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Published June 23, 2023 | Published + Supplemental Material
Journal Article Open

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


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%, enriched in peptides of mitochondrial origin, appears independent of the UPS. A further ~10% of 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 atypical peptides. 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, alternative protein degradation pathways also generate class I peptides when canonical pathways are impaired.

Additional Information

© 2023 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY). We thank K. Beutner for insightful discussions on MHC class I and C. Spahr for help establishing mass spectrometry experiments. We thank S. Garbis, T.-Y. Wang, B. Quan, and T.-F. Chou from Caltech's Proteome Exploration Laboratory for running mass spectrometry experiments. We thank C. Arbelaez and M. 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: Conceptualization: J.L.M., J.A.J., J.R.L., R.V., and R.J.D. Investigation: J.L.M., D.J.S., and J.L. Methodology: A.M., M.J.S., B.L., J.R.C., S.P., and M.K.J. Writing—original draft: J.L.M. Writing—review and editing: D.J.S., J.A.J., R.V., and R.J.D. Supervision: R.J.D. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials, excluding raw mass spectrometry data. Mass spectrometry raw data (*.raw) and peptide spectra identified by ProteomeDiscoverer (*_PSM.txt) files were added to the Mass Spectrometry Interactive Virtual Environment (MassIVE), along with a file ("S1.xlsx") describing the datasets (MSV000091285). Processed mass spectrometry data are available in the Supplementary Materials. 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 laboratory 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. The authors declare no other competing interests.

Attached Files

Published - sciadv.ade7890.pdf

Supplemental Material - sciadv.ade7890_sm.pdf

Supplemental Material - sciadv.ade7890_tables_s1_to_s11.zip


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Additional details

August 22, 2023
October 25, 2023