The ROC-Cullin Family of E3 Ubiquitin Ligases

E3 泛素连接酶的 ROC-Cullin 家族

• 批准号: 7009977
• 负责人: YUE XIONG
• 金额: $ 25.38万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7009977
• 关键词: CHO cells; HeLa cells; cell cycle; cell cycle proteins; cell growth regulation; ligase; molecular chaperones; molecular shape; polymerase chain reaction; protein protein interaction; protein purification; proteomics; southern blotting; ubiquitin; western blottings; yeast two hybrid system; yeasts

DESCRIPTION (provided by applicant): Cullins are a recently identified family of evolutionarily conserved proteins linked to the ubiquitination of a potentially large number of cellular proteins. Characterization of a small number of cullins has identified critical functions in such diverse physiological processes as cell cycle and cell growth control, tumor suppression and animal development. We and others previously discovered a cullin-interacting RING finger protein, ROC1 (also known as Rbx1 or Hrt1), that carries essential function for CUL1-mediated SCF E3 ligases and for cell cycle progression. We have found that ROC1 and its homologue, ROC2, allosterically activates E2 and interacts with all cullin members to constitute many distinct ROC-cullin ubiquitin ligases in vivo. We have recently identified novel interactions between ROC-cullin ligases and two evolutionarily conserved proteins, p 120 TIP120 and p 127 DDB1. Our preliminary studies have led to the hypothesis that DDB 1 may function as a ROC-dependent E3 NEDD8 ligase toward cullin proteins, and that TIP120 proteins may function as assembly factors of various ROC-cullin ligases. We propose an investigation that combines biochemical, proteomic and genetic approaches in mammalian and fission yeast cells to determine the mechanisms and functions of TIP120 and DDB 1 and a poorly characterized CUL4. Through these concerted efforts, we anticipate a great opportunity to better understand the mechanism regulating the ROC-cullin family of ubiquitin ligases and the mechanism underlying the substrate recognition by this family of E3 ligases. Aim I. The function and mechanism of TIP120 1. Regulation of TIP120-cullin association by NEDD8 pathway in vivo2. Genetic analysis of fission yeast TIP120 3. Determination of the in vivo function of TIP120 in mammalian cells 4. Purification and analysis of TIP120 complexes.Aim II. Function of DDB1 as an NEDD8 E3 ligase.1. Biochemical reconstitution of NEDD8 ligation2. Genetic analysis of DDB 1 in fission yeastAim III. Function and complexes of CUL4A1. Determination of the function of human CUL4A2. Functional analysis of CUL4 in fission yeast3. Purification and characterization of CUL4 complexes.

描述（由申请人提供）：Cullin 是最近鉴定的进化保守蛋白家族，与潜在大量细胞蛋白的泛素化相关。对少数库林的表征已经确定了细胞周期和细胞生长控制、肿瘤抑制和动物发育等不同生理过程中的关键功能。我们和其他人之前发现了一种与 cullin 相互作用的环指蛋白 ROC1（也称为 Rbx1 或 Hrt1），它对 CUL1 介导的 SCF E3 连接酶和细胞周期进展具有重要功能。我们发现ROC1及其同源物ROC2变构激活E2并与所有cullin成员相互作用，在体内构成许多不同的ROC-cullin泛素连接酶。我们最近发现了 ROC-cullin 连接酶和两种进化保守蛋白 p 120 TIP120 和 p 127 DDB1 之间的新相互作用。我们的初步研究得出这样的假设：DDB 1 可能充当针对 cullin 蛋白的 ROC 依赖性 E3 NEDD8 连接酶，而 TIP120 蛋白可能充当各种 ROC-cullin 连接酶的组装因子。我们提出一项研究，结合哺乳动物和裂殖酵母细胞中的生化、蛋白质组学和遗传学方法，以确定 TIP120 和 DDB 1 以及特征不明的 CUL4 的机制和功能。通过这些共同努力，我们预计将有一个很好的机会更好地了解泛素连接酶 ROC-cullin 家族的调节机制以及该 E3 连接酶家族底物识别的机制。 目的一、TIP120的功能和机制1.体内NEDD8通路对TIP120-cullin结合的调控2。裂殖酵母TIP120的遗传分析3.哺乳动物细胞中TIP120体内功能的测定4.TIP120复合物的纯化和分析。目的二。 DDB1 作为 NEDD8 E3 连接酶的功能。 1． NEDD8 连接的生化重建2。裂殖酵母中 DDB 1 的遗传分析Aim III。 CUL4A1 的功能和复合物。人CUL4A2功能的测定。裂殖酵母中CUL4的功能分析3。 CUL4 复合物的纯化和表征。