Structural studies of cullin-based ubiquitin ligases

描述（由申请人提供）：基于cullin的泛素连接酶通过促进大量关键调节蛋白的泛素化和随后的降解来调节广谱生物过程，例如细胞周期进程、细胞生长、肿瘤抑制和转录控制。cullin介导的蛋白质泛素化的失调与肿瘤发生、癌症和其他人类疾病有关。在六种人类cullin中，Cul 4A最近被证明与受损DNA结合蛋白DDB 1相互作用，DDB 1在核苷酸切除修复中识别UV损伤的DNA。这两种蛋白共同形成了一种不同于Cul 1 SCF和Cul 2 SCF样复合物的泛素连接酶复合物，并在DNA修复中发挥作用。尽管在亚基组成的差异，所有cullin为基础的泛素连接酶的小泛素样蛋白Nedd 8和可能的CAND 1的调节。例如，在SCF中，CAND 1与Cul 1结合抑制复合物组装，并且抑制作用可以通过Cul 1的Nedd 8修饰来逆转。该建议侧重于最近发现的Cul 4A-DDB 1泛素连接酶的结构-功能研究，以及一般如何基于cullin的泛素连接酶受Nedd 8和CAND 1的调节。目的1：DDB 1和Cul 4A-Rbx 1-DDB 1复合物的结构分析。DDB 1、Cul 4A-DDB 1复合物和DDB 1-DDB 2- DNA复合物的晶体结构将被确定和分析，以阐明Cul 4A-DDB 1泛素连接酶在DNA修复中的活性的结构机制。目的2：Cullin-Rbx 1-CAND 1相互作用的结构分析。CAND 1、CAND 1-CuI 1-Rbx 1复合物和neddylated-Cul 1-Rbx 1复合物的晶体结构将被解析，以揭示CAND 1和Nedd 8如何调节泛素连接酶的组装和活性。

DESCRIPTION (provided by applicant): The cullin-based ubiquitin ligases regulate a broad spectrum of biological processes such as cell cycle progression, cell growth, tumor suppression, and transcriptional control, by promoting ubiquitination and subsequent degradation of a large number of key regulatory proteins. Deregulations of cullin-mediated protein ubiquitination have been implicated in tumorigenesis, cancer, and other human diseases. Among the six human cullins, Cul4A has recently been shown to interact with the damaged-DNA binding protein, DDB1, which recognizes UV-damaged DNA in nucleotide excision repair. Together, the two proteins form a ubiquitin ligase complex distinct from the Cul1-based SCF and Cul2-based SCF-like complexes and play a role in DNA repair. Despite the differences in subunit composition, all cullin-based ubiquitin ligases are regulated by the small ubiquitin-like protein Nedd8 and possibly by CAND1. In SCF, for example, the complex assembly is inhibited by CAND1 binding to Cul1 and the inhibitory effect can be reversed by the Nedd8 modification of Cul1. This proposal focuses on structure-function studies of both the recently identified Cul4A-DDB1 ubiquitin ligase and in general how the cullin-based ubiquitin ligases are regulated by Nedd8 and CAND1. The following specific aims are proposed: Aim 1: Structural analyses of DDB1 and the Cul4A-Rbx1-DDB1 complex. Crystal structures of DDB1, the Cul4A-DDB1 complex, and the DDB1-DDB2- DNA complex will be determined and analyzed to elucidate the structural mechanism underlying the Cul4A-DDB1 ubiquitin ligase activity in DNA repair. Aim 2: Structural analyses of Cullin-Rbx1-CAND1 interactions. Crystal structures of CAND1, the CAND1-CuI1-Rbx1 complex, and the neddylated-Cul1-Rbx1 complex will be solved to reveal how CAND1 and Nedd8 modulate the assembly and activity of the ubiquitin ligase.