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

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• The cullin-RING ubiquitin ligase (CRL) network is crucial for eukaryotic regulation, involving over 300 complexes.
• CRL activation depends on site-specific modification by the ubiquitin-like protein NEDD8.
• Current methods lack probes to survey networks regulated by such modifications.

Purpose of the Study:

• To develop a novel probe for detecting activated CRL complexes.
• To profile cellular repertoires of activated CRLs in response to stimuli.
• To understand the role of neddylated CRLs in cellular regulation and protein degradation.

Main Methods:

• Development of a synthetic antibody recognizing the active conformation of NEDD8-linked cullins.
• Implementation of the antibody for activity-based profiling of CRL networks.
• Profiling of various cell types and activation pathways.

Main Results:

• The synthetic antibody successfully identified activated CRL complexes (CUL1, CUL2, CUL3, CUL4-containing E3s).
• Baseline neddylated CRL repertoires vary significantly across different cell types.
• Differential rewiring of CRL networks was observed in distinct primary cell activation pathways.

Conclusions:

• Conformation-specific probes enable nonenzymatic, activity-based profiling of complex biological networks.
• Neddylated CRLs play a widespread regulatory role and influence the efficiency of targeted protein degradation.
• This approach could aid in the development of novel degrader drugs.