中心体蛋白SAS-4/CPAP及其复合物的结构与功能研究

中心体由一对中心粒和中心粒周质（PCM）组成,中心粒必须成功招募PCM蛋白，才能使中心体发育成功能性细胞器。在PCM招募过程中，SAS-4/CPAP起到关键作用。一方面SAS-4/CPAP通过其N端与alpha/beta-tubulin结合并招募形成PCM复合物，另一方面通过其C端TCP结构域与中心粒蛋白ANA2和Bld10相互作用，将PCM复合物定向转运并锚定在中心粒周围。目前SAS-4/CPAP如何形成超级复合物并协调PCM复合物的定向锚定尚缺少结构信息。我们计划以蛋白质晶体学为主要研究手段，结合生物化学细胞生物学和果蝇遗传学等技术，开展SAS-4/CPAP蛋白自身，及其与alpha/beta-tubulin、ANA2/STIL、Bld10/Cep135等蛋白复合物的结构与功能研究，以期在分子水平上揭示SAS-4/CPAP介导PCM复合物形成及锚定机制，并为相关药物设计工作提供基础。

Centrosomes are macromolecular structures, which is composed of a pair of centrioles surrounded by a multi-protein complexes network of Peri-Centriole Material (PCM) nucleating and organizing microtubules in cytoplasm. In order for centrosomes to form functional organelles as the MTOCs in cells, it must first assemble a centriole and successfully recruit PCM around it. PCM recruitment begins with forming PCM protein complexes in cytoplasm, which are then targeted to centrioles. In this process, SAS-4/CPAP, a conserved centrosomal protein that is essential for centriole formation, scaffolds cytoplasmic PCM protein complexes (SAS-4-PCM complexes) via its N-terminal domain and tethers the components of SAS-4-PCM complexes to the centriole via its conserved C-terminal TCP domain. In this process, tubulin dimer present within the SAS-4-PCM complex (SAS-4-tubulin module) negatively regulates the PCM complexes assembling ability of SAS-4. The TCP domain of SAS-4 then mediates the tethering of the pre-assembled SAS-4-PCM complexes to a centriole by interacting with centriole protein ANA2/STIL and Bld10/Cep135. Although the role of SAS-4 in centrosome biogenesis is critical, little is known about their structures, molecular mechanisms and functions. Therefore, in this proposal, we are asking a fundamental question in cell biology: how SAS-4 involving protein complexes assemble and then coordinate PCM proteins recruitment and anchoring in generating functional centrosomes? To address this question, we propose to solve the crystal structures of SAS-4 and its complexes (SAS-4-tubulin, SAS-4-ANA2 and SAS-4-Bld10), and in combination with biochemistry, cell biology and genetics tools to obtain mechanistic insights into SAS-4/CPAP-mediated functional centrosome biogenesis. This work shall pave the way for SAS-4/CPAP-targeted small molecules screening and optimization.