AUTOPHAGIC CLEARANCE OF INACTIVE PROTEASOMES AND RIBOSOMES AS MODELS FOR PROTEIN QUALITY CONTROL

无活性蛋白酶体和核糖体的自噬清除作为蛋白质质量控​​制的模型

• 批准号: 10063879
• 负责人: RICHARD DAVID VIERSTRA
• 金额: $ 28.98万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-12-11 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10063879
• 关键词: Aging; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Arabidopsis; Autophagocytosis; Autophagosome; Behavior; Binding; Binding Proteins; Biological Assay; Cell Nucleus; Cell model; Cells; Complex; Defect; Deposition; Disease; Eukaryota; Event; Excision; Exposure to; Fluorescence; Goals; Health; Hela Cells; Homeostasis; Human; Human Pathology; Huntington Disease; Impairment; Intervention; Knowledge; Life; Ligase; Light; Maintenance; Malignant Neoplasms; Maps; Mediating; Methodology; Microscopic; Mission; Modeling; Modification; Molecular Chaperones; Nature; Organism; Orthologous Gene; Parkinson Disease; Pathology; Pathway interactions; Plants; Polyubiquitin; Post-Translational Protein Processing; Prions; Process; Proteins; Proteolysis; Quality Control; Research; Ribosomes; Role; Route; Site; Stress; Testing; Translations; Ubiquitin; United States National Institutes of Health; Up-Regulation; Vacuole; Yeasts; cytotoxic; diagnostic biomarker; fitness; improved; inhibitor/antagonist; innovation; multicatalytic endopeptidase complex; mutant; novel; polypeptide; protein aggregation; protein complex; protein degradation; proteostasis; proteotoxicity; receptor; recruit; response; ubiquitin ligase; ubiquitin-protein ligase

PROJECT SUMMARY Maintenance of proteostasis is central to cellular fitness and is achieved through sophisticated protein quality control (PQC) pathways that remove dysfunctional or unwanted proteins and protein complexes that become cytotoxic if allowed to accumulate. In fact, protein aggregation encouraged by defects in PQC is a hallmark of aging, cancer, and numerous human ‘aggregation-prone’ pathologies, including amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s, Huntington’s, and prion-mediated diseases. Consequently, fully understanding PQC could offer new strategies to mitigate protein aggregation and subsequent proteotoxic stress, especially as they relate to these disease states. Recently, ancient and mechanistically conserved PQC pathways were discovered that direct the autophagic elimination of unwanted or inactive proteasomes and ribosomes, termed proteaphagy and ribophagy, respectively. Notably, a selective proteaphagic route triggered by inhibition shares features with amyloidogenic protein removal, including sequestration by the Hsp42 chaperone, ubiquitylation, and subsequent recognition by the autophagic receptors Cue5 or RPN10, suggesting that inhibitor-induced proteaphagy, and likely ribophagy, offer tractable models to dissect features underpinning PQC and protein aggregate clearance. This project aims to characterize proteaphagy and ribophagy induced by inhibition in both Arabidopsis and yeast with the goal of discovering components central to autophagic PQC. Specifically, we will define how, where, and why the yeast ubiquitin (Ub) ligases Hul5, Rsp5, and San1 (and their Arabidopsis orthologs) recognize and ubiquitylate dysfunctional proteasomes, and how this modification triggers autophagy via the Ub-binding Cue5/RPN10 autophagic receptors, using facile confocal fluorescence microscopic and GFP-fusion cleavage assays together with co-localization studies. A special focus will be on the mechanism(s) involving Hsp42 that coalesce inactive proteasomes into large cytoplasmic aggregates similar to those observed for various aggregation-prone proteins. We will determine which proteasome subunits become modified, where the Ubs are attached, which types of poly-Ub chains are assembled, and identify other factors/post- translational modifications that might be important, by mass spectrometric analysis of proteasomes purified before and after inhibition. Using similar methodologies, we will examine how ribosomes are degraded by autophagy after exposure to translation inhibitors, and examine the role(s) of ubiquitylation and the corresponding ligases, Hsp42, and receptors like Cue5/RPN10 in this clearance. We will also confirm proteaphagy in human cells and the involvement of the Cue5 ortholog Tollip in this process, and determine if the pathway(s) used for proteophagy and ribophagy also help clear amyloidogenic proteins. Finally, we will study a new class of autophagic receptors related to RPN10 that might substantially expand the influence of selective autophagy in plants, yeast, and humans. Through this cumulative research, we will define the autophagic routes for proteasome and ribosome clearance that should be relevant to aggregation-associated PQC, and thus will shed light on the roles of various subcellular protein deposits and their effectors in mitigating proteotoxic stress related to numerous aggregation-prone pathologies.

项目摘要 蛋白质的维持是细胞适应性的核心，是通过复杂的蛋白质质量控​​制实现的 （PQC）去除功能失调或不需要的蛋白质和蛋白质复合物的途径，如果允许的话将变为细胞毒性 积累。实际上，PQC缺陷鼓励蛋白质聚集是衰老，癌症和众多的标志 人类“容易发生”病理学，包括肌萎缩性侧面硬化症，阿尔茨海默氏症，帕金森氏症，亨廷顿， 和prion介导的疾病。因此，完全了解PQC可以提供缓解蛋白质的新策略 聚集和随后的蛋白毒性应激，尤其是与这些疾病状态相关的蛋白毒性应激。 最近，发现古代和机械保守的PQC途径，指导自噬消除 分别称为蛋白质和核能的不需要或无活性蛋白酶体和核糖体。值得注意的是，选择性 由抑制均具有去除淀粉样蛋白的抑制作用特征触发的蛋白质路线，包括 自噬受体CUE5或RPN10的HSP42伴侣，泛素化和随后的识别 抑制剂诱导的蛋白质和可能的核能，提供了可拖动的模型，以剖析pqc和 蛋白质骨料清除率。 该项目旨在表征拟南芥和酵母抑制引起的蛋白质磷酸和核能 发现自噬PQC中心的组件的目标。具体来说，我们将定义如何，何处以及为什么酵母 泛素（UB）连接酶HUL5，RSP5和SAN1（及其拟南芥的直系同源物）识别和泛素酯功能失调 蛋白酶体以及这种修饰如何通过使用UB结合CUE5/RPN10自噬受体触发自噬 便捷的共聚焦荧光显微镜和GFP融合裂解测定法以及共定位研究。特别 重点将放在涉及HSP42的机制上 类似于各种易受聚集的蛋白观察到的。我们将确定哪些蛋白酶体的亚基成为 修改了瑞银（UB 通过对蛋白酶体进行质谱分析，可能很重要的翻译修饰 抑制后。使用类似的方法，我们将检查核糖体在暴露后如何通过自噬降解 转化抑制剂，并检查泛素化和相应的连接酶的作用，HSP42和接收器 CUE5/rpn10在此间隙中。我们还将确认人类细胞中的蛋白质和CUE5的参与 在此过程中的直系同源物托里普，并确定用于蛋白质和核能的途径是否有助于清除 淀粉样蛋白。最后，我们将研究一类与RPN10相关的自噬受体 大大扩展了选择性自噬在植物，酵母和人类中的影响。 通过这项累积研究，我们将定义蛋白酶体和核糖体清除的自噬路线，应 与聚集相关的PQC相关，因此会阐明各种亚细胞蛋白沉积物和 它们在缓解蛋白毒性应激中的作用与众多易受聚集的病理相关。