Biochemistry of Anaphase Promoting Complex-mediated Ubiquitination

描述(由申请人提供)：细胞周期的正常运作及其对内外刺激的调节对于正常的生长、分裂和 细胞和组织的分化。对细胞周期进程的主要控制是由泛素介导的蛋白分解，特别是在有丝分裂退出和G1期。入会仪式 染色体分离(后期)和细胞周期的完成需要许多细胞周期调节蛋白的降解，如细胞周期蛋白、Securin(它调节将姐妹染色体结合在一起的蛋白质)和微管马达蛋白。降解这些蛋白质的泛素连接酶(或E3)是后期促进复合体(APC)。APC底物含有降解基序(通常是破坏盒和KEN盒)，并被两种底物识别蛋白CDC20和CDH1靶向APC，这两种底物识别蛋白分别在有丝分裂后期和G1期激活。我们的长期目标是在机械水平上理解APC底物的泛素化。尽管这一进程的组成部分众所周知，但我们在理解上仍存在一些重大差距。我们将使用分子遗传学、体内研究和体外生化分析相结合的方法解决酿酒酵母中的这些问题。我们预计，我们开发的工具以及概念上的进展将为实地带来重大好处。由于APC的功能和调节是高度保守的，我们对酵母系统功能的洞察应该也适用于人类。为了进一步了解APC的功能，我们提出了以下具体目标：1)确定底物是如何从APC释放出来的：泛素化的作用和加工性的起源。含有单一泛素化位点的底物 将用于一次相遇分析，以确定泛素化底物从CDH1和/或APC解离的要求。我们将探索底物处理的性质，并表征底物与APC的doc1亚基之间的相互作用。2)研究脱泛素酶(DUBS)在调节APC底物降解中的作用。与底物泛素化相比，人们对作用于这些蛋白质的去泛素化酶(DUB)知之甚少。我们将识别和确定作用于影响纺锤体检查点的重要APC底物上的DUB的功能，并分析我们假设可能在泛素化过程中发挥校对功能的DUB。APC调控和错调在细胞对多种抗癌药物的应答以及染色体的错配导致非整倍体和肿瘤的发生中起着重要作用。我们的研究将进一步加深我们对这一重要的细胞周期调控过程的理解，并从长远来看，为针对APC本身或作用于其底物的DUBS提供治疗干预的靶点。

DESCRIPTION (provided by applicant): Proper functioning of the cell cycle and its regulation in response to both internal and external stimuli is essential for the normal growth, division, and differentiation of cells and tissues. Major control over cell cycle progression is exerted by ubiquitin-mediated proteolysis, particularly during the exit from mitosis and in G1. The initiation of chromosome separation (anaphase) and completion of the cell cycle require the degradation of a number of cell cycle regulatory proteins such as cyclins, securin (which regulates the protein holding sister chromosomes together), and microtubule motor proteins. The ubiquitin ligase (or E3) for the degradation of these proteins is the Anaphase Promoting Complex (APC). APC substrates contain degradation motifs (typically Destruction Boxes and KEN Boxes) and are targeted to the APC by two substrate recognition proteins, Cdc20 and Cdh1, active during late mitosis and in G1, respectively. Our long-term goal is to understand the ubiquitination of APC substrates at a mechanistic level. Although the components in this process are well known, there are still some significant gaps in our understanding. We will address these points in Saccharomyces cerevisiae using a combination of molecular genetics, studies in vivo, and biochemical assays in vitro. We expect that tools that we develop as well as conceptual advances will be of significant benefit to the field. Since APC function and regulation are highly conserved, insights we gain into the functions of the yeast system should be applicable to humans, as well. To further our understanding of APC function, we propose the following Specific Aims: 1) to determine how substrates are released from the APC: The role of ubiquitination and the origin of processivity. Substrates containing single sites of ubiquitination will be used in single-encounter assays to determine the requirements for dissociation of the ubiquitinated substrate from Cdh1 and/or the APC. We will explore the nature of substrate processivity, and characterize the interaction between a substrate and the Doc1 subunit of the APC. 2) To characterize the roles of deubiquitinating enzymes (DUBs) in regulating APC substrate degradation. Compared to substrate ubiquitination, comparatively little is known about the deubiquitinating enzymes (DUBs) that act on these proteins. We will identify and determine the functions of DUBs acting on important APC substrates affecting the spindle checkpoint and analyze a DUB that we hypothesize may play a proofreading function during ubiquitination. APC regulation and mis-regulation play important roles in the responses of cells to many anti-cancer agents and in the mis-segregation of chromosomes leading to aneuploidy and the development of tumors. Our studies will further our understanding of this essential cell cycle regulatory process and, in the longer term, provide targets for therapeutic intervention, directed towards the APC itself or the DUBs acting on its substrates.