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.
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 new research directions and therapeutic targets for the treatment of cellular dysfunction that is associated with age-related protein aggregation diseases that include Alzheimer's disease and Huntington's disease.
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