Regulation by post-translation modifications in response to stress

 描述(申请人提供)：应激或细胞信号激活的信号通路通过磷酸化、乙酰化和泛素化对现有蛋白质进行翻译后修饰，以迅速改变生理。我们将研究通过翻译后修饰(PTM)对几种形式的应激做出反应的调节，特别关注响应DNA损伤的磷酸化和泛素化的变化。我们对泛素途径的研究主要集中在两个领域：泛素连锁分析和底物鉴定。泛素链是使用泛素的七种赖氨酸中的任何一种在底物上形成的。我们在酵母中采用遗传方法来探索这些链的意义，方法是鉴定与泛素赖氨酸突变体具有合成遗传相互作用的突变体，这些突变体不能形成特定的链类型。为了识别泛素连接酶底物，我们开发了一种称为连接酶捕捉的方法，在这种方法中，我们将一个多泛素结合域融合到泛素连接酶上，从而增加了连接酶与其泛素化底物的亲和力，从而可以通过质谱学来识别底物。我们已经在酵母和人类细胞中进行了这项研究，并将对几个有趣的热门项目进行后续研究。我们特别感兴趣的是泛素介导的蛋白质周转对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个检查点激酶之一磷酸化后。ATR、CHK1和CHK2等检查点蛋白在DNA损伤后被激活，调节着大量的信号转导途径。我们将继续努力确定DNA损伤检查点的底物，重点关注参与细胞周期调节或新陈代谢的靶点。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，通过这种方法，磷酸酶、去泛素酶、HDAC或其他酶可以单独定位到酵母中的每种蛋白质。我们将使用这项技术来识别对未受干扰的细胞的生存能力至关重要的修饰，或者是对诸如DNA损伤之类的压力的反应。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球、功能图景。

 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.