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通路中发挥着专门的作用，使其成为最重要的通路之一。 复杂的信号系统。其中大约 650 个基因编码泛素连接酶 (E3)，可识别 底物并以它们为目标进行降解。我们的实验室开发了几种基于细胞的方法 生物学和亲和方法，以鉴定泛素连接酶的底物，并使用这些方法来鉴定 五种连接酶的两打底物。然而，与磷酸化一样，在许多情况下泛素化会丢失 没有明显的表型。我们利用R35机制将自己彻底重新配置成一个 哺乳动物细胞实验室进行了大规模筛选，以确定对人类知之甚少的表型 E3/DUB。我们创建了针对人类 E3s/DUB 的 CRISPR 文库，并进行了混合 CRISPR-Cas9 结合针对基因组完整性、细胞周期进程的 41 种化合物的筛选和化学抑制， 转录、RNA 加工、翻译、线粒体功能、蛋白质折叠、代谢途径、 运输、细胞骨架等。通过探索 E3/DUB 参与的多种生物过程，我们 能够评估这些相互作用的特异性。总的来说，我们确定了一种或多种特定的相互作用 161 个 E3/DUB（约占所检查的 25%），其中许多以前未经研究。有些基因， 例如 FBXW7，显示出与超过三分之一的化合物存在相互作用。其他人则表现出互动 仅使用单一化合物或一组相关化合物。我们重点抓好四个方面的后续工作 连接酶组：研究不足的 RING 连接酶 RNF25 中的突变体对烷基化极其敏感 剂，但不是其他形式的 DNA 损伤；未研究的 CUL5 适配器 WSB2 中的突变体非常精致 对核输出抑制剂敏感； CUL4 适配器 DCAF7 在维持生存能力方面发挥着作用 CDK4/6 逮捕；特征不明的 F-box 蛋白 FBXO42 在有丝分裂中具有明确的作用。我们将同时使用 遗传筛选和我们建立的质谱方法来识别这些连接酶的相关底物。 此外，我们将继续对泛素连接进行分析。我们进行了一次大型遗传 在酵母中进行相互作用筛选，以确定单个泛素连接的作用。我们将在此屏幕上展开并 将采用补充质谱方法来识别相关底物。