Generation of Naturally Processed Peptide/MHC Class I Complexes Is Independent of the Stability of Endogenously Synthesized Precursors

Proteolysis of endogenously synthesized cellular proteins is essential for constitutive display of processed peptide/MHC class I complexes on the APC surface for stimulating CD8⁺ T cells. However, the extent to which normal protein turnover serves as the source of processed peptides is not clear. To address this question, we used pairs of novel N-end rule substrates that varied in their intracellular stability and served as precursors for generating peptide/MHC class I (OVA257-264/K^b or influenza nucleoprotein 366-374/D^b) complexes. Surprisingly, although each of three precursor pairs tested varied profoundly in their intracellular stability, they were indistinguishable in either T cell stimulation assays, or in the amounts of naturally processed peptides in the APC extracts. Our findings demonstrate that the proteolytic turnover of endogenously synthesized proteins is not directly proportional to the generation of processed antigenic peptide/MHC class I complexes.