Autophagic Clearance of Proteasomes and CDC48 as Models for Amyloidogenic Protein Quality Control.

蛋白酶体和 CDC48 的自噬清除作为淀粉样蛋白质量控制的模型。

• 批准号: 10366935
• 负责人: RICHARD DAVID VIERSTRA
• 金额: $ 30.82万
• 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 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10366935
• 关键词: Aging; Alzheimer' s Disease; Amyloid; Amyotrophic Lateral Sclerosis; Antibodies; Arabidopsis; Autophagocytosis; Autophagosome; Behavior; Binding; Binding Proteins; Biochemical; Catalogs; Cell Cycle; Cell Nucleus; Cell membrane; Cells; Chromosome Mapping; Cytoplasmic Granules; Defect; Disease; Docking; Enzymes; Event; Excision; Family; Fluorescence Microscopy; Funding; Genetic; Goals; Health; Heart; Homeostasis; Human; Huntington Disease; Impairment; Knowledge; Life; Ligase; Light; Liquid substance; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Membrane; Microscopic; Mission; Modeling; Molecular Chaperones; Mutation; Nature; Organism; Orthologous Gene; Outcome; Parkinson Disease; Pathology; Pathway interactions; Physical condensation; Plants; Polymers; Polyubiquitin; Process; Proteins; Proteolysis; Quality Control; Recycling; Research; Role; Route; Signal Transduction; Stress; Surface; Testing; Therapeutic Intervention; Ubiquitin; United States National Institutes of Health; Vesicle; Work; Yeasts; cytotoxic; diagnostic biomarker; familial amyotrophic lateral sclerosis; fitness; improved; information model; innovation; multicatalytic endopeptidase complex; multisystem proteinopathy; nervous system disorder; novel; novel therapeutic intervention; particle; protein aggregation; protein complex; protein degradation; proteostasis; proteotoxicity; receptor; recruit; response; targeted treatment; ubiquitin ligase; valosin-containing protein

PROJECT SUMMARY/ABSTRACT Background − Maintenance of proteostasis is central to cellular fitness and is achieved through sophisticated protein quality control (PQC) pathways that remove dysfunctional and unwanted proteins and protein complexes that become cytotoxic if allowed to accumulate and condense. In fact, protein aggregation encouraged by PQC defects is a hallmark of aging, cancer, and numerous human ‘aggregation- prone’ pathologies, including amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s and Huntington’s diseases, and related multisystem proteinopathies. Consequently, full understandings of PQC could offer new strategies to mitigate protein aggregation and subsequent proteotoxic stress. Previous Work − We discovered mechanistically conserved PQC routes that direct the autophagic elimination of inactive proteasomes and the CDC48 segregase (p97/VCP in humans), which offer experimentally robust models for describing defective protein clearance. Notably, turnover of both protein complexes shares features with amyloidogenic protein removal, including sequestration, ubiquitylation, and subsequent recognition by dedicated autophagic receptors, which for proteasomes also requires a trio of ubiquitin ligases that likely work in concert to assemble appropriate poly-Ub chain topologies. Project Aims − This project proposes to describe in detail the autophagic clearance of proteasomes and CDC48 in both yeast and Arabidopsis, with the goal of discovering aspects central to autophagic PQC. For proteasomes, we will: (i) examine, using genetics, fluorescence microscopy, interaction studies and ubiquitin linkage mapping, where, when, and how the ligases San1, Rsp5 and Hul5 coordinately contribute to dysfunctional proteasome ubiquitylation; (ii) identify ubiquitylation linkages needed to generate autophagy competent substrates; and (iii) deduce how Hsp42-mediated sequestration into cytoplasmic membrane-less aggresomes, versus condensation into proteasome storage granules, contributes to the process. Likewise, studies on CDC48 turnover will confirm that ubiquitylation is a key signal, followed by the identification of relevant ubiquitin ligases and understanding of how CDC48 sequestration contributes to its turnover. Moreover, we will test our hypothesis that the autophagic routes used to clear dysfunctional proteasomes and CDC48 also eliminate amyloidogenic proteins that are at the heart of numerous aggregation-prone pathologies. Finally, we will further define and expand upon a new class of autophagic receptors/adaptors that use a novel interface to dock with ATG8 (LC3 in humans) lining autophagic vesicles, thus helping to increase the known reach of selective autophagy. Outcomes − Through this cumulative research, we hope to define autophagic routes relevant to aggregation-associated PQC, which will shed light on the roles of ubiquitylation, biomolecular condensation, and autophagy in mitigating proteotoxic stress and ultimately inform upon new therapeutic strategies for various amyloidogenic pathologies.

项目总结/摘要 背景-蛋白质稳态的维持是细胞健康的核心，通过以下途径实现： 复杂的蛋白质质量控制（PQC）途径，去除功能失调和不需要的蛋白质， 蛋白质复合物，如果允许其积累和浓缩，则会产生细胞毒性。事实上，蛋白质 由PQC缺陷引起的聚集是衰老、癌症和许多人类聚集的标志， 易发性疾病，包括肌萎缩侧索硬化症、阿尔茨海默氏症、帕金森氏症和亨廷顿氏症 疾病和相关的多系统蛋白质病。因此，充分了解PQC可以提供 减轻蛋白质聚集和随后的蛋白毒性应激的新策略。以前的工作-我们 发现了机制上保守的PQC途径，指导自噬消除非活性 蛋白酶体和CDC 48分离酶（人类p97/VCP），它们提供了实验上可靠的模型 用于描述有缺陷的蛋白质清除。值得注意的是，两种蛋白质复合物的周转率具有相同的特征， 伴随淀粉样蛋白的去除，包括螯合、泛素化和随后的识别 通过专门的自噬受体，蛋白酶体也需要一个泛素连接酶， 协同工作以组装适当的poly-Ub链拓扑。项目目标：本项目提出 为了详细描述酵母和拟南芥中蛋白酶体和CDC 48的自噬清除， 目的是发现自噬PQC的核心方面。对于蛋白酶体，我们将：（i）检查， 使用遗传学，荧光显微镜，相互作用研究和泛素连锁图谱，在哪里，什么时候， 以及连接酶San 1、Rsp 5和Hul 5如何协调地促成功能失调的蛋白酶体 （ii）鉴定产生自噬活性底物所需的泛素化连接;和 (iii)推断Hsp 42介导的隔离如何进入无细胞质膜的侵袭体， 浓缩成蛋白酶体储存颗粒，有助于这一过程。同样，对CDC 48的研究 周转将证实泛素化是一个关键信号，随后是相关泛素的鉴定 连接酶和理解如何CDC 48螯合有助于其营业额。此外，我们将测试 我们的假设是，用于清除功能失调的蛋白酶体和CDC 48的自噬途径也 消除淀粉样蛋白，这是许多聚集倾向的病理学的核心。最后， 我们将进一步定义和扩展一类新的自噬受体/衔接子， 与ATG 8（人类中的LC 3）对接的自噬囊泡，从而有助于增加 已知的选择性自噬的范围。结果-通过这项累积的研究，我们希望定义 与聚集相关的PQC相关的自噬途径，这将阐明 泛素化、生物分子缩合和自噬在减轻蛋白毒性应激和最终 为各种淀粉样病变提供新的治疗策略。