The Role of CRL4-Cdt2 E3 Ubiquitin Ligase in Genomic Stability and Cancer

描述（由申请人提供）：泛素依赖性蛋白水解在多种生理过程中发挥重要作用，包括细胞周期控制和细胞增殖，并且经常是致癌转化的目标。蛋白质底物泛素化的特异性是由许多E3泛素连接酶之一的活性决定的，要么直接决定，要么通过接头蛋白识别底物。其中，基于Cullin 4的E3泛素连接酶（CRL4）正在成为细胞增殖和基因组稳定性的主要调节因子，并参与多种DNA修复过程。Cdt2/DTL是一种含有wd重复的蛋白，与CRL4 （CRL4-Cdt2）相关，并作为底物识别因子，将底物募集到CRL4泛素连接酶的其余部分。CRL4-Cdt2最近被证明在细胞周期的s期和对紫外线照射的反应中促进泛素依赖性的复制起始蛋白Cdt1和周期蛋白依赖性激酶（CDK）抑制剂p21的破坏。值得注意的是，Cdt2在多种人类肿瘤中经常过表达，其表达与肿瘤分级、转移和生存不良相关。本研究旨在了解CRL4-Cdt2如何影响基因组稳定性并促进癌症的发展。具体来说，我将A)鉴定和表征CRL4-Cdt2 E3泛素连接酶复合物的新底物。B)确定Cdt2负调控p21转录的机制；C)检测Cdt2在体内是否表现出致癌活性。使用tap-tandem纯化和质谱分析来自有或没有DNA损伤的人类细胞的cdt2相关蛋白，我将确定可能参与细胞增殖和/或DNA修复的新的CRL4-Cdt2底物。标准生化技术，包括使用纯化的CRL4-Cdt2 E3泛素连接酶复合物进行体内和体外泛素化分析，将验证鉴定的蛋白质是否为真正的底物。我还将从Cre/LoxP构建体中产生过表达Cdt2的转基因小鼠，并在动物模型系统中测试Cdt2过表达是否有助于肿瘤的发展。这些结果将促进我们对如何利用蛋白质泛素化和蛋白质水解的调节过程进行癌症干预的理解。

DESCRIPTION (provided by applicant): Ubiquitin-dependent proteolysis plays a significant role in various physiological processes including cell cycle control and cellular proliferation and is frequently the target of oncogenic transfomation. The specficity of protein substrate ubiquitylation is dictated by the activity of one of many E3 ubiquitin ligases either directly or through substrate recognition through adaptor proteins. Among these, the Cullin 4-based E3 ubiquitin ligase (CRL4) is emerging as a master regulator of cellular proliferation and genomic stability and is involved in multiple DNA repair processes. Cdt2/DTL, a WD-repeat containing protein associates with CRL4 (CRL4-Cdt2) and functions as a substrate recognition factor for recuiting substrates to the rest of the CRL4 ubiquitin ligase. CRL4-Cdt2 has recently been shown to promote the ubiquitin-dependent destruction of the replication initiation protein Cdt1 and the cyclin-dependent kinase (CDK) inhibitor p21, both in S-phase of the cell cycle and in response to UV irradiation. Significantly, Cdt2 is frequently overexpressed in a variety of human tumors and its expression correlates with tumor grade, metastasis and poor survival. This study aims at understanding how CRL4-Cdt2 impacts on genomic stability and contributes to cancer development. Specifically, I will A) Identify and characterize novel substrates for the CRL4-Cdt2 E3 ubiquitin ligase complex. B) Identify the mechanism by which Cdt2 negatively regulates p21 transcription and C) Test whether Cdt2 exhibits oncogenic activity in vivo. Using tap-tandem purification and mass-spectrometry analysis of Cdt2-associated proteins from human cells with or without DNA damage, I will identify new CRL4-Cdt2 substrates that may be involved in cellular proliferation and/or DNA repair. Standard biochemical techniques, including in vivo and in vitro ubiquitylation assays using purfied CRL4-Cdt2 E3 ubiquitin ligase complexes, will verify whether the identified proteins are bona fide substrates. I will also generate transgenic mice overexpressing Cdt2 from Cre/LoxP constructs and test whether Cdt2 overexpression contributes to tumor development in animal model system. The results will advance our understanding of how to exploit the regulated process of protein ubiquitylation and proteolysis for cancer intervention purposes. PUBLIC HEALTH RELEVANCE: One of the fundamental challenges in treating cancer is the lack of a comprehensive understanding of the various players that are important for the survival of cancer cells. This proposal aims at identifying proteins essential for cellular proliferation and DNA repair that are targeted for destruction by a critical enzyme that is involved in the regulation of these processes. It will also test the enzymes' role in tumor development.