Pathway for Degradation of Peptides Generated by Proteasomes

Abstract

The degradation of cellular proteins by proteasomes generates peptides 2-24 residues long, which are hydrolyzed rapidly to amino acids. To define the final steps in this pathway and the responsible peptidases, we fractionated by size the peptides generated by proteasomes from ␤-[ 14 C]casein and studied in HeLa cell extracts the degradation of the 9 -17 residue fraction and also of synthetic deca-and dodecapeptide libraries, because peptides of this size serve as precursors to MHC class I antigenic peptides. Their hydrolysis was followed by measuring the generation of smaller peptides or of new amino groups using fluorescamine. The 14 C-labeled peptides released by 20 S proteasomes could not be degraded further by proteasomes. However, their degradation in the extracts and that of the peptide libraries was completely blocked by o-phenanthroline and thus required metallopeptidases. One such endopeptidase, thimet oligopeptidase (TOP), which was recently shown to degrade many antigenic precursors in the cytosol, was found to play a major role in degrading proteasome products. Inhibition or immunodepletion of TOP decreased their degradation and that of the peptide libraries by 30 -50%. Pure TOP failed to degrade proteasome products 18 -24 residues long but degraded the 9 -17 residue fraction to peptides of 6 -9 residues. When aminopeptidases in the cell extract were inhibited with bestatin, the 9 -17 residue proteasome products were also converted to peptides of 6 -9 residues, instead of smaller products. Accordingly, the cytosolic aminopeptidase, leucine aminopeptidase, could not degrade the 9 -17 residue fraction but hydrolyzed the peptides generated by TOP to smaller products, recapitulating the process in cell extracts. Inactivation of both TOP and aminopeptidases blocked the degradation of proteasome products and peptide libraries nearly completely. Thus, degradation of most 9 -17 residue proteasome products is initiated by endoproteolytic cleavages, primarily by TOP, and the resulting 6 -9 residue fragments are further digested to amino acids by aminopeptidases.

The ER aminopeptidase, ERAP1, trims precursors to lengths of MHC class I peptides by a “molecular ruler” mechanism

“…2 A), these cytosolic aminopeptidases digested the 5-residue QL5 most rapidly and the 9-and 13-mer much more slowly. The finding that leucine aminopeptidase and puromycin-sensitive aminopeptidase degrade shorter peptides preferentially is consistent with the prior findings (35) that cytosolic aminopeptidases, by destroying very short peptides (2-5 residues), catalyze the final steps in the ubiquitinproteasome pathway (34).…”

“…One other metallopeptidase is known that also prefers substrates 8-16 residues long, thimet oligopeptidase. However, it is a cytosolic endopeptidase that cleaves most proteasomal products of this length (34). By destroying cytosolic precursors, this activity limits the supply of antigenic peptides to the ER (40).…”

“…Its strong preference for substrates longer than eight residues clearly distinguishes ERAP1 from the closely homologous members of the M1 family of aminopeptidases [e.g., aminopeptidase B (38) and leukotriene A4 hydrolase (39)], which strongly prefer di-or tripeptides, and from the major cytosolic aminopeptidases (Fig. 4), leucine aminopeptidase and puromycinsensitive aminopeptidase (also a member of the M1 family), and from the several aminopeptidase activities present in mammalian cell extracts (34). These intracellular enzymes strongly prefer peptides shorter than five residues, which is consistent with their carrying out the final steps in the complete degradation of proteins in the cytosol (35).…”

“…Even though no such properties have been reported, we examined whether the major cytosolic aminopeptidases might perhaps show a similar length preference. We therefore incubated QLESIIN-FEKL and analogs with different C-terminal residues with leucine aminopeptidase and puromycin-sensitive aminopeptidase, both of which also have been implicated in trimming some antigenic precursors in the cytosol (16,17) but are probably primarily involved in degrading short peptides to amino acids released by proteasomes (34). Unlike ERAP1, these aminopeptidases degraded the 10-residue peptides at identical slow rates that were independent of their C termini ( Table 2, which is published as supporting information on the PNAS web site).…”

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims MHC class I-presented peptidesin vivoand plays an important role in immunodominance

“…However, in intact cells and mice, LAP appears dispensable for antigen presentation (12), and the importance of BH and PSA in antigen presentation remains to be established. Thimet oligopeptidase in cell lysates has been shown to be important in destroying many peptides (13)(14)(15). It has recently been proposed that most antigenic peptides are generated as very long precursors (Ͼ15 residues) and that tripeptidyl peptidase II (TPPII) is essential for trimming these precursors for antigen presentation (16,17).…”