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: 8706907
• 负责人: MARK J SOLOMON
• 金额: $ 31.64万
• 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/8706907
• 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）：细胞周期的正常运作及其响应内部和外部刺激的调节对于正常生长、分裂和发育至关重要。 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水解作用实现的，特别是在有丝分裂退出期间和 G1 期。启蒙 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（调节将姐妹染色体保持在一起的蛋白质）和微管运动蛋白。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 底物包含降解基序（通常为破坏盒和 KEN 盒），并通过两种底物识别蛋白 Cdc20 和 Cdh1（分别在有丝分裂晚期和 G1 期活跃）靶向 APC。 我们的长期目标是从机械层面了解 APC 底物的泛素化。尽管这个过程的组成部分众所周知，但我们的理解仍然存在一些重大差距。我们将结合分子遗传学、体内研究和体外生化测定来解决酿酒酵母中的这些问题。我们期望我们开发的工具以及概念的进步将为该领域带来重大利益。由于 APC 功能和调节高度保守，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 功能，我们提出以下具体目标：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 Cdh1 和/或 APC 解离的要求。我们将探索底物持续合成能力的本质，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，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 调节和错误调节在细胞对许多抗癌药物的反应以及导致非整倍体和肿瘤发展的染色体错误分离中发挥着重要作用。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 的治疗干预提供目标。