Biochemistry of Anaphase Promoting Complex-mediated Ubiquitination

后期促进复合物介导的泛素化的生物化学

• 批准号: 8435719
• 负责人: MARK J SOLOMON
• 金额: $ 31.38万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8435719
• 关键词: Address; Affect; Anaphase; Aneuploidy; Antineoplastic Agents; Binding; Biochemical; Biochemistry; Biological Assay; Boxing; Cell Cycle; Cell Cycle Completion; Cell Cycle Progression; Cell Cycle Proteins; Cell Differentiation process; Cell division; Cells; Chromosome Segregation; Chromosomes; Cyclins; Deubiquitinating Enzyme; Development; Dissociation; Future; Goals; Growth; Human; In Vitro; Lead; Malignant Neoplasms; Mediating; Microtubules; Mitosis; Modeling; Molecular Genetics; Motor; Nature; Physiological; Play; Process; Proteins; Regulation; Role; Saccharomyces cerevisiae; Signal Transduction; Sister; Site; Stimulus; System; Testing; Therapeutic; Therapeutic Intervention; Tissues; Ubiquitin-mediated Proteolysis Pathway; Ubiquitination; Yeasts; anaphase-promoting complex; daughter cell; genetic regulatory protein; human PTTG1 protein; in vitro Assay; in vivo; insight; protein degradation; public health relevance; research study; response; tool; tumor; ubiquitin ligase

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.

描述（由申请人提供）：细胞周期的适当功能及其对内部和外部刺激的调节对于正常生长，分裂和 细胞和组织的分化。泛素介导的蛋白水解施加了对细胞周期进程的主要控制，尤其是在有丝分裂和G1中退出期间。开始 染色体分离（后期）和细胞周期的完成需要降解许多细胞周期调节蛋白，例如细胞周期蛋白，Securin（调节蛋白质固定姐妹染色体）和微管运动蛋白。这些蛋白质降解的泛素连接酶（或E3）是后期促进复合物（APC）。 APC底物包含降解基序（通常是破坏框和KEN框），并分别由两个底物识别蛋白CDC20和CDH1靶向APC，分别在有丝分裂和G1中活跃。 我们的长期目标是了解机械水平上APC底物的泛素化。尽管此过程中的组成部分是众所周知的，但我们的理解仍然存在一些显着差距。我们将使用分子遗传学，体内研究和体外生化分析的结合在酿酒酵母中解决这些点。我们期望我们开发的工具以及概念上的进步将对该领域带来重大好处。由于APC功能和调节是高度保守的，因此我们获得酵母系统功能的见解也应适用于人类。为了进一步了解APC功能，我们提出了以下特定目的：1）确定底物如何从APC中释放出：泛素化的作用和加工性的起源。包含泛素化单位的底物 将在单次点测定中使用，以确定从CDH1和/或APC中解离的要求。我们将探讨底物加工性的性质，并表征底物与APC的DOC1亚基之间的相互作用。 2）表征去泛素化酶（DUB）在调节APC底物降解中的作用。与底物泛素化相比，对这些蛋白质作用的去泛素化酶（DUB）的了解鲜为人知。我们将识别并确定作用于影响纺锤体检查点的重要APC底物的配音的功能，并分析我们假设的配音在泛素化过程中可能会扮演校对功能。 APC调节和错误调节在细胞对许多抗癌药的反应以及导致非整倍性和肿瘤发育的染色体的错误分离中起着重要作用。我们的研究将进一步了解这一基本细胞周期调节过程，从长远来看，我们提供了针对APC本身或作用于其底物的DUB的治疗干预措施的目标。