Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cDNA coding for a new human matrix metalloproteinase (MMP) has been cloned from a cDNA library derived from a breast tumor. The isolated cDNA contains an open reading frame coding for a polypeptide of 471 amino acids. The predicted protein sequence displays extensive similarity to the previously known MMPs and presents all the structural features characteristic of the members of this protein family, including the well conserved PRCGXPD motif, involved in the latency of the enzyme and the zinc-binding domain (HEXGHXXXXXHS). In addition, this novel human MMP contains in its amino acid sequence several residues specific to the collagenase subfamily (Tyr-214, Asp-235, and Gly-237) and lacks the 9-residue insertion present in the stromelysins. According to these structural characteristics, the MMP described herein has been tentatively called collagenase-3, since it represents the third member of this subfamily, composed at present of fibroblast and neutrophil collagenases. The collagenase-3 cDNA was expressed in a vaccinia virus system, and the recombinant protein was able to degrade fibrillar collagens, providing support to the hypothesis that the isolated cDNA codes for an authentic collagenase. Northern blot analysis of RNA from normal and pathological tissues demonstrated the existence in breast tumors of three different mRNA species, which seem to be the result of the utilization of different polyadenylation sites present in the 3'-noncoding region of the gene. By contrast, no collagenase-3 mRNA was detected either by Northern blot or RNA polymerase chain reaction analysis with RNA from other human tissues, including normal breast, mammary fibroadenomas, liver, placenta, ovary, uterus, prostate, and parotid gland. On the basis of the increased expression of collagenase-3 in breast carcinomas and the absence of detectable expression in normal tissues, a possible role for this metalloproteinase in the tumoral process is proposed.
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PMID:Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas. 820

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is resistant to extremes of temperature and pH. This is thought to be due in part to the presence of six sulfhydryl bridges presumed to maintain the structural integrity of the molecule. As part of a study looking at structure-function relationships, a number of the conserved cysteine residues in TIMP-1 were targeted for replacement with serine. Single and double replacements of these conserved cysteines, as well as replacements around these cysteines, were expressed using a vaccinia virus system and analyzed for functional and structural competence. Analysis by circular dichroism indicated that these mutants maintained secondary structures similar to those of wild-type TIMP-1. Trypsin susceptibility experiments indicated that the tertiary structure of the mutants had not been drastically changed. Analysis of functional competence demonstrated that there were significant changes in some of these mutants. Assays using collagen fibrils or gelatin as substrates indicated that the double mutant C1S/C70S, but not C3S/C99S, had lost inhibitory activity against human fibroblast-type collagenase (FIB-CL) and at high concentrations only had slight activity against Mr 72,000 gelatinase (Mr 72,000 gelatinase). Kinetic analysis of TIMP-1 inhibition of FIB-CL cleavage of a peptide substrate indicated that mutants C1S/C70S, C3S/C99S, and CEEC --> CQQC retained their ability to inhibit FIB-CL in a manner similar to wild-type TIMP-1, while mutants C1S and C70S showed little inhibitory activity. The mutants C99S and C137S could also inhibit FIB-CL cleavage of the peptide substrate. The results indicated that the degree of inhibition by the TIMP-1 mutants varied somewhat depending on the choice of substrates. Interestingly, replacing both cysteines from a disulfide bond in the wild-type molecule resulted in a more competent inhibitor than either of the single site "parent" mutations. Taken together, these experiments indicate that TIMP-1 can be rendered inactive by the loss of a single cysteine.
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PMID:Replacement of conserved cysteines in human tissue inhibitor of metalloproteinases-1. 940 13

Mutants in the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein have been created by site-directed mutagenesis and expressed in HeLa cells, using a recombinant vaccinia virus system. Removal of either or both glycosylation sites yielded proteins which retained wild-type inhibitory activity against both human fibroblast-type collagenase (FIB-CL) and Mr 72000 gelatinase (GL). However, the double glycosylation mutant protein was expressed at a level that was 2-4-fold lower than that of the wild-type or the single site glycosylation mutants. The 'tiny-TIMP' COOH-terminal deletion mutant that lacks the last 57 residues was also inhibitory, but the dose-response curve suggested that the interaction with the Mr 72000 gelatinase had been altered. A number of replacement mutants in the highly conserved NH2-terminal domain, including replacement of P5A and P8A or a double mutation in the VIRAK sequence which is absolutely conserved in all TIMPs in all species (VIRAK to VIAAA), also yielded functional proteins capable of inhibiting FIB-CL and Mr 72000 GL and of forming SDS-resistant complexes with FIB-CL. None of the above manipulations abolished inhibitory function suggesting that binding of the inhibitor by the enzyme involves multiple interactions.
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PMID:Glycosylation and NH2-terminal domain mutants of the tissue inhibitor of metalloproteinases-1 (TIMP-1). 977 3

Sinusoidal entry is the first obligatory process preceding intracellular drug removal in liver. Transport of the angiotensin converting enzyme inhibitor enalapril (1-750 microM with [(3)H]enalapril), a substrate of Oatp1, the sodium-independent organic anion transporting polypeptide 1 cloned from rat liver, was studied in rat hepatocytes isolated from all zones of the liver (homogeneous) and from enriched periportal (PP) and perivenous (PV) hepatocytes prepared by collagenase perfusion and zone-selective destruction with digitonin, respectively. Uptake was linear over 1 min and was concentration-dependent. Transport by the homogeneous hepatocytes (in the presence and absence of Na(+)) and PP and PV cells was described by single saturable components of similar kinetic constants (K(m) values of 344-461 microM and V(max) values of 9.5-11.6 nmol/min/10(6) cells; P >.05, ANOVA). The K(m) value for enalapril uptake in hepatocytes was of the same order of magnitude compared with that for Oatp1 expressed in HeLa cells transfected with cDNA-Oatp1 and Western blot analysis revealed similar levels of immunoreactive Oatp1 expression in PP and PV hepatocytes. However, enalapril was not taken up by Oatp2 nor by the human OATP expressed in recombinant vaccinia systems.
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PMID:Uptake of enalapril and expression of organic anion transporting polypeptide 1 in zonal, isolated rat hepatocytes. 1085 54

Bypass graft surgery is limited by stenosis of vein grafts. Neointimal formation in vein graft stenosis is affected by oxidative stress, acute inflammatory response, and proliferation. Gene therapy offers a novel treatment strategy for vein graft stenosis because gene transfer can be done ex vivo during the graft operation. In this study we used adenovirus-mediated ex vivo gene transfer of extracellular superoxide dismutase (EC-SOD) alone or in combination with tissue inhibitor of metalloproteinase-1 (TIMP-1) or vaccinia virus antiinflammatory protein 35K to prevent vein graft stenosis in a jugular vein graft model in normocholesterolemic New Zealand White rabbits. Vein grafts were analyzed 14 and 28 days after the gene transfer, using histological methods. It was found that at the 2-week time point EC-SOD + 35K and EC-SOD + TIMP-1 combinations delivered by gene transfer were the most efficient treatments in decreasing neointimal formation. At the 4-week time point the effect was seen only in the EC-SOD + TIMP-1 combination group. The combination of antiinflammatory proteins (EC-SOD + 35K) was the most effective in reducing macrophage accumulation, which was still significant at the 4-week time point, but this did not prevent vein graft thickening. In conclusion, oxidative, inflammatory, and proliferative processes are important for neointimal formation in vein graft stenosis. In the rabbit model of vein graft disease, combination gene therapy with antioxidative, antiinflammatory, and antiproliferative genes was effective in decreasing neointimal formation. This may be because two different genes may more efficiently affect different pathogenetic pathways at the early stage of the disease process than gene transfer approaches based on single genes.
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PMID:Extracellular superoxide dismutase with vaccinia virus anti-inflammatory protein 35K or tissue inhibitor of metalloproteinase-1: Combination gene therapy in the treatment of vein graft stenosis in rabbits. 1661 Sep 28