TGF-beta latency and activation

描述（申请人提供）：转化生长因子β（TGF-β）在骨骼和肌肉等组织发育、伤口愈合、免疫调节以及肿瘤细胞生长和抑制中发挥关键作用。TGF-β调节所有组织的修复，包括脑、心脏和骨，以及缺血和中风后。TGF-β 1前结构域中的突变引起Camurati-Engelmann病，其表现为骨生长异常伴肌无力和疼痛。TGF-β 1、2和3被合成为前蛋白，其在内质网中二聚化并与呈递分子、潜在TGF-β结合蛋白（LTBP）或糖蛋白-A重复优势蛋白（GARP）缔合。弗林蛋白酶在前结构域和生长因子结构域之间裂解;然而，前结构域二聚体在前结构域中保持与生长因子二聚体非共价结合。 分泌后的pro-TGF-β。LTBP和GARP分别与前结构域结合并将pro-TGF-β储存在细胞外基质和细胞表面上。虽然储存在组织中，但大的潜在复合物是生物学上无活性的，因为环绕TGF-β二聚体的前结构域阻止与TGF-β受体结合。TGF-β信号传导的关键调节步骤是TGF-β从潜伏期释放。整联蛋白aV β 6和aV β 8结合到一个特定的基序上，该基序包括原TGF-β 1和3中的RGD，并激活它们，但不激活原TGF-β 2。整合素介导的活化需要pro-TGF-β与LTBP或GARP的结合。在这项研究中，我们研究了aV整合素如何结合和激活pro-TGF-β 1和3，pro-TGF-β 1和2之间的结构差异如何与不同的激活机制相关，以及LTBP和GARP如何促进TGF-β 1的潜伏期。结合亲和力、动力学和热力学测量，解析pro-TGF-β 1、整合素α V β 6头部片段单独或与TGF-β 3肽复合、以及整合素α V β 6头部与人pro-TGF-β 1大分子复合的晶体结构，将揭示结合时发生的构象变化以及整合素α V β 6如何以高亲和力和特异性结合pro-TGF-β 1而不结合pro-TGF-β 2的分子基础。潜在的基于结构的治疗方法也正在出现。为了更好地理解TGF-β 1在体内保持潜伏、储存和活化的背景下的结构，我们将研究po-TGF-β 1和2及其与LTBP或GARP的复合物。我们将解决pro-TGF-β 1和2的晶体和SAXS结构，并使用蛋白水解和氢/氘交换方法探测它们在溶液中的动力学。我们将解决至少一个复杂的LTBP或GARP。这些实验将为结合后的构象变化提供关键的结构见解。此外，成熟TGF-β 1和2与游离前结构域和LTBP-或GARP-相关前结构域结合的动力学和亲和力测量将提供它们各自相互作用的定量生化表征。关于pro-TGF-β 1和2以及与LTBP或GARP的复合物的结构信息，将提供对pro-TGF-β 1和2在其潜伏期和激活机制以及新的治疗方法方面如何不同的关键见解。

DESCRIPTION (provided by applicant): Transforming growth factor beta (TGF-ß) plays pivotal roles in development of tissues including bone and muscle, wound healing, immune regulation, and tumor-cell growth and inhibition. TGF-ß regulates repair in all tissues including brain, heart, and bone, and after ischemia and stroke. Mutations in the TGF-ß1 prodomain cause Camurati-Engelmann disease, which manifests as abnormal bone growth with muscle weakness and pain. TGF-ß1, 2 and 3 are synthesized as pro-proteins that dimerize and associate with presenting molecules, latent TGF-ß binding protein (LTBP) or glycoprotein-A repetitions predominant protein (GARP), in the endoplasmic reticulum. Furin cleaves between the prodomain and growth factor domain; however, the prodomain dimer remains noncovalently associated with the growth factor dimer in pro-TGF-ß after secretion. LTBP and GARP bind to the prodomains and store pro-TGF-ßs in the extracellular matrix and on the cell surface, respectively. Although stored in tissues, large latent complexes are biologically inactive because the prodomains encircling the TGF-ß dimer prevent binding to TGF-ß receptors. The key regulatory step in TGF-ß signaling is release of TGF-ß from latency. Integrins aVß6 and aVß8 bind to a specific motif that includes RGD in pro-TGF-ß1 and 3 and activate them, but not pro-TGF-ß2. Integrin-mediated activation requires pro-TGF-ß association with LTBP or GARP. In this grant, we study how aV integrins bind and activate pro-TGF-ß1 and 3, how structural differences between pro-TGF-ß1 and 2 correlate with different activation mechanisms, and how LTBP and GARP contribute to TGF-ß latency. Solving crystal structures of pro-TGF-ß1, integrin aVß6 headpiece alone or in complex with TGF-ß3 peptide, and integrin aVß6 head in complex with human pro-TGF- ß1 macromolecule, coupled with affinity, kinetics, and thermodynamic measurements will reveal conformational changes that occur upon binding and the molecular basis for how integrin aVß6 binds pro-TGF- ß1 with high affinity and specificity, but not pro-TGF-ß2. Potential structure-based therapeutics are also emerging. To better understand TGF-ß structure in the context in which it is kept latent, stored, and activated in vivo, we will study po-TGF-ß1 and 2 and their complexes with LTBP or GARP. We will solve crystal and SAXS structures of pro-TGF-ß1 and 2 and probe their dynamics in solution using proteolysis and hydrogen/deuterium exchange methods. We will solve at least one complex with LTBP or GARP. These experiments will provide key structural insight into conformational changes upon binding. Furthermore, kinetics and affinity measurements on mature TGF-ß1 and 2 binding to free prodomains and LTBP- or GARP-associated prodomains will provide a quantitative, biochemical characterization of their respective interactions. Structural information on pro-TGF-ß1 and 2, and complexes with LTBP or GARP, will provide critical insight into how pro- TGF-ß1 and 2 differ in their latency and activation mechanisms and new therapeutic approaches.