Functional Anatomy of the Cul3 Ubiquitin Ligase

Cul3 泛素连接酶的功能解剖

• 批准号: 7025048
• 负责人: JEFFREY W HARPER
• 金额: $ 29.79万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7025048
• 关键词: RNA interference; SDS polyacrylamide gel electrophoresis; cell cycle; enzyme activity; functional /structural genomics; green fluorescent proteins; human tissue; ligase; polymerase chain reaction; protein binding; protein protein interaction; protein structure function; ubiquitin

DESCRIPTION (provided by applicant): The SCF ubiquitin ligase pathway employs cullin proteins to assemble substrate specificity modules and ubiquitin conjugating enzymes, thereby promoting substrate ubiquitination. Our recent work has revealed that cullin-3 (Cul3) binds a large family of BTB-domain proteins in a manner analogous to the Skpl/F-box complex, which binds Cull. We hypothesize that BTB proteins function as substrate specific adaptors for Cul3. Evidence for this hypothesis comes from our finding that the C. elegans BTB protein Mel-26 interacts with its genetically defined target Mei-1 through its MATH domain. Elimination of Mei-1 protein that accompanies exit from meiosis depends upon Mel-26, Cul-3, and the cullin activator Nedd8, suggesting that the Cul3/Mel-26 complex functions as a ubiquitin ligase to facilitate Mei-1 destruction during this cell cycle transition. In addition, we have recently found that Keap1, which interacts through its BTB domain with Cul3 and through its kelch domain with the transcription factor Nrf2, is required for degradation of Nrf2 in vivo and ubiqutination of Nrf2 in vitro. Here, we seek to further establish the role of BTB proteins in targeted ubiquitintation. In Aim 1, we will elucidate the biochemical requirements for target ubiquitination by two Cul3-based ubiquitin ligases, Mel-26/Cul3 and Keap1/Cul3, and will examine the role of BTB-protein dimerization in ubiquitination activity in vitro and protein turnover in vivo. In aim 2, we will extend our structural analysis of E3s by examining the architecture of the Cul-3/Mel-26/Mei-1 complex through crystallographic and biochemical studies. In aim 3, we will employ newly developed RNAi libraries against BTB proteins to define the role of Cul3/BTB complexes in cell division and monomeric cyclin E turnover. In addition, novel approaches for the identification of ubiquitination targets employing loss-of-function genetics and applicable to genome-wide screens will be developed in the context of the BTB/Cul3 pathway. These experiments will: confirm and extend the hypothesis that BTB proteins function as substrate specific adaptors of Cul3, will reveal the structural basis for Cul-3 and substrate recognition by the Mel-26 BTB protein, and will apply the power of functional genomic approaches to the problem of ubiquitin ligase function and target identification in human cells.

描述（申请人提供）：SCF泛素连接酶途径利用cullin蛋白组装底物特异性模块和泛素结合酶，从而促进底物泛素化。我们最近的工作表明，cullin-3 (Cul3) 以类似于与 Cull 结合的 Skpl/F-box 复合物的方式结合 BTB 结构域蛋白大家族。我们假设 BTB 蛋白作为 Cul3 的底物特异性接头。这一假设的证据来自我们的发现，即秀丽隐杆线虫 BTB 蛋白 Mel-26 通过其 MATH 结构域与其基因定义的目标 Mei-1 相互作用。伴随减数分裂退出的 Mei-1 蛋白的消除依赖于 Mel-26、Cul-3 和 cullin 激活剂 Nedd8，表明 Cul3/Mel-26 复合物作为泛素连接酶发挥作用，以促进该细胞过程中 Mei-1 的破坏循环过渡。此外，我们最近发现Keap1通过其BTB结构域与Cul3相互作用，并通过其kelch结构域与转录因子Nrf2相互作用，是Nrf2体内降解和体外Nrf2泛素化所必需的。在这里，我们寻求进一步确定 BTB 蛋白在靶向泛素化中的作用。在目标 1 中，我们将阐明两种基于 Cul3 的泛素连接酶 Mel-26/Cul3 和 Keap1/Cul3 进行靶标泛素化的生化要求，并将检查 BTB 蛋白二聚化在体外泛素化活性和蛋白质周转中的作用。体内。在目标 2 中，我们将通过晶体学和生化研究检查 Cul-3/Mel-26/Mei-1 复合物的结构，从而扩展 E3 的结构分析。在目标 3 中，我们将采用新开发的针对 BTB 蛋白的 RNAi 文库来定义 Cul3/B​​TB 复合物在细胞分裂和单体细胞周期蛋白 E 周转中的作用。此外，将在 BTB/Cul3 途径的背景下开发采用功能丧失遗传学并适用于全基因组筛选的泛素化靶点识别新方法。这些实验将：证实并扩展 BTB 蛋白作为 Cul3 底物特异性接头的假设，揭示 Cul-3 的结构基础和 Mel-26 BTB 蛋白的底物识别，并将应用功能基因组方法的力量人类细胞中泛素连接酶功能和靶标识别的问题。