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.

项目总结 蛋白质平衡的维持是细胞健康的核心，并通过复杂的蛋白质质量控制来实现 (PQC)去除功能失调或不需要的蛋白质和蛋白质复合体的途径，如果允许，这些蛋白质和蛋白质复合体会变成细胞毒性 积累。事实上，由PQC缺陷引起的蛋白质聚集是衰老、癌症和许多 人类容易聚集的病理，包括肌萎缩侧索硬化症、阿尔茨海默氏症、帕金森氏症、亨廷顿氏症、 以及普恩介导性疾病。因此，充分理解PQC可以为减少蛋白质提供新的策略 聚集和随后的蛋白毒性应激，特别是当它们与这些疾病状态有关时。 最近，古老的和机械保守的PQC通路被发现，它们指导着自噬的消除 不需要的或不活跃的蛋白酶体和核糖体，分别称为嗜蛋白和嗜核糖体。值得注意的是，一个选择性的 由抑制触发的蛋白途径与淀粉样蛋白的去除有相同的特征，包括通过 Hsp42的伴侣，泛素化，以及随后被自噬受体Cue5或RPN10识别，提示 这种抑制剂诱导的蛋白吞噬，以及可能的核吞噬，提供了易于处理的模型，以剖析支撑PQC和PQC的特征 蛋白质聚集清除。 这个项目的目的是研究在拟南芥和酵母中通过抑制而诱导的蛋白吞噬和核吞噬的特征。 目标是发现自噬PQC的核心成分。具体地说，我们将定义酵母如何、在哪里以及为什么 泛素(Ub)连接酶Hul5、Rsp5和San1(及其拟南芥同源基因)识别和泛素化功能障碍 蛋白酶体，以及这种修饰如何通过Ub结合的Cue5/RPN10自噬受体触发自噬 简便的共聚焦荧光显微镜和GFP融合裂解分析以及共定位研究。一份特别的 焦点将集中在涉及HSP42的机制上(S)，该机制涉及到将失活的蛋白酶体聚合成大的细胞质聚集体 与对各种易于聚集的蛋白质观察到的结果相似。我们将确定哪些蛋白酶体亚基成为 修改、瑞银在哪里连接、组装了哪些类型的聚-Ub链，并确定其他因素/发布 可能重要的翻译修饰，通过对之前和之前纯化的蛋白酶体进行质谱分析 在抑制之后。使用类似的方法，我们将研究核糖体在暴露后如何通过自噬而降解。 翻译抑制物，并研究泛素化和相应的连接酶，热休克蛋白42和受体一样的作用(S) 此间隙中的Cue5/RPN10。我们还将证实人类细胞中的蛋白吞噬作用以及Cue5的参与 Tollip在这一过程中的直系同源基因，并确定是否该途径(S)用于蛋白吞噬和核吞噬也有助于清除 淀粉样蛋白。最后，我们将研究与RPN10相关的一类新的自噬受体，它可能 大大扩大选择性自噬在植物、酵母和人类中的影响。 通过这项累积研究，我们将确定蛋白酶体和核糖体清除的自噬途径。 与聚集相关的PQC相关，从而将阐明各种亚细胞蛋白沉积和 它们在减轻蛋白毒性应激方面的效应与许多易于聚集的病理有关。