The Cullin-ROC Family of E3 Ubiquitin Ligases

描述(申请人提供)：这是GM067113的首次竞争性更新，该研究旨在了解E3泛素连接酶(CRL)的cullin环家族的功能和调控。回顾过去几年的历程，我们非常高兴看到我们做出的贡献，更值得注意的是，同事们共同取得的进步。在初步发现ROC1和ROC2之后，我们为实现这一目标做出了几项发现。其中包括：(1)CAND1，一种进化保守的cullin相关和脱氧核糖核酸解离蛋白，控制着所有CRL的底物募集；(2)CUL3与存在于200多个哺乳动物蛋白质中的BTB基序结合，并潜在地组装大量BTB-CUL3-ROC1连接酶；(3)DDB1-CUL4-ROC1连接酶针对复制许可因子CDT1进行降解，以响应DNA损伤；(4)CUL4与大量细胞蛋白相互作用；以及(5)进化保守的WD40蛋白pl70(KIAA080)，通过DDB1与CUL4A大量结合，并以P53依赖的方式在细胞增殖中发挥重要作用。在这一系列发现的基础上，我们建议进一步研究CUL3和CUL4依赖的泛素连接酶的底物募集机制、细胞功能和调控。这项提案中概述的研究结合了计算机建模、蛋白质组学、生化、细胞和遗传学方法，为帮助理解最大的E3泛素连接酶家族提供了一个极好和独特的机会。三个特定的目的是：(1)确定CRL在有丝分裂和转录调控中的功能和底物；(2)确定CRL4连接酶的底物招募机制；(3)确定P170的功能和机制。

DESCRIPTION (provided by applicant): This is the first competitive renewal of GM067113, an investigation aimed at the long term goal of understanding the function and regulation of cullin-RING family of E3 ubiquitin ligases (CRLs). Looking back at the journey of the past several years, we are very pleased to see the contribution we made and more remarkably, the advancements achieved collectively by colleagues. Following the initial discovery of ROC1 and ROC2, we have made several findings toward this goal. These include: (1) that CAND1, an evolutionary conserved cullin-associated and neddylation dissociated protein, controls substrate recruitment to all CRLs, (2) that CUL3 binds to a BTB motif present in more than 200 mammalian proteins and potentially assembles a large number of BTB-CUL3-ROC1 ligases, (3) that DDB1-CUL4-ROC1 ligase target the replication licensing factor CDT1 for degradation in response to DNA damage, and (4) that CUL4 interacts with large number of cellular proteins, and (5) that pl70(KIAA080), an evolutionarily-conserved WD40 protein, binds abundantly with CUL4A via DDB1 and plays an essential role in cellular proliferation in a p53-dependent manner. With these series of findings as the basis, we propose a further research to elucidate the substrate- recruitment mechanism, the cellular function and regulation of CUL3- and CUL4-dependent ubiquitin ligases. The studies outlined in this proposal combine computer modeling, proteomic, biochemical, cellular and genetic approaches, offering an excellent and unique opportunity to help in the understanding of the largest family of E3 ubiquitin ligases. Three specific aims are: (i) to determine the functions and substrates of CRLS in mitosis and in transcriptional regulation, (ii) to determine the substrate recruiting mechanism of CRL4 ligases, and (iii) to determine the function and mechanism of p170.