The degradation of the extracellular matrix requires the coordinated action of enzymes which constitute a family of metalloproteinases. Nowhere is this degradative ability more apparent than in rheumatoid arthritis. In this disease, excessive production of metalloproteinases mediates the degradation of articular cartilage and subchondral bone, resulting in severe deformity. Type V collagenase/gelatinase B is a member of this multigene family of enzymes which exhibits high specificity for denatured collagen, degrades native types V and XI collagen which is a structural component of cartilage. The main objective of the proposed research is to understand the regulation of this enzyme and to define its precise role and significance in rheumatoid arthritis. Towards the achievement of this goal we propose to 1. Investigate the role of hemopexin-like carboxy terminal domain in Ca2+-dependent activation of gelatinase B. This will be done by generating a series of COOH-terminal (CTD) truncated mutant and assessing the effect of truncation on enzyme latency and conformation. The CTD """"""""regulatory peptide"""""""" will be identified by generating a series of GST-CDT peptide and the effect of each peptide on activity of CTD-truncated enzymes will be examined, 2. Substantiate the hypothesis that a salt linkage present in gelatinase B catalytic domain is responsible for Ca2+-independent activity of the enzyme. The NH2- and COOH-terminal regions of catalytic domain will be cross linked and the effect of cross linking on the Ca2+-dependent activity of the enzyme will be assessed, 3. Identify the TIMP-1 binding residues within the active site of gelatinase B by alanine-scanning site-directed mutagenesis, 4. Investigate the role of the fibronectin-like binding domain in substrate specificity of the enzyme by generating deletion mutants. Specific amino acid residues responsible for substrate specificity of the enzyme will be identified by alanine-scanning site directed mutagenesis and finally 5. Region(s) of gelatinase B responsible for protein-protein and protein- matrix membrane interaction will be identified using truncated mutants generated above. These studies will further our understanding on the action of this enzyme and may lead to new therapeutic approaches which would focus on protection of articular tissues from degradation in rheumatoid arthritis.
| Fasciglione, Giovanni Francesco; Gioia, Magda; Tsukada, Hiroki et al. (2012) The collagenolytic action of MMP-1 is regulated by the interaction between the catalytic domain and the hinge region. J Biol Inorg Chem 17:663-72 |
