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Published March 5, 2020 | Supplemental Material
Journal Article Open

PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network


Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development.

Additional Information

© 2019 Elsevier Inc. Received: September 3, 2019; Revised: November 18, 2019; Accepted: December 13, 2019; Published: January 20, 2020. We would like to thank Haibin Mao and Ning Zheng for sharing Cul4A⋅Rbx1 expression constructs. We thank Ruth Huettenhain for sharing CRL4 peptide information at the initiation of this project. We also thank Caltech and Genentech for enabling a unique and fruitful collaboration via the Visiting Scientist Program. Finally, we thank all members of the Deshaies, Wertz, and Kirkpatrick laboratories as well as the Proteome Exploration Laboratory at Caltech and the Department of Microchemistry, Proteomics, and Lipidomics at Genentech for fruitful discussions. The Proteome Exploration Laboratory is supported by the Beckman Institute and NIH 1S10OD02001301. R.J.D. was an Investigator of the Howard Hughes Medical Institute, and this work was supported in part by HHMI. Data Availability: MS Raw data: All mass spectrometry raw files have been deposited into the MassIVE database under the dataset ID MSV000084215 (https://massive.ucsd.edu) (see Table S6 for a file directory). Other unprocessed data: Unprocessed western blot and DNA gel data files are available through Mendeley Data at the following link: https://doi.org/10.17632/48z8gbddzb.1. Author Contributions: Conceptualization: K.M.R., R.J.D., and D.S.K.; Methodology: K.M.R., J.M.R., M.J.S., R.J.D., and D.S.K.; Software: R.S.; Formal Analysis: K.M.R., R.S., M.J.S., T.H., and E.V.; Investigation: K.M.R., J.M.R., and G.P.; Resources: C.M.R., A.M., W.d.B., and N.T.; Writing – Original Draft: K.M.R.; Writing – Review & Editing: K.M.R., J.M.R., M.J.S., A.M., I.E.W., R.J.D., and D.S.K.; Supervision: I.E.W., R.J.D., and D.S.K; Funding Acquisition: I.E.W., R.J.D., and D.S.K. Declaration of Interests: K.M.R. is a Ph.D. candidate at Caltech and an employee of Genentech, Inc., through the Visiting Scientist Program; E.V., C.M.R., W.d.B., I.E.W., and D.S.K. are employees and shareholders of Genentech, Inc.,; J.M.R. is an employee and shareholder of Abbvie Inc.; R.S. is an employee and shareholder of Brystol-Myers Squibb Inc.; R.J.D. is an employee and shareholder of Amgen Inc.

Attached Files

Supplemental Material - 1-s2.0-S1097276519309268-mmc1.pdf

Supplemental Material - 1-s2.0-S1097276519309268-mmc2.xlsx

Supplemental Material - 1-s2.0-S1097276519309268-mmc3.xlsx

Supplemental Material - 1-s2.0-S1097276519309268-mmc4.xlsx

Supplemental Material - 1-s2.0-S1097276519309268-mmc5.xlsx

Supplemental Material - 1-s2.0-S1097276519309268-mmc6.xlsx

Supplemental Material - 1-s2.0-S1097276519309268-mmc7.xlsx


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