-
Journal of Thrombosis and Haemostasis :... Nov 2005Proteases play diverse roles in a variety of essential biological processes, both as non-specific catalysts of protein degradation and as highly specific agents that... (Review)
Review
Proteases play diverse roles in a variety of essential biological processes, both as non-specific catalysts of protein degradation and as highly specific agents that control physiologic events. Here, we review the mechanisms of substrate specificity employed by serine proteases and focus our discussion on coagulation proteases. We dissect the interplay between active site and exosite specificity and how substrate recognition is regulated allosterically by Na+ binding. We also draw attention to a functional polarity that exists in the serine protease fold, which sheds light on the structural linkages between the active site and exosites.
Topics: Allosteric Regulation; Amino Acid Sequence; Animals; Binding Sites; Blood Coagulation; Catalytic Domain; Enzyme Activation; Humans; Molecular Sequence Data; Protein Conformation; Protein Folding; Serine Endopeptidases; Sodium; Substrate Specificity; Thrombin
PubMed: 16241939
DOI: 10.1111/j.1538-7836.2005.01456.x -
Frontiers in Immunology 2022Serine proteases has been considered to be closely associated with the inflammatory response and tumor progression. As a novel serine protease, the biological function...
Serine proteases has been considered to be closely associated with the inflammatory response and tumor progression. As a novel serine protease, the biological function of PRSS23 is rarely studied in cancers. In this study, the prognostic significance of PRSS23 was analyzed in two-independent gastric cancer (GC) cohorts. PRSS23 overexpression was clinically correlated with poor prognosis and macrophage infiltration of GC patients. Loss-of-function study verified that PRSS23 plays oncogenic role in GC. RNA-seq, qRT-PCR, western blotting and ELISA assay confirmed that serine protease PRSS23 positively regulated FGF2 expression and secretion. Single-cell analysis and gene expression correlation analysis showed that PRSS23 and FGF2 were high expressed in fibroblasts, and highly co-expressed with the biomarkers of tumor associated macrophages (TAMs), cancer-associated fibroblasts (CAFs) and mesenchymal cells. Functional analysis confirmed PRSS23/FGF2 was required for TAM infiltration. Rescue assay further verified that PRSS23 promotes GC progression and TAM infiltration through FGF2. Survival analysis showed that high infiltration of M1-macrophage predicted favorable prognosis, while high infiltration level of M2-macrophage predicted poor prognosis in GC. Our finding highlights that PRSS23 promotes TAM infiltration through regulating FGF2 expression and secretion, thereby resulting in a poor prognosis.
Topics: Biomarkers; Fibroblast Growth Factor 2; Humans; Serine Endopeptidases; Serine Proteases; Stomach Neoplasms; Tumor-Associated Macrophages
PubMed: 36189305
DOI: 10.3389/fimmu.2022.955841 -
Development (Cambridge, England) Aug 2016The membrane-anchored serine proteases prostasin (PRSS8) and matriptase (ST14) initiate a cell surface proteolytic pathway essential for epithelial function. Mice...
The membrane-anchored serine proteases prostasin (PRSS8) and matriptase (ST14) initiate a cell surface proteolytic pathway essential for epithelial function. Mice expressing only catalytically inactive prostasin are viable, unlike prostasin null mice, indicating that at least some prostasin functions are non-proteolytic. Here we used knock-in mice expressing catalytically inactive prostasin (Prss8(Ki/Ki)) to show that the physiological and pathological functions of prostasin vary in their dependence on its catalytic activity. Whereas prostasin null mice exhibited partial embryonic and complete perinatal lethality, Prss8(Ki/Ki) mice displayed normal prenatal and postnatal survival. Unexpectedly, catalytically inactive prostasin caused embryonic lethality in mice lacking its cognate inhibitors HAI-1 (SPINT1) or HAI-2 (SPINT2). Proteolytically inactive prostasin, unlike the wild-type protease, was unable to activate matriptase during placentation. Surprisingly, all essential functions of prostasin in embryonic and postnatal development were compensated for by loss of HAI-1, indicating that prostasin is only required for mouse development and overall viability in the presence of this inhibitor. This study expands our knowledge of non-proteolytic functions of membrane-anchored serine proteases and provides unexpected new data on the mechanistic interactions between matriptase and prostasin in the context of epithelial development.
Topics: Animals; Female; Membrane Glycoproteins; Membrane Proteins; Mice; Placentation; Pregnancy; Proteinase Inhibitory Proteins, Secretory; Proteolysis; Serine Endopeptidases; Serine Proteases
PubMed: 27385010
DOI: 10.1242/dev.137968 -
Trends in Biotechnology Nov 2011Trypsin-like proteases (TLPs) are a large family of enzymes responsible for digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis and... (Review)
Review
Trypsin-like proteases (TLPs) are a large family of enzymes responsible for digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis and immunity. A current paradigm posits that the irreversible transition from an inactive zymogen to the active protease form enables productive interaction with substrate and catalysis. Analysis of the entire structural database reveals two distinct conformations of the active site: one fully accessible to substrate (E) and the other occluded by the collapse of a specific segment (E*). The allosteric E*-E equilibrium provides a reversible mechanism for activity and regulation in addition to the irreversible zymogen to protease conversion and points to new therapeutic strategies aimed at inhibiting or activating the enzyme. In this review, we discuss relevant examples, with emphasis on the rational engineering of anticoagulant thrombin mutants.
Topics: Allosteric Regulation; Catalytic Domain; Humans; Models, Molecular; Protein Conformation; Protein Engineering; Serine Endopeptidases; Thrombin
PubMed: 21726912
DOI: 10.1016/j.tibtech.2011.06.001 -
Biochimica Et Biophysica Acta Dec 2013Endopeptidase classification based on catalytic mechanism and evolutionary history has proven to be invaluable to the study of proteolytic enzymes. Such general... (Review)
Review
Endopeptidase classification based on catalytic mechanism and evolutionary history has proven to be invaluable to the study of proteolytic enzymes. Such general mechanistic- and evolutionary- based groupings have launched experimental investigations, because knowledge gained for one family member tends to apply to the other closely related enzymes. The serine endopeptidases represent one of the most abundant and diverse groups, with their apparently successful proteolytic mechanism having arisen independently many times throughout evolution, giving rise to the well-studied soluble chemotrypsins and subtilisins, among many others. A large and diverse family of polytopic transmembrane proteins known as rhomboids has also evolved the serine protease mechanism. While the spatial structure, mechanism, and biochemical function of this family as intramembrane proteases has been established, the cellular roles of these enzymes as well as their natural substrates remain largely undetermined. While the evolutionary history of rhomboid proteases has been debated, sorting out the relationships among current day representatives should provide a solid basis for narrowing the knowledge gap between their biochemical and cellular functions. Indeed, some functional characteristics of rhomboid proteases can be gleaned from their evolutionary relationships. Finally, a specific case where phylogenetic profile analysis has identified proteins that contain a C-terminal processing motif (GlyGly-Cterm) as co-occurring with a set of bacterial rhomboid proteases provides an example of potential target identification through bioinformatics. This article is part of a Special Issue entitled: Intramembrane Proteases.
Topics: Animals; Bacteria; Bacterial Proteins; Cell Membrane; Computational Biology; Evolution, Molecular; Humans; Multigene Family; Phylogeny; Plants; Proteolysis; Sequence Homology, Amino Acid; Serine Endopeptidases; Substrate Specificity
PubMed: 23845876
DOI: 10.1016/j.bbamem.2013.06.031 -
Nature Communications Feb 2023Tailoring of the activity and specificity of proteases is critical for their utility across industrial, medical and research purposes. However, engineering or evolving...
Tailoring of the activity and specificity of proteases is critical for their utility across industrial, medical and research purposes. However, engineering or evolving protease catalysts is challenging and often labour intensive. Here, we describe a generic method to accelerate this process based on yeast display. We introduce the protease selection system A2M that covalently captures protease catalysts by repurposed alpha-2-macroglobulin (A2Ms). To demonstrate the utility of A2M for protease engineering we exemplify the directed activity and specificity evolution of six serine proteases. This resulted in a variant of Staphylococcus aureus serin-protease-like (Spl) protease SplB, an enzyme used for recombinant protein processing, that no longer requires activation by N-terminal signal peptide removal. SCHEMA-based domain shuffling was used to map the specificity determining regions of Spl proteases, leading to a chimeric scaffold that supports specificity switching via subdomain exchange. The ability of A2M to overcome key challenges en route to tailor-made proteases suggests easier access to such reagents in the future.
Topics: Humans; Pregnancy-Associated alpha 2-Macroglobulins; Protein Sorting Signals; Recombinant Proteins; Serine Endopeptidases; Serine Proteases; alpha-Macroglobulins
PubMed: 36765057
DOI: 10.1038/s41467-023-36099-7 -
American Journal of Physiology.... Sep 2016Barrier dysfunction is a characteristic of the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis. Understanding how the tight junction is...
Barrier dysfunction is a characteristic of the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis. Understanding how the tight junction is modified to maintain barrier function may provide avenues for treatment of IBD. We have previously shown that the apical addition of serine proteases to intestinal epithelial cell lines causes a rapid and sustained increase in transepithelial electrical resistance (TER), but the mechanisms are unknown. We hypothesized that serine proteases increase barrier function through trafficking and insertion of tight junction proteins into the membrane, and this could enhance recovery of a disrupted monolayer after calcium switch or cytokine treatment. In the canine epithelial cell line, SCBN, we showed that matriptase, an endogenous serine protease, could potently increase TER. Using detergent solubility-based cell fractionation, we found that neither trypsin nor matriptase treatment changed levels of tight junction proteins at the membrane. In a fast calcium switch assay, serine proteases did not enhance the rate of recovery of the junction. In addition, serine proteases could not reverse barrier disruption induced by IFNγ and TNFα. We knocked down occludin in our cells using siRNA and found this prevented the serine protease-induced increase in TER. Using fluorescence recovery after photobleaching (FRAP), we found serine proteases induce a greater mobile fraction of occludin in the membrane. These data suggest that a functional tight junction is needed for serine proteases to have an effect on TER, and that occludin is a crucial tight junction protein in this mechanism.
Topics: Animals; Cell Line; Dogs; Electric Impedance; Electrophysiological Phenomena; Epithelial Cells; Intestinal Mucosa; Occludin; Protein Transport; Serine Endopeptidases; Serine Proteases; Tight Junction Proteins; Tight Junctions; Trypsin
PubMed: 27492333
DOI: 10.1152/ajpgi.00441.2015 -
Proceedings of the National Academy of... Apr 2022SignificanceClassic serine proteases are synthesized as inactive precursors that are proteolytically processed, resulting in irreversible activation. We report an...
SignificanceClassic serine proteases are synthesized as inactive precursors that are proteolytically processed, resulting in irreversible activation. We report an alternative and reversible mechanism of activation that is executed by an inactive protease. This mechanism involves a protein complex between the serine protease HTRA1 and the cysteine protease calpain 2. Surprisingly, activation is restricted as it improves the proteolysis of soluble tau protein but not the dissociation and degradation of its amyloid fibrils, a task that free HTRA1 is efficiently performing. These data exemplify a challenge for protein quality control proteases in the clearing of pathogenic fibrils and suggest a potential for unexpected side effects of chemical modulators targeting PDZ or other domains located at a distance to the active site.
Topics: Amyloid; Calpain; High-Temperature Requirement A Serine Peptidase 1; Proteolysis; Serine Endopeptidases; Serine Proteases
PubMed: 35349341
DOI: 10.1073/pnas.2113520119 -
The Journal of Clinical Investigation Mar 1990Two 29-kD polypeptides, azurocidin and p29b, were purified to homogeneity from human neutrophils by acid extraction of azurophil granule membrane-associated material...
Two 29-kD polypeptides, azurocidin and p29b, were purified to homogeneity from human neutrophils by acid extraction of azurophil granule membrane-associated material followed by gel filtration and reverse-phase chromatography. Azurocidin and p29b share NH2-terminal sequence homology with each other as well as with elastase, cathepsin G, and other serine proteases. p29b bound [3H]diisopropyl fluorophosphate and hydrolyzed elastin, casein, and hemoglobin. A peptide substrate for p29b could not be identified. Azurocidin neither bound [3H]diisopropyl fluorophosphate nor hydrolyzed any of the proteins, peptides, or esters tested. In microbicidal assays, purified azurocidin was comparable to p29b in activity against Escherichia coli, Streptococcus faecalis, and Candida albicans. The antimicrobial activity of azurocidin was enhanced under mildly acidic conditions, but was inhibited in a dose-dependent manner by NaCl, CaCl2, or serum. Immunoblot analysis with monospecific antibodies localized greater than 90% of the azurocidin and greater than 75% of the p29b to azurophil granule-rich fractions of PMN lysates. Immunoelectron microscopy confirmed the localization of azurocidin to the azurophil granules. Azurocidin associated with the azurophil granule membrane, but did not appear to be an integral membrane protein. Thus, azurocidin and p29b are members of a family of serine protease homologs stored in azurophil granules and may play a role in inflammatory and antimicrobial processes involving PMN.
Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Blood Bactericidal Activity; Blood Proteins; Carrier Proteins; Cathepsin G; Cathepsins; Cytoplasmic Granules; Humans; Hydrogen-Ion Concentration; Neutrophils; Osmolar Concentration; Pancreatic Elastase; Serine Endopeptidases
PubMed: 2312733
DOI: 10.1172/JCI114518 -
Frontiers in Immunology 2023Overactivation of the lectin pathway of complement plays a pathogenic role in a broad range of immune-mediated and inflammatory disorders; mannan-binding...
INTRODUCTION
Overactivation of the lectin pathway of complement plays a pathogenic role in a broad range of immune-mediated and inflammatory disorders; mannan-binding lectin-associated serine protease-2 (MASP-2) is the key effector enzyme of the lectin pathway. We developed a fully human monoclonal antibody, narsoplimab, to bind to MASP-2 and specifically inhibit lectin pathway activation. Herein, we describe the preclinical characterization of narsoplimab that supports its evaluation in clinical trials.
METHODS AND RESULTS
ELISA binding studies demonstrated that narsoplimab interacted with both zymogen and enzymatically active forms of human MASP-2 with high affinity (K 0.062 and 0.089 nM, respectively) and a selectivity ratio of >5,000-fold relative to closely related serine proteases C1r, C1s, MASP-1, and MASP-3. Interaction studies using surface plasmon resonance and ELISA demonstrated approximately 100-fold greater binding affinity for intact narsoplimab compared to a monovalent antigen binding fragment, suggesting an important contribution of functional bivalency to high-affinity binding. In functional assays conducted in dilute serum under pathway-specific assay conditions, narsoplimab selectively inhibited lectin pathway-dependent activation of C5b-9 with high potency (IC ~ 1 nM) but had no observable effect on classical pathway or alternative pathway activity at concentrations up to 500 nM. In functional assays conducted in 90% serum, narsoplimab inhibited lectin pathway activation in human serum with high potency (IC ~ 3.4 nM) whereas its potency in cynomolgus monkey serum was approximately 10-fold lower (IC ~ 33 nM). Following single dose intravenous administration to cynomolgus monkeys, narsoplimab exposure increased in an approximately dose-proportional manner. Clear dose-dependent pharmacodynamic responses were observed at doses >1.5 mg/kg, as evidenced by a reduction in lectin pathway activity assessed that increased in magnitude and duration with increasing dose. Analysis of pharmacokinetic and pharmacodynamic data revealed a well-defined concentration-effect relationship with an EC value of approximately 6.1 μg/mL, which was comparable to the functional potency (IC 33 nM; ~ 5 μg/mL).
DISCUSSION
Based on these results, narsoplimab has been evaluated in clinical trials for the treatment of conditions associated with inappropriate lectin pathway activation, such as hematopoietic stem cell transplantation-associated thrombotic microangiopathy.
Topics: Animals; Humans; Mannose-Binding Protein-Associated Serine Proteases; Lectins; Macaca fascicularis; Serine Endopeptidases
PubMed: 38022610
DOI: 10.3389/fimmu.2023.1297352