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

项目总结/摘要 泛素依赖的蛋白水解在细胞周期等多种生理过程中起着重要作用 控制和细胞增殖，并且经常是致癌性肿瘤的靶点。的特异性 蛋白质底物泛素化由许多E3泛素连接酶之一的活性直接或间接决定。 通过衔接蛋白的底物识别。其中，基于Cullin 4的E3泛素连接酶 CRL 4作为细胞增殖和基因组稳定性的主要调节因子出现，并参与 多重DNA修复过程Cdt 2/DTL，一种含有WD重复序列的蛋白质，与CRL 4（CRL 4-Cdt 2）相关 并作为底物识别因子将底物重新连接到CRL 4遍在蛋白连接酶的其余部分。 CRL 4-Cdt 2最近被证明可以促进复制起始的泛素依赖性破坏， 蛋白Cdt 1和细胞周期蛋白依赖性激酶（CDK）抑制剂p21，无论是在细胞周期的S期， 对UV辐射的反应。值得注意的是，Cdt 2经常在多种人类肿瘤中过表达，并且其表达与肿瘤的发生有关。 表达与肿瘤分级、转移和低生存率相关。这项研究旨在了解如何 CRL 4-Cdt 2影响基因组稳定性并促进癌症发展。具体来说，我将A）确定 并表征CRL 4-Cdt 2 E3泛素连接酶复合物的新底物。B）确定机制 Cdt 2通过其负调节p21转录，和C）测试Cdt 2是否表现出致癌活性， vivo.人Cdt 2相关蛋白的TAP串联纯化及质谱分析 无论有无DNA损伤，我将确定新的CRL 4-Cdt 2底物，可能参与细胞凋亡。 增殖和/或DNA修复。标准生化技术，包括体内和体外泛素化 使用纯化的CRL 4-Cdt 2 E3泛素连接酶复合物的测定将验证所鉴定的蛋白质是否是 真正的基质。我还将从Cre/LoxP构建体产生过表达Cdt 2的转基因小鼠， 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。结果将 推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的理解， 癌症干预的目的。

Project Summary/Abstract 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.