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.

Figure 1. Activity-Based Probes and Proteasome Inhibitors Used in This Study

In addition to the enzyme reactive group (warhead) and targeting sequence of the inhibitors, activity-based probes are equipped with a fluorophore for in-gel detection, a biotin tag for affinity purification or with both.

Figure 2. Activity-Based Profiling, Affinity Purification, and LC-MS Identification of Proteasome β Subunits in Murine Tissues Lysates

(A) In-gel fluorescence detection of active proteasome β subunits in 3-week-old wild-type murine thymus homogenate after labeling with MV151, ABP 1, 2, and 4 (see also Figure S1). M indicates the molecular marker band of 25 kDa. (B) In-gel fluorescence and silver stain detection of active proteasome β subunits in young and adult thymus after labeling with ABP 1, 2, 3, and affinity purification. Protein identification by LC-MS analysis of in-gel digested silver-stained bands (see Tables S1 and S2 for details). (C) In-gel fluorescence detection with ABP 4 of β5t activity in wild-type and absence of activity in the (−/−) β5t knockdown thymus from 3-weeks-old mice. (D) Activity-based proteasome profiling using ABP 4 shows β5t activity in murine thymus (young and adult) but not in heart, lung, liver, spleen, brain, testes, or kidney.

Figure 3. Active-Site Peptide Identification and Determination of Proteasome β Subunits by Affinity Purification, Tryptic Digest, and LC-MS Analysis

(A) Reaction mechanism of biotin-epoxomicin 3 with the catalytically active N-terminal Thr residue of active proteasome β subunits. The morpholino ring formation results in a covalent and irreversible binding. (B) Schematic representation of the biotin-epoxomicin modified, N-terminal active-site tryptic peptide of β5t. Amino acid residues are represented in a three-letter code. (C) LC-MS elution profile of the six unique biotinylated tryptic peptides derived from the active sites. Notice that β5 and β5i active-site peptides are identical (see also Table S3). (D) LC-MS² determination of the β5t active-site fragmentation pattern. The parent ion [m/z (M+2H)²⁺ = 767.44] was fragmented. The b₁, b₂, b₃, and b₄ ions are signature ions of the biotin-epoxomicin N-terminal part. The abundant y₇ ion containing the β5t active-site peptide sequence was selected for further (MS³) fragmentation (see E). (E) LC-MS³ determination of the y₇ ion (MH⁺ = 821.32) revealing the β5t active-site peptide amino acid sequence.

Figure 4. Analysis of β5t Substrate Specificity in Juvenile Murine Thymus Lysates by Competitive Activity-Based Profiling with ABP 4

(A) Lysates were exposed to increasing concentrations Lactacystin or MG132; residual β5t activity was stained with ABP 4 and visualized by in-gel fluorescence detection. The inhibitors are not reactive toward the β5t activity. (B) Bortezomib efficiently inhibits β5, β5i, β1, and β1i activity but not β5t. NC005 specifically targets β5 (indicated by the gray arrow) but not β5t. The mixed inhibitor containing the boronic ester warhead equipped with the epoxomicin tail efficiently blocks β5t. (C) In analogy to (B), installation of the epoxomicin IleIleThrLeu peptide targeting motif to the vinyl sulphone warhead affords a potent inhibitor of the β5t activity.