Regulating the ATR checkpoint through protein ubiquitylation

通过蛋白质泛素化调节 ATR 检查点

• 批准号: 8197327
• 负责人: Richard C. Centore
• 金额: $ 5.39万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-11-18 至 2012-11-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8197327
• 关键词: Biochemical; Biological; Biological Assay; Cancer Patient; Cell Cycle; Cells; Chromatin; DNA Damage; DNA Repair; DNA biosynthesis; Defect; Development; Epitopes; Event; Failure; Fluorescent Antibody Technique; Genome Stability; Human; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Molecular; Molecular Biology; Organism; Pathway interactions; Pharmaceutical Preparations; Phosphotransferases; Post-Translational Protein Processing; Proteasome Inhibitor; Proteins; Proteomics; RNA Interference; Recovery; Recruitment Activity; Regulation; Screening procedure; Signal Pathway; Signal Transduction; Stress; Techniques; Testing; UV induced; Ubiquitin; cancer cell; cancer therapy; multicatalytic endopeptidase complex; mutant; novel; public health relevance; repaired; response; tissue culture; ubiquitin ligase

DESCRIPTION (provided by applicant): The ability to maintain genomic stability is vital to the survival of all organisms. One crucial mechanism to detect and respond to DNA damage involves a signaling pathway mediated by the ATR and Chkl kinases. Defects in this pathway, known as the ATR checkpoint, are associated with a variety of human cancers. Activation of Chkl by ATR is dependent upon a key mediator protein, Claspin. My preliminary results show that Claspin is targeted for ubiquitylation and proteasome-mediated degradation in response to DNA damage. These findings suggest a surprising involvement of protein ubiquitylation in the regulation of ATR checkpoint. My proposal aims to determine the molecular mechanism responsible for, and the biological consequences of damage-induced Claspin degradation. Furthermore, I propose to use proteomic approaches to identify novel proteins that are specifically ubiquitinylated after DNA damage, and to systematically investigate the functions of ubiquitylation in ATR checkpoint response. Specific Aims: 1) Determine the molecular mechanisms responsible for UV-induced Claspin degradation, 2) Investigate the functions of Claspin degradation on ATR checkpoint response and regulation of stressed replication forks, 3) Conduct a proteomic screen to identify novel ubiquitinylated proteins that regulate the ATR checkpoint. Using a combination of molecular biology, mammalian tissue culture, and biochemical techniques, I plan to first test specific mutants of Claspin that are predicted to fail to be degraded by the proteasome. I will also determine how Claspin ubiquitylation is regulated by ATR checkpoint. Once a stabilized Claspin mutant has been identified, I will use it to determine the biological consequences of UV-induced Claspin degradation. I will study the effects of Claspin stabilization on checkpoint recovery, Chkl translocation from chromatin, and DNA repair using biochemical, cell biological, and immunofluorescence techniques. Finally, I propose to conduct a proteomic screen to determine all proteins that are ubiquitinylated in a DNA damage-inducible manner that regulate ATR checkpoint response. Once new proteins have been identified with these techniques, I will use the approaches outlined in Specific Aims 1-2 to determine the mechanism by which these post-translational modifications occur as well as their effects on the cellular DNA damage response. Public Health Relevance: Proteasome inhibitors have emerged as promising drugs for the treatment of cancers. This proposed study may reveal the mechanisms by which proteasome inhibitors eliminate cancer cells, and may facilitate the development of new targeted therapies for cancer patients.

描述（由申请人提供）：维持基因组稳定性的能力对于所有生物体的生存至关重要。检测和响应 DNA 损伤的一个重要机制涉及由 ATR 和 Chk1 激酶介导的信号传导途径。该通路（称为 ATR 检查点）的缺陷与多种人类癌症有关。 ATR 对 Chk1 的激活依赖于关键介质蛋白 Claspin。我的初步结果表明，Claspin 是针对 DNA 损伤而进行泛素化和蛋白酶体介导的降解的。这些发现表明蛋白质泛素化惊人地参与了 ATR 检查点的调节。我的建议旨在确定损伤引起的 Claspin 降解的分子机制和生物学后果。此外，我建议使用蛋白质组学方法来鉴定 DNA 损伤后特异性泛素化的新型蛋白质，并系统地研究泛素化在 ATR 检查点反应中的功能。具体目标：1) 确定导致 UV 诱导 Claspin 降解的分子机制，2) 研究 Claspin 降解对 ATR 检查点响应和应激复制叉调节的功能，3) 进行蛋白质组筛选，以鉴定调节 ATR 检查点的新型泛素化蛋白。我计划结合分子生物学、哺乳动物组织培养和生化技术，首先测试预计无法被蛋白酶体降解的 Claspin 特定突变体。我还将确定 ATR 检查点如何调节 Claspin 泛素化。一旦确定了稳定的 Claspin 突变体，我将用它来确定紫外线诱导的 Claspin 降解的生物学后果。我将使用生化、细胞生物学和免疫荧光技术研究 Claspin 稳定性对检查点恢复、Chkl 染色质易位以及 DNA 修复的影响。最后，我建议进行蛋白质组筛选，以确定所有以 DNA 损伤诱导方式泛素化、调节 ATR 检查点反应的蛋白质。一旦用这些技术鉴定出新的蛋白质，我将使用具体目标 1-2 中概述的方法来确定这些翻译后修饰发生的机制以及它们对细胞 DNA 损伤反应的影响。 公共卫生相关性：蛋白酶体抑制剂已成为治疗癌症的有前途的药物。这项拟议的研究可能揭示蛋白酶体抑制剂消除癌细胞的机制，并可能促进癌症患者新靶向疗法的开发。