Activity-Based Profiling Reveals Reactivity of the Murine Thymoproteasome-Specific Subunit β5t

SummaryEpithelial cells of the thymus cortex express a unique proteasome particle involved in positive T cell selection. This thymoproteasome contains the recently discovered β5t 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 β5t subunit is catalytically active in the murine thymus. A panel of established proteasome inhibitors showed that the broad-spectrum inhibitor epoxomicin blocks the β5t activity and that the subunit-specific antagonists bortezomib and NC005 do not inhibit β5t. We show that β5t has a substrate preference distinct from β5/β5i that might explain how the thymoproteasome generates the MHC class I peptide repertoire needed for positive T cell selection.

► Activity-based profiling with fluorescent epoxomicin shows a new band in murine thymus
► LC-MS3 analysis identified this activity as the thymoproteasome-specific β5t subunit β5t prefers a hydrophilic residue in a hydrophobic stretch shown by competitive ABP
► Thus, β5t is actively involved in positive T cell selection.