Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the course of studies to identify a protease capable of producing a long-lived 50 kDa fragment of bone acidic glycoprotein-75 (BAG-75), it was observed that incubation of matrix metalloproteinase (MMP)-3 (stromelysin 1) with preparations of BAG-75 led to inactivation of proteolytic function, e.g., an inability to fragment 125I-labeled BAG-75 added subsequently. MMP-1 (interstitial collagenase) was also inactivated by exposure to BAG-75 preparations. Investigation of the mechanism revealed that BAG-75 preparations contained millimolar levels of inorganic phosphate which formed hydroxyapatite crystals under digestion conditions. Hydroxyapatite crystals alone and in BAG-75-hydroxyapatite complexes induced the autolytic degradation of both active and precursor forms of MMP-1 and MMP-3. Autolytic degradation in the presence of hydroxyapatite was demonstrated by a loss in catalytic function assayed with peptide and/or protein substrates, and, by fragmentation into polypeptides of <10 kDa. The fate of MMP-3 incubated with hydroxyapatite depends upon the time of incubation, the free calcium concentration, and the concentration of crystals. Specifically, hydroxyapatite-induced autolysis requires a near physiological free calcium concentration of 0.5-1.0 mM. Autolysis was maximal in the presence of 150 microg/ml hydroxyapatite where MMP-3 was only partially bound to crystals. However, autolysis also occurred at higher crystal concentrations where all input MMP-3 was bound (>1000 microg/ml), suggesting that autolysis may be mediated by bound enzyme. The effect of hydroxyapatite appears to be specific for MMP-1 and MMP-3 since the catalytic activity of chymotrypsin, trypsin, papain, and thermolysin remained unchanged after exposure to hydroxyapatite. These results document for the first time a novel catalytic role for hydroxyapatite crystals in vitro and provide an initial biochemical characterization of the intermolecular, autolytic, calcium ion-dependent, matrix metalloproteinase-specific degradative mechanism.
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PMID:Hydroxyapatite induces autolytic degradation and inactivation of matrix metalloproteinase-1 and -3. 984 7

Tissue factor pathway inhibitor-2 (TFPI-2)/matrix-associated serine protease inhibitor (MSPI), a 32- to 33-kDa Kunitz-type serine protease inhibitor, inhibits plasmin and trypsin. Because plasmin and trypsin are involved in the activation of promatrix metalloproteases proMMP-1 and proMMP-3, we investigated the role of TFPI-2/MSPI in the activation of these proenzymes. Both plasmin and trypsin activated proMMP-1 by converting the 53-kDa proenzyme to the partially active 43-kDa polypeptide; this activity was inhibited by TFPI-2/MSPI. Similarly, TFPI-2/MSPI inhibited the conversion of 66-kDa proMMP-3 to the activated 45- and 30-kDa polypeptides by plasmin and trypsin. Because plasmin is involved in the physiological activation of proMMP-3, we tested whether TFPI-2/MSPI inhibits the activation of proMMP-3 by HT-1080 fibrosarcoma cells and urokinase-charged HeLa cells. We found that the inhibitor inhibited proMMP-3 activation by HT-1080 cells and urokinase-charged HeLa cells. Collectively, our results suggest that TFPI-2/MSPI indirectly regulates MMP-1- and MMP-3-catalyzed matrix proteolysis by regulating the activation of proMMP-1 and proMMP-3.
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PMID:Regulation of ProMMP-1 and ProMMP-3 activation by tissue factor pathway inhibitor-2/matrix-associated serine protease inhibitor. 1008 61

C1q, a subunit of the first component (C1) of the classical complement pathway, binds to neutrophils via its collagen-like region (C1q-CLR) stimulating superoxide production. We previously identified a region of C1q-CLR, defined by fragments generated by trypsin and endoLys-C digestion, that was required for triggering this respiratory burst. To further localize that critical site, purified human C1q was digested with pepsin to generate C1q-CLR, and subsequently cleaved with the matrix metalloproteinases, MMP-1, MMP-2, MMP-3, and MMP-9. Digestion of C1q-CLR with any of these MMPs did not alter the circular dichroism spectra, demonstrating that the fragments generated had maintained the secondary structure observed in the native molecule. All fragments retained the ability to trigger superoxide production by neutrophils. Analysis of the amino acid sequences of the purified cleavage products (none of which are identical to the published cleavage site specificities for these enzymes) demonstrated that it is the C-chain, but not the A-chain of C1q, that is critical for stimulating this activity, and thus may be a target for future therapeutic intervention.
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PMID:Digestion of C1q collagen-like domain with MMPs-1,-2,-3, and -9 further defines the sequence involved in the stimulation of neutrophil superoxide production. 1049 11

There is compelling in vitro and in vivo evidence to implicate mast cells in the development of fibrosis. However, an important question remains as to the mechanisms by which mast cells mediate fibrosis. Recent evidence from our laboratory (Gruber et al., 1997, J. Immunol. , 158:2310-2317) has revealed that tryptase, the unique and abundant serine protease of human mast cells, is capable of activating fibroblasts by stimulating chemotaxis, proliferation, and procollagen mRNA synthesis. Regulation of matrix metalloproteinase (MMP) expression is another key step in connective tissue remodeling. Therefore, the effect of tryptase on fibroblast MMP expression was investigated. Proteolytically active tryptase did not alter the cellular mRNA levels for fibroblast MMP-1, MMP-2, MMP-3, and MMP-9 as detected by RNase protection assays. Moreover, tryptase did not alter the basal levels of MMP-1, MMP-2, MMP-3, MMP-9, or the tissue inhibitor of MMP-1 (TIMP-1) in fibroblast conditioned media as detected by specific enzyme-linked immunosorbent assay (ELISA). These results indicate that tryptase does not increase MMP expression in normal dermal fibroblasts. Moreover, these data strengthen the potential role of this unique serine protease as a potent fibrogenic factor.
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PMID:Mast cell tryptase does not alter matrix metalloproteinase expression in human dermal fibroblasts: further evidence that proteolytically-active tryptase is a potent fibrogenic factor. 1049 10

Progestin-only contraceptives are associated with menstrual bleeding disturbances; a major reason why these agents are discontinued. The pathogenesis of such abnormal uterine bleeding associated with progestin-only contraceptives remains ill-defined. Matrix metalloproteinases (MMP)s and mast cells (MC)s are postulated to be involved in endometrial breakdown observed in normal menstruation. In this study comparisons were made of the immunolocalization of MMP-1 and -3 and MC in endometrium from women using Norplant or depot medroxyprogesterone acetate (DMPA) with normal controls. Positive MMP immunostaining was observed focally in stromal cells and adjacent extracellular matrix. Quantitative assessment revealed significantly higher MMP-1 immunostaining associated with the use of Norplant compared with DMPA or menstrual phase controls. Endometrial MMP-1 immunostaining in DMPA users was similar to that in menstrual controls. Positive MMP-3 immunolocalization was observed in a minority of endometrial samples. Activated MC, shown by the presence of extracellular MC tryptase, predominated in the endometrium of Norplant and DMPA users as also observed in menstrual phase controls. There was no correlation between MMP immunostaining, number of MC and number of bleeding days reported. These results indicate that in women using progestin-only contraceptives, endometrial MMP-1, -3 and MC demonstrate similarities to menstrual phase controls but also variation with different progestins.
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PMID:Matrix metalloproteinase-1 and -3 and mast cells are present in the endometrium of women using progestin-only contraceptives. 1061 Dec

Degradation of the extracellular matrix occurs under physiological and pathological conditions, thought to be principally mediated by a family of neutral proteolytic enzymes termed the matrix metalloproteinases (MMPs). The present study was initiated to determine whether mast cells have the ability to produce these proteases in diseased and normal human tissue. Immunohistochemistry and in situ hybridization was performed to localize interstitial collagenase protein and mRNA transcripts in diseased human tissue. The human mast cell line HMC-1 was cultured under serum free conditions, stimulated with phorbol mystrate acetate (PMA) and supernatants analyzed by Western blotting and zymography to determine the profile of secreted MMPs. The dog mast cell line BR, known to secrete gelatinolytic enzymes, was used in parallel studies. Total RNA was extracted and analyzed by RT-PCR for the expression of tissue inhibitors of MMP (TIMPs). Collagenase-1 protein and mRNA were expressed by tryptase and chymase positive human mast cells in all tissue analyzed. This proteinase was also detected in the cytoplasm and conditioned media of HMC-1 cells. PMA induced gelatinolytic activity in both mast cell lines examined. TIMP-1 immunoreactivity was detected and TIMP-1, and -2 (but not TIMP-3) mRNA transcripts were amplified from HMC-1 cells. This is the first demonstration of the expression of collagenase-1 by human mast cells in both inflamed and normal tissues, and by a human mast cell line. MMPs secreted by these cells could contribute to the extensive matrix lysis characteristic of diseases such as rheumatoid arthritis and inflammatory ocular disorders. Alternatively collagenase-1 production by mast cells may play a critical role in cell invasion and migration into sites of inflammation.
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PMID:In vitro and in vivo expression of interstitial collagenase/MMP-1 by human mast cells. 1109 7

Interstitial collagen is degraded by members of the matrix metalloproteinase (MMP) family, including MMP-1. Previous work has shown that the region of MMP-1 coded for by exon 5 is implicated both in substrate specificity and inhibitor selectivity. We have constructed a chimeric enzyme, the exon 5 chimera, consisting primarily of MMP-1, with the region coded for by exon 5 replaced with the equivalent region of MMP-3, a noncollagenolytic MMP. Unlike MMP-3, the exon 5 chimera is capable of cleaving type I collagen, but the activity is only 2.2% of trypsin-activated MMP-1. 'Superactivation' of the chimera has no discernible effect, suggesting that the salt bridge formed in 'superactive' MMP-1 is not present. The kinetics for exon 5 chimera cleavage of two synthetic substrates display an MMP-3 phenotype, however, cleavage of gelatin is slightly impaired as compared to the parent enzymes. The K(iapp) values for the exon 5 chimera complexed with synthetic inhibitors and N-terminal TIMP-2 also show a more MMP-3-like behaviour. However, the k(on) values for N-terminal TIMP-1 and N-terminal TIMP-2 are more comparable to those for MMP-1. These data show that the region of MMP-1 coded for by exon 5 is involved in both substrate specificity and inhibitor selectivity and the structural basis for our findings is discussed.
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PMID:The role of exon 5 in fibroblast collagenase (MMP-1) substrate specificity and inhibitor selectivity. 1124 10

The endometrium displays characteristic cyclical changes involving proliferation and differentiation. The differentiation that takes place requires major tissue remodelling involving the matrix metalloproteinase (MMP) family as key enzymes in this process. Mast cells, containing the tryptase and chymase enzymes that are capable of stimulating the MMP cascade, have been identified in the endometrium, but their role is still unclear. In this study, we observed that the majority of mast cells in the uterus reside in the myometrium and that they co-express mast cell tryptase and MMP-1 in the same intracellular granules. In endometrium exposed to synthetic progestogen via an intrauterine levonorgestrel system a significant increase in mast cells numbers was observed in women experiencing breakthrough bleeding compared to those in women with no reported bleeding. We conclude that mast cells contain MMP-1 and we postulate a potential role for mast cells in breakthrough bleeding.
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PMID:Co-localization of matrix metalloproteinase-1 and mast cell tryptase in the human uterus. 1138 11

Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, non-fluorogenic and fluorogenic triple-helical peptide models of MMP-1 cleavage sites in interstitial collagens have been constructed. Triple-helical peptides were assembled by either (a) covalent branching or (b) self-association driven by hydrophobic interactions. Fluorogenic triple-helical peptide (fTHP) substrates contained the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P5 and P5' positions, respectively. Investigation of MMP family hydrolysis of THPs showed kcat/Km values in the order of MMP-13 > MMP-1 approximately MMP-1(delta243-450) approximately MMP-2 >> MMP-3. Studies on the effect of temperature on fTHP and an analogous fluorogenic single-stranded peptide (fSSP) hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. The general proteases trypsin and thermolysin were also studied for triple-helical peptidase activity. Both of these enzymes exhibited similar activation energies to MMP-1 for hydrolysis of fTHP versus fSSP. These results suggest that 'triple-helical peptidase' activity can be distinguished from 'collagenolytic' activity, and that mechanistically distinct enzymes convergently evolved to develop collagenolytic activity.
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PMID:Triple-helical peptide analysis of collagenolytic protease activity. 1243 92

A critical step in cancer growth and metastasis is the dissolution of the extracellular matrix surrounding the malignant tumor, which leads to tumor cell invasion and dissemination. Type I collagen degradation involves the initial action of collagenolytic matrix metalloproteinases (MMP-1, -8, and -13) activated by MMP-3 (stromelysin-1). The role of interactive matrix serine proteinases (MSPs), including tumor-associated trypsinogens, has been unclear in collagenolysis. Now, we provide evidence that the major isoenzyme of human tumor-associated trypsinogens, trypsin-2, can directly activate three collagenolytic proMMPs as well as proMMP-3. These proMMP activations are inhibited by tumor-associated trypsin inhibitor (TATI). Furthermore, we demonstrate that trypsin-2 efficiently degrades native soluble type I collagen, which can be inhibited by TATI. However, cell culture studies showed that trypsin-2 transfection into the HSC-3 cell line did not result in MMP-1, -3, -8, and -13 activation but affected MMP-3 and -8 production at the protein level. These findings indicate that human trypsin-2 can be regarded as a potent tumor-associated matrix serine protease capable of being the initial activator of the collagenolytic MMP activation network as well as directly attacking type I collagen.
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PMID:Tumor-associated trypsinogen-2 (trypsinogen-2) activates procollagenases (MMP-1, -8, -13) and stromelysin-1 (MMP-3) and degrades type I collagen. 1273 83


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