title: Activity-based profiling reveals reactivity of the murine thymoproteasome-specific subunit beta5t.
abstract: Epithelial cells of the thymus cortex express a unique proteasome particle involved in positive T cell selection. This thymoproteasome contains the recently discovered beta5t subunit that has an uncharted activity, if any. We synthesized fluorescent epoxomicin probes that were used in a chemical proteomics approach, entailing activity-based profiling, affinity purification, and LC-MS identification, to demonstrate that the beta5t subunit is catalytically active in the murine thymus. A panel of established proteasome inhibitors showed that the broad-spectrum inhibitor epoxomicin blocks the beta5t activity and that the subunit-specific antagonists bortezomib and NC005 do not inhibit beta5t. We show that beta5t has a substrate preference distinct from beta5/beta5i that might explain how the thymoproteasome generates the MHC class I peptide repertoire needed for positive T cell selection.
authors.affiliation: Leiden Institute of Chemistry and Netherlands Proteomics Centre, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands. b.florea@chem.leidenuniv.nl
authors.forename: Bogdan I
authors.initials: BI
authors.lastname: Florea
authors.forename: Martijn
authors.initials: M
authors.lastname: Verdoes
authors.forename: Nan
authors.initials: N
authors.lastname: Li
authors.forename: Wouter A
authors.initials: WA
authors.lastname: van der Linden
authors.forename: Paul P
authors.initials: PP
authors.lastname: Geurink
authors.forename: Hans
authors.initials: H
authors.lastname: van den Elst
authors.forename: Tanja
authors.initials: T
authors.lastname: Hofmann
authors.forename: Arnoud
authors.initials: A
authors.lastname: de Ru
authors.forename: Peter A
authors.initials: PA
authors.lastname: van Veelen
authors.forename: Keiji
authors.initials: K
authors.lastname: Tanaka
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Sasaki
authors.forename: Shigeo
authors.initials: S
authors.lastname: Murata
authors.forename: Hans
authors.initials: H
authors.lastname: den Dulk
authors.forename: Jaap
authors.initials: J
authors.lastname: Brouwer
authors.forename: Ferry A
authors.initials: FA
authors.lastname: Ossendorp
authors.forename: Alexei F
authors.initials: AF
authors.lastname: Kisselev
authors.forename: Herman S
authors.initials: HS
authors.lastname: Overkleeft
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002849:Chromatography, Gas; D002853:Chromatography, Liquid; D051379:Mice; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*
chemical_list: D021122:Protein Subunits; D046988:Proteasome Endopeptidase Complex
references.citation: Chembiochem. 2008 Jul 21;9(11):1735-8
references.pmid: 18600815
references.citation: Annu Rev Biochem. 2008;77:383-414
references.pmid: 18366325
references.citation: Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):6629-34
references.pmid: 9192616
references.citation: Science. 2007 Jun 1;316(5829):1349-53
references.pmid: 17540904
references.citation: Chem Biol. 2006 Nov;13(11):1217-26
references.pmid: 17114003
references.citation: Anal Biochem. 1984 Apr;138(1):141-3
references.pmid: 6731838
references.citation: Chem Biol. 2009 Dec 24;16(12):1278-89
references.pmid: 20064438
references.citation: Bioorg Med Chem Lett. 1998 Feb 17;8(4):333-8
references.pmid: 9871680
references.citation: Annu Rev Immunol. 1999;17:739-79
references.pmid: 10358773
references.citation: Cell. 2005 Jul 29;122(2):247-60
references.pmid: 16051149
references.citation: Nat Protoc. 2006;1(6):2856-60
references.pmid: 17406544
references.citation: Curr Opin Immunol. 2008 Apr;20(2):192-6
references.pmid: 18403190
references.citation: Nature. 2008 Sep 4;455(7209):64-71
references.pmid: 18668037
references.citation: Trends Immunol. 2008 Jun;29(6):251-5
references.pmid: 18472300
references.citation: Curr Opin Immunol. 2004 Feb;16(1):76-81
references.pmid: 14734113
references.citation: Methods Enzymol. 2005;398:364-78
references.pmid: 16275343
references.citation: Mol Cell Proteomics. 2005 Dec;4(12):2010-21
references.pmid: 16249172
references.citation: Nat Protoc. 2007;2(8):1896-906
references.pmid: 17703201
references.citation: Cell. 1998 Feb 6;92(3):367-80
references.pmid: 9476896
references.citation: Org Biomol Chem. 2007 May 7;5(9):1416-26
references.pmid: 17464411
references.citation: Cell. 1994 Jan 28;76(2):275-85
references.pmid: 7904901
references.citation: Nature. 2007 Oct 18;449(7164):819-26
references.pmid: 17943118
references.citation: Science. 1995 May 5;268(5211):726-31
references.pmid: 7732382
references.citation: Adv Immunol. 2008;99:59-94
references.pmid: 19117532
references.citation: Chem Biol. 2001 Sep;8(9):913-29
references.pmid: 11564559