title: Total conversion of tissue inhibitor of metalloproteinase (TIMP) for specific metalloproteinase targeting: fine-tuning TIMP-4 for optimal inhibition of tumor necrosis factor-{alpha}-converting enzyme.
abstract: Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases, the ADAMs (a disintegrin and metalloproteinase) and the ADAM-TS (ADAM with thrombospondin repeats) proteinases. There are four mammalian TIMPs (TIMP-1 to -4), and each TIMP has its own profile of metalloproteinase inhibition. TIMP-4 is the latest member of the TIMPs to be cloned, and it has never been reported to be active against the tumor necrosis factor-alpha-converting enzyme (TACE, ADAM-17). Here we examined the inhibitory properties of the full-length and the N-terminal domain form of TIMP-4 (N-TIMP-4) with TACE and showed that N-TIMP-4 is a far superior inhibitor than its full-length counterpart. Although full-length TIMP-4 displayed negligible activity against TACE, N-TIMP-4 is a slow tight-binding inhibitor with low nanomolar binding affinity. Our findings suggested that the C-terminal subdomains of the TIMPs have a significant impact over their activities with the ADAMs. To elucidate further the molecular basis that underpins TIMP/TACE interactions, we sculpted N-TIMP-4 with the surface residues of TIMP-3, the only native TIMP inhibitor of the enzyme. Transplantation of only three residues, Pro-Phe-Gly, onto the AB-loop of N-TIMP-4 resulted in a 10-fold enhancement in binding affinity; the K(i) values of the resultant mutant were almost comparable with that of TIMP-3. Further mutation at the EF-loop supported our earlier findings on the preference of TACE for leucine at this locus. Drawing together our previous experience in TACE-targeted mutagenesis by using TIMP-1 and -2 scaffolds, we have finally resolved the mystery of the selective sensitivity of TACE to TIMP-3.
authors.affiliation: Department of Oncology, Cambridge University, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Cambridge CB2 2XY, United Kingdom.
authors.forename: Meng-Huee
authors.initials: MH
authors.lastname: Lee
authors.forename: Magdalini
authors.initials: M
authors.lastname: Rapti
authors.forename: Gillian
authors.initials: G
authors.lastname: Murphy
mesh_terms: D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D019714:Tissue Inhibitor of Metalloproteinases / Q000235:genetics / Q000378:metabolism*; D000097792:Tissue Inhibitor of Metalloproteinase-4
chemical_list: D051722:ADAM Proteins; D000072198:ADAM17 Protein; D008666:Metalloendopeptidases; D019714:Tissue Inhibitor of Metalloproteinases; D000097792:Tissue Inhibitor of Metalloproteinase-4; C000606462:ADAM17 protein, human