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A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates.

Urbi MukhopadhyaySophie LevantovskyTeresa Maria CarusoneSarah GharbiFrank SteinChristian BehrendsSagar Bhogaraju
Published in: Science advances (2024)
Over 600 E3 ligases in humans execute ubiquitination of specific target proteins in a spatiotemporal manner to elicit desired signaling effects. Here, we developed a ubiquitin-specific proximity-based labeling method to selectively biotinylate substrates of a given ubiquitin ligase. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich bona fide substrates of a ligase using a one-step streptavidin pulldown under denaturing conditions. We applied our method, which we named Ub-POD, to the really interesting new gene (RING) E3 ligase RAD18 and identified proliferating cell nuclear antigen and several other critical players in the DNA damage repair pathway. Furthermore, we successfully applied Ub-POD to the RING ubiquitin ligase tumor necrosis factor receptor-associated factor 6 and a U-box-type E3 ubiquitin ligase carboxyl terminus of Hsc70-interacting protein. We anticipate that our method could be widely adapted to all classes of ubiquitin ligases to identify substrates.
Keyphrases
  • dna damage
  • small molecule
  • binding protein
  • rheumatoid arthritis
  • oxidative stress
  • single cell
  • transcription factor
  • protein protein
  • stem cells
  • dna methylation
  • bone marrow
  • protein kinase
  • genome wide identification