Structure and Function of Spindle Checkpoint Proteins

描述（申请人提供）：纺锤体检查点是一种细胞周期监测系统，可防止有丝分裂中姐妹染色单体过早分离，并确保染色体遗传的准确性。姐妹染色单体的分离需要一种多亚基泛素连接酶复合物，称为后期促进复合物或细胞核小体（APC/C）。纺锤体检查点蛋白Mad 2与APC/C的有丝分裂激活剂Cdc 20结合，并抑制APC/CCdc 20，从而延迟后期的开始。Mad 2受Mad 1的正调控，受p31 comet的抑制。在有丝分裂中，Mad 1-Mad 2核心复合物通过Mad 2构象二聚化将胞质Mad 2招募到动粒，并将Mad 2转化为更适合Cdc 20结合的中间构象异构体（I-Mad 2），从而促进检查点激活。在检查点失活过程中，p31彗星与Mad 1或Cdc 20结合的Mad 2结合，从而阻止Mad 2活化并促进Cdc 20自泛素化和Mad 2从Cdc 20解离。我们以前已经确定了结构的潜在和积极的构象的人Mad 2使用核磁共振（NMR）光谱。在未发表的初步结果中，我们已经确定了对称Mad 2二聚体和Mad 2-p31彗星复合物的晶体结构。这些结构为Mad 2调控提供了关键见解。在本研究中，我们将进一步研究Mad 1和p31 comet对Mad 2的调控。在目的1中，我们将通过NMR确定中间体Mad 2（I-Mad 2）的结构。在目标2中，我们将描述Mad 1的C-末端结构域（CTD）的结构和功能。在目标3中，我们将对p31 comet-Mad 2-Cdc 20复合物进行结构和生化分析。纺锤体检查点的缺陷导致非整倍性，这是人类癌症中基因组不稳定性的普遍形式。这项研究将有助于阐明纺锤体检查点的分子机制，并帮助我们了解非整倍体的根本原因。公共卫生相关性：在本研究中，我们将结合结构、生物化学和细胞生物学方法，进一步研究Mad 1和p31 comet对纺锤体检查点Mad 2的调控。纺锤体检查点的缺陷导致非整倍性（染色体数目异常），这是人类癌症中基因组不稳定性的普遍形式。这项研究将有助于阐明纺锤体检查点的分子机制，并帮助我们了解非整倍体的根本原因。

DESCRIPTION (provided by applicant): The spindle checkpoint is a cell-cycle surveillance system that prevents premature sister-chromatid separation in mitosis and ensures the accuracy of chromosome inheritance. A multisubunit ubiquitin ligase complex called the anaphase-promoting complex or cyclosome (APC/C) is required for sister-chromatid separation. The spindle checkpoint protein Mad2 binds to Cdc20, the mitotic activator of APC/C, and inhibits APC/CCdc20, thus delaying the onset of anaphase. Mad2 is positively regulated by Mad1 and inhibited by p31comet. In mitosis, the Mad1-Mad2 core complex recruits cytosolic Mad2 to kinetochores through Mad2 conformational dimerization and converts Mad2 to an intermediate conformer (I-Mad2) more amenable to Cdc20 binding, thus facilitating checkpoint activation. During checkpoint inactivation, p31comet binds to Mad1- or Cdc20-bound Mad2, thereby preventing Mad2 activation and promoting Cdc20 autoubiquitination and the dissociation of Mad2 from Cdc20. We have previously determined the structures of both latent and active conformers of human Mad2 using nuclear magnetic resonance (NMR) spectroscopy. In unpublished preliminary results, we have determined the crystal structures of the symmetric Mad2 dimer and the Mad2-p31comet complex. These structures have provided key insights into Mad2 regulation. In this proposal, we will further investigate the regulation of Mad2 by Mad1 and p31comet. In Aim 1, we will determine the structure of intermediate Mad2 (I-Mad2) by NMR. In Aim 2, we will characterize the structure and function of the C-terminal domain (CTD) of Mad1. In Aim 3, we will perform both structural and biochemical analysis of the p31comet-Mad2-Cdc20 complex. Defects of the spindle checkpoint cause aneuploidy, which is a prevalent form of genomic instability in human cancers. The proposed research will shed light on the molecular mechanism of the spindle checkpoint and help us understand the root causes of aneuploidy. PUBLIC HEALTH RELEVANCE: In this proposal, we will further investigate the regulation of spindle checkpoint Mad2 by Mad1 and p31comet with a combination of structural, biochemical and cell biological approaches. Defects of the spindle checkpoint cause aneuploidy (abnormal numbers of chromosomes), which is a prevalent form of genomic instability in human cancers. The proposed research will shed light on the molecular mechanism of the spindle checkpoint and help us understand the root causes of aneuploidy.