Regulation by post-translation modifications in response to stress

通过翻译后修饰来应对压力的调节

• 批准号: 9982380
• 负责人: David Paul Toczyski
• 金额: $ 69.67万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982380
• 关键词: Acetylation; Acute; Affinity; Alleles; Area; Attention; Binding; Biology; CHEK1 gene; CHEK2 gene; Cell Cycle Regulation; Cell division; Cell physiology; Cells; Cues; DNA Damage; DNA damage checkpoint; Enzymes; Genetic; Histone Deacetylase; Human; Individual; Ligase; Lysine; Mass Spectrum Analysis; Mediating; Metabolism; Methods; Modification; Normal Cell; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiology; Polyubiquitin; Post-Translational Protein Processing; Proteins; Regulation; Signal Pathway; Stress; Technology; Translations; Ubiquitin; Ubiquitination; Yeasts; follow-up; genetic approach; genetic linkage analysis; interest; mutant; protein degradation; public health relevance; response; ubiquitin isopeptidase; ubiquitin ligase

 DESCRIPTION (provided by applicant): Signaling pathways activated by stress or cellular cues modify existing proteins post-translationally by phosphorylation, acetylation and ubiquitination, in order to rapidly alter physiology. We will examine regulation through post-translational modifications (PTMs) in response to several forms of stress, paying special attention to alterations in phosphorylation and ubiquitination in response to DNA damage. Our examination of the ubiquitin pathway has two primary areas of focus: ubiquitin linkage analysis and substrate identification. Ubiquitin chains are formed on substrates using any of ubiquitin's seven lysines. We take a genetic approach in yeast to explore the significance of these chains by identifying mutants that have synthetic genetic interactions with ubiquitin lysine mutants unable to form particular chain types. To identify ubiquitin ligase substrates, we developed a method called Ligase Trapping, in which we fuse a poly-ubiquitin binding domain onto a ubiquitin ligase, which increases the affinity of the ligase with its ubiquitinated substrate, allowing substrate identification via mass spectroscopy. We have carried this out in both yeast and human cells, and will follow-up on several interesting hits. We are particularly interested in ubiquitin-mediated protein turnover in response to DNA damage. DNA damage-regulated protein turnover typically occurs after a substrate is phosphorylated by one of several checkpoint kinases. Checkpoint kinases, such as ATR, CHK1 and CHK2 are activated upon DNA damage and regulate a large number of pathways. We will continue our effort to identify substrates of the DNA damage checkpoint, focusing on targets involved in either cell cycle regulation or metabolism. As with our examination of ubiquitin ligase substrates, we will generate alleles that cannot be modified and examine their effects on cellular physiology. Finally, we have developed a method by which phosphatases, de-ubiquitinases, HDACs, or other enzymes can be localized individually to each protein in yeast. We will use this technology to identify modifications that are essential for viability either in unperturbed cells, or in respose to stresses such as DNA damage. Together with our substrate identification studies, this will allow us to generate a global, functional picture of protein modification.

 描述（由申请人提供）：由压力或细胞信号激活的信号通路通过磷酸化、乙酰化和泛素化对现有蛋白质进行翻译后修饰，以便快速改变生理学，我们将通过翻译后修饰（PTM）来检查对应激反应的调节。多种形式的应激，特别关注 DNA 损伤引起的磷酸化和泛素化的变化。我们对泛素通路的检查有两个主要领域：焦点：泛素连锁分析和底物鉴定。我们在酵母中采用遗传方法，通过鉴定与泛素赖氨酸突变体不具有合成遗传相互作用的突变体来探索这些链的重要性。为了识别泛素连接酶底物，我们开发了一种称为连接酶捕获的方法，其中我们将多聚泛素结合域融合到泛素连接酶，它增加了连接酶与其泛素化底物的亲和力，从而可以通过质谱法进行底物鉴定。我们已经在酵母和人类细胞中进行了这项研究，并将对几个有趣的结果进行后续研究。我们对泛素特别感兴趣。 DNA 损伤介导的蛋白质更新通常在底物被检查点激酶（例如 ATR、CHK1 和 CHK2）之一磷酸化后发生。在 DNA 损伤时被激活并调节大量途径，我们将继续努力识别 DNA 损伤检查点的底物，重点关注涉及细胞周期调节或代谢的靶标，就像我们对泛素连接酶底物的检查一样。生成无法修改的等位基因并检查它们对细胞生理学的影响最后，我们开发了一种方法，可以将磷酸酶、去泛素酶、HDAC 或其他酶单独定位到酵母中的每种蛋白质。我们将利用这项技术来识别对于未受干扰的细胞的生存能力或应对 DNA 损伤等应激所必需的修饰，结合我们的底物识别研究，这将使我们能够生成蛋白质修饰的全局功能图。