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

项目摘要/摘要 泛素依赖的蛋白分解在包括细胞周期在内的各种生理过程中发挥着重要作用。 控制和细胞增殖，并经常是癌基因转化的目标。的特殊性 蛋白质底物泛素化是由许多E3泛素连接酶中的一种直接或 通过接头蛋白进行底物识别。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，并参与了 多个DNA修复过程。CDT2/DTL，一种与CRL4(CRL4-CDT2)相关的WD重复蛋白 并作为底物识别因子将底物回收到CRL4泛素连接酶的其余部分。 CRL4-CDT2最近被证明促进泛素依赖的复制起始的破坏 蛋白CDT1和细胞周期蛋白依赖性激酶抑制因子p21，在细胞周期的S期和在 对紫外线照射的反应。值得注意的是，CDT2在多种人类肿瘤中经常过表达，其 其表达与肿瘤分级、转移和不良生存有关。这项研究的目的是了解 CRL4-CDT2影响基因组稳定性并促进癌症的发展。具体地说，我将A)确定 并表征了CRL4-CDT2 E3泛素连接酶复合体的新型底物。B)确定机制 通过CDT2负性调节p21转录和C)检测CDT2是否显示出致癌活性 活着。人CDT2相关蛋白的TAP-串联纯化和质谱分析 有或没有DNA损伤的细胞，我将鉴定新的CRL4-CDT2底物，这些底物可能参与细胞 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CRL4-CD2 E3泛素连接酶复合体的分析将验证鉴定的蛋白质是否 真正的衬底。我还将从Cre/loxP结构中产生过表达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生。结果将会是 提高我们对如何利用蛋白质泛素化和蛋白质降解的调控过程的理解 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.