title: A FRET-based approach for identification of proteasome catalytic subunit composition.
abstract: Mammalian cells have two main types of proteasomes, the constitutive proteasome and the immunoproteasome, each containing a distinct set of three catalytic subunits. Recently, additional proteasome subtypes containing a non-standard mixture of catalytic subunits have gained increasing attention, especially due to their presence in cancer settings. However, practical methods for identifying proteasome subtypes have been lacking. Here, we report the development of the first fluorescence resonance energy transfer (FRET)-based strategy that can be utilized to identify different proteasome subtypes present within cells. We have developed FRET donor- and acceptor-probes that are based on previously reported peptide epoxyketones and selectively target individual proteasome catalytic subunits. Using the purified proteasome and cancer cell lysates, we demonstrate the feasibility of a FRET-based approach for determining the catalytic subunit composition of individual 20S proteasome subtypes. Ultimately, this approach may be utilized to study the functions of individual proteasome subtypes in cells.
authors.affiliation: Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky, USA 40536-0596. kbkim2@uky.edu.
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Park
authors.forename: Ying
authors.initials: Y
authors.lastname: Wu
authors.forename: Kimberly Cornish
authors.initials: KC
authors.lastname: Carmony
authors.forename: Zachary
authors.initials: Z
authors.lastname: Miller
authors.forename: Lalit Kumar
authors.initials: LK
authors.lastname: Sharma
authors.forename: Do-Min
authors.initials: DM
authors.lastname: Lee
authors.forename: Doo-Young
authors.initials: DY
authors.lastname: Kim
authors.forename: Wooin
authors.initials: W
authors.lastname: Lee
authors.forename: Kyung-Bo
authors.initials: KB
authors.lastname: Kim
mesh_terms: D020134:Catalytic Domain; D015180:Electrophoresis, Gel, Two-Dimensional; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D005456:Fluorescent Dyes; D046988:Proteasome Endopeptidase Complex