title: Elucidating the catalytic subunit composition of distinct proteasome subtypes: a crosslinking approach employing bifunctional activity-based probes.
abstract: In addition to two well-recognized proteasome subtypes-constitutive proteasomes and immunoproteasomes-mounting evidence also suggests the existence of intermediate proteasome subtypes containing unconventional mixtures of catalytic subunits. Although they appear to play unique biological roles, the lack of practical methods for detecting distinct proteasome subtypes has limited functional investigations. Here, we report the development of activity-based probes that crosslink two catalytic subunits within intact proteasome complexes. Identification of the crosslinked subunit pairs provides direct evidence of the catalytic subunit composition of proteasomes. Using these probes, we found that U266 multiple myeloma cells contain intermediate proteasomes comprising both β1i and β2, but not β1 and β2i, consistent with previous findings with other cell types. Our bifunctional probes can be utilized in functional investigations of distinct proteasome subtypes in various biological settings.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536-0596 (USA).
authors.forename: Kimberly
authors.initials: K
authors.lastname: Cornish Carmony
authors.forename: Lalit Kumar
authors.initials: LK
authors.lastname: Sharma
authors.forename: Do-Min
authors.initials: DM
authors.lastname: Lee
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Park
authors.forename: Wooin
authors.initials: W
authors.lastname: Lee
authors.forename: Kyung-Bo
authors.initials: KB
authors.lastname: Kim
mesh_terms: D020134:Catalytic Domain; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000737:chemistry / Q000494:pharmacology; D009101:Multiple Myeloma / Q000201:enzymology; D011092:Polyethylene Glycols / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000737:chemistry / Q000494:pharmacology; D013912:Threonine / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D015335:Molecular Probes; D061988:Proteasome Inhibitors; D013912:Threonine; D011092:Polyethylene Glycols; D046988:Proteasome Endopeptidase Complex
keywords: constitutive proteasome; crosslinking agents; immunoproteasome; intermediate proteasome; proteasome subtype