On-going antigenic peptide presentation during selective inhibition of protein degradation 3208

Abstract Description Antigenic peptides presented by MHC class I molecules at the surface of the cell are often derived from proteins degraded by the ubiquitin-proteasome system. Here we examined the relationship between protein ubiquitination and peptide presentation by chemically inhibiting the action of Cullin-Ring-Ligases (CRLs), a large family of ubiquitin ligases, and determining the fate of peptides presented from proteins targeted for ubiquitination and degradation by CRLs. Hela cells expressing secreted HLA-A*11:01 molecules were treated with MLN4924, an inhibitor of NEDDylation, which is essential for CRL functional activity. Peptides were isolated from secreted HLA molecules and identified by mass spectrometry. Approximately 2/3rd of source-proteins presented was largely unchanged following MLN4924 treatment, and, interestingly, changes to the identity of the immunopeptidome could not be explained by alterations to the cellular transcriptome. More than half of the peptides derived from substrates of CRLs were still presented after treatment with MLN4924. Western blot analysis demonstrated that numerous CRL substrates were spared from degradation following MLN4924 treatment. These data indicate that despite inhibiting ubiquitination of many proteins targeted for degradation, peptides are still liberated for MHC class I antigen presentation, perhaps due to alternative degradation pathways or defective forms of the protein degraded independently of their canonical ubiquitin ligase. Funding Sources NIH R01AI130059 Topic Categories Antigen and Dendritic Cell Processing, Presentation, and Biology (AGDC)