2. Academic Validation
  3. Activity-based profiling of cullin-RING E3 networks by conformation-specific probes

Activity-based profiling of cullin-RING E3 networks by conformation-specific probes

  • Nat Chem Biol. 2023 Aug 31. doi: 10.1038/s41589-023-01392-5.
Lukas T Henneberg # 1,  Jaspal Singh # 2,  David M Duda # 3 4,  Kheewoong Baek 1,  David Yanishevski 3,  Peter J Murray 5,  Matthias Mann 6 7,  Sachdev S Sidhu 8,  Brenda A Schulman 9 10
Affiliations

Affiliations

  • 1 Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany.
  • 2 School of Pharmacy, University of Waterloo, Waterloo, Ontario, Canada.
  • 3 Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.
  • 4 Siduma Therapeutics, New Haven, CT, USA.
  • 5 Immunoregulation, Max Planck Institute of Biochemistry, Martinsried, Germany.
  • 6 Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
  • 7 NNF Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
  • 8 School of Pharmacy, University of Waterloo, Waterloo, Ontario, Canada. sachdev.sidhu@uwaterloo.ca.
  • 9 Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany. schulman@biochem.mpg.de.
  • 10 Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA. schulman@biochem.mpg.de.
  • # Contributed equally.
PMID: 37653169 DOI: 10.1038/s41589-023-01392-5
Abstract

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.

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