Structure and Function of Spindle Checkpoint Proteins

描述(申请人提供)：纺锤体检查点是一个细胞周期监测系统，防止有丝分裂中过早的姐妹染色单体分离，并确保染色体遗传的准确性。姐妹染色单体的分离需要一个多亚基泛素连接酶复合体，称为后期促进复合体或环体(APC/C)。纺锤体检查点蛋白MAD2与APC/C的有丝分裂激活剂CDC20结合，抑制APC/CCdc20，从而延缓后期的开始。MAD2受MAD1正向调节，受p31彗星抑制。在有丝分裂中，MAD1-MAD2核心复合体通过MAD2构象二聚化将胞质中的MAD2招募到动点，并将MAD2转化为更容易与Cdc20结合的中间构象(I-MAD2)，从而促进检查点激活。在检查点失活过程中，p31comet与MAD1或Cdc20结合的MAD2结合，从而阻止MAD2的激活，促进Cdc20的自素化和MAD2与Cdc20的解离。我们以前已经用核磁共振波谱确定了人MAD2的潜在和活性构象的结构。在未发表的初步结果中，我们确定了对称的MAD2二聚体和MAD2-p31彗星复合体的晶体结构。这些结构为MAD2的调控提供了关键的见解。在本提案中，我们将进一步研究Mad1和p31comet对MAD2的调控。在目标1中，我们将通过核磁共振确定中间体MAD2(I-MAD2)的结构。在目标2中，我们将表征Mad1的C末端结构域(CTD)的结构和功能。在目标3中，我们将对p31comet-MAD2-Cdc20复合体进行结构和生化分析。纺锤体检查点的缺陷导致非整倍体，这是人类癌症基因组不稳定的一种普遍形式。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：在这项提案中，我们将结合结构、生化和细胞生物学方法，进一步研究Mad1和p31comet对纺锤体检查点MAD2的调节。纺锤体检查点的缺陷会导致非整倍体(染色体数量异常)，这是人类癌症基因组不稳定的一种普遍形式。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 In unpublished preliminary results, we have determined the crystal structures of the symmetric Mad2 dimer and the Mad2-p31comet complex.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.