Deubiquitinases in Cell Cycle Control

细胞周期控制中的去泛素酶

• 批准号: 10582033
• 负责人: Michael James Emanuele
• 金额: $ 6.56万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10582033
• 关键词: Address; Cell Culture Techniques; Cell Cycle; Cell Cycle Progression; Cell Cycle Proteins; Cell Cycle Regulation; Cell division; Cell physiology; Cells; Cellular Morphology; Deubiquitinating Enzyme; Deubiquitination; Enzymes; Eukaryota; Funding; Future; Genome; Goals; Image; Knock-out; Maintenance; Malignant Neoplasms; Methods; Microscope; National Institute of General Medical Sciences; Normal Cell; Parents; Patients; Peptide Hydrolases; Phase; Process; Proliferating; Protein Dynamics; Proteins; Proteome; Request for Proposals; Research; Role; System; Transfection; Ubiquitin; Ubiquitination; cancer cell; cellular imaging; human disease; imaging system; improved; instrument; knock-down; multicatalytic endopeptidase complex; protein degradation; therapeutic target; tissue culture; virtual

Project Summary Abstract The focus of our current NIGMS funded R01 proposal (R01GM134231) is to study the role of deubiquitinating enzymes in proliferation, cell cycle control and genome maintenance. The ubiquitin proteasome system is the major regulator of protein degradation in all eukaryotes. Ubiquitination is implicated in virtually all aspects of cell physiology and human disease. The post-translational conjugation of ubiquitin onto specific substrates requires the concerted action of three enzymes, termed E1, E2 and E3. However, substrate ubiquitination and degradation is determined not only by the addition of ubiquitin onto substrates, but also by the action of catalytic proteases termed deubiquitinases (DUBs), which remove ubiquitin from substrates proteins, thereby antagonizing degradation. The major goals of the parent R01 are to determine substrates and mechanisms of deubiquitination during cell division, mechanisms of DUB action in the cell cycle, and the identity of additional DUBs involved in cell cycle. The unifying theme of this research is the study of protein dynamics in cell cycle progression. Determining the abundance of proteins in specific phases of the cell cycle is therefore vital to determining the mechanisms underlying proteome remodeling and connecting enzymes with cognate substrates. This analysis has significant technical challenges. First, many enzymes that control cell cycle progression are themselves dynamically regulated, and their manipulation (e.g., knockdown or knockout) can perturb cell cycle progression. Since the abundance of many cell cycle proteins, and particularly cell cycle regulated ubiquitin substrates, is regulated as cells proliferate, their abundance can be altered by alterations to the cell cycle. Thus, the ability to examine the abundance of specific proteins in specific cell cycle phases, without the need for synchronization, is a tremendous advantage. We address these challenges through single cell imaging. This current proposal requests support for the purchase of a Revolve R4 Echo Imaging System. This instrument would allow our lab to perform imaging analysis in-house and would significantly improve the capabilities of our current day-to-day tissue culture microscope, which lacks the ability of capture images, precluding on-the-fly, quantitative analysis of transfection efficiencies or cell morphology. This purchase would expand our current capabilities, facilitate orthogonal analysis methods that are easy to use and accessible, and provide everyday access to improved cell culture processes.

项目摘要摘要 我们目前由NIGMS资助的R01提案(R01GM134231)的重点是研究去泛素化的作用 酶在增殖、细胞周期控制和基因组维持中的作用。泛素蛋白酶体系统是 所有真核生物中蛋白质降解的主要调节因子。泛素化几乎涉及到 细胞生理学和人类疾病。泛素在特定底物上的翻译后结合 需要三种酶的协同作用，分别称为E1、E2和E3。然而，底物泛素化和 降解不仅由添加到底物上的泛素决定，也由 被称为脱泛素酶(DUBS)的催化酶，它能从底物蛋白质中去除泛素，从而 对抗退化。亲本R01的主要目标是确定底物和机制 细胞分裂过程中的去泛素化作用，DUB在细胞周期中的作用机制，以及其他 参与细胞周期的DUBS。这项研究的统一主题是研究细胞周期中的蛋白质动力学。 进步。因此，确定细胞周期特定阶段的蛋白质丰度对 蛋白质组重塑机制的确定及其与同源酶的连接 底物。这一分析具有重大的技术挑战。首先，许多控制细胞周期的酶 进程本身是动态调节的，它们的操作(例如，击倒或敲除)可以 扰乱细胞周期进程。由于许多细胞周期蛋白，特别是细胞周期蛋白的丰富 调节的泛素底物，随着细胞的增殖而调节，它们的丰度可以通过改变 细胞周期。因此，能够在特定的细胞周期阶段检测特定蛋白质的丰度， 不需要同步，是一个巨大的优势。我们通过单一的 细胞成像。目前的提案要求支持购买Revolve R4回声成像系统。 这台仪器将允许我们的实验室在内部进行成像分析，并将显著提高 我们现在的日常组织培养显微镜的能力，它缺乏捕捉图像的能力， 排除了对转染率或细胞形态的实时、定量分析。这笔收购将 扩展我们现有的能力，促进易于使用和访问的正交分析方法，以及 提供改进的细胞培养过程的日常访问。