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: 10609884
• 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/10609884
• 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; Dedications; 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; Nuclear Export; Pathway interactions; Phenotype; Phosphorylation; Post-Translational Protein Processing; Post-Translational Regulation; RNA Processing; Role; Signal Transduction; Specificity; Stress; System; Translations; Ubiquitin; Ubiquitination; Work; Yeasts; 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.

摘要 超过1000个人类基因在泛素途径中发挥作用，使其成为最重要的基因之一。 复杂的信号系统这些基因中约有650个编码泛素连接酶（E3），其识别 底物并将其作为降解目标。我们的实验室已经开发了几种方法，基于两种细胞 生物和亲和方法，以确定底物的泛素连接酶，并已使用这些来确定 五种连接酶的二十多种底物。然而，像磷酸化一样，在许多情况下， 没有明显的表型。我们使用R35机制将自己完全重新配置为 哺乳动物细胞实验室，并进行了大屏幕，以确定表型为知之甚少的人类 E3/DUBs。我们创建了针对人E3 s/DUB的CRISPR文库，并进行了合并的CRISPR-Cas9 筛选与化学抑制相结合的41种化合物，靶向基因组完整性，细胞周期进程， 转录，RNA加工，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 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 E3/DUBs（约占检查的25%），其中许多以前未研究过。一些基因， 例如FBXW 7，显示出与超过三分之一的化合物的相互作用。另一些则显示了 只有一种化合物或一组相关化合物。我们正在集中努力， 连接酶的集合：研究不足的RING连接酶RNF 25的突变体对烷基化非常敏感。 试剂，但不是其他形式的DNA损伤;在未研究的CUL 5衔接子WSB 2突变体是精致的， 对核输出的抑制剂敏感; CUL 4衔接子DCAF 7在维持细胞存活过程中起作用。 CDK 4/6停滞;而特征不佳的F-box蛋白FBXO 42在有丝分裂中具有明确的作用。我们将使用两者 遗传筛选和我们建立的质谱方法来鉴定这些连接酶的相关底物。 此外，我们将继续我们的泛素连接的分析。我们已经进行了一次大型的基因 在酵母中进行相互作用筛选以鉴定单个泛素连接作用。我们将在这个屏幕上扩展， 将采用互补质谱法确定相关底物。