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泛素化和随后大量关键调节蛋白的降解，调节广泛的生物过程，如细胞周期进程，细胞生长，肿瘤抑制和转录控制。cullin介导的蛋白泛素化调控的解除与肿瘤发生、癌症和其他人类疾病有关。在六种人类cullins中，Cul4A最近被证明与受损DNA结合蛋白DDB1相互作用，DDB1在核苷酸切除修复中识别紫外线损伤的DNA。这两种蛋白共同形成一个泛素连接酶复合物，不同于基于cul1的SCF和基于cul2的SCF样复合物，并在DNA修复中发挥作用。尽管亚基组成不同，但所有基于cullin的泛素连接酶都受小泛素样蛋白Nedd8和可能受CAND1的调节。例如，在SCF中，CAND1与Cul1的结合抑制了复合物的组装，而Cul1的Nedd8修饰可以逆转这种抑制作用。本研究主要关注最近发现的Cul4A-DDB1泛素连接酶的结构-功能研究，以及基于cullin的泛素连接酶如何受到Nedd8和CAND1的调节。提出以下具体目标：目标1:DDB1和Cul4A-Rbx1-DDB1复合物的结构分析。我们将对DDB1、Cul4A-DDB1复合物和DDB1- ddb2 - DNA复合物的晶体结构进行测定和分析，以阐明Cul4A-DDB1泛素连接酶在DNA修复中活性的结构机制。目的2:Cullin-Rbx1-CAND1相互作用的结构分析。通过解析CAND1、CAND1- cui1 - rbx1复合物和木化- cul1 - rbx1复合物的晶体结构，揭示CAND1和Nedd8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.