title: Peptide from the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) inhibits membrane activation of matrix metalloproteinase-2 (MMP-2).
abstract: Cellular activation of latent matrix metalloproteinase-2 (proMMP-2) requires formation of a cell membrane-associated activation complex that involves specific binding between the hemopexin domain of proMMP-2 (PEX) and the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (C-TIMP-2). In this study, we tested the feasibility of inhibiting activation of proMMP-2 by exogenous inhibitors, which block the binding between PEX and TIMP-2. The recombinant C-TIMP-2 and synthetic peptides from C-TIMP-2 were used as inhibitors for proMMP-2 activation. Recombinant C-TIMP-2 bound specifically to both the catalytically inactive MMP-2(E404A) and the C-terminal domain of MMP-2 (PEX) in a concentration dependent manner with apparent K(d) of 3.9×10(-7)M and 1.7×10(-7)M, respectively. Moreover, C-TIMP-2 competed the binding between MMP-2(E404A) and full-length TIMP-2. Finally, activity assays showed that addition of C-TIMP-2 to HT-1080 fibrosarcoma cells inhibited proMMP-2 activation in a concentration-dependent manner. We then designed a synthetic peptide, P175L, consisting of 20 residues from the PEX-binding tail region of C-TIMP-2. P175L bound PEX and inhibited cell membrane-mediated activation of proMMP-2 in a concentration dependent manner. Deletion of the last 9 tail residues of C-TIMP-2 in P175L abrogated the inhibitory activities of the peptide showing that these residues were essential for function. Overall, these experiments have demonstrated that proMMP-2 activation can be inhibited by exogenous inhibitors which points to a potential strategy for MMP-2 specific inhibition.
authors.affiliation: Department of Periodontics, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA. xux@uthscsa.edu
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Xu
authors.forename: Margarita
authors.initials: M
authors.lastname: Mikhailova
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Chen
authors.forename: Sanjay
authors.initials: S
authors.lastname: Pal
authors.forename: Trista K
authors.initials: TK
authors.lastname: Robichaud
authors.forename: Eileen M
authors.initials: EM
authors.lastname: Lafer
authors.forename: Sam
authors.initials: S
authors.lastname: Baber
authors.forename: Bjorn
authors.initials: B
authors.lastname: Steffensen
mesh_terms: D001665:Binding Sites; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D060326:Chemistry Techniques, Synthetic; D017077:Culture Media, Conditioned; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism*
chemical_list: D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D061965:Matrix Metalloproteinase Inhibitors; D011994:Recombinant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D020778:Matrix Metalloproteinase 2