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)

Plasmin inhibited the biosynthesis of tissue-type plasminogen activator (tPA) antigen by human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. The amount of tPA antigen found in the 24-h conditioned medium of cells treated with 100 nM plasmin for 1 h was 20-30% of that in the control group. However, in contrast to tPA, such treatment led to a 3-fold increase in plasminogen activator inhibitor (PAI) activity, whereas the amount of PAI type 1 antigen was unchanged. The effects of plasmin on HUVEC were binding- and catalytic activity-dependent and were specifically blocked by epsilon-aminocaproic acid. Microplasmin, which has no kringle domains, was less effective in reducing tPA antigen biosynthesis or enhancing PAI activity in HUVEC. Kringle domains of plasmin affected neither tPA antigen nor PAI activity of the cells. Other proteases including chymotrypsin, trypsin, and collagenase at comparable concentrations did not have a significant effect on the biosynthesis of tPA antigen or PAI activity of HUVEC. Thrombin stimulated the biosynthesis of tPA and PAI-1 antigens by HUVEC. Thrombin also stimulated an increase in the protein kinase activity in HUVEC, whereas plasmin inhibited the protein kinase activity of the cells. It is possible that plasmin regulates the biosynthesis of tPA in HUVEC through the signal transduction pathway involving protein kinase.
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PMID:Plasmin and the regulation of tissue-type plasminogen activator biosynthesis in human endothelial cells. 138 68

Migrating cells degrade pericellular matrices and basement membranes. For these purposes cells produce a number of proteolytic enzymes. Mast cells produce two major proteinases, chymase and tryptase, whose physiological functions are poorly known. In the present study we have analyzed the ability of purified human mast cell tryptase to digest pericellular matrices of human fibroblasts. Isolated matrices of human fibroblasts and fibroblast conditioned medium were treated with tryptase, and alterations in the radiolabeled polypeptides were observed in autoradiograms of sodium dodecyl sulphate polyacrylamide gels. It was found that an M(r) 72,000 protein was digested to an M(r) 62,000 form by human mast cell tryptase while the plasminogen activator inhibitor, PAI-1, was not affected. Cleavage of the M(r) 72,000 protein could be partially inhibited by known inhibitors of tryptase but not by aprotinin, soybean trypsin inhibitor, or EDTA. Fibroblastic cells secreted the M(r) 72,000 protein into their medium and it bound to gelatin as shown by analysis of the medium by affinity chromatography over gelatin-Sepharose. The soluble form of the M(r) 72,000 protein was also susceptible to cleavage by tryptase. Analysis using gelatin containing polyacrylamide gels showed that both the intact M(r) 72,000 and the M(r) 62,000 degraded form of the protein possess gelatinolytic activity after activation by sodium dodecyl sulphate. Immunoblotting analysis of the matrices revealed the cleavage of an immunoreactive protein of M(r) 72,000 indicating that the protein is related to type IV collagenase. Further analysis of the pericellular matrices indicated that the protease sensitive extracellular matrix protein fibronectin was removed from the matrix by tryptase in a dose-dependent manner. Fibronectin was also susceptible to proteolytic degradation by tryptase. The data suggest a role for mast cell tryptase in the degradation of pericellular matrices.
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PMID:Pericellular substrates of human mast cell tryptase: 72,000 dalton gelatinase and fibronectin. 146 68

Evidence has accumulated that invasion and metastasis in solid tumors require the action of tumor-associated proteases, which promote the dissolution of the surrounding tumor matrix and the basement membranes. Receptor-bound urokinase-type plasminogen activator (uPA) appears to play a key role in these events. uPA converts plasminogen into plasmin and thus mediates pericellular proteolysis during cell migration and tissue remodeling under physiological and pathophysiological conditions. uPA is secreted as an enzymatically inactive proenzyme (pro-uPA) by tumor cells and stroma cells. uPA exerts its proteolytic function on normal cells and tumor cells as an ectoenzyme after having bound to a high-affinity cell surface receptor. After binding, pro-uPA is activated by serine proteases (e.g. plasmin, trypsin or plasma kallikrein) and by the cysteine proteases cathepsin B or L, resp. Receptor-bound enzymatically active uPA converts plasminogen to plasmin which is bound to a different low-affinity receptor on tumor cells. Plasmin then degrades components of the tumor stroma (e.g. fibrin, fibronectin, proteoglycans, laminin) and may activate procollagenase type IV which degrades collagen type IV, a major part of the basement membrane. Hence receptor-bound uPA will promote plasminogen activation and thus the dissolution of the tumor matrix and the basement membrane which is a prerequisite for invasion and metastasis. Tissues of primary cancer and/or metastases of the breast, ovary, prostate, cervix uteri, bladder, lung and of the gastrointestinal tract contain elevated levels of uPA compared to benign tissues. In breast cancer uPA and PAI-1 antigen in tumor tissue extracts are independent prognostic factors for relapse-free and overall survival.
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PMID:Tumor-associated urokinase-type plasminogen activator: biological and clinical significance. 151 91

The binding of type 1 plasminogen activator inhibitor (PAI-1) to the extracellular matrix (ECM) of cultured bovine aortic endothelial cells was investigated using purified 125I-labeled or L-[35S]methionine-labeled PAI-1 as probes. Little specific binding of latent PAI-1 to ECM previously depleted of endogenous PAI-1 could be demonstrated. In contrast, the guanidine-activated form of PAI-1 bound to ECM in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 60 nM by Scatchard analysis, and approximately 6 pmol of activated PAI-1 was bound per cm2 of ECM. Binding was relatively specific since unlabeled, activated PAI-1 competed with 35S-labeled PAI-1 for binding to ECM, but latent PAI-1 did not. Moreover, PAI-2, protein C inhibitor (i.e. PAI-3), protease nexin-1, and alpha 2-antiplasmin were not able to compete. Tissue-type plasminogen activator (tPA) also inhibited binding, but diisopropyl fluorophosphate-inactivated tPA did not. Pretreatment of ECM with tPA, urokinase-type PA, or thrombin had no effect on its ability to subsequently bind PAI-1, whereas trypsin, plasmin, and elastase pretreatment greatly reduced its ability to bind PAI-1. Guanidine-activated, radiolabeled PAI-1 resembled active endogenous PAI-1 since it was unstable in solution but stable when bound to ECM. In addition, it formed complexes with tPA that had a relatively low affinity for ECM. These data suggest that ECM of bovine aortic endothelial cells contains a protease-sensitive structure that binds active PAI-1 tightly and relatively selectively and that this association stabilizes PAI-1 against the spontaneous loss of activity that occurs in solution.
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PMID:Binding of type 1 plasminogen activator inhibitor to the extracellular matrix of cultured bovine endothelial cells. 249 80

Tissue-type plasminogen activator (t-PA) converts the inactive zymogen, plasminogen, into the powerful protease, plasmin, which then degrades the fibrin meshwork of thrombi. To prevent systemic activation of plasminogen, plasma contains several inhibitors of t-PA, the most important of which is plasminogen activator inhibitor-1 (PAI-1), a member of the serpin superfamily. As the ability to produce serpin-resistant variants of t-PA could increase the potential of this enzyme as a thrombolytic agent, we have used the known three-dimensional structure of the complex between trypsin and bovine pancreatic trypsin inhibitor (BPTI) to model the interactions between the active site of human t-PA and PAI-1. On the basis of this model we then altered by site-directed mutagenesis those amino acids of t-PA predicted to make contact with PAI-1 but not with the substrate plasminogen. We report here that although the resulting mutants have enzymatic properties similar to those of wild-type t-PA, they display significant resistance to inhibition by PAI-1. For example, following incubation with an amount of the serpin that completely inhibits the wild-type enzyme, one variant retains 95% of its initial activity. This mutant is also resistant to inhibition by the complex mixture of serpins present in human plasma.
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PMID:Serpin-resistant mutants of human tissue-type plasminogen activator. 250 May 99

The serpin plasminogen activator inhibitor-1 (PAI-1) spontaneously adopts an inactive or latent conformation by inserting the N-terminal part of the reactive center loop as strand 4 into the major beta-sheet (sheet A). To examine factors that may regulate reactive loop insertion in PAI-1, we determined the inactivation rate of the inhibitor in the pH range 4.5-13. Below pH 9, inactivation led primarily to latent PAI-1, and one predominant effect of pH on the corresponding rate constant could be observed. Protonation of a group exhibiting a pKa of 7.6 (25 degrees C, ionic strength = 0.15 M) reduced the rate of formation of latent PAI-1 by a factor of 35, from 0.17 h-1 at pH 9 to about 0.005 h-1 below pH 6. The ionization with a pKa 7.6 was found to have no effect on the rate by which PAI-1 inhibits trypsin and is therefore unlikely to change the flexibility of the loop or the orientation of the reactive center. The peptides Ac-TEASSSTA and Ac-TVASSSTA (cf. P14-P7 in the reactive loop of PAI-1) formed stable complexes with PAI-1 and converted the inhibitor to a substrate for tissue type plasminogen activator. We found that peptide binding and formation of latent PAI-1 are mutually exclusive events, similarly affected by the pKa 7.6 ionization. This is direct evidence that external peptides can substitute for strand 4 in beta-sheet A of PAI-1 and that the pKa 7.6 ionization regulates insertion of complementary, internal or external, strands into this position. A model that accounts for the observed pH effects is presented, and the identity of the ionizing group is discussed based on the structure of latent PAI-1. The group is tentatively identified as His-143 in helix F, located on top of sheet A.
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PMID:The acid stabilization of plasminogen activator inhibitor-1 depends on protonation of a single group that affects loop insertion into beta-sheet A. 749 70

Active PAI-1 (plasminogen activator inhibitor 1) is bound to vitronectin in plasma and in the extracellular matrix. In this study we aimed at identifying the PAI-1 binding site in vitronectin, which at present is a matter of dispute. Vitronectin was cleaved with trypsin and the fragments were tested for inhibitory effect on the PAI-1/vitronectin interaction using vitronectin-coated microtiter plates. Intact vitronectin and the tryptic digest of vitronectin both caused a 50% reduction in PAI-1 binding at a concentration of about 2 nmol/I. Gel-filtration on Sephadex G-50 superfine of the tryptic peptides resulted in one main peak of inhibitory activity. The elution volume, Kav, was 0.55 indicating (a) medium-size peptide(s). The peptide was further purified by reverse-phase HPLC. Structural analysis revealed that it constituted the 45 NH2-terminal amino-acid residues in vitronectin. The NH2-terminal vitronectin peptide caused a 50% decrease in PAI-1 binding to the vitronectin-coated microtiter plates at a concentration of about 13 nmol/l. Thus, the peptide is a little less effective in this respect than intact vitronectin. Reduced and S-carboxymethylated peptide had no effect on the interaction. The NH2-terminal vitronectin fragment increased the stability of active PAI-1 by about 60%, which is a little less than with intact vitronectin. The peptide also prevented PAI-1 from oxidation with chloramine T. The half-life was prolonged about 4-fold as compared to about 30-fold with intact vitronectin.
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PMID:Identification of a PAI-1 binding site in vitronectin. 752 53

Protein C inhibitor (PCI) is a heparin-binding plasma serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C. We are using recombinant PCI (rPCI) to study structural determinants of target proteinase specificity. A cDNA encoding full-length PCI has been expressed as a fully active proteinase inhibitor using Autographa californica nuclear polyhedrosis virus (baculovirus). rPCI was expressed maximally 4 days after infection and could be expressed either in Sf9 or High-Five cells. rPCI bound heparin and was conveniently purified with heparin-Sepharose (eluting > 0.5 M NaCl). The rPCI formed sodium dodecyl sulfate-polyacrylamide gel electrophoresis-stable complexes with thrombin and activated protein C (APC). The inhibitory properties of wild-type rPCI and plasma-derived PCI are essentially the same either in the absence or presence of heparin with thrombin, APC, trypsin, and urokinase. The residues Phe353-Arg354-Ser355 (P2-P1-P1') constitute part of the reactive site loop of PCI with the Arg-Ser peptide bond being cleaved by the proteinase. Using site-directed mutagenesis we studied the contribution of the reactive site FRS for proteinase inhibition in rPCI. Changing the P1 residue Arg354-->Met generated a reactive site similar to alpha 1-proteinase inhibitor which was a much poorer inhibitor of thrombin, APC, trypsin, and urokinase. Changing the P2 residue Phe353-->Gly generated a mutant with a reactive site like antithrombin which was better at inhibiting thrombin or urokinase, but was much less active with APC or trypsin. Changing the P1' residue Ser355-->Met generated a reactive site like plasminogen activator inhibitor-1 and this protein inhibits all the proteinases essentially like wild-type rPCI. These results show the importance of PCI's Phe353 (P2) and Arg354 (P1) in target proteinase specificity, and they further support the concept of reactive site sequences determining serpin function.
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PMID:Mutagenesis of recombinant protein C inhibitor reactive site residues alters target proteinase specificity. 820 90

Tissue-type plasminogen activator (t-PA), a multidomainal serine proteinase of the trypsin-family, catalyses the rate-limiting step in fibrinolysis, the activation of plasminogen to the fibrin-degrading proteinase plasmin. Trigonal crystals have been obtained of the recombinant catalytic domain of human-two-chain t-PA, consisting of a 17 residue A chain and the 252 residue B chain. Its X-ray crystal structure has been solved applying Patterson and isomorphous replacement methods, and has been crystallographically refined to an R-value of 0.184 at 2.3 A resolution. The chain fold, active-site geometry and Ile276-Asp477 salt bridge are similar to that observed for trypsin. A few surface-located insertion loops differ significantly, however. The disulfide bridge Cys315-Cys384, practically unique to the plasminogen activators, is incorporated without drastic conformational changes as the insertion loop preceding Cys384 makes a bulge on the molecular surface. The unique basic insertion loop Lys296-Arg304 flanking the primed subsites, which has been shown to be of importance for PAI-1 binding and for fibrin specificity, is partially disordered; it can therefore freely adapt to proteins docking to the active site. The S1 pocket of t-PA is almost identical to that of trypsin, whereas the S2 site is considerably reduced in size by the imposing Tyr368 side-chain, in agreement with the measured preference for P1 Arg and P2 Gly residues. The neighbouring S3-S4 hydrophobic groove is mainly hydrophobic in nature. The structure of the proteinase domain of two-chain t-PA suggests that the formation of a salt bridge between Lys429 and Asp477 may contribute to the unusually high catalytic activity of single-chain t-PA, thus stabilizing the catalytically active conformation without unmasking the Ile276 amino terminus. Modeling studies show that the covalently bound kringle 2 domain in full-length t-PA could interact with an extended hydrophobic groove in the catalytic domain; in such a docking geometry its "lysine binding site" and the "fibrin binding patch" of the catalytic domain are in close proximity.
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PMID:The 2.3 A crystal structure of the catalytic domain of recombinant two-chain human tissue-type plasminogen activator. 861 82

Plasminogen activation is observed in the human epidermis during reepithelialization of epidermal defects and under certain pathological conditions. The activation reaction depends on keratinocyte-associated plasminogen activators (PAs), which convert the ubiquitous proenzyme plasminogen into the active trypsin-like serine proteinase plasmin. The PAs are controlled by PA inhibitors (PAIs), of which two major types are known: PAI-1 and PAI-2. In vitro and in vivo keratinocytes express both PAIs. In the current study, we have addressed the possible function of PAI-2 in regulating extracellular PA activity in cultured normal human epidermal keratinocytes (NHEK), the human keratinocyte cell line (HaCaT), and a Ha-ras transfected HaCaT variant (HaRas). PAI-2 was detected intracellularly in all three cell types. Whereas only the NHEK and the HaCaT cells secreted detectable levels of PAI-2 into the culture medium, all three cell types released urokinase-type PA (uPA) into the supernatants. When comparing HaCaT and HaRas cells, we found that the cell lines secreted comparable levels of uPA antigen, whereas the levels of uPA activity were low in the presence of PAI-2, indicating that PAI-2 serves to regulate uPA activity. This assumption was supported by the findings that PAI-2 formed complexes with secreted uPA and that uPA/PAI-2 complexes were present at the surface of the PAI-2-secreting HaCaT cells but not at the surface of PAI-2 nonsecreting HaRas cells. Finally, PAI-2 was found to counteract the uPA-dependent and plasmin-mediated detachment of cultured HaCaT cells. Taken together, our findings indicate that secreted PAI-2 serves to regulate the activity of extracellular uPA in keratinocytes.
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PMID:Plasminogen activator inhibitor type-2 (PAI-2) in human keratinocytes regulates pericellular urokinase-type plasminogen activator. 863


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