Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.17 (MMP-3)
 3,419 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).
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PMID:Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties. 140 Apr 81

E1A genes deficient in the carboxy-terminal exon can cooperate with activated ras oncogenes to induce transformation of rat embryo fibroblasts. However, the resulting transformed foci show a distinct appearance characterized by a decreased adhesion of the cells to the substrate. Here, we demonstrate that cell lines derived from foci showing the variant morphology are defective in down-regulation of stromelysin 1 metalloprotease expression and show an increased invasive propensity compared with cells transformed by wild-type E1A. The altered focus morphology, the high invasive propensity and the elevated stromelysin 1 expression were abrogated by glucocorticoid treatment. Our results show that E1A functions necessary for transformation and inhibition of invasive properties may be separated, and indicate that a 23 amino acid serine/threonine-rich region within the E1A carboxy-terminal exon is required for efficient repression of metalloprotease expression in transformed cells.
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PMID:Enhanced invasive properties of rat embryo fibroblasts transformed by adenovirus E1A mutants with deletions in the carboxy-terminal exon. 153 46

Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDS/PAGE with Mr = 72,000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-Asp-Val-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with Mr = 67,000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-Gln-Ile. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,10-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.
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PMID:Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties. 226 96

Soluble recombinant human fibroblast collagenase catalytic domain was highly expressed and purified from Escherichia coli. The expression construct utilized the T7 gene 10 promoter for transcription of a two-cistron messenger RNA which encoded the ubiquitin-collagenase catalytic domain fusion protein as the second cistron. The ubiquitin domain was attached to the collagenase catalytic domain with the linker sequences Gly-Gly-Thr-Gly-Asp-Val-Ala-Gln (wild type) or Gly-Gly-Thr-Gly-Asp-Val-Gly-His (mutant) which served as cleavage sites for in vitro activation. The last four residues of the linker were included based on the crystal structure of human prostromelysin-1 catalytic domain. Soluble fusion proteins purified from E. coli retained the proteolytic activity of the collagenase catalytic domain. The collagenase catalytic domain was released by either autoproteolytic or stromelysin-1-catalyzed cleavage, purified to homogeneity, and separately possess Phe-81, Val-82, or Leu-83 as the amino-terminal residue. Very similar kcat/Km values were determined for the Phe-81 and Val-82 forms using continuous fluorogenic and chromogenic peptide cleavage assays.
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PMID:Characterization of the Phe-81 and Val-82 human fibroblast collagenase catalytic domain purified from Escherichia coli. 767 41

In addition to the known 94-kd gelatinase (matrix metalloproteinase 9, MMP-9), HL-60 leukemia cells release a hither-to undescribed 45-kd metalloproteinase into the culture medium. This enzyme cleaves the synthetic substrate Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg, which represents the cleavage site for collagenases in collagen type I not between isoleucine and alanine--the typical cleavage site for collagenases--but between alanine and glycine. The enzymatic activity was purified through a combination of zinc-chelate-Sepharose column chromatography, precipitation with Fractogel TSK-AF Red and gelatin-Sepharose, and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Microsequence analysis of the NH2-terminus of the purified 45-kd proteinase revealed the sequence Asp-Ile-Ser-Lys-Tyr-Thr-Thr-Thr-, which could not be found in other proteins when searched in several protein data bases. Incubation of the enzyme immobilized on nitrocellulose membranes with polyclonal antibodies to collagenase and stromelysin or gelatinases revealed no cross-reactivity. The proteolytic activity was not increased by treatment with trypsin, 8M urea, acid, or organomercurials. The proteinase, which was inhibited by chemical inhibitors of metalloproteinases, such as phenanthrolene or EDTA, is able to degrade several matrix constituents, such as collagen type IV, fibronectin, gelatin, and proteoglycans. In contrast to all known MMPs, the proteolytic activity of the 45-kd enzyme was not abolished upon incubation with recombinant tissue inhibitors of matrix metalloproteinases (TIMP) 1 or 2. Thus, the novel enzyme may influence extracellular matrix (ECM) turnover in vivo because its activity is not influenced by specific inhibitors of MMPs.
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PMID:Leukemic cells (HL-60) produce a novel extracellular matrix-degrading proteinase that is not inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs). 782 72

Matrix metalloproteinase 7 (MMP-7) has been purified as an inactive zymogen of M(r) 28,000 (proMMP-7) from the culture medium of CaR-1 human rectal carcinoma cells. The NH2-terminal sequence of proMMP-7 is Lys-Pro-Lys-Pro-Gln-Glu, which is identical to that of matrilysin. The zymogen is activated by 4-aminophenylmercuric acetate (APMA), yielding an intermediate form of M(r) 21,000 and an active species of M(r) 19,000 which shows the new NH2-terminal sequence of Tyr78-Ser-Leu-Phe-Pro-Asn-Ser. Although trypsin fully activates the zymogen, the activation rate by plasmin or leukocyte elastase is confined to approximately 50%. ProMMP-7 can be activated by MMP-3 (stromelysin 1) to its full activity in a single-step mechanism and generates the same NH2 terminus obtained by APMA activation, whereas MMP-1 (tissue collagenase), MMP-2 (gelatinase A), and MMP-9 (gelatinase B) do not have such an effect. On the other hand, proMMP-1 is activated by MMP-7 to an activity similar to that obtained by APMA and the activation by MMP-7 is enhanced up to approximately 6.5 fold in the presence of APMA. This enhanced activity is donated by specific cleavage at the Gln80-Phe81 bond of proMMP-1. MMP-7 can also activate proMMP-9 up to approximately 50% of the full activity with a new NH2 terminus of Leu16-Arg-Thr-(Asn)-Leu. Incubation of proMMP-2 or proMMP-3 with MMP-7 results in no activation of these proMMPs. MMP-7 degrades type IV collagen, laminin-1, fibronectin, proteoglycan, type I gelatin, and insoluble elastin. These results suggest that in vivo MMP-7 may play a role in degradation of extracellular matrix macromolecules in concert with MMP-1, -3, and -9 under pathological conditions.
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PMID:Matrix metalloproteinase 7 (matrilysin) from human rectal carcinoma cells. Activation of the precursor, interaction with other matrix metalloproteinases and enzymic properties. 789 11

The neuron-like differentiation of PC12 cells is induced by nerve growth factor (NGF) through stimulation of a membrane-bound protooncoprotein signaling pathway containing the NGF receptor Trk, the tyrosine kinase Src, and the GTP-binding protein Ras. The Raf-1 and B-raf protooncogenes encode cytoplasmic serine/threonine kinases that are stimulated by NGF in a Ras-dependent manner. To investigate the possible roles of cytoplasmic Raf kinases in eliciting neuronal differentiation, we have expressed the activated Raf-1 oncogene in PC12 cells. Expression of the raf oncogene results in the elaboration of a neuron-like phenotype, including neurite growth and the induction of the NGF-responsive genes NGFI-A and transin. The actions of activated Raf-1 and NGF are not additive. Furthermore, activated Raf-1 oncoprotein can prime cells for transcription-independent neurite growth by NGF and can elicit rapid neurite growth from NGF-primed cells. Our data indicate that the pathways utilized by NGF and activated raf to effect PC12 differentiation overlap and lead to the suggestion that cellular raf kinase activities play significant roles in transducing the differentiating signals of neuronal growth factors.
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PMID:The cytoplasmic raf oncogene induces a neuronal phenotype in PC12 cells: a potential role for cellular raf kinases in neuronal growth factor signal transduction. 838 63

Matrix metalloproteinases (MMPs) are a group of enzymes with the potential to degrade extracellular matrix proteins. One of the MMPs, stromelysin-1 (MMP-3) has been localized to extracellular matrix vesicles in growth plate chondrocyte cultures, suggesting involvement of this enzyme in remodeling of the extracellular matrix during endochondral development, a process which is regulated by the vitamin D metabolites, 1,25-(OH)2D3 and 24,25-(OH)2D3. To determine whether stromelysin-1 is regulated by vitamin D as well, confluent cultures of cells derived from growth zone (GC) and resting zone (RC) rat costochondral cartilage were treated with 1 alpha, 25-(OH)2D3 (1,25) and 24R,25-(OH)2D3 (24,25), respectively, and the effect on stromelysin-1 assessed by casein gel zymography and Western blots. Although stromelysin-1 activity was enriched in the matrix vesicle fraction, only the plasma membrane enzyme was affected by the treatment; 1, 25 and 24,25 caused a marked decrease in plasma membrane stromelysin-1 activity in their target cells. Since plasma membrane protein kinase C (PKC) activity is stimulated by 1,25 and 24,25, we hypothesized that stromelysin-1 activity was regulated by the vitamin D metabolites via PKC-dependent phosphorylation. To test this, membrane fractions (containing endogenous PKC alpha and zeta as well as stromelysin-1) were incubated in the presence of purified rat brain PKC and/or recombinant human (rh) stromelysin-1 and [gamma 32 P]-ATP and anti-stromelysin-1 immunoprecipitates were analyzed by autoradiography and Western blots. Immuno-phospho-stromelysin-1 was localized to a 52-kDa band in the plasma membrane fraction only; no phosphorylation was observed in the matrix vesicle fraction. Selective inhibitors of PKC activity demonstrated that phosphorylation was inhibited by H7 and low concentrations of H8, but not by HA1004, indicating that PKC, not PKA, was responsible. Protein phosphatase 2A1 (PP2A), a serine/threonine-specific phosphatase, selectively removed the radiolabel in a time-dependent manner, providing further support for a PKC-dependent phosphorylation mechanism. Incubation of resting zone cell plasma membranes with 24,25 but not 1, 25, resulted in phosphorylation of stromelysin-1, demonstrating that the nongenomic effect was metabolite-specific. This suggests that this may be one mechanism by which vitamin D metabolites regulate stromelysin-1 activity and that PKC-dependent phosphorylation inhibits the metalloproteinase.
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PMID:Vitamin D3 regulation of stromelysin-1 (MMP-3) in chondrocyte cultures is mediated by protein kinase C. 881 11

Matrix metalloproteinases (MMPs) are zinc endopeptidases that are required for the degradation of extracellular matrix components during normal embryo development, morphogenesis and tissue remodelling. Their proteolytic activities are precisely regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in diseases such as arthritis, atherosclerosis, tumour growth and metastasis. Here we report the crystal structure of an MMP-TIMP complex formed between the catalytic domain of human stromelysin-1 (MMP-3) and human TIMP-1. TIMP-1, a 184-residue protein, has the shape of an elongated, contiguous wedge. With its long edge, consisting of five different chain regions, it occupies the entire length of the active-site cleft of MMP-3. The central disulphide-linked segments Cys 1-Thr 2-Cys 3-Val 4 and Ser 68-Val 69 bind to either side of the catalytic zinc. Cys 1 bidentally coordinates this zinc, and the Thr-2 side chain extends into the large specificity pocket of MMP-3. This unusual architecture of the interface between MMP-3 and TIMP-1 suggests new possibilities for designing TIMP variants and synthetic MMP inhibitors with potential therapeutic applications.
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PMID:Mechanism of inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1. 928 70

Matrix metalloproteinases (MMP) are synthesized as inactive zymogens (proMMP) and subsequently activated by many factors to degrade the extracellular matrix (ECM). In the present study, we have examined the intermolecular activation mechanisms of proMMP by MMP-10 (stromelysin 2). ProMMP-10 was purified from the culture media of OSC-20 human oral squamous carcinoma cells stimulated with 12-O-tetradecanoylphorbol 13-acetate. The final products are partially activated (approximately 38% of the full activity) during the purification steps and contain proMMP-10 of Mr 56,000 with minor protein bands of Mr 47,000, 24,000 and 22,000. The zymogen is activated by 4-aminophenylmercuric acetate and processed to the active forms of Mr 47,000 and 24,000. The NH2-terminal sequence of the 47,000- and 24,000-Mr species is Phe82-Ser-Ser-Phe-Pro-Gly, which is identical to that of stromelysin 2. ProMMP-9 (progelatinase B) is activated by MMP-10 to its full activity and processed to the low-Mr species of Mr 81,000, 65,000, 57,000 and 55,000, the former two of which show proteolytic activity on a gelatin zymography. The NH2-terminal sequence analysis indicates that the 81,000-, 65,000- and 57,000-M, species have the identical sequence of Phe88-Gln-Thr-Phe-Glu-Gly, suggesting the cleavage of the Arg87-Phe88 peptide bond for activation and both NH2-terminal and COOH-terminal truncation in the 65,000- and 57,000-Mr forms. MMP-10 also activates proMMP-7 (promatrilysin) up to about 60% of the full activity and generates the same active species of Mr 19,000 as that obtained by activation with 4-aminophenylmercuric acetate. Incubation of proMMP-2 (progelatinase A) or proMMP-3 with MMP-10 does not result in activation of these proMMP. These results indicate that in addition to the previously reported activation of proMMP-1 (tissue procollagenase) and proMMP-8 (neutrophil procollagenase), MMP-10 can also activate proMMP-9 and proMMP-7, and suggest the possibility that MMP-10 may replace a role of MMP-3 in the ECM degradation in concert with other MMP under various pathological conditions.
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PMID:Activation of the precursor of human stromelysin 2 and its interactions with other matrix metalloproteinases. 957 62


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