E2-Ub-R74G strategy reveals E2-specific ubiquitin conjugation profiles in live cells 

 - Nature Chemical Biology (2025). https://doi.org/10.1038/s41589-024-01809-9 .
<br/><br/> Artículo 
<br/><br/> The E2 ubiquitin (Ub)-conjugating enzyme primarily determines Ub conjugation as Ub-isopeptide (lysine), Ub-oxyester (serine/threonine) or Ub-thioester (cysteine). However, E2-specifc Ub conjugation profles within cells remain elusive. Here we developed the fusion E2-Ub-R74G profling (FUSEP) strategy to access E2-specifc Ub conjugation profles in cells with amino acid resolution. The probe-specifc leucine-glycine-glycine-glycine-modifed Ub remnant enables systematic studies of non-lysine Ub conjugation and provides site-specifc information. Multiple E2 enzymes were found to be involved in non-lysine ubiquitination. Profling with UBE2D3-Ub-R74G probes identifed a post-translational modifcation, tyrosine ubiquitination, in human Cullin-1, a scafold protein for Cullin-RING E3 Ub ligases. This modifcation is distinct from lysine ubiquitination. A single-pass membrane-bound E3 ligase, RNF149, was identifed to pair with UBE2D3 to regulate pyroptosis by ubiquitinating apoptosis-associated speck-like protein ASC. The availability of this toolbox paves the way for uncovering E2-specifc Ub conjugation profles and identifying previously unknown E3 Ub ligases for potential therapeutic applications. 
<br/><br/><label>Subjects--Topical Terms: </label><br/>UB-R74G STRATEGY REVEALS E2-SPECIFIC UBIQUITIN CONJUGATION PROFILES IN LIVE CELLS