title: Proteomic characterization of aggregating proteins after the inhibition of the ubiquitin proteasome system.
abstract: Protein aggregation, which is associated with the impairment of the ubiquitin proteasome system, is a hallmark of many neurodegenerative diseases. To better understand the contribution of proteasome inhibition in aggregation, we analyzed which proteins may potentially localize in chemically induced aggregates in human neuroblastoma tissue culture cells. We enriched for proteins in high-density structures by using a sucrose gradient in combination with stable isotope labeling with amino acids in cell culture (SILAC). The quantitative analysis allowed us to distinguish which proteins were specifically affected by the proteasome inhibition. We identified 642 potentially aggregating proteins, including the p62/sequestosome 1 and NBR1 ubiquitin-binding proteins involved in aggregation. We also identified the ubiquitin-associated protein 2 like (UBAP2L). We verified that it cofractionated with ubiquitin in the high-density fraction and that it was colocalized in the ubiquitin-containing aggregates after proteasome inhibition. In addition, we identified several chaperone proteins and used data from protein interaction networks to show that they potentially interact with distinct subgroups of proteins within the aggregating structures. Several other proteins associated with neurodegenerative diseases, like UCHL1, were identified, further underlining the potential of our analysis to better understand the aggregation process and proteotoxic stress caused by proteasome inhibition.
authors.affiliation: Department of Biochemistry and Molecular Biology, Centre for High-Throughput Biology, University of British Columbia, 2125 East Mall, Vancouver, British Columbia, V6T 1Z4, Canada.
authors.forename: Inga B
authors.initials: IB
authors.lastname: Wilde
authors.forename: Maria
authors.initials: M
authors.lastname: Brack
authors.forename: Jason M
authors.initials: JM
authors.lastname: Winget
authors.forename: Thibault
authors.initials: T
authors.lastname: Mayor
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D061988:Proteasome Inhibitors; D011506:Proteins; D020543:Proteome; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex