The Final N-Terminal Trimming of a Subaminoterminal Proline-Containing HLA Class I-Restricted Antigenic Peptide in the Cytosol Is Mediated by Two Peptidases

Abstract

The proteasome produces MHC class I-restricted antigenic peptides carrying N-terminal extensions, which are trimmed by other peptidases in the cytosol or within the endoplasmic reticulum. In this study, we show that the N-terminal editing of an antigenic peptide with a predicted low TAP affinity can occur in the cytosol. Using proteomics, we identified two cytosolic peptidases, tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, that trimmed the N-terminal extensions of the precursors produced by the proteasome, and led to a transient enrichment of the final antigenic peptide. These peptidases acted either sequentially or redundantly, depending on the extension remaining at the N terminus of the peptides released from the proteasome. Inhibition of these peptidases abolished the CTL-mediated recognition of Ag-expressing cells. Although we observed some proteolytic activity in fractions enriched in endoplasmic reticulum, it could not compensate for the loss of tripeptidyl peptidase II/puromycin-sensitive aminopeptidase activities.

An integrative approach to CTL epitope prediction: A combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions

“…The major cytosolic protease associated with the generation of antigenic peptides -in particular the C-terminal end of the peptides -is the proteasome [2][3][4][5][6]. After protea-somal cleavage the peptides may be trimmed at the Nterminal end by other peptidases in the cytosol [7]. The next step is the translocation of the peptides from the cytosol to the interior of the ER.…”

Re-evaluating the Generation of a “Proteasome-Independent” MHC Class I-Restricted CD8 T Cell Epitope

“…Despite several observations that suggest peptides are available for MHC I folding in adapted cells and that up-regulated TPP II can prevent the accumulation of ubiquitinylated proteins, the processing and presentation of specific epitopes in proteasome-inhibitor adapted cells has not been fully examined. In addition, although TPP II can trim protein fragments under cellfree conditions to generate antigenic peptides (26,32), the precise Ag processing role of this protease in intact cells remains unclear. L-K d cells were adapted to grow in the presence of the irreversible inhibitor Z-L 3 -VS essentially as described by Glas et al (25).…”

“…These results suggest that an AAF-cmk-sensitive proteolytic activity can contribute to the processing of NP 147-155 in normal cells and NP 147-155 and other epitopes in proteasome inhibitor adapted cells. TPP II is an obvious candidate for this processing activity given its role in compensating for chronic proteasome inhibition (25,26,29) and studies reporting a trimming (31,32) or proteasome-independent (30) processing role for this enzyme. However, AAF-cmk may affect other proteases and despite the potent inhibition of TPP II activity by AAF-cmk, epitope presentation was blocked by only ϳ10 -50% in adapted cells.…”

“…However, the capacity of cells with chronically impaired proteasomes to present specific peptides to CD8 T cells has not been completely addressed, and the contribution of up-regulated protease activities, including TPP II, to Ag processing under these conditions requires additional investigation. TPP II has been proposed to perform proteasome-independent processing (30) or proteasome-dependent trimming (31,32). Therefore, TPP II could account for the first steps in epitope generation under conditions of proteasome inhibition, or, alternatively, TPP II and/or additional proteases may trim proteasomal products to liberate precise epitopes and this activity may be more important when proteasome function is suboptimal.…”

“…In addition to TPP II, several reports have demonstrated that other nonproteasomal cytosolic proteases may contribute to the generation or destruction of antigenic peptides via post proteasomal trimming. These include LA (33), thymet oligopeptidase (TO) (34,35), bleomycin hydrolase (BH), and puromycin-sensitive aminopeptidase (PSA) (32,36). In addition to these cytosolic proteases, ER-resident amino peptidase (ERAAP or ERAP1 in the mouse, ERAP1/2 complex in the human) can further trim peptides at the N terminus once they have been transported via TAP (10 -13).…”