Recent reports concluded that tripeptidyl peptidase (TPPII) is essential for MHC class I Ag presentation and that the proteasome in vivo mainly releases peptides 16 residues or longer that require processing by TPPII. However, we find that eliminating TPPII from human cells using small interfering RNA did not decrease the overall supply of peptides to MHC class I molecules and reduced only modestly the presentation of SIINFEKL from OVA, while treatment with proteasome inhibitors reduced these processes dramatically. Purified TPPII digests peptides from 6 to 30 residues long at similar rates, but eliminating TPPII in cells reduced the processing of long antigenic precursors (14-17 residues) more than short ones (9-12 residues). Therefore, TPPII appears to be the major peptidase capable of processing proteasome products longer than 14 residues. However, proteasomes in vivo (like purified proteasomes) release relatively few such peptides, and these peptides processed by TPPII require further trimming in the endoplasmic reticulum (ER) by ER aminopeptidase 1 for presentation. Taken together, these observations demonstrate that TPPII plays a specialized role in Ag processing and one that is not essential for the generation of most presented peptides. Moreover, these findings reveal that three sequential proteolytic steps (by proteasomes, TPPII, and then ER aminopepsidase 1) are required for the generation of a subset of epitopes.

Most antigenic peptides presented on MHC class I molecules are generated by proteasomes during protein breakdown. It is unknown whether these peptides are protected from destruction by cytosolic peptidases. In cytosolic extracts, most antigenic peptides are degraded by the metalloendopeptidase, thimet oligopeptidase (TOP). We therefore examined whether TOP destroys antigenic peptides in vivo. When TOP was overexpressed in cells, class I presentation of antigenic peptides was reduced. In contrast, TOP overexpression didn't reduce presentation of peptides generated in the endoplasmic reticulum or endosomes. Conversely, preventing TOP expression with siRNA enhanced presentation of antigenic peptides. TOP therefore plays an important role in vivo in degrading peptides released by proteasomes and is a significant factor limiting the extent of antigen presentation.

Most of the MHC class I peptides presented to the immune system are generated during the course of protein breakdown by the proteasome. However, the precise role of the proteasome, e.g., whether this particle or some other protease generates the carboxyl (C) and amino (N) termini of the presented 8- to 10-residue peptides, is not clear. Here, we show that presentation on Db of ASNENMETM, a peptide from influenza nucleoprotein, and on Kb of FAPGNYPAL, a peptide from Sendai virus nucleoprotein, was blocked by the proteasome inhibitor, lactacystin. Using plasmid minigene constructs encoding oligopeptides of various lengths, we found that presentation of ASNENMETM from C-terminally extended peptides that contain this antigenic peptide plus three or five additional amino acids and presentation of FAPGNYPAL from a peptide containing FAPGNYPAL plus one additional C-terminal residue required the proteasome. In contrast, the proteasome inhibitor did not reduce presentation of cytosolically expressed ASNENMETM or FAPGNYPAL or N-terminally extended versions of these peptides, suggesting involvement of aminopeptidase(s) in trimming these N-extended variants. Accordingly, when the N termini of these 3N-extended peptides were blocked by acetylation, they were resistant to hydrolysis by cellular aminopeptidases and pure leucine aminopeptidase. Moreover, if introduced into the cytosol, Ag presentation of these peptides occurred to a much lesser extent than from their nonacetylated counterparts. Thus, the proteasome is essential for the generation of ASNENMETM and FAPGNYPAL peptides from the full-length nucleoproteins. Although it generates the C termini of these presented peptides, distinct aminopeptidase(s) can trim the N termini of these presented peptides to their proper size.