Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.23 (MMP)
 4,246 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Metalloproteinase inhibitors were surveyed with the culture media of 19 kinds of human tumor cell lines, using transin (rat stromelysin) as the target enzyme. This survey showed that most of the cell lines more or less secreted inhibitor activity, and that a human hepatoma cell line, HLE, secreted an extremely high inhibitor activity into the culture medium. Two kinds of metalloproteinase inhibitors were purified from the serum-free conditioned medium of HLE cells. The major inhibitor, which showed a single protein band with a molecular weight (Mr) of 21,000 (21k) (nonreduced) or 24k (reduced) on SDS-polyacrylamide gel electrophoresis, was identified as TIMP-2 (tissue inhibitor of metalloproteinases-2) by the analysis of its N-terminal amino acid sequence. The other was immunologically identified as TIMP. Purified TIMP-2 inhibited the activities of transin, matrin (pump-1), Mr 72k gelatinase, and interstitial collagenase with 1:1 stoichiometry. When the latent precursor form (Mr 57k) of transin was incubated with p-aminophenylmercuric acetate as an activating reagent, TIMP-2 inhibited the conversion of the intermediate form (Mr 45k) into the mature enzyme (Mr 42k). This indicated that TIMP-2 regulates not only the activity of the mature enzyme but also the autolytic processing of the proenzyme. TIMP-2 also inhibited in vitro tumor invasion through reconstituted basement membrane (matrigel) in chemotaxis chambers, showing that the metalloproteinase inhibitors as well as the extracellular matrix metalloproteinases are involved in tumor invasion through basement membrane and other extracellular matrices.
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PMID:Efficient purification of TIMP-2 from culture medium conditioned by human hepatoma cell line, and its inhibitory effects on metalloproteinases and in vitro tumor invasion. 166 1

A truncated form of the membrane-type matrix metalloproteinase-1 [(Ala21-Ile318)proMT1-MMP] lacking the hemopexin-like and trans-membrane domain was produced in E. coli. We demonstrate that the recombinant proenzyme was autoproteolytically processed to a fully active catalytic domain with N-terminal Ile114. The catalytic domain of MT1-MMP initiated the activation of progelatinase A and progelatinase A complexed with tissue inhibitor of metalloproteinases-2 (TIMP-2). As a typical soluble metalloproteinase it was able to cleave physiologic as well as synthetic substrates. Our kinetic data demonstrate that TIMP-2 is a potent inhibitor for the recombinant enzyme.
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PMID:The recombinant catalytic domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) induces activation of progelatinase A and progelatinase A complexed with TIMP-2. 895 63

Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed on cancer cell membranes and activates the zymogen of MMP-2 (gelatinase A). We have recently isolated MT1-MMP complexed with tissue inhibitor of metalloproteinases 2 (TIMP-2) and demonstrated that MT1-MMP exhibits gelatinolytic activity by gelatin zymography (Imai, K., Ohuchi, E., Aoki, T., Nomura, H., Fujii, Y., Sato, H., Seiki, M., and Okada, Y. (1996) Cancer Res. 56, 2707-2710). In the present study, we have further purified to homogeneity a deletion mutant of MT1-MMP lacking the transmembrane domain (DeltaMT1) and native MT1-MMP secreted from a human breast carcinoma cell line (MDA-MB-231 cells) and examined their substrate specificities. Both proteinases are active, without any treatment for activation, and digest type I (guinea pig), II (bovine), and III (human) collagens into characteristic 3/4 and 1/4 fragments. The cleavage sites of type I collagen are the Gly775-Ile776 bond for alpha1(I) chains and the Gly775-Leu776 and Gly781-Ile782 bonds for alpha2(I) chains. DeltaMT1 hydrolyzes type I collagen 6.5- or 4-fold more preferentially than type II or III collagen, whereas MMP-1 (tissue collagenase) digests type III collagen more efficiently than the other two collagens. Quantitative analyses of the activity of DeltaMT1 and MMP-1 indicate that DeltaMT1 is 5-7.1-fold less efficient at cleaving type I collagen. On the other hand, gelatinolytic activity of DeltaMT1 is 8-fold higher than that of MMP-1. DeltaMT1 also digests cartilage proteoglycan, fibronectin, vitronectin and laminin-1 as well as alpha1-proteinase inhibitor and alpha2-macroglobulin. The activity of DeltaMT1 on type I collagen is synergistically increased with co-incubation with MMP-2. These results indicate that MT1-MMP is an extracellular matrix-degrading enzyme sharing the substrate specificity with interstitial collagenases, and suggest that MT1-MMP plays a dual role in pathophysiological digestion of extracellular matrix through direct cleavage of the substrates and activation of proMMP-2.
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PMID:Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. 899 57

To measure matrix metalloproteinase (MMP) activity in a large number of samples it is advisable to use easily automated methods. We have evaluated and compared the activity of stromelysin-1 (MMP-3), matrilysin (MMP-7), 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) by zymogram analysis and fluorescent substrate degradation assays. FITC-casein and the fluorogenic peptide Dnp-Pro-beta-cyclo-hexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-Abz)-NH 2 were used as fluorescent substrates. FITC-casein was more efficiently degraded than the fluorogenic peptide by all MMPs tested except MMP-9. MMP-2 was not significantly able to degrade the fluorogenic peptide. Gelatin zymography was the most sensitive method to detect the activity of both gelatinases but quantitation problems compromise its use. The degradation of fluorogenic substrates by MMPs could be inhibited by the chelating agent EDTA and by the tissue inhibitor of metalloproteinases 2 (TIMP-2), an MMP-specific inhibitor. Fluorometric methods represent a good alternative for MMP activity measurement, especially when a large number of samples must be processed.
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PMID:Evaluation of fluorometric and zymographic methods as activity assays for stromelysins and gelatinases. 900 3

Lung cancer is a heterogeneous tumor in terms of clinical and biological behavior, and its aggressiveness depends on its invasive and metastatic properties. Matrix metalloproteinases and serine proteinases are believed to play a crucial role in invasion and metastasis of malignant tumor cells. In the present study, the authors evaluated immunohistochemically the expression of gelatinase A; tissue inhibitor of metalloproteinases-2 (TIMP-2), an inhibitor of gelatinase A; matrilysin; and trypsin(ogen) in 67 lung tumors from a variety of histological types including 17 squamous cell carcinomas, 16 adenocarcinomas, 15 small cell carcinomas, and 12 carcinoids. Interestingly, normal bronchial, bronchiolar, and alveolar epithelial cells expressed gelatinase A, TIMP-2, matrilysin, and trypsin(ogen) at varying frequencies and intensities. Bronchial smooth muscle cells and cartilage cells expressed gelatinase A alone, whereas endothelial cells, fibroblasts, and macrophages expressed gelatinase A and TIMP-2. Gelatinase A was expressed at high levels in most lung tumors examined (47% to 80%). TIMP-2 was also expressed at high levels except in the small cell carcinomas, which showed TIMP-2 expression at a lower frequency (60%) compared with other types of lung tumors (80% to 100%). Although matrilysin was expressed by tumor cells of all the histological types at various frequencies (13% to 63%), its expression was most common in adenocarcinomas. Expression of trypsin(ogen) was observed almost exclusively in adenocarcinomas (56%); other types of lung tumors expressed trypsin(ogen) far less frequently (0% to 12%). The present results, taken together with those of previous studies, suggest that gelatinase A is associated with malignant behavior of all the types of lung tumors, whereas its activity may be controlled by the endogenous inhibitor TIMP-2. The aggressive clinical behavior of small cell carcinoma may be attributable, at least in part, to a loss of the inhibitory effect of TIMP-2, as a significant proportion of these tumors showed negative or low levels of TIMP-2 expression. Matrilysin and trypsin(ogen) expressions are unlikely to be correlated with the aggressiveness of lung tumors. The expression of trypsin (ogen) may rather reflect the differentiation of adenocarcinoma cells toward normal airway epithelial cells.
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PMID:Expression of gelatinase A, tissue inhibitor of metalloproteinases-2, matrilysin, and trypsin(ogen) in lung neoplasms: an immunohistochemical study. 915 11

The treatment of human uterine cervical fibroblasts with concanavalin A (ConA), or a specific calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) or trifluoperazine resulted in accumulation of an active form of matrix metalloproteinase 2 (MMP-2, gelatinase A). In contrast, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), a weaker antagonist of calmodulin, did not modulate the activation of proMMP-2. The activation of proMMP-2 was confirmed by the enhanced activity on gelatin and the conversion of proMMP-2 to a 62-kDa form by zymography and western blotting. The plasma membrane, but not the conditioned medium, of the W-7- or trifluoperazine-treated cells activated proMMP-2; this activation was blocked by membrane-type-1 MMP (MT1-MMP) antibody and EDTA. The plasma membrane from trifluoperazine- or ConA-treated cells contained MT1-MMP and tissue inhibitor of metalloproteinases 2. Both trifluoperazine treatment and ConA treatment increased the steady-state levels of MT1-MMP mRNA and proMMP-2 mRNA. These results, together with our previous observations on the production of proMMP-1 (interstitial procollagenase) and proMMP-3 (prostromelysin 1) [Ito, A., Sato, T., Ojima, Y., Chen, L.-C., Nagase, H. & Mori, Y. (1991) J. Biol. Chem. 266, 13598-13601], suggest that calmodulin negatively regulates the matrix turnover by suppressing the production of a number of proMMPs including proMMP-1, proMMP-3 and MT1-MMP, and the activation of proMMP-2 in human uterine cervical fibroblasts.
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PMID:Calmodulin antagonists increase the expression of membrane-type-1 matrix metalloproteinase in human uterine cervical fibroblasts. 949 4

The high resolution structure of the N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP-2) in solution has been determined using multidimensional heteronuclear NMR spectroscopy, with the structural calculations based on an extensive set of constraints, including 3132 nuclear Overhauser effect-based distance constraints, 56 hydrogen bond constraints, and 220 torsion angle constraints (an average of 26.9 constraints/residue). The core of the protein consists of a five-stranded beta-barrel that is homologous to the beta-barrel found in the oligosaccharide/oligonucleotide binding protein fold. The binding site for the catalytic domain of matrix metalloproteinases-3 (N-MMP-3) on N-TIMP-2 has been mapped by determining the changes in chemical shifts on complex formation for signals from the protein backbone (15N, 13C, and 1H). This approach identified a discrete N-MMP-3 binding site on N-TIMP-2 composed of the N terminus of the protein and the loops between beta-strands AB, CD, and EF. The beta-hairpin formed from strands A and B in N-TIMP-2 is significantly longer than the equivalent structure in TIMP-1, allowing it to make more extensive binding interactions with the MMP catalytic domain. A detailed comparison of the N-TIMP-2 structure with that of TIMP-1 bound to N-MMP-3 (Gomis-Ruth, F.-X., Maskos, K., Betz, M., Bergner, A., Huber, R., Suzuki, K., Yoshida, N., Nagase, H. , Brew, K., Bourne, G. P., Bartunik, H. & Bode, W. (1997) Nature 389, 77-80) revealed that the core beta-barrels are very similar in topology but that the loop connecting beta-strands CD (P67-C72) would need to undergo a large conformational change for TIMP-2 to bind in a similar manner to TIMP-1.
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PMID:High resolution structure of the N-terminal domain of tissue inhibitor of metalloproteinases-2 and characterization of its interaction site with matrix metalloproteinase-3. 970 10

Matrix metalloproteinase 2 (MMP-2)/gelatinase A plays an important role in tumour invasion and metastasis. Since MMP-2 is secreted as an inactive form (proMMP-2) from tumour and neighbouring stroma cells, the activation process is necessary to express the enzymic activity for degradation of extracellular matrix components. We herein reported that the activation of proMMP-2 was induced in human squamous carcinoma cells co-cultured with normal human dermal fibroblasts. When A431 cells were co-cultured with human fibroblasts at various cell ratios, 72-kDa proMMP-2 was converted to a 62-kDa active form through the appearance of a 64-kDa intermediate. The activation of proMMP-2 by co-culture was also observed in other carcinoma cell lines, HSC-4 and SAS, but not in normal human keratinocytes. We characterized by in vitro invasion assay that A431 cells in co-culture preferentially invaded through Matrigel and the increased invasive activity was inhibited by exogenously adding tissue inhibitor of metalloproteinases 2. The augmented proMMP-2 activation by co-culture was achieved by the increase in membrane type 1-MMP (MT1-MMP) production along with that of its mRNA level. The predominant appearance of MT1-MMP was immunologically observed in A431 cells, but not human fibroblasts of the co-culture. Furthermore, epidermal growth factor (EGF) enhanced the co-culture-mediated proMMP-2 activation by increasing the production and gene expression of MT1-MMP, and thereby tumour invasive activity was further augmented. These results suggest that the cell-cell contact between carcinoma cells and normal fibroblasts enhances the production of MT1-MMP followed by sequential activation of proMMP-2 on the tumour cell surface, which may be closely implicated in tumour invasion in vivo.
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PMID:Enhancement of membrane-type 1-matrix metalloproteinase (MT1-MMP) production and sequential activation of progelatinase A on human squamous carcinoma cells co-cultured with human dermal fibroblasts. 1037 63

Membrane type 3 matrix metalloproteinase (MT3-MMP), an activator for the zymogen of MMP-2 (proMMP-2, or progelatinase A), is known to be expressed in human placenta, brain, lung and rat vascular smooth muscle cells, but information about its biochemical properties is limited. In the present study, we expressed and purified a truncated form of MT3-MMP lacking the transmembrane and intracytoplasmic domain (DeltaMT3) and characterized the enzyme biochemically. DeltaMT3 digested type III collagen into characteristic 3/4- and 1/4-fragments by cleaving the Gly781-Ile782 and Gly784-Ile785 bonds of alpha1(III) chains. Although DeltaMT3 did not have such an activity against type I collagen, it attacked the Gly4-Ile5 bond of the triple helical portion of alpha2(I) chains, leading to removal of the crosslink containing N-terminal telopeptides. By quantitative analyses of the activities of DeltaMT3 and a similar deletion mutant of MT1-MMP (DeltaMT1), DeltaMT3 was approximately fivefold more efficient at cleaving type III collagen. DeltaMT3 also digested cartilage proteoglycan, gelatin, fibronectin, vitronectin, laminin-1, alpha1-proteinase inhibitor and alpha2-macroglobulin into almost identical fragments to those given by DeltaMT1, although carboxymethylated transferrin digestion by DeltaMT3 generated some extra fragments. The activity of DeltaMT3 was inhibited by tissue inhibitor of metalloproteinases-2 (TIMP-2) and TIMP-3 in a 1 : 1 stoichiometry, but not by TIMP-1. ProMMP-2 was partially activated by DeltaMT3 to give the intermediate form. These results indicate that, like MT1-MMP, MT3-MMP exhibits proteolytic activities against a wide range of extracellular matrix molecules. However, differences in the proMMP-2 activation and tissue distribution suggest that MT3-MMP and MT1-MMP play different roles in the pathophysiological digestion of extracellular matrix.
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PMID:Characterization of a truncated recombinant form of human membrane type 3 matrix metalloproteinase. 1041 55

Matrix metalloproteinase-2 (MMP-2) is involved in extracellular matrix remodeling. It is secreted as a proenzyme and activated by membrane type-MMPs (MT-MMP), such as MT1-MMP. In liver fibrosis, MMP-2 is highly expressed in myofibroblasts and may have a profibrogenic role. The mechanisms of its activation in the liver are still unclear. The aim of this work was to show that pro-MMP-2 is efficiently activated in human fibrotic liver and to investigate the role of cell-matrix interactions in this process. Liver specimens obtained from patients with active cirrhosis were compared to normal liver specimens. Human hepatic myofibroblasts were cultured either on plastic, fibronectin, laminin, or on collagen I gels. MMP-2 activity was visualized by gelatin zymography. MMP-2 active form (59 kd) was detected in active cirrhosis but not in normal liver. Myofibroblasts cultured on plastic, fibronectin, or laminin predominantly expressed inactive pro-MMP-2 (66 kd). In contrast, myofibroblasts cultured on collagen I markedly activated the enzyme. Similar results were obtained using membrane fractions from cells previously cultured on collagen or plastic. Activation was inhibited by the tissue inhibitor of metalloproteinases-2 but not by tissue inhibitor of metalloproteinases-1, implicating a MT-MMP-mediated process. Culture on collagen I up-regulated MT1-MMP protein detected by Western blotting, but decreased MT1-MMP mRNA. This study shows that MMP-2 is activated in fibrotic liver. It suggests that interactions between collagen I and myofibroblasts promote this process through a post-translational increase of MT1-MMP expression in these cells.
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PMID:Matrix metalloproteinase-2 activation in human hepatic fibrosis regulation by cell-matrix interactions. 1049 46


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