SYSTEMATIC ANALYSIS OF PROTEOLYSIS PATHWAYS FOR CULLIN TARGETS

描述(申请人提供)：基于库林环的泛素连接酶(CRL)是哺乳动物细胞中最大的E3泛素连接酶之一，然而这些E3的调节机制以及它们的底物最终如何被蛋白酶体降解的许多方面仍不清楚。在之前的资助周期中，我们已经通过结构、机制和遗传方法探索了CUL3-BTB蛋白和CUL4-DDB1DCAF E3s的功能。在这次更新中，我们试图研究泛素化领域中的两个新兴主题。主题1涉及一个问题，即如何定义发出底物泛素化信号所需的整个基因谱系，执行泛素化事件，然后将蛋白质靶向蛋白酶体。这个问题是在复制许可蛋白CDT1及其通过CUL4-DDB1CDt2E3依赖DNA损伤的周转的背景下进行分析的。我们最近完成了全基因组范围的基因筛选，筛选其被RNAi耗尽阻止UV依赖的CDT1周转的基因。这一筛选显示了目前正在验证的600个候选基因，包括直接参与CDT1泛素化和蛋白降解的已知基因的近饱和。一些已知的和候选的DNA损伤基因被鉴定出来，使我们能够将这些基因放置在CDT1泛素化的上游和下游的假设途径中。通过一系列二次测试，Aim 1将把有效的基因排序到一个信号通路中，在哺乳动物细胞和非洲爪哇卵提取液中的实验将确定在该通路中不同点作用的一组基因所涉及的机制，包括可能将泛素化的CDT1连接到蛋白酶体的组件。主题2解决泛素化底物如何靶向蛋白酶体的一般问题。该领域越来越多的证据表明，促进剂蛋白，包括具有各种泛素结合域的蛋白，促进蛋白酶体识别泛素化的蛋白。我们采用并进一步开发了新兴技术，使我们能够在存在和不存在针对降解机制组件的RNAi的情况下测量单个底物的相对周转率。在SCF2-TRCP复合体的一组底物的背景下使用这项技术，AIM 2将对候选的“促进剂”蛋白进行组合遗传分析，以揭示特定底物的相关和潜在冗余的“促进剂”蛋白，从而揭示底物招募到蛋白酶体的潜在特异性。使用全球蛋白质稳定性(GPS)系统，我们将从表达12,000个人类基因的细胞库中识别与蛋白酶体相关的Rpn10“促进器”蛋白质的靶标，从而定义需要Rpn10进行周转的泛素化靶标的库。体外重建实验将试图总结体外环境中促进剂的体内特异性。与公共健康相关：泛素修饰蛋白质构成了细胞中蛋白质调节的主要模式，并构成了多种信号通路的基础。许多人类疾病反映了控制细胞蛋白质周转的途径的中断。这项提议试图更详细地了解控制蛋白质泛素化的途径，以及底物如何针对蛋白酶体。

DESCRIPTION (provided by applicant): Cullin-ring based ubiquitin ligases (CRLs) constitute one of the largest classes of E3 ubiquitin ligases in mammalian cells, yet many aspects of the mechanisms by which these E3s are regulates and how they their substrates ultimately are degraded by the proteasome remain unknown. In the previous funding cycle, we have explored the functions of Cul3-BTB protein and Cul4-Ddb1DCAF E3s through structural, mechanistic, and genetic approaches. In this renewal, we seek to examine two emerging themes in the ubiquitination field. Theme 1 concerns the question of how one goes about defining the entire repertoire of genes that are require to signal ubiquitination of a substrate, perform the ubiquitination event, and then target the protein to the proteasome. This question is being analyzed in the context of the replication licensing protein Cdt1 and its DNA damage-dependent turnover via the Cul4-Ddb1Cdt2 E3. We have recently completed a genome wide screen for genes whose depletion by RNAi blocks UV-dependent Cdt1 turnover. This screen has revealed 600 candidate genes currently being validated, including near saturation of the known genes involved directly in Cdt1 ubiquitination and proteolysis. A number of known and candidate DNA damage genes were identified, allowing us to place these genes in a hypothetic pathway upstream and downstream of Cdt1 ubiquitination. Through a series of secondary assays, Aim 1 will order validated genes into a signaling pathway, and experiments in mammalian cells and in Xenopus egg extracts will define the mechanisms involved for a subset of genes acting at distinct points in the pathway, including components that may link ubiquitinated Cdt1 to the proteasome. Theme 2 addresses the general question of how ubiquitinated substrates are targeted to the proteasome. There is accumulating evidence in the field indicating that "facilitator" proteins, including proteins with various sorts of ubiquitin binding domains, promote the recognition of ubiquitinated proteins by the proteasome. We have adapted and further developed emerging technologies which allow us to measure the relative rates of turnover of individual substrate in the presence and absence of RNAi targeting components of the degradation machinery. Using this technology in the context of a panel of substrates of the SCF2-TRCP complex, Aim 2 will perform a combinatorial genetic analysis of candidate "facilitator" proteins in order to uncover relevant and potentially redundant "facilitator" proteins for particular substrates, thereby revealing the underlying specificity in substrate recruitment to the proteasome. Using the Global Protein Stability (GPS) system, we will identify targets of the proteasome-associated Rpn10 "facilitator" protein from a library of cells expressing 12,000 human genes, thereby defining the repertoire of ubiquitination targets that require Rpn10 for their turnover. In vitro reconstitution experiments will attempt to recapitulate in vivo specificity of facilitators in an in vitro setting. PUBLIC HEALTH RELEVANCE: Modification of proteins by ubiquitin constitutes a primary mode of protein regulation in cells and underlies diverse signaling pathways. Many human diseases reflect disruption in the pathways that control turnover of cellular proteins. This proposal seeks to understand in greater detail the pathways that control protein ubiquitination and how substrates are targeted to the proteasome.