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

Reichermeier, Kurt M. and Straube, Ronny and Reitsma, Justin M. and Sweredoski, Michael J. and Rose, Christopher M. and Moradian, Annie and den Besten, Willem and Hinkle, Trent and Verschueren, Erik and Petzold, Georg and Thomä, Nicolas H. and Wertz, Ingrid E. and Deshaies, Raymond J. and Kirkpatrick, Donald S. (2020) PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network. Molecular Cell, 77 (5). pp. 1092-1106. ISSN 1097-2765. https://resolver.caltech.edu/CaltechAUTHORS:20200122-081543072

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Abstract

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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.molcel.2019.12.013DOIArticle
https://massive.ucsd.eduRelated ItemMassIVE Database
https://doi.org/10.17632/48z8gbddzb.1Related ItemData
ORCID:
AuthorORCID
Sweredoski, Michael J.0000-0003-0878-3831
Moradian, Annie0000-0002-0407-2031
Deshaies, Raymond J.0000-0002-3671-9354
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.
Funders:
Funding AgencyGrant Number
Caltech Beckman InstituteUNSPECIFIED
NIH1S10OD02001301
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Subject Keywords:ubiquitin; Nedd8; CRL4; DCAFs; cullin-RING ligase; targeted protein degradation; mass spectrometry; quantitative proteomics; QconCAT; PIKES
Issue or Number:5
Record Number:CaltechAUTHORS:20200122-081543072
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200122-081543072
Official Citation:Kurt M. Reichermeier, Ronny Straube, Justin M. Reitsma, Michael J. Sweredoski, Christopher M. Rose, Annie Moradian, Willem den Besten, Trent Hinkle, Erik Verschueren, Georg Petzold, Nicolas H. Thomä, Ingrid E. Wertz, Raymond J. Deshaies, Donald S. Kirkpatrick, PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network, Molecular Cell, Volume 77, Issue 5, 2020, Pages 1092-1106.e9, ISSN 1097-2765, https://doi.org/10.1016/j.molcel.2019.12.013. (http://www.sciencedirect.com/science/article/pii/S1097276519309268)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:100825
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:22 Jan 2020 17:16
Last Modified:09 Mar 2020 17:45

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