Dataset Information

Quantitative Measurement of the Requirement of Diverse Protein Degradation Pathways in MHC Class I Peptide Presentation

ABSTRACT: 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 peptide antigens. Our results suggest that generation of MHC Class I/peptide complexes is more complex than previously recognized, with UPS-dependent and UPS-independent components; paradoxically, a smaller number of atypical peptides increase in presentation when canonical pathways are impaired. -------------------------------------------------------------------------------------------------------------Please see supplementary file S1.xlsx for a detailed file description.

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ray Deshaies

PROVIDER: MSV000091285 | MassIVE | Wed Feb 15 06:52:00 GMT 2023

REPOSITORIES: MassIVE

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Quantitative measurement of the requirement of diverse protein degradation pathways in MHC class I peptide presentation.

Mamrosh Jennifer L JL Sherman David J DJ Cohen Joseph R JR Johnston James A JA Joubert Marisa K MK Li Jing J Lipford J Russell JR Lomenick Brett B Moradian Annie A Prabhu Siddharth S Sweredoski Michael J MJ Vander Lugt Bryan B Verma Rati R Deshaies Raymond J RJ

Science advances 20230623 25

Peptides from degradation of intracellular proteins are continuously displayed by major histocompatibility complex (MHC) class I. To better understand origins of these peptides, we performed a comprehensive census of the class I peptide repertoire in the presence and absence of ubiquitin-proteasome system (UPS) activity upon developing optimized methodology to enrich for and quantify these peptides. Whereas most class I peptides are dependent on the UPS for their generation, a surprising 30%, en ...[more]

PMID: 37352349

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Project description:The majority of peptides presented by MHC class I results from proteasomal protein turnover. The specialized immunoproteasome, which is induced during inflammation, plays a major role in antigenic peptide generation. However, other cellular proteases can, either alone or together with the proteasome, contribute peptides for MHC class I loading non-canonically. We used an im-munopeptidomics workflow combined with a prediction software for proteasomal cleavage probabilities to analyze how inflammatory conditions affect proteasomal processing of immune epitopes presented by A549 cells. Treatment of A549 cells with IFNγ enhanced proteasomal cleavage probability of MHC class I ligands for both, the constitutive proteasome and the im-munoproteasome. Furthermore, IFNγ alters the contribution of the different HLA allotypes to the immunopeptidome. When we calculated HLA allotype-specific proteasomal cleavage probabili-ties for MHC class I ligands, peptides presented by HLA-A*30:01 showed characteristics hinting at a reduced C-terminal proteasomal cleavage probability independently of the type of proteasome. This was confirmed by HLA-A*30:01 ligands from the immune epitope database, which also showed this effect. Furthermore, two additional HLA allotypes, namely HLA-A*03:01 and HLA-A*11:01, presented peptides with a markedly reduced C-terminal proteasomal cleavage probability. Peptides eluted from all three HLA allotypes shared a peptide binding motif with a C-terminal lysine residue suggesting that this lysine residue impairs proteasome-dependent HLA ligand production and might, in turn, favor peptide generation by other cellular proteases. The mass spectrometry raw files used in this study were originally produced for a previous publication, where they were utilized to address the influence of cigarette smoke on the antiviral T-cell immune response. Here, we have reanalyzed the data to investigate processing of MHC class I ligands by c20S or i20S, respectively. Original publication: Chen, J.; Wang, X.; Schmalen, A.; Haines, S.; Wolff, M.; Ma, H.; Zhang, H.; Stoleriu, M.G.; Nowak, J.; Nakayama, M.; et al. Antiviral CD8(+) T cell immune responses are impaired by cigarette smoke and in COPD. Eur Respir J 2023, doi:10.1183/13993003.01374-2022.