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.

项目摘要/摘要 背景 - 维持蛋白质的人是细胞适应性的核心，可以通过 软化蛋白质质量控​​制（PQC）途径，消除功能失调和不需要的蛋白质， 如果允许积聚并凝结，蛋白质复合物会变成细胞毒性。实际上，蛋白质 PQC缺陷鼓励的聚集是衰老，癌症和许多人类聚集的标志 - 俯卧的病理，包括肌萎缩性侧面硬化症，阿尔茨海默氏症，帕金森氏症和亨廷顿 疾病和相关的多系统蛋白质病。因此，对PQC的充分理解可以提供 减轻蛋白质聚集和随后的蛋白质应激的新策略。以前的工作 - 我们 发现了机械保守的PQC路线，该路由指导自噬消除无效 蛋白酶体和cdc48分离酶（人类中的p97/vcp），它们提供实验性强大的模型 用于描述有缺陷的蛋白质清除率。值得注意的是，两种蛋白质复合物的营业额具有共享特征 去除淀粉样蛋白的蛋白质，包括会话，泛素化和随后的识别 由专用的自噬受体，对于蛋白酶体而言，也需要三个可能的泛素连接酶 协同工作以组装合适的聚链链拓扑。项目的目的 - 该项目建议 要详细描述酵母和拟南芥中蛋白酶体和cdc48的自噬清除， 目的是发现自噬PQC中心的方面。对于蛋白酶，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接映射，其中，何时， 以及连接酶SAN1，RSP5和HUL5如何协调促进功能失调的蛋白酶体 泛素化； （ii）确定生成自噬统一底物所需的泛素化链接；和 （iii）推断出HSP42介导的疗程如何将无细胞质膜脂肪介导，而不是 凝结成蛋白酶体储存颗粒，有助于该过程。同样，对CDC48的研究 营业额将确认泛素化是一个关键信号，然后确定相关的泛素 连接酶和对CDC48隔离如何有助于其营业额的理解。而且，我们将测试 我们的假设是，用于清除功能失调蛋白酶和CDC48的自噬路线也 消除淀粉样蛋白的核心蛋白，这些蛋白质是众多易受聚集的病理的核心。最后， 我们将进一步定义和扩展使用新型的自噬受体/适配器，这些受体/适配器使用小说 与ATG8（人类的LC3）衬里自噬蔬菜的接口，从而有助于增加 选择性自噬的已知范围。结果 - 通过这项累积研究，我们希望定义 与聚合相关的PQC相关的自噬路线，该路由将阐明 泛素化，生物分子凝结和自噬在减轻蛋白质毒性应激和最终 告知各种淀粉样蛋白原病理学的新治疗策略。