Biochemistry of Anaphase Promoting Complex-mediated Ubiquitination

描述（由申请人提供）：细胞周期的正常功能及其对内部和外部刺激的响应调节对于正常生长、分裂和增殖是必不可少的。 细胞和组织的分化。泛素介导的蛋白水解作用是细胞周期进程的主要控制因素，特别是在有丝分裂和G1期。启动 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，例如细胞周期蛋白、securin（其调节将姐妹染色体保持在一起的蛋白）和微管马达蛋白。用于降解这些蛋白质的泛素连接酶（或E3）是后期促进复合物（APC）。APC底物含有降解基序（通常是Destruction Box和KEN Box），并通过两种底物识别蛋白Cdc 20和Cdh 1靶向APC，这两种蛋白分别在有丝分裂晚期和G1期有活性。 我们的长期目标是在机械水平上了解APC底物的泛素化。虽然这一过程的组成部分是众所周知的，但我们的理解仍然存在一些重大差距。我们将在酿酒酵母中使用分子遗传学，体内研究和体外生化测定相结合来解决这些问题。我们希望我们开发的工具以及概念上的进步将对该领域产生重大影响。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了解也应该适用于人类。为了进一步了解APC的功能，我们提出了以下具体目标：1）确定底物如何从APC中释放：泛素化的作用和持续合成能力的起源。含有单一泛素化位点的底物 将用于单次接触测定，以确定泛素化底物从Cdh 1和/或APC解离的要求。我们将探索基板的持续合成能力的性质，并表征基板和APC的Doc 1亚基之间的相互作用。 2)探讨去泛素化酶（DUBs）在调节APC底物降解中的作用。与底物泛素化相比，对作用于这些蛋白质的去泛素化酶（DUBs）知之甚少。我们将确定和确定DUB的功能作用于重要的APC基板影响纺锤体检查点和分析DUB，我们假设可能发挥校对功能，在泛素化。 APC的调节和错误调节在细胞对许多抗癌药物的反应中以及在导致非整倍体和肿瘤发展的染色体错误分离中起重要作用。我们的研究将进一步加深我们对这一重要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DUB的治疗干预提供靶点。

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.