The ROC-Cullin Family of E3 Ubiquitin Ligases

描述（由申请人提供）：Cullins是最近发现的一个进化保守蛋白家族，与潜在的大量细胞蛋白的泛素化有关。少量cullins的鉴定已经确定了在细胞周期和细胞生长控制、肿瘤抑制和动物发育等多种生理过程中的关键功能。我们和其他人之前发现了一种cullin相互作用的RING finger蛋白ROC1（也称为Rbx1或Hrt1），它在cul1介导的SCF E3连接酶和细胞周期进程中具有重要功能。我们发现ROC1及其同系物ROC2在体内变构激活E2并与所有cullin成员相互作用，形成许多不同的ROC-cullin泛素连接酶。我们最近发现了ROC-cullin连接酶与两个进化上保守的蛋白p120 TIP120和p127 DDB1之间的新相互作用。我们的初步研究假设ddb1可能作为roc依赖的E3 NEDD8连接酶对cullin蛋白起作用，TIP120蛋白可能作为各种ROC-cullin连接酶的组装因子。我们建议在哺乳动物和分裂酵母细胞中结合生化、蛋白质组学和遗传学方法来确定TIP120和ddb1以及一个尚未被充分描述的CUL4的机制和功能。通过这些共同努力，我们期望有一个很好的机会来更好地了解ROC-cullin家族泛素连接酶的调节机制以及该家族E3连接酶识别底物的机制。

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.