- MMPs play a central role in extracellular matrix breakdown in disease processes such as arthritis, glomerulonephritis, tissue ulceration, and tumor cell metastasis. MMP activities are specifically regulated by a family of protein inhibitors designated TIMPs of which three forms (TIMPs-1, -2 and -3) are known. Recent work has identified mutations in TIMP-3 as the cause of a dominant early onset macular degeneration, Sorsby's fundus dystrophy (SFD), in humans. The principal investigator's long-term goals are to elucidate the structural basis and mechanisms by which TIMPs regulate MMP activities and how this can be manipulated to enhance its specificity or is disturbed in pathological condition.
The specific aims of the proposal are as follows: (1) Investigation of the inhibition mechanisms of TIMPs. This will be carried out by site-directed mutagenesis using the N-terminal domain of TIMP-1 (N-TIMP-1) as a prototype. The design of mutants will be based on the information obtained from biochemical studies and a forthcoming crystal structure of the TIMP-1-MMP-3 complex that is being determined by a collaborator, Dr. Wolfram Bode at Max-Planck-Institut in Germany. (2) Isolation of selective inhibitors for specific MMPs. The functional sites identified will be subjected to saturation mutagenesis in N-TIMP-1 displayed on M13 phage and inhibitors with enhanced specificity will be isolated by selection with immobilized MMPS. (3) Determination of the functional properties of TIMP-3 and its domains. TIMP-3 and its two domains will be expressed and characterized with respect to inhibitory action on MMPs and binding to extracellular matrix components. The effect of the SFD mutation on these properties, including possible new covalent interactions, will be assessed. (4) Characterization of TIMP-2 binding to the cell surface, a phenomenon that is related to the activation of progelatinase A (proMMP-2). This will be investigated by characterizing a novel TIMP-2 binding protein expressed on the cell surface. This protein will be isolated, cloned and sequenced. (5) Generation of proteins, mutants and enzyme-inhibitor complexes to provide to collaborators for structural studies of TIMP-MMP interactions by X-ray crystallography and multinuclear NMR spectroscopy. These studies will yield mechanistic and structural information about the interaction of TIMPs and MMPs and new insights into the role of TIMP-2 in regulation of pericellular proteolysis in the extracellular matrix.
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