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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Collagenases, divided into metallocollagenases and serine collagenases, are the only proteases that cleave the triple helix of collagen under physiological conditions. In the present work, the serine protease collagenase purified from Hypoderma lineatum larvae is studied. From crystals grown in the International Microgravity Laboratory (IML2), a data set was collected at 1.7 A using synchrotron radiation. Although the resolution is not very different, the signal-to-noise ratio and the quality of the electron density are much improved. Alternate conformations were revealed for several residues, in particular Tyr99, suggesting a gate mechanism of recognition.
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PMID:1.7 A x-ray structure of space-grown collagenase crystals. 1071 32

We have identified and partially characterized several gelatinase activities associated with the sea urchin extraembryonic matrix, the hyaline layer. A previously identified 41-kDa collagenase/gelatinase activity was generally not found to be associated with isolated hyaline layers but was dissociated from the surface of 1-h-old embryos in the absence of Ca2+ and Mg2+. While hyaline layers, freshly prepared from 1-h-old embryos, were devoid of any associated gelatinase activities, upon storage at 4 degrees C for 4 days, a number of gelatin-cleavage activities appeared. Comparative analysis of these activities with the 41-kDa collagenase/gelatinase revealed that all species were inhibited by ethylenediamine tetraacetic acid but were refractory to inhibition with the serine protease inhibitors, phenylmethyl sulfonyl fluoride and benzamidine. In contrast, the largely Zn2+ specific chelator 1,10-phenanthroline had markedly different effects on the gelatinase activities. While several of the storage-induced, hyaline-layer-associated gelatinase activities were inhibited, the 41-kDa collagenase/gelatinase was refractory to inhibition as was a second gelatinase species with an apparent molecular mass of 45 kDa. We also examined the effects of a series of divalent metal ions on the gelatin-cleavage activities. In both qualitative and quantitative assays, Ca2+ was the most effective activator while Mn2+, Cu2+, Cd2+, and Zn2+ were all inhibitory. In contrast, Mg2+ had a minimal inhibitory effect on storage-induced gelatinase activities but significantly inhibited the 41-kDa collagenase/gelatinase. These results identify several distinct gelatin-cleavage activities associated with the sea urchin extraembryonic hyaline layer and point to diversity in the biochemical nature of these species.
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PMID:Identification and characterization of gelatin-cleavage activities in the apically located extracellular matrix of the sea urchin embryo. 1101 84

We report here the continued characterization of a 41-kDa protease expressed in the early stage of the sea urchin embryo. This protease was previously shown to possess both a gelatin-cleavage activity and an echinoderm-specific collagen-cleavage activity. In the experiments reported here, we have explored the biochemical nature of this proteolytic activity. Pepstatin A (an acidic protease inhibitor), 1,10-phenanthroline (a metalloprotease inhibitor), and E-64 (a thiol protease inhibitor) were without effect on the gelatin-cleavage activity of the 41-kDa species. Using a gelatin substrate gel zymographic assay, the serine protease inhibitors phenylmethylsulfonyl fluoride and benzamide appeared to partially inhibit gelatin-cleavage activity. This result was confirmed in a quantitative gelatin-cleavage assay using the water soluble, serine protease inhibitor [4-(2-aminoethyl)benzenesulfonylfluoride]. The biochemical character of this protease was further explored by examining the effects of calcium and magnesium, the major divalent cations present in sea water, on the gelatin-cleavage activity. Calcium and magnesium competed for binding to the 41-kDa collagenase/gelatinase, and prebound calcium was displaced by magnesium. Cleavage activity was inhibited by magnesium, and calcium protected the protease against this inhibition. These results identify calcium and magnesium as antagonistic agents that may regulate the proteolytic activity of the 41-kDa species.
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PMID:Effects of calcium and magnesium on a 41-kDa serine-dependent protease possessing collagen-cleavage activity. 1102 61

A collagenolytic protease was purified to homogeneity from thermophilic Bacillus sp. strain MO-1. The protease from strain MO-1 showed high activity toward type I and IV collagens and gelatin. However, peptide substrates (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg and 2-furylacryloyl-Leu-Gly-Pro-Ala) for collagenases were inert as substrates. The collagenolytic protease cleaved oxidized insulin B-chain at 11 sites and degraded type I and IV collagens into anonymous small pieces, suggesting that the protease digests collagens at multiple sites. The collagenolytic protease was far more thermostable than a mesophilic Clostridium histolyticum collagenase. The collagenolytic protease possesses two salient features: (1) it has a very large molecular mass, 210 kDa, and consists of two, identical 105-kDa subunits; (2) it belongs to a serine protease group. The high molecular mass is unique among serine proteases but common for collagenases. The features of the enzyme from strain MO-1 suggest that it is a new collagenolytic protease which is distinct from previously reported collagenases and serine proteases.
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PMID:A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1. 1169 5

Fibroblast activation protein (FAP) is a type II integral membrane glycoprotein belonging to the serine protease family. Human FAP is selectively expressed by tumor stromal fibroblasts in epithelial carcinomas, but not by epithelial carcinoma cells, normal fibroblasts, or other normal tissues. FAP has been shown to have both in vitro dipeptidyl peptidase and collagenase activity, but its biological function in the tumor microenvironment is unknown. The modeled structure of murine FAP consists of a short cytoplasmic tail, a single hydrophobic transmembrane region, and a large extracellular domain. A seven-bladed beta-propeller domain is situated on top of the catalytic triad and may serve as a "gate" to selectively filter protein access to the catalytic site. HEK293 cells transfected to constitutively express murine FAP, when xenografted into scid mice, were 2-4 times more likely to develop s.c. tumors and showed a 10-40-fold enhancement of tumor growth compared with mock-transfected HEK293 cells. Rabbits immunized with recombinant murine FAP developed polyclonal anti-FAP antibodies that significantly inhibited murine FAP dipeptidyl peptidase activity in vitro. HT-29 xenografts treated with these inhibitory anti-FAP antisera exhibited attenuated growth compared with tumors treated with preimmunization rabbit antisera. These data demonstrate the ability of FAP to potentiate tumor growth in an animal model. Moreover, tumor growth is attenuated by antibodies that inhibit the proteolytic activity of FAP. These findings suggest a possible therapeutic role for functional inhibition of FAP activity.
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PMID:Promotion of tumor growth by murine fibroblast activation protein, a serine protease, in an animal model. 1218 36

A serine protease, subtilisin YaB, produced by alkalophilic Bacillus YaB, shows promises as a potent meat tenderizer, because its substrate specificity is for small amino acids, which are found at high levels in meat connective tissue proteins. Substrate specificity engineering of the substrate binding pockets was used to generate more suitable meat-tenderizing mutants, G124A, G124V, G159A, and G159S, derived from recombinant wild subtilisin YaB and expressed in Bacillus subtilis DB104. The characteristics of these recombinant enzymes were studied to evaluate their usefulness as improved meat tenderizers. The proteolytic activities of recombinant subtilisin YaB, engineered subtilisin YaBs, and commercially available papain, bromelain, collagenase, and elastase were compared using elastin, collagen, casein, and myofibrillar proteins as substrates. Hydrolysis of beef proteins was evaluated using the myofibrillar fragmentation index and collagen solubility. The results demonstrated that recombinant mutant G159A was the most improved meat tenderizer and can be used in the meat pH range of 5.5-6.0 and the temperature range of 10-50 degrees C. Contrary to the result obtained from artificial substrate, mutant enzymes engineered on G124 residues did not exhibit better tenderizing ability when elastin, collagen, or meat was used as substrate, suggesting the necessity of evaluation by real substrate before protein-engineered enzymes are applied commercially.
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PMID:Application potency of engineered G159 mutants on P1 substrate pocket of subtilisin YaB as improved meat tenderizers. 1235 2

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

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCys-uPA19-31) directed against cysteine proteases and the urokinase-type plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)-binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the N-terminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCys-uPA19-31 or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OV-MZ-6#8). Additionally, OV-MZ-6#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitor-producing cells was significantly reduced compared to vector-transfected control cells. Thus, these novel, compact, and small-size inhibitors directed against up to three different tumor-associated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis.
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PMID:Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems. 1295 25

The fibrous cap of a lipid-containing atherosclerotic plaque consists of collagen produced by arterial smooth muscle cells (SMCs) of synthetic phenotype. A thick cap protects the lipid-rich core, whereas a thin cap predisposes it to rupture, with ensuing acute clinical complications, such as myocardial infarction. Among the pathological mechanisms leading to plaque weakening and rupture, one possibility is loss of the matrix-synthesizing SMCs. Indeed, caps of ruptured coronary plaques contain a reduced number of SMCs. In contrast, in such lesions, the number of activated inflammatory cells, such as mast cells, is increased, suggesting that they may regulate the SMC number. We have shown that heparin proteoglycans secreted by activated mast cells can efficiently inhibit proliferation of SMCs in vitro and reduce their ability to produce collagen. Chymase, a neutral serine protease secreted by activated mast cells, can also inhibit SMC-mediated collagen synthesis by a transforming growth factor-beta-dependent and -independent mechanism, and moreover, cause degradation of the collagen matrix by activating latent interstitial collagenase (MMP-1). Furthermore, chymase can induce SMC apoptosis by degrading the extracellular matrix component fibronectin necessary for SMC adhesion, with subsequent disruption of focal adhesions and loss of outside-in survival signaling. Thus, activated mast cells may participate in the weakening and rupture of atherosclerotic plaques by secreting mediators, such as heparin proteoglycans and chymase, which affect the growth, function and death of arterial SMCs.
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PMID:Regulation of smooth muscle cell growth, function and death in vitro by activated mast cells--a potential mechanism for the weakening and rupture of atherosclerotic plaques. 1455 26

Intracerebral hemorrhage (ICH) causes morbidity and mortality and commonly follows the reperfusion after an ischemic event. Tissue plasminogen activator (tPA), a fibrinolytic serine protease, is routinely given for the treatment of stroke. However, tPA also can promote neuronal death, suggesting that caution should be exercised when using it. Furthermore, tPA upon brain injury mediates microglial activation and modulates neuronal survival. To investigate the role of tPA and microglia during brain hemorrhage, we induced experimentally ICH by intracerebral injection of collagenase. Seven days after the introduction of ICH, it persisted in tPA-deficient (tPA(-/-)) mice but is drastically reduced in size in wild-type mice. Three weeks after ICH, there are still red blood cells in tPA(-/-) but not in wild-type animals. Activated microglia persist around the injury site. When microglial activation is inhibited by tuftsin fragment 1-3 macrophage/microglial inhibitory factor (MIF), the stroke injury volume is significantly reduced, and the neurobehavioral deficits exhibited by the mice are improved. Our results suggest that endogenous tPA assists in the clearance of intracerebral hemorrhage, presumably by affecting microglial activation, and MIF could be a valuable neuroprotective agent for the treatment of ICH.
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PMID:Protective role of tuftsin fragment 1-3 in an animal model of intracerebral hemorrhage. 1459 55


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