有丝分裂中Cdc20与相关调控蛋白的复合体结构与功能研究

项目名称： 有丝分裂中Cdc20与相关调控蛋白的复合体结构与功能研究

项目编号： No.31470763

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 田伟

作者单位： 华中科技大学

项目金额： 85万元

中文摘要： 细胞有丝分裂所必须的关键蛋白Cdc20，在有丝分裂中期激活APC/C，识别并促进底物Securin和CyclinB的泛素化降解而引起姐妹染色体分离。纺锤体组装检测点主要调控Cdc20活性从而保证姐妹染色体的正确分离。纺锤体组装检测点蛋白（如BubR1和Bub1）的异常会导致非整倍体和肿瘤发生。我们之前解析了Cdc20的WD40结构域及其复合物的晶体结构，提出了Cdc20采用非协同的多位点的分子机制识别底物。但上游调控蛋白如何调控Cdc20活性，Cdc20如何选择性识别底物仍然不清楚，有待分子水平的机制研究。 基于申请人的前期研究结果，已建立的研究方法，本项目拟采用结构生物学、蛋白间相互作用，体外泛素化活性检测和体内细胞周期调控等技术方法，在结构与功能上研究Cdc20与上游调控蛋白以及下游底物的互作关系和分子调控机制，为进一步认识有丝分裂调控机制提供依据。

中文关键词： 结构生物学；细胞周期调控；蛋白质相互作用；泛素化；晶体结构

英文摘要： In mitosis,as an essential cell-cycle regulator, Cdc20 binds to and activates APC/C, which degrades Securin and Cyclin B to allow sister-chromatid separation. Cdc20 recognizes the APC/C degrons called the D-box or the KEN-box commonly found in substrates. Cdc20 is also a major target of the spindle checkpoint, a cellular surveillance system that ensures the accuracy of chromosome segregation. The defects in the spindle checkpoint proteins(Bub1 and BubR1) promote aneuploidy and tumorigenesis. We has determined the crystal structure of human Cdc20 WD40 domain and complex with KEN-box. We also showed that Cdc20 utilizes a novel low-cooperativeity and multisite binding mechanism to recognize APC/C substrates.But, the molecular mechanism of how Cdc20 was regulated by upstream regulator, and how Cdc20 recognizes different substrates had remained unclear. Based on our current works and discoveries, this proposal will focus on the structural and functional study of Cdc20 complex with related proteins including substrates or regulators during mitosis. This research will unveil the molecular mechanism of how Cdc20 recognize substrate and is regulated by upstream checkpoint proteins, and explicates how APC/C-Cdc20 drive mitosis and cell cycle oscillations, and how spindle checkpoint proteins regulate mitosis progression. The experimental methods that we are using and have developed include structure biology, protein-protein interaction,in vitro ubiquitination assay and in-vivo cell-cycle regulation assays.

英文关键词： structure biology;cell-cycle regulation;protein-protein interaction;ubiquitination;crystallography