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: 8519202
• 负责人: Eugene Drokhlyansky
• 金额: $ 2.96万
• 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/8519202
• 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.

在正常生理条件下，连续的合成后蛋白质损伤和错误折叠产生异常蛋白质，其可在细胞内形成错误和毒性相互作用。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是必不可少的，这对于在衰老期间保护细胞以及对抗与年龄相关的蛋白质聚集病症（诸如神经退行性疾病，包括阿尔茨海默病和亨廷顿病）至关重要。具体而言，UPS负责清除细胞整个生命周期中产生的大多数受损和错误折叠的蛋白质。UPS底物（包括异常蛋白质）是泛素化的，其靶向它们通过26 S蛋白酶体进行不可逆的ATP依赖性水解。然而，遍在蛋白化底物传递到26 S蛋白酶体的分子途径和机制尚未得到很好的表征。先前的研究支持p97（在后生动物中称为VCP，在酵母中称为Cdc 48）对于受损和错误折叠的蛋白质的降解是重要的，可能通过防止它们的聚集并将它们从异常复合物和聚集物中提取出来。p97/VCP是一种重要的保守AAA [ATP酶与各种细胞活性相关]蛋白，形成同源六聚体环，与多种决定p97功能的衔接蛋白相关。迄今为止，特定的p97-含有复合物的组合物和它们的分子作用机制没有得到很好的表征。 本研究的目标是：（1）使用亲和蛋白纯化策略和定量质谱来表征特定的内源性p97复合物的组成，并阐明p97-辅因子相互作用与衰老和多聚谷氨酸扩增疾病模型系统中是否发生变化。(2)使用细胞生物学试验研究正常细胞条件下p97在短寿命蛋白（主要是受损和错误折叠的蛋白）降解中的作用。(3)使用蛋白质生物化学方法阐明p97和相关辅因子在细胞中发挥功能的作用机制。具体而言，将使用体外系统来测试纯化的含p97的复合物是否可以直接解折叠泛素化的模型底物。 该提案的完成将为细胞用于防止受损和错误折叠蛋白质的积累和聚集的保护机制提供见解。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的蛋白质聚集疾病相关的细胞功能障碍提供线索。为了实现这些目标，哈佛医学院的环境和Goldberg教授和其他实验室成员的专业知识为我提供了广泛的培训，用于分离和研究大型AAA蛋白复合物及其相关蛋白质的技术。