Activity-based profiling reveals reactivity of the murine thymoproteasome-specific subunit beta5t.
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.
Florea
Bogdan I
BI
Leiden Institute of Chemistry and Netherlands Proteomics Centre, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands. b.florea@chem.leidenuniv.nl
Verdoes
Martijn
M
Li
Nan
N
van der Linden
Wouter A
WA
Geurink
Paul P
PP
van den Elst
Hans
H
Hofmann
Tanja
T
de Ru
Arnoud
A
van Veelen
Peter A
PA
Tanaka
Keiji
K
Sasaki
Katsuhiro
K
Murata
Shigeo
S
den Dulk
Hans
H
Brouwer
Jaap
J
Ossendorp
Ferry A
FA
Kisselev
Alexei F
AF
Overkleeft
Herman S
HS
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*
D021122:Protein Subunits; D046988:Proteasome Endopeptidase Complex
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