有丝分裂中Cdc20与相关调控蛋白的复合体结构与功能研究

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.

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。

Cdc20是有丝分裂所必须的关键调控蛋白。在有丝分裂中期，Cdc20激活并辅助APC/C 泛素化一系列底物，促使染色体的正确体分离。细胞内Cdc20的活性是被严格调控的。在有丝分裂中期，当任一染色体或与纺锤体微管没有建立正确连接时，纺锤体组装检测点蛋白Bub1，BubR1和Mad2抑制Cdc20的活性，阻止染色体分离，引发周期阻滞。我们在有丝分裂调控蛋白BubR1和Bub1中研究发现一段保守序列phe-box基团可以特异性结合Cdc20，进而发现并鉴定了BubR1中的Phe-box结合Cdc20的位点以及抑制APC/C泛素化能力，并在细胞中揭示了Bub1和BubR1中的Phe-box基团在维持纺锤体检查点功能上发挥着重要的作用。.有丝分裂中，染色体的运动、排列与分离以及均匀分配都依赖于染色体着丝粒两侧的重要的蛋白质复合体——动粒。动粒(Kinetochore)也称为着丝点或动粒，是由十几种蛋白与染色质组成的生物大分子复合体。CENP-H/I/K复合体在动粒亚基中处于中间位置，紧密联系上下游分子，是动粒组装所必须的结构单元与重要组成。CENP-H/I/K三者通过直接相互作用形成CENP-H/I/K亚复合体，对整个动点的结构组装和发挥功能有着重要意义。最新研究发现CENP-H/I/K参与招募Cdc20与纺锤体检测点蛋白，调控微管连接以及纺锤体检测点活性。我们解析了染色体动粒亚基CENP-H/I/K三元复合物的晶体结构，并利用生物化学和细胞生物学方法揭示了动粒亚基的分子组装模式及其激活Cdc20功能的分子机制。该成果于2018年以通讯作者发表在Nucleic Acids Research杂志上。