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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

基本信息

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
项目类别：
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

项目摘要

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）：细胞周期的正常功能及其对内部和外部刺激的调节对正常生长，分裂和生长至关重要