The role of p97 in protein degradation during aging and disease

在正常的生理条件下，持续的蛋白质合成后损伤和错误折叠会产生异常蛋白质，从而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)是确保蛋白质质量控制的关键，蛋白质质量控制对于保护细胞在衰老过程中以及对抗与年龄相关的蛋白质聚集障碍至关重要，如阿尔茨海默病和亨廷顿病等神经退行性疾病。具体地说，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)被泛素化，这使它们成为26S蛋白酶体不可逆的依赖于ATP的水解酶的靶标。然而，泛素化底物被运送到26S蛋白酶体的分子途径和机制还没有得到很好的描述。以前的研究支持p97(在后生动物中称为VCP，在酵母中称为CDC48)对受损和错误折叠的蛋白质的降解起重要作用，可能是通过阻止它们聚集并从异常的复合体和聚集体中提取它们。P97/VCP是一种必需和保守的AAA[与各种细胞活动相关的ATPase]蛋白，它形成一个同源六聚体环，与决定p97功能的多种接头蛋白结合。到目前为止，含有p97的特定络合物的组成和它们的分子作用机制还没有得到很好的表征。 这项研究的目标是：(1)使用亲和蛋白纯化策略和定量质谱学来表征特定的内源性p97复合体的组成，并阐明p97辅因子与衰老的相互作用是否发生变化，以及在多谷氨酸扩张性疾病模型系统中是否发生变化。(2)用细胞生物学方法研究p97在正常细胞条件下降解短寿命蛋白(主要是损伤和错误折叠的蛋白)中的作用。(3)用蛋白质生物化学方法阐明p97及其相关辅助因子在细胞中的作用机制。具体地说，体外系统将被用来测试纯化的含有p97的复合体是否可以直接展开泛素化的模型底物。 这项提议的完成将提供对细胞用来防止受损和错误折叠的蛋白质积累和聚集的保护机制的洞察。阐明细胞如何实现高保真的蛋白质质量控制，最终可能为缓解与年龄相关的蛋白质聚集性疾病相关的细胞功能障碍提供线索。为了实现这些目标，哈佛医学院的环境以及Goldberg教授和其他实验室成员的专业知识为我提供了用于分离和研究大型AAA蛋白质复合体及其相关蛋白质的技术方面的广泛培训。

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.