The overall long-term goals of this project are to determine the molecular basis for functions associate with the type 1 repeats (TSRs) of thrombospondin-1. These activities include (1) modulation of endothelial cell phenotype through CD36 and other receptors, (2) activation of transforming growth factor beta (TGFbeta) and (3) inhibition of extracellular proteolysis. Specific focus during the proposed period of support will be placed in the following areas.
Specific Aim 1. Determination of the structure of the TSRs. We hypothesize that the TSRs and the TSR-binding site of CD36 fold into stable three-dimensional structures in such a way that amino acids that are essential for particular interactions are clustered into well-defined binding sites. We plan to use X-ray crystallographic approaches to solve the structure of the TSRs and the TSR-binding domain of CD36 individually and in combination.
Specific Aim 2. Identification of key amino acids for the interaction of the TSRs with other proteins. We hypothesize that (1) the interaction of the TSRs with other proteins involves distinct amino acids and that mutation of these amino acids will inhibit specific TSR-dependent functions and (2) post- translational modifications of specific amino acids will inhibit some TSR interactions. We plan to identify key amino acids within the TSRs for the binding of TSP-1 to CD36, MMP-2, MMP-9, plasmin, laminin-1 and fibronectin. The structural data obtained in Specific Aim 1 and the previously published data obtained with synthetic peptides will be used to design mutations.
Specific Aim 3. Determine molecular mechanisms for modulation of tumor growth by TSP-1. TSP-1 is a potent inhibitor of tumor growth in vivo. We hypothesize that the inhibition of tumor growth by TSP-1 involves multiple activities including the activation of TGFbeta, inhibition of angiogenesis and induction of apoptosis. In addition, we hypothesize that some domains of TSP-1 stimulate tumor growth. We plan to use recombinant domains of TSP-l to map regions with both positive and negative effects on tumor growth. Whereas the domains of TSP-1 have been systematically compared in angiogenesis assays, they have not be assayed for inhibition of tumor growth in vivo. Mutant forms of the TSRs that are characterized in Specific Aim 2 will be used to evaluate the relative importance of the various TSR-dependent activities for inhibition of angiogenesis and tumor growth.
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