Characterization of proteins associated with polyglutamine aggregates: a novel approach towards isolation of aggregates from protein conformation disorders.
The common feature of many neurodegenerative diseases is emergence of protein aggregates. Identifying their composition can provide valuable insights into the cellular mechanisms of protein aggregation and neuronal death. No reliable method for identification of the aggregate-associated proteins has been available. Here we describe a method for characterization of protein aggregates based on sedimentation of immunocomplexes without involvement of a solid support. As a model, we used the aggregates formed in yeast by a polyglutamine-containing segment of mutant huntingtin. Sixteen proteins associated with the isolated aggregates were identified with 2-D gel electrophoresis followed by mass spectrometry. We found that the aggregates in cells lacking Rnq1 prion recruited lesser amounts of chaperones than those in the wild-type cells. The method can be utilized for characterization of various types of aggregates, prions and very large protein complexes under mild conditions that preserve associated proteins.
Wang
Yan
Y
Department of Biochemistry, Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Meriin
Anatoli B
AB
Costello
Catherine E
CE
Sherman
Michael Y
MY
D006801:Humans; D000071058:Huntingtin Protein; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019636:Neurodegenerative Diseases / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011328:Prions / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
C086055:HTT protein, human; D000071058:Huntingtin Protein; D018832:Molecular Chaperones; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011328:Prions; C404807:RNQ1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C097188:polyglutamine