Targeted protein degradation via NEDD8 conjugation does not enhance direct MHC class I antigen presentation

MHC class I molecules present peptide antigens derived from proteins synthesized and degraded within the antigen presenting cell. An understanding the protein degradation pathways that contribute to peptide generation is necessary for developing next generation immunotherapies. We recently discovered that conjugation of the ubiquitin-like molecule NEDD8 to the N-terminus of a protein can result in the rapid degradation of the target protein by either the ubiquitin-proteasome system (UPS) and the autophagy pathway. To determine if protein NEDDylation increased peptide presentation, we fused NEDD8 to the N-terminus of a cytosolic form of ovalbumin (OVA) and measured presentation of the SIINFEKL peptide via the murine MHC class I molecule K^b using a monoclonal antibody specific for the K^b-SIINFEKL complex. Like other constructs we have characterized, NEDDylated OVA was rapidly degraded by the UPS and autophagy pathways. While proteasome inhibition prevented presentation of SIINFEKL peptides, inhibiting autophagy did not affect antigen presentation. We then compared presentation of NEDDylated OVA to ubiquitin conjugated OVA and found that ubiquitin conjugation was far more effective at generating antigenic peptides. These data demonstrate that rapid degradation per se of an antigenic protein is not sufficient to enhance antigen presentation but rather the precise manner in which a protein is degraded governs the efficiency of peptide generation. Our findings are consistent with the idea that protein degradation is organized in some way to optimize generation the immunopeptidome for immunosurveillance.