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，持续的合成后蛋白损伤和错误折叠会产生异常蛋白，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，它对于在衰老过程中保护细胞和防止与年龄相关的蛋白质聚集疾病（如阿尔茨海默氏病和亨廷顿氏病）至关重要。具体来说，UPS负责清除细胞一生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）被泛素化，其目标是26S蛋白酶体不可逆的atp依赖性水解。然而，泛素化底物传递到26S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Cdc48）可能通过阻止它们的聚集和从异常复合物和聚集体中提取它们来降解受损和错误折叠的蛋白质。p97/VCP是一种重要且保守的AAA[与各种细胞活动相关的atp酶]蛋白，它形成一个同型六聚体环，与多种决定p97功能的适配器蛋白相关。迄今为止，含p97的特异性配合物的组成及其作用的分子机制尚未得到很好的表征。