Activity-based profiling of cullin–RING E3 networks by conformation-specific probes
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The cullin–RING ubiquitin ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking. We developed a synthetic antibody recognizing the active conformation of NEDD8-linked cullins. Implementing the probe to profile cellular networks of activated CUL1-, CUL2-, CUL3- and CUL4-containing E3s revealed the complexes responding to stimuli. Profiling several cell types showed their baseline neddylated CRL repertoires vary, and prime efficiency of targeted protein degradation. Our probe also unveiled differential rewiring of CRL networks across distinct primary cell activation pathways. Thus, conformation-specific probes can permit nonenzymatic activity-based profiling across a system of numerous multiprotein complexes, which in the case of neddylated CRLs reveals widespread regulation and could facilitate the development of degrader drugs. [Figure not available: see fulltext.]
Originalsprog | Engelsk |
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Tidsskrift | Nature Chemical Biology |
Vol/bind | 19 |
Sider (fra-til) | 1513-1523 |
ISSN | 1552-4450 |
DOI | |
Status | Udgivet - 2023 |
Bibliografisk note
Funding Information:
We thank D. Scott, D. Miller, S. Bozeman, J. Kellermann, J.R. Prabu, I. Paron, T. Heymann, K-P. Wu and the MPIB Biochemistry Facility for assistance, reagents and helpful discussions. This work was funded by ERC Advanced Grant (Nedd8Activate, Grant agreement ID: 789016), a Leibniz Prize from the DFG (SCHU3196/1-1), St. Jude Children’s Research Hospital and NIH P30CA021765 (to B.A.S.); Canadian Institutes of Health grant MOP-93684 (to S.S.S.); Max-Planck-Gesellschaft (to P.J.M., M.M. and B.A.S). Figure 3 and Extended Data Fig. 4a–d were prepared using UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from the National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. Figure 6f was made with assets from Biorender.com.
Publisher Copyright:
© 2023, The Author(s).
ID: 367909175