Elucidation of E3 ubiquitin ligase specificity through proteome-wide internal degron mapping

The ubiquitin-proteasome system plays a critical role in biology by regulating protein degradation. Despite their importance, precise recognition specificity is known for a few of the 600 E3s. Here, we establish a two-pronged strategy for identifying and mapping critical residues of internal degrons on a proteome-scale in HEK-293T cells. We employ global protein stability profiling combined with machine learning to identify 15,800 peptides likely to contain sequence-dependent degrons. We combine this with scanning mutagenesis to define critical residues for over 5,000 predicted degrons. Focusing on Cullin-RING ligase degrons, we generated mutational fingerprints for 219 degrons and developed DegronID, a computational algorithm enabling the clustering of degron peptides with similar motifs. CRISPR analysis enabled the discovery of E3-degron pairs, of which we uncovered 16 pairs that revealed extensive degron variability and structural de-terminants. We provide the visualization of these data on the public DegronID data browser as a resource for future exploration.

Automated summary

This study establishes a strategy for identifying and mapping critical residues of internal degrons on a proteome-scale using global protein stability profiling and machine learning. By combining this with scanning mutagenesis, the researchers determined the critical residues for over 5,000 predicted degrons. They focused on Cullin-RING ligase degrons and generated mutational fingerprints for 219 degrons, developing the DegronID algorithm for clustering degron peptides with similar motifs. The study also uncovered 16 E3-degron pairs with extensive degron variability and structural determinants.