Anaphase Promoting Complex-mediated Proteolysis

描述(由申请人提供)：有丝分裂过程中的一个关键事件是后期促进复合体(APC；也称为环体)对细胞周期蛋白和其他关键蛋白的泛素依赖的降解。APC底物含有降解基序，如破坏框和KEN框，这是它们泛素化和与APC激活剂CDC20和CDH1结合所必需的。这两个基序都是有效降解所必需的，但其中任何一个都足以与CDh1p结合。我们提出了一种组装APC-CDH1P-底物复合体的方法，其中不含APC的CDH1P首先与底物结合。CDh1p与底物破坏盒的结合可能是通过CDh1p的构象变化来刺激CDh1p与APC的结合。目前的研究旨在加深我们对APC介导的芽殖酵母蛋白分解的理解。特别是，我们希望寻找新的APC底物，了解APC-CDH1p/CDC20P-底物复合体是如何组装和拆解的，并了解当染色体没有正确连接到有丝分裂纺锤体时，纺锤体组装检查点如何利用Ken盒来关闭APC介导的蛋白质降解。为了实现这些目标，我提出了以下具体目标：1)寻找新的APC底物。2)评估APC-CDH1p-底物组装途径的一般性。3)确定D-Box参与如何刺激CDh1p与APC结合：4)确定保守的CDh1p基序和磷酸化在APC-CDh1p-底物组装中的作用。5)确定APC-Cdh1p-底物复合体的解离是否是一个活性过程。6)确定主轴组件检查点中MadSp Ken Box的功能。关键蛋白质的降解是细胞分裂所必需的。因此，了解这种降解对于了解正常的细胞增殖以及这个过程在疾病状态下是如何出错的，特别是癌症，是很重要的。

DESCRIPTION (provided by applicant): A key event in the progression through and exit from mitosis is the ubiquitin-dependent degradation of cyclins and other key proteins by the Anaphase Promoting Complex (APC; also called the cyclosome). APC substrates contain degradation motifs such as the Destruction Box and the KEN Box that are required for their ubiquitination and for their binding to the APC activators, Cdc20 and Cdh1. Both motifs are required for efficient degradation, but either one alone suffices for binding to Cdh1p. We have proposed a pathway for the assembly of the APC-Cdh1p-substrate complex in which APC-free Cdh1p first binds a substrate. Engagement of the substrate Destruction Box by Cdh1p stimulates the binding of Cdh1p to the APC, presumably via a conformation change in Cdh1p. The current studies are aimed at furthering our understanding of APC-mediated proteolysis in budding yeast. In particular, we wish to identify novel APC substrates, to understand how APC-Cdh1p/Cdc20p-substrate complexes are assembled and disassembled, and to understand how the spindle assembly checkpoint makes use of a KEN box to turn off APC-mediated proteolysis when chromosomes are not properly attached to the mitotic spindle. To achieve these goals, I propose the following Specific Aims: 1) To Identify Novel APC Substrates. 2) To Assess the Generality of the APC-Cdh1p-Substrate Assembly Pathway. 3) To Determine How D-Box Engagement Stimulates Cdh1p Binding to the APC: 4) To Determine the Roles of Conserved Cdh1p Motifs and of Phosphorylation in APC-Cdh1p-Substrate Assembly. 5) To Determine Whether Disassembly of the APC-Cdh1p-Substrate Complex is an Active Process. 6) To Determine the Function of the MadSp KEN Box in the Spindle Assembly Checkpoint. Degradation of key proteins is essential for cell division. Understanding this degradation is thus important for understanding normal cell proliferation, and how this process goes awry in disease states, particularly cancers.