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Published October 19, 2021 | Submitted
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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.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. Version 1 - October 8, 2021; Version 2 - July 26, 2022. We thank Karl Beutner for insightful discussions on MHC Class I and Chris Spahr for help establishing mass spectrometry experiments. We thank Spiros Garbis and Tsui-Fen Chou from Caltech's Proteome Exploration Laboratory for running mass spectrometry experiments. We thank Carlos Arbelaez and Mahshid Amini for help with peptide immunogenicity assays. J.L.M. was previously supported by a Life Sciences Research Foundation postdoctoral fellowship funded by Astellas Pharma and is currently supported by Amgen's Postdoctoral Fellows Program. At the outset of this work R.J.D. was an Investigator of the Howard Hughes Medical Institute and was supported therefrom. Author contributions: J.L.M., J.A.J., J.R.L., R.V., and R.J.D. conceptualized the study. J.L.M., D.J.S., J.R.C., J.L., and S.P. performed experiments. J.R.C., M.K.J., and S.P. provided specialized expertise in immunology assays and B.L., A.M., and M.J.S. provided specialized expertise in mass spectrometry. J.L.M., D.J.S., J.R.C., J.A.J., M.K.J., J.R.L., B.L., M.J.S., B.V.L., R.V., and R.J.D. interpreted results. J.L.M. wrote the original manuscript. D.J.S., J.A.J., R.V., and R.J.D. revised the manuscript. R.J.D. directed the study. Competing interests: J.L.M., D.J.S., J.R.C., J.A.J., M.K.J., J.L., J.R.L., S.P., B.V.L., R.V., and R.J.D. are or were employees of Amgen, Inc., although this study was initiated while J.L.M. was a postdoctoral fellow in the lab of R.J.D. at California Institute of Technology. J.R.C holds patent for CAR T cell technology which is commercially licensed from City of Hope. Data and materials availability: Raw mass spectrometry and PSM analysis files from ProteomeDiscoverer are available from (PRIDE link; not yet public). Processed mass spectrometry data is available in the Supplementary Materials.

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Submitted - 2021.10.07.463289v2.full.pdf


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August 20, 2023
October 23, 2023