Changes in Cereblon substrates landscape upon molecular glue treatment

The specificity of the ubiquitination process is mediated by the E3 ligases. Discriminating specific substrates of E3s from interacting proteins is one of the major challenges in the field. We previously developed BioE3, a biotin-based approach that uses BirA-E3 fusions together with ubiquitin fused to a low-affinity AviTag to obtain a site-specific and proximity-dependent biotinylation of the target proteins. We proved the suitability of BioE3 to identify targets of RING and HECT-type E3 ligases. Here we demonstrate that the system is suitable for the multi-protein complex Cullin-RING E3s, choosing as proof of principle the substrate receptor CRBN. We identified both CRBN endogenous substrates and new neosubstrates upon pomalidomide treatment, including CSDE1 which contains a new motif involved in the binding to CRBN in presence of pomalidomide. Importantly, we observed a major rearrangement of the endogenous ubiquitination landscape upon this molecular glue treatment. The ability of our system to detect changes in the specificity of the E3s in response to drugs may be of interest in the development of targeted protein degradation strategies.