A Systematic Interrogation of MHC Class I Peptide Presentation Identifies Constitutive and Compensatory Protein Degradation Pathways

The adaptive immune system distinguishes self from non-self by surveying peptides generated from degradation of intracellular proteins that are loaded onto MHC Class I molecules for display on the cell surface. While early studies reported that the bulk of cell surface MHC Class I complexes require the ubiquitin-proteasome system (UPS) for their generation, this conclusion has been challenged. To better understand MHC Class I peptide origins, we sought to carry out a comprehensive, quantitative census of the MHC Class I peptide repertoire in the presence and absence of UPS activity. We introduce optimized methodology to enrich for authentic Class I-bound peptides in silico and then quantify by mass spectrometry their relative amounts upon perturbation of the ubiquitin-proteasome system. Whereas most peptides are dependent on the proteasome and ubiquitination for their generation, a surprising 30% of the MHC Class I repertoire, enriched in peptides of mitochondrial origin, appears independent of these pathways. A further ∼10% of Class I-bound peptides were found to be dependent on the proteasome but independent of ubiquitination for their generation. Notably, clinically achievable partial inhibition of the proteasome resulted in display of novel peptides antigens, at least one of which promotes immune system activation. Our results suggest that generation of MHC Class I•peptide complexes is more complex than previously recognized and also provide evidence for compensatory peptide-generating pathways when canonical pathways are impaired.