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（由应用程序提供）：由应力或细胞提示激活的信号传导途径通过磷酸化，乙酰化和泛素化来改变现有的蛋白质，以快速改变生理学。我们将通过翻译后修饰（PTM）对几种形式的压力进行调查，特别注意响应DNA损伤的磷酸化和泛素化的改变。我们对泛素途径的检查具有两个主要重点领域：泛素链接分析和底物鉴定。使用泛素的七个歌词中的任何一个，在底物上形成了泛素链。我们在酵母中采用一种遗传方法来探索这些链的重要性，通过识别与无法形成特定链类型的泛素遗传相互作用的突变体。为了鉴定泛素连接酶底物，我们开发了一种称为连接酶诱捕的方法，在该方法中，我们将多泛素结合结构域融合到泛素蛋白连接酶上，从而增加了连接酶与其泛素化的底物的亲和力，从而可以通过质谱鉴定底物鉴定。我们已经在酵母和人类细胞中进行了此操作，并将在几次有趣的命中进行跟进。我们对泛素介导的蛋白质更新特别感兴趣，以应对DNA损伤。 DNA损伤调节的蛋白质更新通常是在底物被几种检查点激酶之一磷酸化之后发生的。检查点激酶（例如ATR，CHK1和CHK2）在DNA损伤时被激活并调节大量途径。我们将继续努力确定DNA损伤检查点的底物，重点关注细胞周期调节或代谢涉及的靶标。与我们对泛素连接酶底物的检查一样，我们将产生无法修改的等位基因并检查其对细胞生理的影响。最后，我们开发了一种方法，通过这种方法，磷酸酶，去偶联酶，HDACS或其他酶可以单独地定位于酵母中的每种蛋白质。我们将使用这项技术来识别对不受干扰的细胞中生存力至关重要的修改，或者在抗DNA损伤之类的压力中均可进行修改。与我们的底物鉴定研究一起，这将使我们能够产生蛋白质修饰的全局功能图像。