Regulating the ATR checkpoint through protein ubiquitylation

通过蛋白质泛素化调节 ATR 检查点

• 批准号: 7881733
• 负责人: Richard C. Centore
• 金额: $ 5.13万
• 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/7881733
• 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的生存至关重要。一种检测和响应DNA损伤的关键机制涉及由ATR和CHKL激酶介导的信号通路。该途径中的缺陷被称为ATR检查点，与各种人类癌症有关。 ATR激活CHKL取决于关键的介体蛋白Claspin。我的初步结果表明，claspin是针对泛素化和蛋白酶体介导的响应DNA损伤的降解的。这些发现表明，蛋白质泛素化在ATR检查点的调节中引起了惊人的参与。我的提议旨在确定负责损伤引起的扣发降解的分子机制以及生物学后果。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ATR检查点响应中泛素化的功能。具体目的：1）确定负责紫外线诱导的链蛋白降解的分子机制，2）研究链球蛋白降解对ATR检查点响应的功能，并调节应力复制叉的调节，3）进行蛋白质组学筛选以鉴定调节ATR检查点的新型泛素化蛋白。使用分子生物学，哺乳动物组织培养和生化技术的结合，我计划首先测试蛋白酶的特定特定突变体，这些突变体预计不会被蛋白酶体降解。我还将确定如何通过ATR检查点调节claspin泛素化。一旦鉴定出稳定的链蛋白突变体，我将使用它来确定紫外线诱导的链蛋白降解的生物学后果。我将使用生化，细胞生物学和免疫荧光技术研究固定稳定对检查点恢复，CHKL易位以及DNA修复的影响。最后，我建议进行蛋白质组学筛选，以确定以DNA损伤诱导方式调节ATR检查点响应的所有泛素蛋白。一旦使用这些技术鉴定出新的蛋白质，我将使用特定目的1-2中概述的方法来确定这些翻译后修饰的机制以及它们对细胞DNA损伤响应的影响。 公共卫生相关性：蛋白酶体抑制剂已成为癌症治疗的有前途的药物。这项拟议的研究可能揭示了蛋白酶体抑制剂消除癌细胞的机制，并可能促进为癌症患者开发新的靶向疗法。