CHARACTERIZATION OF POLYGLUTAMINE AGGREGATES BY MASS SPECTROMETRY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Polyglutamine diseases are inherited neurodegenerative diseases caused by the expansion of polyglutamine tract in the disease-causing mutant proteins. A unifying pathological feature of polyglutamine disorders is the presence of microscopically discernible intracellular inclusions in the affected neurons. It has been proposed that expanded polyglutamine tract formed-aggregates participate in inappropriate protein-protein interactions that lead to cell death. To date, there is still no reliable method to isolate highly purified aggregates which are usually morphologically heterogeneous. We have developed a new purification method and the proteomics procedure was carried out to analyze the aggregate-interacting proteins by mass spectrometry. Mass spectrometry provides a powerful and direct analysis of aggregate proteins in tiny quantities and will help us understand the nativity of aggregates in the polyglutamine disorders. We have obtained mass spectral data that indicates the presence of several expected proteins in the aggregate and others that would not have been easily predicted. Immunochemical approaches have confirmed these aggregate interacting proteins. A manuscript describing our novel purification method was recently published. Recent experiments have provided evidence that the yeast cells form aggresomes from some isoforms of the Huntington proteins; this is the first indication that aggresomes can form in yeast. The manuscript describing the identification of proteins that are required for aggresome formation and presenting our new model for aggresome formation has been published in FASEB journal. Extensive details are included in the PhD thesis of Y. Wang, completed in June 2008. In the current phase of the research, human cells are being employed. The components of the complex are again being identified using proteomic approaches. In addition, their spatial relationships to one another will be probed using the top-down crosslinking methods being developed within a TRD project.