Degron Miner · High-throughput discovery of degrons encoded by posttranslational modifications

The proteins of a cell are constantly turned over to adapt to changing environments or to eliminate damaged protein species. 80% of protein degradation is mediated via the ubiquitin-proteasome system (UPS). Specificity of the UPS is conveyed by E3 ubiquitin ligases (E3s), which transfer ubiquitin chains onto substrates to mark them for proteasomal degradation. E3s recognize their substrates via motifs termed degrons. Identifying degrons of all E3s would boost our understanding of proteostasis and would help designing pharmacologic strategies to modulate E3 activity. However, the “degron logic” of most of the >600 E3s encoded in the human genome remains elusive. This gap in our understanding of E3-degron relationships is related to the fact that degrons can be encoded by chemical modifications/posttranslational modifications (PTMs), which are not amenable to previously developed high-throughput degron discovery approaches via functional genomics. Here, we propose the Degron Miner platform as a high-throughput approach to systematically investigate the impact of PTMs on target protein stability. To this end, we will generate a 9000-member DegTag library consisting of modified peptides linked to a ligand that binds a nano-luciferase-tagged target protein. This library can be screened in a high-throughput fashion in live cells to select DegTags that efficiently degrade the target protein. To unravel the underlying mechanism and match E3s to the identified PTM-degron, I will employ FACS-based CRISPR/Cas9-KO screens and proteomics-based interactomics approaches. Further, I will leverage the identified PTM-degrons in chemical biology and FACS-based CRISPR/Cas9-KO screens to identify the endogenous substrates of E3s and the mechanisms leading to PTM deposition.Ultimately, the Degron Miner platform will deorphanize E3-substrate relationships and biological pathways involved in the regulation of protein degradation and will additionally provide tools to modulate E3s.