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We have reported previously that reactive-site mutants of N-TIMP-3 [N-terminal inhibitory domain of TIMP-3 (tissue inhibitor of metalloproteinases 3)] modified at the N-terminus, selectively inhibited ADAM17 (a disintegrin and metalloproteinase 17) over the MMPs (matrix metalloproteinases). The primary aggrecanases ADAMTS (ADAM with thrombospondin motifs) -4 and -5 are ADAM17-related metalloproteinases which are similarly inhibited by TIMP-3, but are poorly inhibited by other TIMPs. Using a newly developed recombinant protein substrate based on the IGD (interglobular domain) of aggrecan, gst-IGD-flag, these reactive-site mutants were found to similarly inhibit ADAMTS-4 and ADAMTS-5. Further mutations of N-TIMP-3 indicated that up to two extra alanine residues can be attached to the N-terminus before the Ki (app) for ADAMTS-4 and ADAMTS-5 increased to over 100 nM. No other residues tested at the [-1] position produced inhibitors as potent as the alanine mutant. The mutants N-TIMP-3(T2G), [-1A]N-TIMP-3 and [-2A]N-TIMP-3 were effective inhibitors of aggrecan degradation, but not of collagen degradation in both IL-1α (interleukin-1α)-stimulated porcine articular cartilage explants and IL-1α with oncostatin M-stimulated human cartilage explants. Molecular modelling studies indicated that the [-1A]N-TIMP-3 mutant has additional stabilizing interactions with the catalytic domains of ADAM17, ADAMTS-4 and ADAMTS-5 that are absent from complexes with MMPs. These observations suggest that further mutation of the residues of N-TIMP-3 which make unique contacts with these metalloproteinases may allow discrimination between them.

Original publication

DOI

10.1042/BJ20100725

Type

Journal article

Journal

Biochem J

Publication Date

01/10/2010

Volume

431

Pages

113 - 122

Keywords

ADAM Proteins, ADAMTS4 Protein, Aggrecans, Animals, Cartilage, Articular, Catalytic Domain, Cells, Cultured, Humans, Mutation, Procollagen N-Endopeptidase, Swine, Tissue Inhibitor of Metalloproteinase-3