Regulation by post-translation modifications in response to stress

摘要 超过一千个人类基因在泛素途径中扮演着特定的角色，使其成为最多的 复杂的信号系统。其中约650个基因编码泛素连接酶(E3)，泛素连接酶识别 底物，并针对它们进行降解。我们的实验室已经开发出几种基于这两种细胞的方法 生物学和亲和力方法，以确定泛素连接酶的底物，并使用这些方法来识别 五种连接酶的二十多种底物。然而，像磷酸化一样，在许多情况下泛素化的丧失 没有明显的表型。我们已经使用R35机制将我们自己完全重新配置为 哺乳动物细胞实验室，并进行了一次大筛选，以确定鲜为人知的人类的表型 E3S/Dubs。我们创建了一个针对人类E3S/DUB的CRISPR文库，并执行了一个池CRISPR-Cas9 筛选结合化学抑制的41种化合物，以基因组完整性、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， 运输、细胞骨架等。通过探索E3/DUB参与的一系列不同的生物学过程，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交互 对于161个E3/DUB(约占检查的25%)，其中许多以前没有研究过。一些基因， 如FBXW7，显示出与超过三分之一的化合物相互作用。其他人则显示出互动 仅与单一化合物或一组相关化合物一起使用。我们正在集中力量跟进四个问题 连接酶组：研究甚少的环连接酶RNF25的突变体对烷基化极其敏感 毒剂，但不是其他形式的DNA损伤；在未研究的CUL5接头WSB2中的突变体被巧妙地 对核出口抑制物敏感；CUL4接头DCAF7在维持 CDK4/6停滞；而特征不佳的F-box蛋白FBXO42在有丝分裂中具有明显的作用。我们将使用这两种方法 基因筛查和我们建立的质谱学方法来鉴定这些连接酶的相关底物。 此外，我们还将继续分析泛素之间的联系。我们已经进行了一项大规模的基因改造 在酵母中进行相互作用筛选以确定个体泛素连接的作用。我们将在此屏幕上展开，并 将采用互补的质谱学方法来鉴定相关底物。

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.