Uncovering the substrate recognition mechanisms of the E3 ligase adaptor cereblon - PROJECT SUMMARY The immunomodulatory drugs thalidomide, lenalidomide, and pomalidomide exert their therapeutic effects by tailoring the substrate specificity of cereblon, a component of the ubiquitin proteasome system, resulting in altered substrate recruitment, ubiquitylation, and degradation. However, the native substrate specificity and biological functions of cereblon are elusive, despite the conservation of cereblon across species, its association with neurological development, and the potential impact that therapeutic engagement of cereblon would have on these substrates and pathways. To address the urgent need for better characterization of the native role cereblon plays in biological regulation, we undertook a novel approach to discover the substrate recognition mechanism of cereblon. We used a targeted protein degradation approach to systematically screen physiologically relevant ligands for functional engagement of cereblon in cells and discovered that we could replace thalidomide with peptides bearing a C-terminal glutarimide or aspartimide, which are post-translational modifications derived from cyclization of glutamine or asparagine, respectively. Endogenously, these C-terminal cyclic imides may act as molecular glues to recruit substrates to cereblon by tailoring substrate recognition, analogous to thalidomide, or may be generated directly on the substrate during protein aging and damage response or in response to signaling events. To address these two hypotheses, we will functionally characterize the recognition of these C-terminal cyclic imides by cereblon in cells in the context of two models: as peptide- based metabolites that alter substrate recognition or as part of a novel degron found at the C-terminus of proteins after protein aging or during a signaling event. We will first fully evaluate the recognition of peptide-based ligands in the context of bifunctional degraders for targeted protein degradation to characterize the scope and ligandability of cereblon by peptides and evaluate their ability to act as molecular glues for substrate recruitment in a manner analogous to thalidomide and lenalidomide. Next, we will assess whether C-terminal cyclic imides act as degrons that promote substrate recognition directly on engineered and endogenous proteins in biological systems. To facilitate the study of these modifications, we will develop orthogonal chemical labeling strategies to detect and map where and when these modifications occur in cells. Finally, we will investigate the formation of these modifications in cells to characterize the conditions, pathways, and “writers” that generate the C-terminal cyclic imides recognized by cereblon. The definition of C-terminal glutarimide and aspartimide modifications as the key recognition elements used to recruit endogenous substrates to cereblon and the associated studies constitutes a significant advance that will open new investigations into the biological regulation of proteins through these post-translational modifications and the effects on these pathways during cereblon engagement by small molecules, which will inform the use and development of therapeutics that engage cereblon in the clinic.
