CHARACTERIZATION OF HUNTINGTON AND PARKINSON AGGREGATES BY MASS SPECTROMETRY

Boston University Medical Campus, Boston MA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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 developed a new purification method and the proteomics procedure was carried out to analyze the aggregate-interacting proteins by mass spectrometry. MS 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 obtained MS data that indicates the presence of several expected proteins in the aggregate and others that would not have been easily predicted. Immunochemical approaches confirmed these aggregate interacting proteins;these results have been published. later experiments provided evidence that the yeast cells form aggresomes from some isoforms of the Huntington proteins;this was 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. Abnormal protein aggregates can cause cell toxicity leading to various other pathologies, including Parkinson disease (PD). Synphilin-1 is one of the major components of these inclusions, and is implicated in the pathogenesis of PD. Expression of Synphilin-1 led to cytoplasmic aggregates, which were transported to form aggresomes upon proteasome inhibition. Deletion of Synphilin-1 functional domains revealed that unknown signaling proteins must be associated with Synphilin-1 to trigger aggresome formation [N. Zaarur, et al., J Biol Chem, 2008, 283, 27575-27584]. The Sherman group has recently reported identification and quantification of these unknown protein candidates, and investigation of their relevant functions and pathways. in the current studies, Synphilin-1 was constructed to carry a His-tag and a GFP-tag at the N- and C- termini, respectively. A plasmid containing this recombinant synphinlin-1 was produced and transfected into HEK293T cells. Synphilin-1 and associated proteins were purified from cells treated with proteasome inhibitor or na[unreadable]ve cells. Purified synphilin-1 protein complexes were digested by trypsin in solution, analyzed by data-dependent nano-capillary reversed-phase LC-MS/MS using an LTQ-Orbitrap MS (Thermo Scientific) in triplicate. Label-free quantification to determine relative protein expression levels was performed using Progenesis LCMS (Nonlinear Dynamics). Proteins were identified by searching against the Swiss-Prot human protein database using MASCOT (Matrix Science). To address nonspecific interactors, vector transfected HEK293T cells were treated identically and analyzed in parallel as a negative control. Significantly regulated proteins (p<0.05, fold change>2) were subjected to network, pathway and function analyses using Ingenuity pathway analysis (Ingenuity) to determine interacting partner candidates and their involvement in pathways and cellular functions. We determined that the Synphinlin-1 complex was isolated with high efficiency. Network, pathway, and function analyses of significant regulated proteins (p<0.05, fold change>2) determined by label-free quantification (Figure 1) were performed. Network analysis uncovered potential protein candidates for further Western blot validations. Pathway analysis revealed regulated proteins involved in the protein ubiquitination pathway, which is critical for degradation of abnormal protein aggregates. Functional analysis showed that regulated proteins mainly affected protein synthesis and RNA post-transcriptional modification, which impact on formation of abnormal proteins. These results indicate that Synphilin-1 and its interactors are involved in both protein synthesis and degradation to regulate formation of protein aggregates and aggresomes.