Ubiquitin-specific proximity labeling for the identification of E3 ligase substrates

Author: Huang, Hai-Tsang; Lumpkin, Ryan J.; Tsai, Ryan W.; Su, Shuyao; Zhao, Xu; Xiong, Yuan; Chen, James; Mageed, Nada; Donovan, Katherine A.; Fischer, Eric S.; Sellers, William R.

Description: Protein ubiquitylation controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in disease. Moreover, small-molecule degraders that redirect ubiquitylation activities toward disease targets are an emerging and promising therapeutic class. Over 600 E3 ubiquitin ligases are expressed in humans, but their substrates remain largely elusive, necessitating the development of new methods for their discovery. Here we report the development of E3-substrate tagging by ubiquitin biotinylation (E-STUB), a ubiquitin-specific proximity labeling method that biotinylates ubiquitylated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitylated targets of protein degraders, including collateral targets and ubiquitylation events that do not lead to substrate degradation. It also detects known substrates of E3 ligase CRBN and VHL with high specificity. With the ability to elucidate proximal ubiquitylation events, E-STUB may facilitate the development of proximity-inducing therapeutics and act as a generalizable method for E3-substrate mapping.

Subject headings: Ubiquitin-Protein Ligases; Humans; Ubiquitination; Ubiquitin; Substrate Specificity; Biotinylation; HEK293 Cells; Von Hippel-Lindau Tumor Suppressor Protein; Proteolysis

Publication year: 2024

Journal or book title: Nature Chemical Biology

Volume: 20

Issue: 9

Pages: 1227-1236

Find the full text: https://www.nature.com/articles/s41589-024-01590-9

Find more like this one (cited by): https://scholar.google.com/scholar?cites=12938877122588066095&as_sdt=1000005&sciodt=0,16&hl=en

Serial number: 3968

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