Nuclear protein quality control mechanisms during genotoxic stress

基因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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760764
• 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.

细胞应激反应可以以精心策划的方式改变细胞周期、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性。 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，作为多级 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。酵母细胞是了解 DNA 损伤反应过程中蛋白质动态变化的有用模型。基因毒性应激可以引发几个空间和功能上不同的蛋白质区室，包括 DNA 修复灶、应激颗粒和称为核内质量控制位点 (INQ) 的核仁周围聚集结构。 INQ 区室在基因毒性应激反应中的功能尚不清楚。 INQ 的形成受到分子伴侣和蛋白酶体的调节，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，人类核仁已被确定为相分离的蛋白质质量控​​制区室。因此，阐明 DNA 损伤反应中 INQ 的形成和功能将有助于我们了解空间核 PQC 及其在跨物种应激反应中的作用。强大的应激反应有助于健康老龄化、工业生物过程以及具有重要农业意义的生物体。因此，了解应激生物学的基本原理最终将影响研究和工业的许多领域。我们的目标是了解 INQ 上蛋白质隔离的调控，并开发 Rpd3 INQ 定位作为新模型。我们将通过三个具体目标来实现这一目标： 目标 1. 识别调节 INQ 隔离的蛋白质的翻译后修饰：首先将重点关注 INQ 驻留蛋白的 SUMO 或泛素修饰。目标2.剖析INQ解析分子伴侣的功能：我们将直接测试Cdc48及其辅因子或Apj1-Hsp70-Hsp110系统在INQ底物蛋白周转中的作用。目标 3. 定义 Rpd3 在 INQ 处隔离的功能：我们假设 Rpd3 赖氨酸脱乙酰酶在 INQ 处隔离以控制晚期激发 DNA 复制起点的染色质状态。总体而言，该项目将从翻译后修饰和分子伴侣的角度揭示DNA损伤反应过程中核蛋白聚集体形成的机制。它还将直接测试 INQ 蛋白隔离的功能后果，首先直接测试 Rpd3 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基因毒性应激的反应的 DNA 修复和转录重塑活动相结合。