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包括细胞周期控制和细胞增殖在内的各种生理过程中起重要作用，并且经常是致癌性肿瘤的靶点。蛋白质底物泛素化的特异性由许多E3泛素连接酶之一的活性直接或通过接头蛋白的底物识别决定。其中，基于Cullin 4的E3泛素连接酶（CRL 4）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。Cdt 2/DTL是一种含有WD重复序列的蛋白质，与CRL 4（CRL 4-Cdt 2）结合，并作为底物识别因子将底物重新结合到CRL 4泛素连接酶的其余部分。CRL 4-Cdt 2最近被证明可以促进复制起始蛋白Cdt 1和细胞周期蛋白依赖性激酶（CDK）抑制剂p21的泛素依赖性破坏，无论是在细胞周期的S期还是在对UV照射的响应中。值得注意的是，Cdt 2经常在多种人类肿瘤中过表达，并且其表达与肿瘤分级、转移和不良存活相关。这项研究旨在了解CRL 4-Cdt 2如何影响基因组稳定性并促进癌症发展。具体地，我将A）鉴定和表征CRL 4-Cdt 2 E3泛素连接酶复合物的新底物。B）鉴定Cdt 2负调节p21转录的机制和C）测试Cdt 2是否在体内表现出致癌活性。使用自来水串联纯化和质谱分析Cdt 2相关蛋白从人类细胞或没有DNA损伤，我将确定新的CRL 4 Cdt 2基板，可能参与细胞增殖和/或DNA修复。标准的生物化学技术，包括使用纯化的CRL 4-Cdt 2 E3泛素连接酶复合物的体内和体外泛素化测定，将验证所鉴定的蛋白质是否是真正的底物。我还将从Cre/LoxP构建体中产生过表达Cdt 2的转基因小鼠，并在动物模型系统中测试Cdt 2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。 公共卫生相关性：治疗癌症的根本挑战之一是缺乏对癌细胞存活重要的各种参与者的全面了解。该提案旨在确定细胞增殖和DNA修复所必需的蛋白质，这些蛋白质被参与这些过程的调控的关键酶靶向破坏。它还将测试酶在肿瘤发展中的作用。

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.