Adaptive exchange sustains cullin–RING ubiquitin ligase networks and proper licensing of DNA replication
Abstract
Cop9 signalosome (CSN) regulates the function of cullin–RING E3 ubiquitin ligases (CRLs) by deconjugating the ubiquitin-like protein NEDD8 from the cullin subunit. To understand the physiological impact of CSN function on the CRL network and cell proliferation, we combined quantitative mass spectrometry and genome-wide CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens to identify factors that modulate cell viability upon inhibition of CSN by the small molecule CSN5i-3. CRL components and regulators strongly modulated the antiproliferative effects of CSN5i-3, and in addition we found two pathways involved in genome integrity, SCF^(FBXO5)–APC/C–GMNN and CUL4^(DTL)–SETD8, that contribute substantially to the toxicity of CSN inhibition. Our data highlight the importance of CSN-mediated NEDD8 deconjugation and adaptive exchange of CRL substrate receptors in sustaining CRL function and suggest approaches for leveraging CSN inhibition for the treatment of cancer.
Additional Information
© 2022 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). We thank Novartis for providing the CSN5i-3 compound; Marc Payton and Grace Chung for their help with cell cycle and apoptosis analysis; Sandy Cole for her help with flow cytometry setup and maintenance; Jeanette G. Cook, Julie Bailis, and Paul Hughes for their critical reading and helpful comments of the manuscript; and all members of the R.J.D. laboratory for helpful discussions. Next-generation sequencing was in part performed in the University of California San Francisco Center for Advanced Technology. M.J. was supported by NIH grant K99 GM130964. J.S.W. is a Howard Hughes Medical Institute investigator. Data, Materials, and Software Availability: Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (PXD027862) (78). The data will be openly available upon publication. Author contributions: Y.Z., J.R.L., and R.J.D. designed research; Y.Z., M.J., R.A.P., D.L., and J.L. performed research; Y.Z., M.J., R.A.P., D.L., J.L., B.L., S.D.G., C.-M.L., and J.S.W. contributed new reagents/analytic tools; Y.Z., M.J., D.L., B.L., S.D.G., C.-M.L., and J.S.W. analyzed data; Y.Z. and R.J.D. wrote the paper; and R.J.D. supervised the research. Competing interest statement: Y.Z. is an employee of Amgen. D.L., C.-M. L., and R.J.D. are employees and shareholders of Amgen. J.L. was an employee of Amgen. J.R.L. was an employee and shareholder of Amgen. M.J. consults for Maze Therapeutics and Gate Biosciences. S.D.G. is the founder, president, CEO, and CTO of Proteas Bioanalytics, Inc. The work described in the paper does not have any direct financial implications for Amgen.Attached Files
Published - pnas.2205608119.pdf
Supplemental Material - pnas.2205608119.sapp.pdf
Supplemental Material - pnas.2205608119.sd01.xlsx
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Supplemental Material - pnas.2205608119.sd03.xlsx
Supplemental Material - pnas.2205608119.sd04.xlsx
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Additional details
- PMCID
- PMC9456757
- Eprint ID
- 122519
- Resolver ID
- CaltechAUTHORS:20230725-706009000.22
- NIH
- K99 GM130964
- Howard Hughes Medical Institute (HHMI)
- Created
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2023-08-11Created from EPrint's datestamp field
- Updated
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2023-10-20Created from EPrint's last_modified field