinguished critical from bystander cleavages, but it was limited to three time points. Therefore, it did not allow recording kinetic profiles of the relative abundance of N-terminal peptides that are required for determination of apparent kinetic parameters for processing events. Agard et al. elegantly overcame this limitation by use of selected reaction monitoring (SRM)¹ in combination with a positive N-terminal enrichment platform and determined apparent catalytic efficiencies for hundreds of caspase cleavage events in parallel (13). In a similar approach the same group characterized cellular responses to pro-apoptotic cancer drugs by recording time-courses for caspase-generated neo-N termini (14). Although very powerful and highly accurate in quantification, this method strongly exploited the canonical cleavage specificity of caspases after aspartate residues and required a two-stage process involving two types of mass spectrometers. Hence, it would be desirable to monitor the time-resolved generation of neo-N termini in complex proteomes in a single experiment by a simple and robust workflow in an unbiased manner.The development of such an analysis platform would require a reliable method for the system-wide characterization of protein N termini that is easy to perform, fast and highly multiplexible. All these criteria are met by iTRAQ-terminal amine isotopic labeling of substrates (TAILS), a multiplex N-terminome analysis technique that has been applied in 2plex and 4plex experiments to map the matrix metalloproteinase (MMP) 2 and MMP9 substrate degradomes in vitro (15) and most recently to quantitatively analyze the proteome and N-terminome of inflamed mouse skin in the presence or absence of the immune-modulatory protease MMP2 in vivo (8).Here, we exploited the multiplex capabilities of iTRAQ-TAILS by use of 8plex-iTRAQ reagents to monitor the generation of neo-N-terminal peptides by a test protease in complex samples over time. First, using GluC as a test protease with canonical cleavage specificity, we established a workflow for time-resolved substrate degradomics. Recording kinetic profiles significantly increased the confidence in identified cleavage events compared with binary systems and categorized primary cleavage specificities as well as secondary structure elements based on clusters of processing events with different efficiencies. By including data from before N-terminal enrichment, we extended our analysis to neo-C-terminal peptides and concomitantly monitored the generation of neo-N termini and neo-C termini as well as the decrease in abundance of the tryptic peptides spanning the cleavage sites in the same experiment. Next, we applied this approach to the time-resolved analysis of the hardly elucidated substrate degradome of matrix metalloproteinase 10 (MMP10). This important wound- and tumor-related protease is secreted by proliferating and migrating keratinocytes at the wound edge in close proximity to dermal fibroblasts and is also highly expressed in aggressive tumor cells (16–18). Our analysis revealed MMP10-dependent shedding of the platelet-derived growth factor receptor alpha (PDGFRα), processing of ADAMTS-like protein 1 (ADAMTSL1) and multiple cleavages of type I collagen, which could be validated and classified by time-resolved abundance profiles of their corresponding neo-N termini.     

Metadegradomics: toward in vivo quantitative degradomics of proteolytic post-translational modifications of the cancer proteome

Post-translational modifications enable extra layers of control of the proteome, and perhaps the most important is proteolysis, a major irreversible modification affecting every protein. The intersection of the protease web with a proteome sculpts that proteome, dynamically modifying its state and function. Protease expression is distorted in cancer, so perturbing signaling pathways and the secretome of the tumor and reactive stromal cells. Indeed many cancer biomarkers are stable proteolytic fragments. It is crucial to determine which proteases contribute to the pathology versus their roles in homeostasis and in mitigating cancer. Thus the full substrate repertoire of a protease, termed the substrate degradome, must be deciphered to define protease function and to identify drug targets. Degradomics has been used to identify many substrates of matrix metalloproteinases that are important proteases in cancer. Here we review recent degradomics technologies that allow for the broadly applicable identification and quantification of proteases (the protease degradome) and their activity state, substrates, and interactors. Quantitative proteomics using stable isotope labeling, such as ICAT, isobaric tags for relative and absolute quantification (iTRAQ), and stable isotope labeling by amino acids in cell culture (SILAC), can reveal protease substrates by taking advantage of the natural compartmentalization of membrane proteins that are shed into the extracellular space. Identifying the actual cleavage sites in a complex proteome relies on positional proteomics and utilizes selection strategies to enrich for protease-generated neo-N termini of proteins. In so doing, important functional information is generated. Finally protease substrates and interactors can be identified by interactomics based on affinity purification of protease complexes using exosite scanning and inactive catalytic domain capture strategies followed by mass spectrometry analysis. At the global level, the N terminome analysis of whole communities of proteases in tissues and organs in vivo provides a full scale understanding of the protease web and the web-sculpted proteome, so defining metadegradomics.     

A Tobacco Etch Virus Protease with Increased Substrate Tolerance at the P1' position

Site-specific proteases are important tools for in vitro and in vivo cleavage of proteins. They are widely used for diverse applications, like protein purification, assessment of protein–protein interactions or regulation of protein localization, abundance or activity. Here, we report the development of a procedure to select protease variants with altered specificity based on the well-established Saccharomyces cerevisiae adenine auxotrophy-dependent red/white colony assay. We applied this method on the tobacco etch virus (TEV) protease to obtain a protease variant with altered substrate specificity at the P1’ Position. In vivo experiments with tester substrates showed that the mutated TEV protease still efficiently recognizes the sequence ENLYFQ, but has almost lost all bias for the amino acid at the P1’ Position. Thus, we generated a site-specific protease for synthetic approaches requiring in vivo generation of proteins or peptides with a specific N-terminal amino acid.     

In vivo enzymatic digestion of HRV 3C protease cleavage sites-containing proteins produced in a silkworm-baculovirus expression system

Baculovirus expression vector system (BEVS) has been recognized as a potent protein expression system in engineering valuable enzymes and vaccines. Various fusion tags facilitate protein purification, leaving the potential risk to influence the target protein''s biological activity negatively. It is of great interest to consider removing the additional tags using site-specific proteases, such as human rhinoviruses (HRV) 3C protease. The current study validated the cleavage activity of 3C protease in Escherichia coli and silkworm-BEVS systems by mixing the cell or fat body lysates of 3C protein and 3C site containing target protein in vitro. Further verification has been performed in the fat body lysate from co-expression of both constructs, showing remarkable cleavage efficiency in vivo silkworm larvae. We also achieved the glutathione-S-transferase (GST) tag-cleaved product of the VP15 protein from the White spot syndrome virus after purification, suggesting that we successfully established a coinfection-based recognition-and-reaction BEVS platform for the tag-free protein engineering.     

Expression Profile of the Schistosoma japonicum Degradome Reveals Differential Protease Expression Patterns and Potential Anti-schistosomal Intervention Targets

Blood fluke proteases play pivotal roles in the processes of invasion, nutrition acquisition, immune evasion, and other host-parasite interactions. Hundreds of genes encoding putative proteases have been identified in the recently published schistosome genomes. However, the expression profiles of these proteases in Schistosoma species have not yet been systematically analyzed. We retrieved and culled the redundant protease sequences of Schistosoma japonicum, Schistosoma mansoni, Echinococcus multilocularis, and Clonorchis sinensis from public databases utilizing bioinformatic approaches. The degradomes of the four parasitic organisms and Homo sapiens were then comparatively analyzed. A total of 262 S. japonicum protease sequences were obtained and the expression profiles generated using whole-genome microarray. Four main clusters of protease genes with different expression patterns were identified: proteases up-regulated in hepatic schistosomula and adult worms, egg-specific or predominantly expressed proteases, cercaria-specific or predominantly expressed proteases, and constantly expressed proteases. A subset of protease genes with different expression patterns were further validated using real-time quantitative PCR. The present study represents the most comprehensive analysis of a degradome in Schistosoma species to date. These results provide a firm foundation for future research on the specific function(s) of individual proteases and may help to refine anti-proteolytic strategies in blood flukes.     

Development of selective protease inhibitors via engineering of the bait region of human α2-macroglobulin

Human α₂-macroglobulin (A2M) is an abundant protease inhibitor in plasma, which regulates many proteolytic processes and is involved in innate immunity. A2M’s unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible “bait region.” As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the “tabula rasa” bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer.     

PACMANS: A bioinformatically informed algorithm to predict,design, and disrupt protease‐on‐protease hydrolysis

Multiple proteases in a system hydrolyze target substrates, but recent evidence indicates that some proteases will degrade other proteases as well. Cathepsin S hydrolysis of cathepsin K is one such example. These interactions may be uni‐ or bi‐directional and change the expected kinetics. To explore potential protease‐on‐protease interactions in silico, a program was developed for users to input two proteases: (1) the protease‐ase that hydrolyzes (2) the substrate, protease. This program identifies putative sites on the substrate protease highly susceptible to cleavage by the protease‐ase, using a sliding‐window approach that scores amino acid sequences by their preference in the protease‐ase active site, culled from MEROPS database. We call this PACMANS, Protease‐Ase Cleavage from MEROPS ANalyzed Specificities, and test and validate this algorithm with cathepsins S and K. PACMANS cumulative likelihood scoring identified L253 and V171 as sites on cathepsin K subject to cathepsin S hydrolysis. Mutations made at these locations were tested to block hydrolysis and validate PACMANS predictions. L253A and L253V cathepsin K mutants significantly reduced cathepsin S hydrolysis, validating PACMANS unbiased identification of these sites. Interfamilial protease interactions between cathepsin S and MMP‐2 or MMP‐9 were tested after predictions by PACMANS, confirming its utility for these systems as well. PACMANS is unique compared to other putative site cleavage programs by allowing users to define the proteases of interest and target, and can also be employed for non‐protease substrate proteins, as well as short peptide sequences.     

Quantifying the Proteolytic Release of Extracellular Matrix-Sequestered VEGF with a Computational Model

Background

VEGF proteolysis by plasmin or matrix metalloproteinases (MMPs) is believed to play an important role in regulating vascular patterning in vivo by releasing VEGF from the extracellular matrix (ECM). However, a quantitative understanding of the kinetics of VEGF cleavage and the efficiency of cell-mediated VEGF release is currently lacking. To address these uncertainties, we develop a molecular-detailed quantitative model of VEGF proteolysis, used here in the context of an endothelial sprout.

Methodology and Findings

To study a cell''s ability to cleave VEGF, the model captures MMP secretion, VEGF-ECM binding, VEGF proteolysis from VEGF₁₆₅ to VEGF₁₁₄ (the expected MMP cleavage product of VEGF₁₆₅) and VEGF receptor-mediated recapture. Using experimental data, we estimated the effective bimolecular rate constant of VEGF₁₆₅ cleavage by plasmin to be 328 M⁻¹s⁻¹ at 25°C, which is relatively slow compared to typical MMP-ECM proteolysis reactions. While previous studies have implicated cellular proteolysis in growth factor processing, we show that single cells do not individually have the capacity to cleave VEGF to any appreciable extent (less than 0.1% conversion). In addition, we find that a tip cell''s receptor system will not efficiently recapture the cleaved VEGF due to an inability of cleaved VEGF to associate with Neuropilin-1.

Conclusions

Overall, VEGF₁₆₅ cleavage in vivo is likely to be mediated by the combined effect of numerous cells, instead of behaving in a single-cell-directed, autocrine manner. We show that heparan sulfate proteoglycans (HSPGs) potentiate VEGF cleavage by increasing the VEGF clearance time in tissues. In addition, we find that the VEGF-HSPG complex is more sensitive to proteases than is soluble VEGF, which may imply its potential relevance in receptor signaling. Finally, according to our calculations, experimentally measured soluble protease levels are approximately two orders of magnitude lower than that needed to reconcile levels of VEGF cleavage seen in pathological situations.     

Proteomic approaches to uncover MMP function

Proteomics has revolutionized protease research and particularly contributed to the identification of novel substrates and their sites of cleavage as key determinants of protease function. New technologies and rapid advancements in development of powerful mass spectrometers allowed unprecedented insights into activities of matrix metalloproteinases (MMPs) within their complex extracellular environments. Mass spectrometry-based proteomics extended our knowledge on MMP cleavage specificities and will help to develop more specific inhibitors as new therapeutics. Quantitative proteomics and N-terminal enrichment strategies have revealed numerous novel MMP substrates and shed light on their modes of action in vitro and in vivo. In this review, we provide an overview of current proteomic technologies in protease research and their application to the functional characterization of MMPs.     

Epistasis as a Determinant of the HIV-1 Protease's Robustness to Mutation

The robustness of phenotypes to mutation is critical to protein evolution; robustness may be an adaptive trait if it promotes evolution. We hypothesised that native proteins subjected to natural selection in vivo should be more robust than proteins generated in vitro in the absence of natural selection. We compared the mutational robustness of two human immunodeficiency virus type 1 (HIV-1) proteases with comparable catalytic efficiencies, one isolated from an infected individual and the second generated in vitro via random mutagenesis. Single mutations in the protease (82 and 60 in the wild-type and mutant backgrounds, respectively) were randomly generated in vitro and the catalytic efficiency of each mutant was determined. No differences were observed between these two protease variants when lethal, neutral, and deleterious mutations were compared (P = 0.8025, chi-squared test). Similarly, average catalytic efficiency (−72.6% and −64.5%, respectively) did not significantly differ between protease mutant libraries (P = 0.3414, Mann Whitney test). Overall, the two parental proteins displayed similar mutational robustness. Importantly, strong and widespread epistatic interactions were observed when the effect of the same mutation was compared in both proteases, suggesting that epistasis can be a key determinant of the robustness displayed by the in vitro generated protease.     

Fluorescent and bioluminescent nanoprobes for in vitro and in vivo detection of matrix metalloproteinase activity

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade the extracellular matrix (ECM) and regulate the extracellular microenvironment. Despite the significant role that MMP activity plays in cell-cell and cell-ECM interactions, migration, and differentiation, analyses of MMPs in vitro and in vivo have relied upon their abundance using conventional immunoassays, rather than their enzymatic activities. To resolve this issue, diverse nanoprobes have emerged and proven useful as effective activity-based detection tools. Here, we review the recent advances in luminescent nanoprobes and their applications in in vitro diagnosis and in vivo imaging of MMP activity. Nanoprobes with the purpose of sensing MMP activity consist of recognition and detection units, which include MMP-specific substrates and luminescent (fluorescent or bioluminescent) nanoparticles, respectively. With further research into improvement of the optical performance, it is anticipated that luminescent nanoprobes will have great potential for the study of the functional roles of proteases in cancer biology and nanomedicine. [BMB Reports 2015; 48(6): 313-318]     

Dysregulation of Protease and Protease Inhibitors in a Mouse Model of Human Pelvic Organ Prolapse

Mice deficient for the fibulin-5 gene (Fbln5^−/−) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5^−/− mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5^−/− mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5^−/− and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5^−/− epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.     

Analyzing protease specificity and detecting in vivo proteolytic events using tandem mass spectrometry

Although trypsin remains the most commonly used protease in MS, other proteases may be employed for increasing peptide coverage or generating overlapping peptides. Knowledge of the accurate specificity rules of these proteases is helpful for database search tools to detect peptides, and becomes crucial when label‐free MS is used to discover in vivo proteolytic cleavages. Since in vivo cleavages are inferred by subtracting digestion‐induced cleavages from all observed cleavages, it is important to ensure that the specificity rule used to identify digestion‐induced cleavages are broad enough to capture even minor cleavages produced in digestion, to avoid erroneously identifying them as in vivo cleavages. In this study, we describe MS‐Proteolysis, a software tool for identifying putative sites of in vivo proteolytic cleavage using label‐free MS. The tool is used in conjunction with digestion by trypsin and three other proteases, whose specificity rules are revised and extended before inferring proteolytic cleavages. Finally, we show that comparative analysis of multiple proteases can be used to detect putative in vivo proteolytic sites on a proteome‐wide scale.     

The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity

The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. Prostasin null (Prss8^−/−) mice lack barrier formation and display fatal postnatal dehydration. In sharp contrast, mice homozygous for a point mutation in the Prss8 gene, which causes the substitution of the active site serine within the catalytic histidine-aspartate-serine triad with alanine and renders prostasin catalytically inactive (Prss8^{Cat−/Cat−} mice), develop barrier function and are healthy when followed for up to 20 weeks. This striking difference could not be explained by genetic modifiers or by maternal effects, as these divergent phenotypes were displayed by Prss8^−/− and Prss8^{Cat−/Cat−} mice born within the same litter. Furthermore, Prss8^{Cat−/Cat−} mice were able to regenerate epidermal covering following cutaneous wounding. This study provides the first demonstration that essential in vivo functions of prostasin are executed by a non-enzymatic activity of this unique membrane-anchored serine protease.     

Development and binding characteristics of phosphonate inhibitors of SplA protease from Staphylococcus aureus

Staphylococcus aureus is responsible for a variety of human infections, including life-threatening, systemic conditions. Secreted proteome, including a range of proteases, constitutes the major virulence factor of the bacterium. However, the functions of individual enzymes, in particular SplA protease, remain poorly characterized. Here, we report development of specific inhibitors of SplA protease. The design, synthesis, and activity of a series of α-aminoalkylphosphonate diaryl esters and their peptidyl derivatives are described. Potent inhibitors of SplA are reported, which may facilitate future investigation of physiological function of the protease. The binding modes of the high-affinity compounds Cbz-Phe^P-(OC₆H₄−4-SO₂CH₃)₂ and Suc-Val-Pro-Phe^P-(OC₆H₅)₂ are revealed by high-resolution crystal structures of complexes with the protease. Surprisingly, the binding mode of both compounds deviates from previously characterized canonical interaction of α-aminoalkylphosphonate peptidyl derivatives and family S1 serine proteases.     

Solar Ultraviolet Irradiation Induces Decorin Degradation in Human Skin Likely via Neutrophil Elastase

Exposure of human skin to solar ultraviolet (UV) irradiation induces matrix metalloproteinase-1 (MMP-1) activity, which degrades type I collagen fibrils. Type I collagen is the most abundant protein in skin and constitutes the majority of skin connective tissue (dermis). Degradation of collagen fibrils impairs the structure and function of skin that characterize skin aging. Decorin is the predominant proteoglycan in human dermis. In model systems, decorin binds to and protects type I collagen fibrils from proteolytic degradation by enzymes such as MMP-1. Little is known regarding alterations of decorin in response to UV irradiation. We found that solar-simulated UV irradiation of human skin in vivo stimulated substantial decorin degradation, with kinetics similar to infiltration of polymorphonuclear (PMN) cells. Proteases that were released from isolated PMN cells degraded decorin in vitro. A highly selective inhibitor of neutrophil elastase blocked decorin breakdown by proteases released from PMN cells. Furthermore, purified neutrophil elastase cleaved decorin in vitro and generated fragments with similar molecular weights as those resulting from protease activity released from PMN cells, and as observed in UV-irradiated human skin. Cleavage of decorin by neutrophil elastase significantly augmented fragmentation of type I collagen fibrils by MMP-1. Taken together, these data indicate that PMN cell proteases, especially neutrophil elastase, degrade decorin, and this degradation renders collagen fibrils more susceptible to MMP-1 cleavage. These data identify decorin degradation and neutrophil elastase as potential therapeutic targets for mitigating sun exposure-induced collagen fibril degradation in human skin.