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: 10388393
• 负责人: David Paul Toczyski
• 金额: $ 73.67万
• 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 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10388393
• 关键词: Affinity; Alkylating Agents; Biological; Biological Process; CDK4 gene; CRISPR library; CRISPR screen; CUL5 gene; Cell Cycle Progression; Cells; Chemicals; Complex; Cytoskeleton; DNA Damage; F-Box Proteins; FBXW7 gene; Genes; Genetic; Genetic Screening; Genetic Transcription; Human; Individual; Laboratories; Ligase; Mammalian Cell; Mass Spectrum Analysis; Metabolic Pathway; Methods; Mitochondria; Mitosis; Modification; Nuclear Export; Pathway interactions; Phenotype; Phosphorylation; RNA Processing; Regulation; Role; Signal Transduction; Specificity; Stress; System; Translations; Ubiquitin; Ubiquitination; Work; Yeasts; base; genome integrity; inhibitor; mutant; protein folding; response; ubiquitin ligase

Abstract Over a thousand human genes have dedicated roles in the ubiquitin pathway, making it one of the most complex signaling systems. About 650 of these genes encode ubiquitin ligases (E3s), which recognize substrates and target them for degradation. Our laboratory has developed several methods, based on both cell biological and affinity approaches, to identify substrates of ubiquitin ligases, and have used these to identify over two dozen substrates of five ligases. However, like phosphorylation, in many cases loss of ubiquitination has no obvious phenotype. We have used the R35 mechanism to completely re-configure ourselves into a mammalian cell laboratory and undertaken a large screen to identify phenotypes for poorly understood human E3s/DUBs. We created a CRISPR library against human E3s/DUBs and performed a pooled CRISPR-Cas9 screen combined with chemical inhibition of 41 compounds targeting genome integrity, cell cycle progression, transcription, RNA processing, translation, mitochondrial function, protein folding, metabolic pathways, transport, cytoskeleton, etc. By probing a diverse set of biological processes for E3/DUB involvement, we were able to assess the specificity of these interactions. Overall, we identified one or more specific interactions for 161 E3/DUBs (about 25% of those examined), many of which were previously unstudied. Some genes, such as FBXW7, showed interactions with more than one-third of the compounds. Others showed interactions only with a single compound or a set of related compounds. We are focusing our efforts on following up four sets of ligases: mutants in the poorly studied RING ligase RNF25 were extremely sensitive to alkylating agents, but not other forms of DNA damage; mutants in the unstudied CUL5 adaptor WSB2 were exquisitely sensitive to inhibitors of nuclear export; the CUL4 adaptor DCAF7 has a role in maintaining viability during a CDK4/6 arrest; and the poorly-characterized F-box protein FBXO42 has clear roles in mitosis. We will use both genetic screens and our established mass spec approaches to identify the relevant substrates of these ligases. In addition, we will continue with our analysis of ubiquitin linkages. We have carried out a large genetic interaction screen in yeast to identify roles of individual ubiquitin linkages. We will expand upon this screen and will carry out complementary mass spectrometry approaches to identify relevant substrates.

抽象的 超过一千个人类基因在泛素途径中扮演着专门的角色，使其成为最多的人之一 复杂的信号系统。这些基因中约有650个编码泛素连接酶（E3S），它们识别 底物并将其靶向降解。我们的实验室已经开发了几种基于两个细胞的方法 生物学和亲和力方法，以识别泛素连接酶的底物，并使用它们来识别 超过五个连接酶的二十个底物。但是，像磷酸化一样，在许多情况下，泛素化丧失 没有明显的表型。我们已经使用R35机制将自己彻底重新配置为 哺乳动物的细胞实验室并进行了大屏幕，以识别人类知识不足的表型 E3S/DUB。我们为人类E3/配音创建了一个CRISPR库，并进行了汇总CRISPR-CAS9 筛选结合了靶向基因组完整性，细胞周期进程的41种化合物的化学抑制作用， 转录，RNA处理，翻译，线粒体功能，蛋白质折叠，代谢途径， 通过探测E3/DUB参与的各种生物学过程，我们的运输，细胞骨架等，我们 能够评估这些相互作用的特异性。总体而言，我们确定了一个或多个特定的互动 对于161个E3/DUB（约有25％的检查），其中许多以前是未研究的。一些基因， 例如FBXW7，显示出与三分之一以上化合物的相互作用。其他人则表现出互动 仅具有单个化合物或一组相关化合物。我们正在专注于跟进四个 连接酶集：研究不足的环连接酶RNF25中的突变体对烷基化极为敏感 代理，但没有其他形式的DNA损伤；未研究的Cul5适配器WSB2中的突变体是精美的 对核出口抑制剂敏感； Cul4适配器DCAF7在维持生存能力中起作用 CDK4/6逮捕；并且特征较差的F-box蛋白FBXO42在有丝分裂中具有明显的作用。我们将两者都使用 遗传筛选和我们既定的质量规格方法，以识别这些连接酶的相关底物。 此外，我们将继续对泛素链接的分析。我们进行了大遗传 酵母中的相互作用屏幕以识别单个泛素链接的作用。我们将在此屏幕上扩展 将采用互补的质谱方法来识别相关的底物。