Nuclear protein quality control mechanisms during genotoxic stress

遗传毒性应激期间的核蛋白质量控制机制

• 批准号: RGPIN-2020-04360
• 负责人: Stirling, Peter
• 金额: $ 4.23万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=716418
• 关键词: Nuclear; protein; quality; control; mechanisms

Cellular stress responses can alter cell cycle, gene expression, and protein homeostasis in an orchestrated way that promotes cell survival and future stress tolerance. This is true of DNA damaging stress in which proteins relocalize to different subcellular compartments as part of a multi-level DNA damage response including DNA repair, transcriptional changes and proteome remodeling. Yeast cells have been a useful model for understanding dynamic changes of proteins during the DNA damage response. Genotoxic stress can elicit several spatially and functionally distinct protein compartments, including DNA repair foci, stress granules, and a peri-nucleolar aggregate structure called the intranuclear quality control site (INQ). The function of the INQ compartment in genotoxic stress responses is not known. INQ formation is regulated by molecular chaperones and the proteasome, suggesting it is a site of protein quality control (PQC). Additionally, the INQ sits adjacent to the nucleolus, and human nucleoli have been identified as phase-separated protein quality control compartments. Thus, elucidating INQ formation and function in the DNA damage response will inform our understanding of spatial nuclear PQC and its role in stress responses across species. Robust stress responses contribute to healthy aging, industrial biological processes, and in organisms of agricultural importance. Therefore, understanding fundamental principles of stress biology will ultimately impact many sectors of research and industry. Our goal is to understand the regulation of protein sequestration at the INQ and develop Rpd3 INQ localization as a new model. We will address this goal with three specific aims: Aim 1. Identify regulatory post-translational modifications on proteins that regulate INQ sequestration: This will initially focus on SUMO or ubiquitin modifications of INQ resident proteins. Aim 2. Dissect the function of INQ resolving molecular chaperones: We will directly test the role of Cdc48 and its cofactors, or the Apj1-Hsp70-Hsp110 system in turnover of INQ substrate proteins. Aim 3. Define the function of Rpd3 sequestration at INQ: We hypothesize that the Rpd3 lysine deacetylase is sequestered at INQ to control the chromatin state of late firing DNA replication origins. Overall, this project will reveal the mechanisms of nuclear protein aggregate formation during the DNA damage response from the perspective of post-translational modifications and molecular chaperones. It will also directly test the functional consequences of INQ protein sequestration starting with direct tests of Rpd3 histone deacetylase function in the DNA damage response. These data will reveal how proteome remodeling and quality control contribute to DNA damage tolerance and recovery. Together these outcomes will elucidate a novel nuclear PQC pathway that is integrated with the DNA repair and transcriptional remodeling activities known to accompany cellular responses to genotoxic stress.

细胞应激反应可以改变细胞周期、基因表达和蛋白质稳态，从而促进细胞存活和未来的应激耐受。DNA损伤应激也是如此，其中蛋白质重新定位到不同的亚细胞区室，作为多水平DNA损伤反应的一部分，包括DNA修复、转录变化和蛋白质组重塑。酵母细胞是理解DNA损伤反应过程中蛋白质动态变化的有用模型。遗传毒性应激可以引起几种空间和功能不同的蛋白质区室，包括DNA修复灶，应激颗粒和称为核内质量控制位点（INQ）的核仁周围聚集体结构。INQ隔室在遗传毒性应激反应中的功能尚不清楚。INQ的形成受分子伴侣和蛋白酶体的调节，这表明它是蛋白质质量控制（PQC）的一个位点。此外，INQ位于核仁附近，并且人类核仁已被鉴定为相分离的蛋白质质量控制区室。因此，阐明INQ的形成和功能的DNA损伤反应将告知我们的理解空间核PQC及其在跨物种的应激反应中的作用。强大的压力反应有助于健康的老龄化，工业生物过程，并在农业生物的重要性。因此，了解压力生物学的基本原理将最终影响许多研究和工业部门。我们的目标是了解蛋白质螯合在INQ的调节，并开发Rpd 3 INQ定位作为一个新的模型。我们将通过三个具体目标来实现这一目标： 目标1。鉴定调节INQ螯合的蛋白质上的调节性翻译后修饰：这将首先集中在INQ驻留蛋白的SUMO或泛素修饰上。 目标2.剖析INQ解析分子伴侣的功能：我们将直接测试Cdc 48及其辅因子或Apj 1-Hsp 70-Hsp 110系统在INQ底物蛋白周转中的作用。 目标3。定义Rpd 3螯合在INQ的功能：我们假设Rpd 3赖氨酸脱乙酰基酶被螯合在INQ，以控制晚发DNA复制起点的染色质状态。 总之，本项目将从翻译后修饰和分子伴侣的角度揭示DNA损伤反应中核蛋白聚集体形成的机制。 它还将直接测试INQ蛋白螯合的功能后果，首先直接测试Rpd 3组蛋白脱乙酰酶在DNA损伤反应中的功能。这些数据将揭示蛋白质组重塑和质量控制如何有助于DNA损伤耐受和恢复。这些结果将共同阐明一种新的核PQC途径，该途径与已知伴随细胞对遗传毒性应激反应的DNA修复和转录重塑活动相结合。