Structure and Function of Thrombospondin-1

该项目的总体长期目标是确定与血小板反应蛋白-1的1型重复序列（TSRs）相关的功能的分子基础。这些活性包括（1）通过CD 36和其他受体调节内皮细胞表型，（2）活化转化生长因子β（TGF β）和（3）抑制细胞外蛋白水解。拟议支助期间的具体重点将放在以下领域。具体目标1。确定TSR的结构。我们假设，TSRs和CD 36的TSRs结合位点折叠成稳定的三维结构，使得对特定相互作用至关重要的氨基酸聚集成明确的结合位点。 我们计划使用X射线晶体学方法来解决的TSR和TSR结合域的CD 36单独和组合的结构。具体目标2。鉴定TSR与其他蛋白质相互作用的关键氨基酸。 我们假设（1）TSR与其他蛋白质的相互作用涉及不同的氨基酸，这些氨基酸的突变将抑制特定的TSR依赖性功能，（2）特定氨基酸的翻译后修饰将抑制某些TSR相互作用。 我们计划确定TSP-1与CD 36、MMP-2、MMP-9、纤溶酶、层粘连蛋白-1和纤连蛋白结合的TSR内的关键氨基酸。 Specific Aim 1中获得的结构数据和先前发表的合成肽数据将用于设计突变。具体目标3。确定TSP-1调节肿瘤生长的分子机制。TSP-1是体内肿瘤生长的有效抑制剂。 我们推测TSP-1抑制肿瘤生长涉及多种活性，包括激活TGF β、抑制血管生成和诱导细胞凋亡。 此外，我们假设TSP-1的某些结构域刺激肿瘤生长。 我们计划使用TSP-1的重组结构域来定位对肿瘤生长具有积极和消极影响的区域。尽管TSP-1的结构域已在血管生成测定中进行了系统比较，但尚未测定它们对体内肿瘤生长的抑制。 特异性目的2中表征的TSR突变形式将用于评价各种TSR依赖性活性对抑制血管生成和肿瘤生长的相对重要性。

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.