Structure and Function of Thrombospondin-1

Thrombospondin-1 的结构和功能

• 批准号: 6625601
• 负责人: John W LAWLER
• 金额: $ 37.94万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6625601
• 关键词: X ray crystallography; adenocarcinoma; angiogenesis inhibitors; apoptosis; binding sites; collagenase; disease /disorder model; fibronectins; gene mutation; genetically modified animals; human tissue; laboratory mouse; laminin; lung neoplasms; melanoma; neoplastic cell; neoplastic growth; plasmin; posttranslational modifications; protein protein interaction; protein structure function; proteolysis; receptor; thrombospondins; transforming growth factors; vascular endothelium

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.

该项目的总体长期目标是确定与血小板传播1的1型重复（TSR）相关的功能的分子基础。这些活性包括（1）通过CD36和其他受体调节内皮细胞表型，（2）转化生长因子β（TGFBETA）的激活和（3）抑制细胞外蛋白水解。在拟议的支持期间的特定重点将放在以下领域。特定目的1。确定TSR的结构。我们假设CD36的TSR和TSR结合位点折叠成稳定的三维结构，以使特定相互作用必不可少的氨基酸聚集到定义明确的结合位点中。 我们计划使用X射线晶体学方法来解决TSR的结构和CD36的TSR结合域单独和组合。特定目标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，这些氨基酸的突变会抑制特定的TSR依赖性功能，并且（2）特定氨基酸的转化后修饰将抑制某些TSR相互作用。 我们计划鉴定TSR中的键氨基酸，以结合TSP-1与CD36，MMP-2，MMP-9，质蛋白，粘胶蛋白，层粘连蛋白1和纤连蛋白的结合。 在特定目标1中获得的结构数据以及先前使用合成肽获得的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。 我们假设TSP-1对肿瘤生长的抑制涉及多种活性，包括激活TGFBETA，抑制血管生成和凋亡诱导。 此外，我们假设TSP-1的某些结构域刺激了肿瘤的生长。 我们计划使用TSP-L的重组结构域来绘制对肿瘤生长的阳性和负面影响的区域。尽管在血管生成测定中已系统地比较了TSP-1的结构域，但尚未对它们抑制体内肿瘤生长进行测定。 在特定目标2中表征的TSR的突变形式将用于评估各种TSR依赖性活性对抑制血管生成和肿瘤生长的相对重要性。