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

CRL4-Cdt2 E3 泛素连接酶在基因组稳定性和癌症中的作用

基本信息

批准号：
8721855
负责人：
TAREK A. ABBAS
金额：
$ 24.15万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8721855
关键词：
Adaptor Signaling Protein Animal Model BRCA1 Mutation Binding Biochemical Biological Assay Biological Models Breast Cancer Cell Bypass Cancer Intervention Cancer cell line Cell Cycle Cell Cycle Progression Cell Cycle Regulation Cell Proliferation Cells Complex Cre-LoxP Cullin Proteins Cyclin-Dependent Kinase Inhibitor Cytokinesis DNA DNA Damage DNA Repair DNA biosynthesis Databases Development Dose Enzyme Tests Enzymes Exhibits Genes Genetic Transcription Genome Stability Human In Vitro Lesion Licensing Factor Malignant Neoplasms Mammalian Cell Manuscripts Mass Spectrum Analysis Messenger RNA Modification Molecular Profiling Neoplasm Metastasis Neuronal Differentiation Oncogenic Patients Physiological Processes Play Polymerase Process Proliferating Property Proteins Proteolysis RNA Recruitment Activity Regulation Replication Initiation Replication Licensing Rest Role S Phase Techniques Testing Transcript Transgenic Mice Translations Tretinoin Ubiquitin WD Repeat abstracting base cancer cell in vivo malignant breast neoplasm novel oncoprotein p21 overexpression response segregation tumor tumorigenesis ubiquitin ligase ubiquitin-protein ligase ultraviolet irradiation

项目摘要

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.

项目摘要/摘要泛素依赖性蛋白水解在包括细胞周期在内的各种生理过程中起着重要作用对照和细胞增殖，通常是致癌转移的靶标。特定的蛋白质底物泛素化是由许多E3泛素连接酶之一的活性或直接或通过辅助蛋白通过底物识别。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剂，并参与多个DNA修复过程。 CDT2/DTL，一种与CRL4（CRL4-CDT2）的WD重复相关的WD重复并用作将基材恢复到其余CRL4泛素连接酶的底物识别因子。最近已显示CRL4-CDT2促进复制起始的泛素依赖性破坏蛋白CDT1和细胞周期蛋白依赖性激酶（CDK）抑制剂P21，均在细胞周期的S期和中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表达与肿瘤等级，转移和存活不良相关。这项研究旨在了解如何 CRL4-CDT2对基因组稳定性有影响，并有助于癌症的发展。具体来说，我将a）确定并表征了CRL4-CDT2 E3泛素连接酶复合物的新型底物。 b）确定机制 CDT2负调控P21转录，C）测试CDT2是否在体内。使用Tap-Tandem纯化和来自人类CDT2相关蛋白的质谱分析有或没有DNA损伤的细胞，我将确定可能参与细胞的新的CRL4-CDT2底物增殖和/或DNA修复。标准生化技术，包括体内和体外泛素化技术使用净化的CRL4-CDT2 E3泛素连接酶配合物的测定将验证确定的蛋白是否是真正的基板。我还将生成从CRE/LOXP构建体过表达CDT2的转基因小鼠，并且测试CDT2过表达是否有助于动物模型系统的肿瘤发展。结果将促进我们对如何利用蛋白质泛素化和蛋白水解的调节过程的理解癌症干预目的。