SYSTEMATIC ANALYSIS OF PROTEOLYSIS PATHWAYS FOR CULLIN TARGETS

描述（由申请人提供）：基于Cullin环的泛素连接酶（CRL）构成哺乳动物细胞中最大类别的E3泛素连接酶之一，但这些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然未知。在上一个资助周期中，我们通过结构、机制和遗传方法探索了Cul3-BTB蛋白和Cul4-Ddb1DCAF E3的功能。在这次更新中，我们试图研究两个新兴的主题在泛素化领域。主题1涉及的问题是，如何定义整个基因库，这些基因需要发出底物泛素化的信号，执行泛素化事件，然后将蛋白质靶向蛋白酶体。这个问题正在分析的背景下，复制许可蛋白Cdt1和它的DNA损伤依赖性营业额通过Cul4-Ddb1Cdt2 E3。我们最近完成了一个基因组范围内的筛选基因，其消耗的RNAi块紫外线依赖Cdt1营业额。该筛选揭示了目前正在验证的600个候选基因，包括直接参与Cdt1泛素化和蛋白水解的已知基因的接近饱和。一些已知的和候选的DNA损伤基因被确定，使我们能够将这些基因在Cdt1泛素化的上游和下游的一个假设的途径。通过一系列的二次检测，目标1将命令验证的基因进入一个信号通路，在哺乳动物细胞和非洲爪蟾卵提取物中的实验将定义在通路中的不同点上起作用的基因子集所涉及的机制，包括可能将泛素化Cdt1连接到蛋白酶体的成分。主题2解决了泛素化底物如何靶向蛋白酶体的一般问题。越来越多的证据表明，“促进剂”蛋白，包括具有各种泛素结合结构域的蛋白质，促进蛋白酶体对泛素化蛋白的识别。我们已经适应并进一步开发了新兴技术，这些技术使我们能够在存在和不存在RNAi靶向降解机制组分的情况下测量单个底物的相对周转率。在SCF 2-TRCP复合物的一组底物的背景下使用该技术，Aim 2将对候选"促进剂"蛋白进行组合遗传分析，以发现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蛋白酶体相关Rpn 10“促进剂”蛋白的目标，从而定义需要Rpn 10进行营业额的泛素化目标的库。体外重建实验将试图在体外环境中概括促进剂的体内特异性。公共卫生相关性：泛素对蛋白质的修饰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。该提案旨在更详细地了解控制蛋白质泛素化的途径以及底物如何靶向蛋白酶体。

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.