The role of p97 in protein degradation during aging and disease

p97 在衰老和疾病过程中蛋白质降解中的作用

• 批准号: 8366219
• 负责人: Eugene Drokhlyansky
• 金额: $ 2.95万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2014-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8366219
• 关键词: 26S proteasome; ATP phosphohydrolase; Accounting; Adaptor Signaling Protein; Affinity; Aging; Alzheimer' s Disease; Biochemical; Biological Assay; Biological Models; Brain; Cell Culture Techniques; Cell physiology; Cells; Cellular biology; Complex; Data; Disease; Disease model; Ensure; Environment; Equilibrium; Eukaryota; Functional disorder; Glutamates; Goals; Heat Shock 70kD Protein Binding Protein; Heavy Metals; Homo; Huntington Disease; Hydrolysis; In Vitro; Laboratories; Life; Mammals; Mass Spectrum Analysis; Mitochondria; Modeling; Molecular; Molecular Chaperones; Molecular Mechanisms of Action; Muscle; Neurodegenerative Disorders; Pathogenesis; Pathway interactions; Physiological; Play; Process; Protein Biochemistry; Proteins; Quality Control; Rattus; Reactive Oxygen Species; Research; Research Proposals; Role; Skeletal Muscle; Stress; System; Techniques; Testing; Training; Ubiquitin; Yeasts; adapter protein; age related; aged; base; cofactor; design; disease-causing mutation; human disease; insight; medical schools; member; multicatalytic endopeptidase complex; prevent; professor; protein aggregate; protein aggregation; protein complex; protein degradation; protein folding; protein misfolding; protein purification; synthetic protein; therapeutic target; ubiquitin ligase; ubiquitin-protein ligase

Under normal physiologic conditions continuous post-synthetic protein damage and misfolding generate aberrant proteins that can form erroneous and toxic interactions within the cell. In eukaryotes, the ubiquitin proteasome system (UPS) is essential for ensuring protein quality control, which is crucial for protecting cells during aging and against age-related protein aggregation disorders such as neurodegenerative diseases including Alzheimer's and Huntington's disease. Specifically, the UPS is responsible for the clearance of most damaged and misfolded proteins that are produced throughout the lifetime of a cell. UPS substrates (including aberrant proteins) are ubiquitinated, which targets them for irreversible ATP-dependent hydrolysis by the 26S proteasome. However, the molecular pathways and mechanisms by which ubiquitinated substrates are delivered to the 26S proteasome are not well characterized. Previous studies support that p97 (known as VCP in metazoans and Cdc48 in yeast) is important for the degradation of damaged and misfolded proteins possibly by preventing their aggregation and extracting them from aberrant complexes and aggregates. p97/VCP is an essential and conserved AAA [ATPase associated with various cellular activities] protein that forms a homo-hexameric ring, which associates with a wide variety of adapter proteins that determine p97 function. To date, the compositions of specific p97-containing complexes and their molecular mechanism of action are not well characterized. The goals of this research proposal are: (1) To use an affinity protein purification strategy and quantitative mass spectrometry to characterize the composition of specific endogenous p97 containing complexes, and to elucidate whether changes occur in p97-cofactor interactions with aging and in a poly-glutamate expansion disease model system. (2) To use cell biology assays to investigate the role of p97 in the degradation of short- lived proteins (which are mainly damaged and misfolded proteins) under normal cellular conditions. (3) To use a protein biochemistry approach to elucidate the mechanism of action by which p97 and associated co-factors function in the cell. Specifically, an in vitro system will be used to test whether purified p97-containg complexes can directly unfold an ubiquitinated model substrate. The completion of this proposal will provide insights into the protective mechanisms that cells use to prevent the accumulation and aggregation of damaged and misfolded proteins. Elucidating how cells achieve high-fidelity protein quality control may ultimately provide clues for the mitigation of cellular dysfunction that is associated with age-related protein aggregation diseases. To achieve these goals, the environment of Harvard Medical School and the expertise of Professor Goldberg and fellow laboratory members have provided me with extensive training in both the techniques used to isolate and study large AAA protein complexes and their associated proteins.

在正常的生理条件下，连续合成后蛋白损伤和错误折叠会产生异常蛋白质，这些蛋白质可能在细胞内形成错误和有毒的相互作用。在真核生物中，泛素蛋白酶体系统（UPS）对于确保蛋白质质量控​​制至关重要，蛋白质质量控​​制对于在衰老过程中保护细胞以及与年龄相关的蛋白质聚集障碍至关重要，例如神经退行性疾病，包括阿尔茨海默氏病和亨廷顿氏病。具体而言，UPS负责清除整个细胞生命周期产生的最受损和错误折叠的蛋白质。 UPS底物（包括异常蛋白）是泛素化的，它针对26S蛋白酶体的不可逆ATP依赖性水解。但是，将泛素化底物传递到26S蛋白酶体的分子途径和机制尚未很好地表征。先前的研究支持p97（在酵母中称为VCP，在酵母中称为VCP）对于可能通过防止其聚集并从异常的复合物和聚集物中提取损伤和错误折叠蛋白的降解至关重要。 p97/vcp是形成同型杂物环的蛋白质必不可少的AAA [与各种细胞活性相关的ATPase [ATPase]，该蛋白与确定p97功能的多种衔接蛋白相关联。迄今为止，特定含P97的复合物的组成及其分子作用机理的表征还没有很好地表征。 该研究建议的目标是：（1）使用亲和力蛋白纯化策略和定量质谱法来表征特定内源性p97含有复合物的组成，并阐明在p97-cactors与衰老相互作用中是否发生了变化，并且在多 - 谷氨酸盐扩张疾病模型模型系统中是否发生了变化。 （2）使用细胞生物学测定法研究p97在正常细胞条件下降解在短寿命蛋白（主要受损和错误折叠蛋白）中的作用。 （3）使用蛋白质生物化学方法来阐明p97和相关的副因素在细胞中起作用的作用机理。具体而言，体外系统将用于测试是否可以直接展开泛素化模型底物。 该提案的完成将提供有关细胞用于防止受损和错误折叠蛋白的积累和聚集的保护机制的见解。阐明细胞如何实现高保真蛋白质质量控​​制可能最终为缓解与年龄相关蛋白质聚集疾病相关的细胞功能障碍的缓解提供线索。为了实现这些目标，哈佛医学院的环境以及戈德堡教授和实验室成员的专业知识为我提供了对隔离和研究大型AAA蛋白复合物及其相关蛋白质的技术的广泛培训。