膜锚定状态Rab11的构象变化与功能关系研究

项目名称： 膜锚定状态Rab11的构象变化与功能关系研究

项目编号： No.31500632

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 生物科学

项目作者： 马培翔

作者单位： 上海科技大学

项目金额： 20万元

中文摘要： 随着我国进入人口老龄化，癌症、糖尿病等慢性病已经成为我国居民的主要死因。这些疾病的发生发展与囊泡运输及其介导的受体和粘连分子的细胞表面分布有关，小G蛋白11（Rab11）正是调控这一过程的关键蛋白之一，因此对Rab11活性调控的研究至关重要。Rab11碳末端的异戊二烯化修饰影响其膜锚定状态，而这一状态是Rab11发挥功能的必要条件之一。但是由于体外类膜环境的不均一性和不稳定性，难以进行相关生物化学及结构生物学研究，目前对其膜锚定状态下的构象变化知之甚少。本项目拟结合近年发展起来的纳米磷脂盘技术，将Rab11的碳末端与磷脂偶联并锚定在磷脂膜上，通过核磁共振等技术研究膜锚定状态下影响Rab11构象和功能的重要位点及其对Rab11解离常数的影响。进而结合定点突变技术，在细胞内验证这些位点对其定位和生理功能的影响，以此完善Rab11依赖的囊泡运输的调控机制，为防治Rab11相关的疾病提供理论依据。

中文关键词： 膜锚定蛋白质；核磁共振；纳米磷脂盘；蛋白质相互作用；构象变化

英文摘要： Chronic diseases such as diabetes and cancer have become major threats to the Chinese public health. The occurrence and development of these diseases are mediated by the vesicle trafficking, which regulates the cellular surface expression of the receptor and adhesion molecules. Small monomeric GTPases 11 (Rab11) is one of the key molecules to regulate these processes via the recycling of endosomes. Rab11 undergoes prenylation and anchored on the membrane for its functional activity. As a result of the heterogeneity and instability of the conventional membrane mimicking system, the membrane anchored state has quite limited structural and biochemical study. This study will take advantage of the stability and homogeneity of the newly developed technology nanodiscs. Rab11 will covalently attach to the lipid moiety in vitro and attach on the nanodiscs, therefore mimicking the post-translational modification in vitro. NMR will be applied to investigate the conformational exchange upon membrane anchor. The important residues responsible for the conformation exchange upon membrane anchor will be mutated and investigated in the mammalian cells in order to link the structural conformation exchange and its cellular function. This study will improve the Rab11 dependent vesicle trafficking theory and provide novel targets for the therapeutic invention.

英文关键词： membrane-anchored protein;NMR;nanodisc;protein-protein interaction;conformational exchange