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)

Gelatinase B (MMP-9), a member of the matrix metalloproteinase family, is a zinc- and calcium-dependent endopeptidase that is known to play a role in tumor cell invasion and in destruction of cartilage in arthritis. It contains a conserved sequence. 400His-(X)3-His-(X)28-Asp-Asp-(X)2-436Gly, the function of which is under investigation. The conserved Asp-432 and Asp-433 residues were individually replaced with Gly; these substitutions reduced the gelatinolytic activity of the enzyme to 23% and 0%, respectively. Replacing Asp-433 with Glu, however, decreased the gelatinolytic activity of the enzyme by 93% and proteolytic activity of the enzyme for the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 substrate by 79%. The wild-type and D432G and D433E, mutant enzymes had similar Km values for the synthetic substrate and similar Ki values for the competitive inhibitor, GM6001. The kcat/Km values for D432G and D433E mutant enzymes, however, were reduced by a factor of approximately 4 and their KaCa values were increased by four- and sixfold, respectively. The significance of His-400 in the activity of the enzyme was assessed by replacing this residue with Ala and Phe. Both H400A and H400F mutants were inactive toward gelatin substrate. These data demonstrate that Asp-432, Asp-433, and His-400 residues are important for the activity of gelatinase B. His-400 may act as a zinc-binding ligand similar to the His-197 in interstitial collagenase (MMP-7) and Asp-432 and Asp-433 residues are probably involved in stabilization of the active site of the enzyme. The His-400 and Asp-433 residues are conserved in all members of the MMP family. Therefore, our results are relevant to this group as a whole.
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PMID:Role of the conserved histidine and aspartic acid residues in activity and stabilization of human gelatinase B: an example of matrix metalloproteinases. 856 49

Chemotaxis of human T lymphoblastoma cells of the Tsup-1 line, which migrate similarly to blood T cells, through a layer of basement membrane-like Matrigel on a polycarbonate micropore filter was evoked by vasoactive intestinal peptide (VIP; concentration for a maximal response, 10(-7)M), IL-2 (10(-9)M), and the chemokines RANTES (10(-10)M) and macrophage inflammatory protein-1 alpha (10(-10)M). Chemotactic concentrations of each factor increased Tsup-1 cell secretion of matrix metalloproteinase-9 (MMP-9), with significant responses by 4 h for VIP, IL-2, and IL-4, but only after 24 h for macrophage inflammatory protein-1 alpha and RANTES, as quantified by Western blots and zymography. 3H-Labeled type IV human collagen incorporated in the Matrigel layer was degraded by migrating Tsup-1 cells, as assessed by release of radioactive fragments of the collagen. The in situ degradation of type IV collagen in Matrigel by migrating Tsup-1 cells was enhanced most significantly by VIP, IL-2, and IL-4 after 4 h at concentrations that increased the secretion of MMP-9 optimally, but only after 24 h by macrophage inflammatory protein-1 alpha and RANTES. The specific MMP inhibitor GM6001 suppressed Tsup-1 cell MMP activity evoked by all stimuli, as determined by zymography and in situ degradation of 3H-Labeled type IV human collagen. The chemotactic migration of Tsup-1 cells through Matrigel, but not through a filter alone, in response to optimal concentrations of VIP, IL-2, and IL-4, but not the chemokines, was inhibited by GM6001, with a concentration dependence similar to that for suppression of MMP activity. Thus elicitation of T cell chemotactic migration through a model basement membrane by stimuli that increase MMP activity early in the response depends on degradation of matrix proteins by MMP, whereas stimuli that recruit MMP late may rely on early activation of other proteases.
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PMID:Stimulus specificity of matrix metalloproteinase dependence of human T cell migration through a model basement membrane. 859 57

We examined the anti-invasive activity of ursolic acid (UA) on the highly metastatic HT1080 human fibrosarcoma cell line. UA reduced tumor cell invasion through a reconstituted basement membrane in a transwell chamber. A significant down-regulation of matrix metalloproteinase-9 [MMP-9; Mr 92,000 gelatinase/type IV collagenase (gelatinase B)] by UA was detected by Northern blot analysis. However, MMP-2 [Mr 72,000 gelatinase/type IV collagenase (gelatinase A)] and membrane-type MMP were constantly expressed, and the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 also was not changed after 3 and 6 days of treatment with UA. Quantitative gelatin-based zymography confirmed a markedly reduced expression of MMP-9 but not MMP-2 after treatment with UA. To confirm the UA-induced down-regulation of MMP-9 expression, we constructed a secreted alkaline phosphatase (SEAP) reporter vector including MMP-9 promoter. After transfection of MMP-9/SEAP reporter vector into HT1080 cells, reduced SEAP activity was detected after treatment with UA. These results suggest that down-regulation of MMP-9 contributes to the anti-invasive activity of UA in HT1080 cells.
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PMID:Anti-invasive activity of ursolic acid correlates with the reduced expression of matrix metalloproteinase-9 (MMP-9) in HT1080 human fibrosarcoma cells. 862 99

Induction of stromelysin and collagenase mRNAs in response to phorbol myristate acetate (PMA) and autocrine factors (CAF) was compared in primary cultures of lapine synovial fibroblasts and an immortalized line of these cells known as HIG-82. In both cell types, message induction was quicker for CAF than for PMA. Appearance of both stromelysin and collagenase mRNAs occurred earlier in HIG-82 cells and, unlike primary cells, HIG-82 cells partially resisted inhibition by cycloheximide. To determine whether differences in AP-1 activity could account for these observations, the induction of c-fos and c-jun mRNAs was studied in conjunction with gel shift assays for AP-1 binding. Both inducers increased the abundance of c-fos mRNA, although the response was weaker in HIG-82 cells. However, the increase in c-jun mRNA was more marked in HIG-82 cells; furthermore, this increase was sustained for over 6 h. Gel shift assays confirmed that in both types of cells PMA and CAF increased AP-1 binding activity. In primary cells, this activity was sensitive to cycloheximide, but in HIG-82 cells, there was only partial sensitivity to cycloheximide. The gel shift analyses and data from experiments using an AP-1-CAT reporter construct revealed, in many cultures, constitutive AP-1 activity in the absence of stromelysin and collagenase expression, suggesting that AP-1 alone is insufficient for matrix metalloproteinase induction. Antisense oligonucleotides to c-fos and c-jun strongly inhibited the induction of stromelysin mRNA in primary cells treated with PMA, but was only weakly active against message induction in HIG-82 cells. In neither primary cells nor HIG-82 cells did antisense oligonucleotides strongly inhibit stromelysin induction in response to CAF. These data suggest there may exist an AP-1-independent route to message induction or that factors other than c-FOS and c-JUN may be used in certain circumstances. Western blot analyses detected no marked difference between HIG-82 cells and primary cells in their resting levels of c-FOS and c-JUN. Thus the differences reported here between HIG-82 cells and primary cells in their resting levels of c-FOS and c-JUN. Thus the differences reported here between HIG-82 cells and primary cells in the kinetics and cycloheximide sensitivity of MMP induction may reside in their abilities to modify posttranslationally the relevant transcription factors.
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PMID:Effects of immortalization upon the induction of matrix metalloproteinases in rabbit synovial fibroblasts. 863 83

Gelatinase A is secreted as a proenzyme (progelatinase A) which is activated and bound on the surface of tumor and normal cells. We have reported that the expression of a membrane-type-1-matrix metalloproteinase (MT1-MMP) induces activation of progelatinase A. Here we demonstrate that the expression of MT1-MMP in COS-1 cells induces cell-surface binding of progelatinase A which is consequently processed to an intermediate form. Processing from the intermediate to the fully active form is dependent on the gelatinase A concentration. These results suggest that the cell-surface binding concentrates the gelatinase A intermediate form locally to allow autoproteolytic processing to the fully active form.
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PMID:Cell surface binding and activation of gelatinase A induced by expression of membrane-type-1-matrix metalloproteinase (MT1-MMP). 864 59

Human matrix metalloproteinase-7 (MMP-7 = matrilysin) was overproduced in Escherichia coli as a recombinant zymogen (31 kDa), the C-terminus of which bears artificial hexa-histidines. Most of the enzyme was isolated from the insoluble fraction of the cell lysate and purified by a single step using Ni-NTA resin after solubilization of the precipitates with 8 M urea solution. The resin-bound recombinant protein was refolded into a form that is activatable by p-amino-phenylmercuric acetate in an autocatalytic manner. The activated enzyme cleaved a synthetic peptide substrate at the reported site for MMP-7. Digestion of carboxymethylated transferrin (a natural substrate of MMP-7) by the recombinant proteinase generated fragments with the same peptide map as in the case of native purified MMP-7. The autocatalytic activation and enzyme reaction were entirely dependent on the presence of calcium and zinc ions. The enzyme activity to cleave carboxymethylated transferrin was inhibited by tissue inhibitors of metalloproteinases-1 and -2, MMP-specific inhibitors. The activity of the recombinant MMP-7 was also inhibited by a synthetic peptide derived from a part of the cysteine switch that maintains the zymogen in an inactive state. Thus, we report here a simple means of preparing a large quantity of recombinant proMMP-7 that can be used to study the activation mechanism and to screen synthetic inhibitors.
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PMID:Purification and refolding of recombinant human proMMP-7 (pro-matrilysin) expressed in Escherichia coli and its characterization. 874 67

92-kDa type IV collagenase/gelatinase (matrix metalloproteinase-9; MMP-9; gelatinase B) expression and secretion has been shown to correlate with the invasive and metastatic potential of various malignant cells. MMP activity is tightly controlled by specific tissue inhibitors of metalloproteinases (TIMPs). We found the leukemic cell line HL-60 constitutively to release a 94-kDa gelatinase which we identified as MMP-9 shortened by nine amino acids at its N-terminal end. An additional gelatinolytic activity was present in small amounts and identified as a 63-kDa fragment of MMP-9 generated by autocatalytical processing. Both enzymes were identical regarding their N-terminus, indicating C-terminal truncation for the former. Incubation of cells with phorbol ester resulted in elevated amounts of both enzymes in conditioned media and in the secretion of TIMP-1. Both gelatinases were shown to be activated by trypsin and organomercurials and to possess similar activities towards various substrates. However, the 63-kDa enzyme differed from the 94-kDa enzyme in a significantly reduced inhibition by recombinant TIMP-1 and TIMP-2. Thus, the 63-kDa fragment of MMP-9 once activated may escape the regulatory influence of its specific inhibitors and may thereby promote matrix degradation during invasion of leukemic cells.
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PMID:HL-60 leukemia cells produce an autocatalytically truncated form of matrix metalloproteinase-9 with impaired sensitivity to inhibition by tissue inhibitors of metalloproteinases. 875 73

Membrane type 1-matrix metalloproteinase (MT1-MMP) initiates the activation of the zymogen progelatinase A/ 72-kDa type IV collagenase by cleavage of the Asn66-Leu peptide bond. We previously pointed out that MT1-MMP possesses a unique amino acid sequence Arg-Arg-Lys-Arg111 which is a potential recognition sequence for furin-like proteases (Nature, 370 (1994) 61-65). Here, using a recombinant MT1-MMP expressed in Escherichia coli we demonstrated that furin specifically cleaves MT1-MMP between Arg111-Tyr in vitro, which resulted in a stimulation of progelatinase A-activation function. Tissue inhibitor of metalloproteinases (TIMP)-2 inhibited activation of progelatinase A by forming a stable complex with activated MT1-MMP.
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PMID:Activation of a recombinant membrane type 1-matrix metalloproteinase (MT1-MMP) by furin and its interaction with tissue inhibitor of metalloproteinases (TIMP)-2. 880 34

Matrilysin (PUMP-1) is a member of the matrix metalloproteinase (MMP) family of extracellular matrix degrading enzymes that has been found to be overexpressed in human prostate cancer. The rat ventral prostate (RVP) following castration has been used as a model for both tissue involution and apoptosis. Northern analysis and in situ hybridization were used to determine the time course and localization of matrilysin during 8 days of RVP involution. Northern analysis revealed that the 1.2 kb matrilysin mRNA was undetectable in normal RVP. An increase in the steady-state levels of matrilysin mRNA was observed 5 days after castration, and the levels began to decline by 8 days after castration. The mRNAs for tissue inhibitor of metalloproteinase-1 and urokinase-type plasminogen activator also showed a time-dependent induction during the course of involution. Localization of matrilysin by in situ hybridization indicated that the mRNA was produced by epithelial cells of the involuting RVP. The matrilysin message was observed in a small number of glands within the whole RVP. Matrilysin protein was present in the RVP and peaked 3 days after castration. The combination of proteinase genes expressed in the RVP following castration indicate that the MMP and serine protease families of enzymes may interact during tissue remodeling of the RVP following castration.
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PMID:Matrilysin expression in the involuting rat ventral prostate. 882 84

Previous immunolabeling studies have indicated that increased expression of the matrix metalloproteinase-2 (MMP-2) zymogen is associated with an increased Gleason score for human prostate cancer. In the accompanying paper, we have found by immunoblotting and ELISA that the MMP-2 enzyme (termed MMP-2a) is expressed in prostate cancer and that increased expression is associated with progression. Monoclonal antibodies specific for MMP-2a were used to investigate the expression of MMP-2a in human prostate tissue sections of benign and malignant cancers. Immunohistochemistry indicated that MMP-2a expression was undetectable in fetal (n = 4), benign (n = 11), and low Gleason score 4 (n = 8) tissue. MMP-2a was faintly expressed (+) in cancer assigned Gleason scores 5 (n = 20) and 6 (n = 13). In comparison, MMP-2a was expressed at an intermediate level (++) in tissues of Gleason score 7 (n = 24), and at a intense level ( to +) in tissues of score 8 (n = 48), 9 (n = 9) and 10 (n = 35) and in lymph node metastases (n = 10). These observations were confirmed by quantitative Computer Assisted Imaging Analysis. In general, MMP-2a was primarily expressed by the glandular epithelial cells, and in high Gleason score 10 specimens (n = 25/35) there was clear evidence of MMP-2a localization at the cell surface. These data suggest that increased MMP-2a expression may be associated with malignant progression and metastases.
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PMID:Immunohistochemical studies of activated matrix metalloproteinase-2 (MMP-2a)expression in human prostate cancer. 885 76


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