Proteomics of polyglutamine aggregates.
In nine members of polyglutamine (polyQ) diseases, CAG repeat expansions of their responsible genes are observed. The disease is considered to be caused by the formation of polyQ aggregates that sequester proteins essential for cell viability. To understand the pathological process of polyQ diseases, a proteomic approach was used to identify aggregate interacting proteins (AIPs). Constructs were designed to express EGFP-fused, CAG-expanded (150 Q) huntingtin exon1 under the control of an ecdysone-inducible promoter and either lacking or containing a nuclear localization signal (NLS). After induction of a stably transfected Neuro 2A cell line with ecdysone, aggregates form in either the cytoplasm or the nucleus. The aggregates in these two different compartments were isolated with different methods. Cytoplasmic aggregate particles were purified using a fluorescence-activated cell sorter (FACS) by monitoring EGFP fluorescence, whereas nuclear aggregates were purified by using the detergent insoluble nature of aggregates. The resulting highly pure aggregates were subjected to SDS-PAGE followed by Coomassie blue staining. Bands containing AIP candidates were excised, and, after in-gel digestion with trypsin, were analyzed by mass spectrometry to identify the proteins. Novel candidates were confirmed as AIPs by immunocytological analysis to observe colocalization with polyQ aggregates. This chapter describes methods for the establishment of stable mutant cells, the purification of polyQ aggregates, and sample preparation for mass spectrometry analysis in detail.
Mitsui
Kenichi
K
Research Resources Center, Riken Brain Science, Institute, Saitama 351-0198, Japan.
Doi
Hiroshi
H
Nukina
Nobuyuki
N
D000818:Animals; D001921:Brain / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D013058:Mass Spectrometry; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics; D009447:Neuroblastoma; D009687:Nuclear Proteins / Q000235:genetics; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D019680:Trinucleotide Repeat Expansion / Q000235:genetics
C086055:HTT protein, human; C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C097188:polyglutamine