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: 9071173
• 负责人: David Paul Toczyski
• 金额: $ 54.54万
• 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/9071173
• 关键词: Acetylation; 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; Human; 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）：应激或细胞信号激活的信号通路通过磷酸化、乙酰化和遍在蛋白质翻译后修饰现有的蛋白质，以快速改变生理学。我们将通过翻译后修饰（PTM）来研究对几种形式的应激的调节，特别注意对DNA损伤的磷酸化和泛素化的改变。我们对泛素途径的研究主要集中在两个方面：泛素连接分析和底物鉴定。泛素链是利用泛素的七个赖氨酸中的任何一个在底物上形成的。我们在酵母中采取遗传方法，通过鉴定与不能形成特定链类型的泛素赖氨酸突变体具有合成遗传相互作用的突变体来探索这些链的意义。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，在该方法中，我们将多聚泛素结合结构域融合到泛素连接酶上，这增加了连接酶与其泛素化底物的亲和力，从而允许通过质谱进行底物识别。我们已经在酵母和人类细胞中进行了这一研究，并将跟踪几个有趣的结果。我们特别感兴趣的是泛素介导的蛋白质周转在DNA损伤的反应。DNA损伤调节的蛋白质周转通常发生在底物被几种检查点激酶之一磷酸化之后。检查点激酶如ATR、CHK1和CHK2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，重点关注参与细胞周期调节或代谢的靶点。正如我们对泛素连接酶底物的研究一样，我们将产生不能被修饰的等位基因，并研究它们对细胞生理学的影响。最后，我们开发了一种方法，通过该方法，磷酸酶、去泛素化酶、HDAC或其他酶可以单独定位于酵母中的每种蛋白质。我们将使用这种技术来识别在未受干扰的细胞中或在应对诸如DNA损伤等应激时对生存力至关重要的修饰。结合我们的底物鉴定研究，这将使我们能够生成蛋白质修饰的全局功能图。