Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of methylglyoxal on the plasminogen-plasmin system is studied. Treatment of plasminogen with methylglyoxal at a 20-fold molar excess results in covalent modification of the molecule as evidenced by the decreased number of NH(2) side chains, arginine side chain residues and the new band in the non-tryptophan dependent fluorescent spectrum. This structural modification is associated with profound functional alterations: the rate of activation by streptokinase, tissue-type plasminogen activator, urokinase-type plasminogen activator and trypsin decreases and the amidolytic activity of the generated plasmin is impaired. Plasmin treatment with methylglyoxal on the other hand does not alter its steady-state kinetic parameters on a peptidyl-anilide synthetic substrate, indicating that modification susceptible side chains are sensitive to methylglyoxal only in the zymogen. Our data suggest that in vivo fibrinolysis could be impaired under pathological conditions, e.g. increased methylglyoxal formation in diabetes mellitus.
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PMID:Modulation of plasminogen activation and plasmin activity by methylglyoxal modification of the zymogen. 1089 32

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. Thrombin selectively cleaves PAR1, PAR3, and PAR4 to induce activation of platelets and vascular cells, while PAR2 is preferentially cleaved by trypsin. In pathological situations, other proteolytic enzymes may be generated in the circulation and could modify the responses of PARs by cleaving their extracellular domains. To assess the ability of such proteases to activate or inactivate PARs, we designed a strategy for locating cleavage sites on the exofacial NH(2)-terminal fragments of the receptors. The first extracellular segments of PAR1 (PAR1E) and PAR2 (PAR2E) expressed as recombinant proteins in Escherichia coli were incubated with a series of proteases likely to be encountered in the circulation during thrombosis or inflammation. Kinetic and dose-response studies were performed, and the cleavage products were analyzed by MALDI-TOF mass spectrometry. Thrombin cleaved PAR1E at the Arg41-Ser42 activation site at concentrations known to induce cellular activation, supporting a native conformation of the recombinant polypeptide. Plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3 cleaved at multiple sites and would be expected to disable PAR1 by cleaving COOH-terminal to the activation site. Cleavage specificities were further confirmed using activation site defective PAR1E S42P mutant polypeptides. Surface plasmon resonance studies on immobilized PAR1E or PAR1E S42P were consistent with cleavage results obtained in solution and allowed us to determine affinities of PAR1E-thrombin binding. FACS analyses of intact platelets confirmed the cleavage of PAR1 downstream of the Arg41-Ser42 site. Mass spectrometry studies of PAR2E predicted activation of PAR2 by trypsin through cleavage at the Arg36-Ser37 site, no effect of thrombin, and inactivation of the receptor by plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3. The inhibitory effect of elastase was confirmed on native PAR1 and PAR2 on the basis of Ca(2+) signaling studies in endothelial cells. It was concluded that none of the main proteases generated during fibrinolysis or inflammation appears to be able to signal through PAR1 or PAR2. This strategy provides results which can be extended to the native receptor to predict its activation or inactivation, and it could likewise be used to study other PARs or protease-dependent processes.
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PMID:Proteolysis of the exodomain of recombinant protease-activated receptors: prediction of receptor activation or inactivation by MALDI mass spectrometry. 1097 67

GMP-33 is a platelet membrane associated protein that is recognised by RUU-SP 1.77, an antibody raised against activated platelets. GMP-33 is predominantly associated with the membrane of platelet alpha-granules and it is translocated to the plasma membrane upon platelet activation (Metzelaar et al. Blood 1992; 79: 372-9). In this study we have isolated the protein by immunoaffinity chromatography. The N-terminus was sequenced and was identical to the N-terminal sequence of human thrombospondin. The protein was N-glycosylated and bound to heparin as would be expected of the N-terminal part of thrombospondin. RUU-SP 1.77 reacted only with reduced thrombospondin. Plasmin and trypsin digestion of thrombospondin yielded fragments of approximately the same size as GMP 33 that reacted with RUU-SP 1.77 after reduction. No evidence for alternative splicing was found. We postulate that GMP 33 is an N-terminal proteolytic fragment of thrombospondin that is membrane associated.
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PMID:The 33-kDa platelet alpha-granule membrane protein (GMP-33) is an N-terminal proteolytic fragment of thrombospondin. 1158 23

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. We have reported earlier that primary cultures of rat brain capillary endothelial (RBCE) cells express at least two receptors for thrombin: PAR-1 and PAR-3. In the present study we show that PAR-2 activation by trypsin or by the PAR-2 agonist peptide (SLIGRL) evokes [Ca(2+) ](i) signal in RBCE cells. Taking advantage of RBCE cells expressing PAR-1 and PAR-2, we show that trypsin activates both receptors. The relative agonist activity of trypsin and thrombin on PARs of RBCE cells compared with that of SLIGRL were 112% and 48%, respectively, whereas the potency of trypsin was 10(5) -fold higher than that of SLIGRL. Because under pathological conditions other proteases such as plasmin or leukocyte elastase may reach the cells of the blood-brain barrier, we investigated the effect of these proteases on RBCE cells. Elastase evoked a small increase in [Ca(2+) ](i) but preincubation of cells with elastase dose-dependently reduced the trypsin-induced [Ca(2+) ](i) signal. Plasmin had a 30% inhibitory effect on the trypsin-induced response, and reduced the SLIGRL signal by 20%. It is concluded that PAR-2 is functional in brain capillary endothelium, and that the main fibrinolytic proteases, plasmin and elastase, may regulate PAR-2 signalling under pathological conditions.
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PMID:Protease-activated receptor-2 (PAR-2) in brain microvascular endothelium and its regulation by plasmin and elastase. 1194 37

Many eukaryotic and prokaryotic cells bind plasminogen in a specific and saturable manner. When plasminogen is bound to cell-surface proteins with C-terminal lysines via its lysine binding sites, its activation to plasmin is accelerated, and cell-bound plasmin is protected from inactivation by natural inhibitors. Plasmin mediates direct or indirect degradation of the extracellular matrix, and bound plasmin is used by cells to facilitate migration through extracellular matrices. Since cell migration and tissue remodelling are the underpinnings of many physiological and pathological responses, the modulation of plasminogen receptors may serve as a primary regulatory mechanism for control of many cellular responses. Specific examples of cell types on which plasminogen receptors undergo modulation include: fibroblasts, where modulation may contribute to cartilage and bone destruction in rheumatoid arthritis; leukemic cells, where enhanced plasminogen binding may contribute to the heightened fibrinolytic state in the patients; other tumor cells, where up-regulation may support invasion and metastasis; bacteria, where enhanced plasminogen binding may facilitate tissue destruction and invasion; platelets, where up-regulation of plasminogen binding may play a role in regulating clot lysis; and adipocytes, where the modulation of plasminogen receptor expression may regulate cell differentiation and fat accumulation. Two pathways for modulation of plasminogen receptors have been characterized: A protease-dependent pathway can either up-regulate or down-regulate plasminogen binding to cells by changing the availability of plasminogen-binding proteins with C-terminal lysines. New receptors may be generated by trypsin-like proteases, including plasmin, which create new C-terminal lysines; other enzymes may expose existing membrane proteins by altering the cell surface; or receptor function may be lost by removal of C-terminal lysines. The basic carboxypeptidases of blood carboxypeptidase N and plasma carboxypeptidase B (TAFI) mediate such down-regulation. A non-protease dependent pathway for modulation of plasminogen receptors may be initiated by growth factors, chemokines or cytokines that alter the cell membrane and/or cytoskeleton architectures to expose plasminogen binding sites. Many examples of the modulation of plasminogen receptors have been demonstrated in vitro, and the development of knock-out mice may soon lead to incisive evaluations of the significance of the regulation of plasminogen receptors in vivo.
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PMID:Regulation of plasminogen receptors. 1245 18

Suspension of cultured human foreskin keratinocytes (HKs) with trypsin phosphorylates tyrosine residues on an 80-kDa membrane glycoprotein, p80 (Xia, Y., Gil, S. G., and Carter, W. G. (1996) J. Cell Biol. 132, 727-740). Readhesion dephosphorylates p80. Sequencing of a p80 cDNA established identity to CUB domain-containing protein 1 (CDCP1), a gene elevated in carcinomas. CDCP1/p80 cDNA encodes three extracellular CUB domains, a transmembrane domain, and two putative cytoplasmic Tyr phosphorylation sites. Treatment of adherent HKs with suramin, a heparin analogue, or inhibitors of phosphotyrosine phosphatases (PTPs; vanadate or calpeptin) increases phosphorylation of p80 and a novel 140-kDa membrane glycoprotein, gp140. Phosphorylated gp140 was identified as a trypsin-sensitive precursor to p80. Identity was confirmed by digestion and phosphorylation studies with recombinant gp140-GFP. Plasmin, a serum protease, also converts gp140 to p80, providing biological significance to the cleavage in wounds. Phosphorylation of gp140 and p80 are mediated by Src family kinases at multiple Tyr residues including Tyr(734). Dephosphorylation is mediated by PTP(s). Conversion of gp140 to p80 prolongs phosphorylation of p80 in response to suramin and changes in adhesion. This distinguishes gp140 and p80 and explains the relative abundance of phosphorylated p80 in trypsinized HKs. We conclude that phosphorylation of gp140 is dynamic and balanced by Src family kinase and PTPs yielding low equilibrium phosphorylation. We suggest that the balance is altered by conversion of gp140 to p80 and by adhesion, providing a novel transmembrane phosphorylation signal in epithelial wounds.
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PMID:Adhesion or plasmin regulates tyrosine phosphorylation of a novel membrane glycoprotein p80/gp140/CUB domain-containing protein 1 in epithelia. 1473 93

alpha2-Macroglobulin (alpha2M) is a protease inhibitor that has separate binding sites for transforming growth factor-beta (TGF-beta) and beta-amyloid peptide (Abeta), both of which have been identified in the beta2M sequence. In the 3D-structure of alpha2M, TGF-beta occupies the alpha2M central cavity, overlapping with the space that can accommodate up to two molecules of protease. As a result, ternary alpha2M-protease complexes (2 mol protease/mol alpha2M) have been reported to not bind TGF-beta. The goal of the present study was to test whether binding of Abeta to alpha2M is controlled by steric constraints imposed by associated proteases, similarly to TGF-beta. We confirmed that binary alpha2M-trypsin complex (1 mol trypsin/mol alpha2M) binds increased amounts of TGF-beta1, compared with native alpha2M, while ternary alpha2M-trypsin complex binds substantially decreased amounts of TGF-beta1. By contrast, Abeta-binding to binary and ternary alpha2M trypsin complex was equivalent. In both cases, binding was substantially increased compared with the negligible level observed with native alpha2M. Plasmin is a large protease (Mr approximately 82,000) that substantially occupies the alpha2M central cavity; however, alpha2M-plasmin complex also bound increased amounts of Abeta, compared with native alpha2M. We conclude that Abeta accesses its binding site, in alpha2M, from outside the alpha2M central cavity. The TGF-beta- and Abeta-binding sites are spatially separated not only in the primary sequence of alpha2M, but also in the 3D-structure.
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PMID:Beta-amyloid peptide binds equivalently to binary and ternary alpha2-macroglobulin-protease complexes. 1600 50

1. Fibrinolysin-activated lysin factor and chloroform-activated serum protease of serum and plasma are one and the same enzyme, differing only in their mode of activation. 2. The enzyme as it normally occurs in serum or plasma is not inactive because of combination with serum inhibitor. It is present as an inactive precursor or zymogen and may be activated from this state by streptococcal fibrinolysin. 3. The activation of serum protease by streptococcal fibrinolysin is a catalytic reaction, analogous to the kinase activation of trypsinogen by enterokinase. Treatment of serum or plasma with chloroform apparently results in removal of serum inhibitor which may allow autocatalytic activation of the serum protease. 4. The serum enzyme differs from trypsin in its pH of optimum activity, in its reactions with specific protease inhibitors, and in its action on casein. 5. A revised nomenclature for the serum enzyme system is suggested which more accurately describes its properties than the terms in current use.
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PMID:A PROTEOLYTIC ENZYME OF SERUM: CHARACTERIZATION, ACTIVATION, AND REACTION WITH INHIBITORS. 1987 36

Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ_0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ_0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ_0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ_0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ_0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ_0125 and a source of amino acids for growth of M. hyopneumoniae. This unique interaction represents a new paradigm in microbial pathogenesis.
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PMID:MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae. 2359 79

A plasmin inhibitor, named tenerplasminin-1 (TP1), was isolated from Micrurus tener tener (Mtt) venom. It showed a molecular mass of 6542Da, similarly to Kunitz-type serine peptidase inhibitors. The amidolytic activity of plasmin (0.5nM) on synthetic substrate S-2251 was inhibited by 91% following the incubation with TP1 (1nM). Aprotinin (2nM) used as the positive control of inhibition, reduced the plasmin amidolytic activity by 71%. Plasmin fibrinolytic activity (0.05nM) was inhibited by 67% following incubation with TP1 (0.1nM). The degradation of fibrinogen chains induced by plasmin, trypsin or elastase was inhibited by TP1 at a 1:2, 1:4 and 1:20 enzyme:inhibitor ratio, respectively. On the other hand, the proteolytic activity of crude Mtt venom on fibrinogen chains, previously attributed to metallopeptidases, was not abolished by TP1. The tPA-clot lysis assay showed that TP1 (0.2nM) acts like aprotinin (0.4nM) inducing a delay in lysis time and lysis rate which may be associated with the inhibition of plasmin generated from the endogenous plasminogen activation. TP1 is the first serine protease plasmin-like inhibitor isolated from Mtt snake venom which has been characterized in relation to its mechanism of action, formation of a plasmin:TP1 complex and therapeutic potential as anti-fibrinolytic agent, a biological characteristic of great interest in the field of biomedical research. They could be used to regulate the fibrinolytic system in pathologies such as metastatic cancer, parasitic infections, hemophilia and other hemorrhagic syndromes, in which an intense fibrinolytic activity is observed.
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PMID:Purification and characterization of tenerplasminin-1, a serine peptidase inhibitor with antiplasmin activity from the coral snake (Micrurus tener tener) venom. 2641 85


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