The ROC-Cullin Family of E3 Ubiquitin Ligases

描述（由申请人提供）：Cullin是最近鉴定的进化上保守的蛋白质家族，与潜在大量细胞蛋白质的泛素化相关。对少量cullin的表征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。我们和其他人以前发现了一个cullin相互作用的环指蛋白，ROC 1（也称为Rbx 1或Hrt 1），它携带CUL 1介导的SCF E3连接酶和细胞周期进程的基本功能。我们已经发现，ROC 1和它的同源物，ROC 2，变构激活E2和相互作用的所有cullin成员，构成许多不同的ROC-cullin泛素连接酶在体内。我们最近已经确定了新的ROC-cullin连接酶和两个进化上保守的蛋白质，p120 TIP 120和p127 DDB 1之间的相互作用。我们的初步研究已经导致的假设，DDB 1可能作为一个ROC依赖性E3 NEDD 8连接酶对cullin蛋白，和TIP 120蛋白可能作为各种ROC-cullin连接酶的装配因子。我们提出了一项调查，结合生物化学，蛋白质组学和遗传学的方法在哺乳动物和裂殖酵母细胞，以确定的机制和功能的TIP 120和DDB 1和一个不好的特点CUL 4。通过这些共同的努力，我们期待一个很好的机会，以更好地了解调控的ROC-cullin家族的泛素连接酶的机制和E3连接酶的这个家庭的底物识别的机制。 艾姆岛TIP 120的功能和机制 1.通过体内NEDD 8途径调节TIP 120-cullin结合2.裂殖酵母TIP 120的遗传分析3. TIP 120在哺乳动物细胞中的体内功能的测定4. TIP 120复合物的纯化和分析。DDB 1作为NEDD 8 E3连接酶的功能。NEDD 8连接的生化重建2.裂殖酵母DDB 1基因的遗传分析CUL 4A 1的功能和复合物。人CUL 4A 2的功能的测定。CUL 4在裂变酵母中的功能分析3. CUL 4复合物的纯化和表征。

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.