No abstract

Peptides displayed on the cell surface by major histocompatibility class I molecules (MHC class I) are generated by proteolytic processing of protein-antigens in the cytoplasm. Initially, antigens are degraded by the 26 S proteasome, most probably following ubiquitination. However, it is unclear whether this proteolysis results in the generation of MHC class I ligands or if further processing is required. To investigate the role of the 26 S proteasome in antigen presentation, we analyzed the processing of an intact antigen by purified 26 S proteasome. A recombinant ornithine decarboxylase was produced harboring the H-2K b -restricted peptide epitope, derived from ovalbumin SIINFEKL (termed ODC-ova). Utilizing recombinant antizyme to target the antigen to the 26 S proteasome, we found that proteolysis of ODCova by the 26 S proteasome resulted in the generation of the K b -ligand. Mass spectrometry analysis indicated that in addition to SIINFEKL, the N-terminally extended ligand, HSIINFEKL, was also generated. Production of SIINFEKL was linear with time and directly proportional to the rate of ODC-ova degradation. The overall yield of SIINFEKL was approximately 5% of the amount of ODC-ova degraded. The addition of PA28, the 20 S, or the 20 S-PA28 complex to the 26 S proteasome did not significantly affect the yield of the antigenic peptide. These findings demonstrate that the 26 S proteasome can efficiently digest an intact physiological substrate and generate an authentic MHC class I-restricted epitope. Cells display foreign and altered intracellular antigens to cytotoxic T lymphocytes (CTL)1 through MHC class I molecules. Antigenic peptides presented through class I molecules are generated in the cytoplasm by proteolytic degradation of endogenously synthesized antigens. Suitable peptides are then translocated through specialized peptide transporters (termed TAP) to the lumen of the endoplasmic reticulum, where they bind and subsequently stabilize newly synthesized MHC class I molecules. Assembled class I molecules then migrate to the cell surface for recognition by T cells (1).There is now substantial evidence implicating the proteasome in antigen processing. When membrane-permeable inhibitors of proteasomes were added to cells, they severely inhibited proteasome activity in vitro, the cellular turnover of short and long lived proteins, and assembly of class I molecules as well as presentation of ovalbumin (OVA) introduced into the cytoplasm (2, 3).Proteasomes are multicatalytic complexes that constitute the major proteolytic activity in the cytosol and nucleus of all eukaryotes. Proteasomes are found in the cytoplasm as 20 and 26 S particles. The 20 S proteasome is a barrel-shaped complex consisting of four stacked rings, each composed of seven related subunits. The outer rings are formed by noncatalytic ␣ subunits, whereas catalytic ␤ subunits occupy the inner two rings. The 20 S proteasome is an ATP-independent protease that in vitro cleaves only peptides. It can also digest several unfolded proteins, but onl...

Peptides presented to cytotoxic T lymphocytes by the class I major histocompatability complex are 8 -11 residues long. Although proteasomal activity generates the precise C termini of antigenic epitopes, the mechanism(s) involved in generation of the precise N termini is largely unknown. To investigate the mechanism of N-terminal peptide processing, we used a cell-free system in which two recombinant ornithine decarboxylase (ODC) constructs, one expressing the native H2-K b -restricted ovalbumin (ova)-derived epitope SIINFEKL (ODC-ova) and the other expressing the extended epitope LESIINFEKL (ODC-LEova), were targeted to degradation by 26 S proteasomes followed by import into microsomes. We found that the cleavage specificity of the 26 S proteasome was influenced by the N-terminal flanking amino acids leading to significantly different yields of the final epitope SIINFEKL. Following incubation in the presence of purified 26 S proteasome, ODCLEova generated largely ESIINFEKL that was efficiently converted to the final epitope SIINFEKL following translocation into microsomes. The conversion of ESIINFEKL to SIINFEKL was strictly dependent on the presence of H2-K b and was completely inhibited by the metalloaminopeptidase inhibitor 1,10-phenanthroline. Importantly, the converting activity was resistant to a stringent salt/EDTA wash of the microsomes and was only apparent when transport of TAP, the transporter associated with antigen processing, was facilitated. These results strongly suggest a crucial role for a luminal endoplasmic reticulum-resident metalloaminopeptidase in the N-terminal trimming of major histocompatability complex class I-associated peptides. MHC1 class I molecules associate with peptides of 8 -11 amino acids derived from the proteolytic degradation of intracellular protein antigens and present them to CD8ϩ T cells on the cell surface (1-3). Although the vast majority of class I ligands are translocated from the cytosol into the endoplasmic reticulum (ER) by the ATP-dependent transporter associated with antigen processing (TAP) (3), alternative pathways have been described including the liberation of minigene-encoded peptides from ER signal sequences (4 -6), TAP-independent processing of signal sequences (7,8), and processing of membrane-associated or soluble proteins in the secretory pathway (9 -12). Peptides that are not retained in the ER by binding to class I or glycosylation are released back into the cytosol through the Sec61 channel (13-15). Studies using membrane-permeable inhibitors of proteasomes have indicated that the proteasome is the major proteolytic activity responsible for the generation of antigenic peptides (16 -21), although the incomplete inhibition of antigen processing by proteasome inhibitors has also demonstrated the involvement of nonproteasomal cytosolic proteases (22-28).The proteasome is an abundant cytosolic multisubunit protease consisting of the proteolytic 20 S core particle that associates with PA700 regulatory complexes to form 26 S proteasomes or with PA28 comp...