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)

The tight regulation of extracellular matrix remodeling and degradation is of great importance in physiological processes like development and morphogenesis, as well as in pathological situations like tumor invasion and metastasis. Tissue inhibitors of metalloproteinases (TIMPs) are the naturally occuring inhibitors of matrix metalloproteinases, which are involved in matrix turnover. In this report we describe the cloning of human TIMP-4 from a human adenocarcinoma and an osteosarcoma cell line and the expression of the inhibitory domain in the methylotrophic yeast Pichia pastoris. The inhibition of MMP-8, -9, -12, -13 and -14 by the N-terminal domain of TIMP-4 was analysed. Using a fluorescent MCA-peptide, Ki values for each subclass of MMPs were determined. With dissociation constants in the nanomolar range, TIMP-4 seems to be a good inhibitor for all classes of MMPs without remarkable preference for special MMPs.
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PMID:Characterization of C-terminally truncated human tissue inhibitor of metalloproteinases-4 expressed in Pichia pastoris. 1150 66

MMPs, part of a family of enzymes with >35 known members, play an important role in tissue remodeling and repair, in the biology of neoplasia, and during development. Hydroxamic and carboxylic acid inhibitors of these proteases have long been available, but their specificities are poor and there still exists a desire to find novel chemical structures, which could be modified to optimize specificity and biocompatibility. Established methods for measuring MMP activity are based on the cleavage of MCA-PLGL-A2pr(DNP)-AR, which provides a prompt fluorescent signal when cleaved; however, its absorption/emission properties (325/400 nm) are not best suited for HTS assays. We describe an HTS-compatible method using the peptide substrate PLGLAARK, labeled at N- and C-termini with CyDye fluors Cy3 and Cy5Q, respectively, which is cleavable by MMP-1, -2, -3, -7, -9, and -13. HTS assays for MMP-13 and MMP-9 inhibitors were set up in approximately 20 microl in 384-well plates as a prompt fluorescence readout (excitation/emission = 540/570 nm) using the LEADseeker homogenous imaging system. These assays yielded IC(50) values comparable to standard methods, but with a faster, very sensitive, and normalized readout, thus conserving compound, enzyme (approximately 1.5 ng/well), and time (20 s read/plate). Data quality (Z' approximately 0.9) was such that hit-picking to -25% change in primary screening could be performed with confidence, and the subsequent rate of confirmation and validation in IC(50) determinations of the picked compounds was >60%. Parallel screening of related proteases also permitted immediate specificity comparisons, including evaluation of inactive or weakly active compounds.
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PMID:Development of an assay suitable for high-throughput screening to measure matrix metalloprotease activity. 1509 Jan 79

Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.
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PMID:Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface. 1789 65


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