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<p>The E2 ubiquitin (Ub)-conjugating enzyme primarily determines Ub conjugation as Ub-isopeptide (lysine), Ub-oxyester (serine/threonine) or Ub-thioester (cysteine). However, E2-specific Ub conjugation profiles within cells remain elusive. Here we developed the fusion E2–Ub-R74G profiling (FUSEP) strategy to access E2-specific Ub conjugation profiles in cells with amino acid resolution. The probe-specific leucine-glycine-glycine-glycine-modified Ub remnant enables systematic studies of non-lysine Ub conjugation and provides site-specific information. Multiple E2 enzymes were found to be involved in non-lysine ubiquitination. Profiling with UBE2D3–Ub-R74G probes identified a post-translational modification, tyrosine ubiquitination, in human Cullin-1, a scaffold protein for Cullin-RING E3 Ub ligases. This modification is distinct from lysine ubiquitination. A single-pass membrane-bound E3 ligase, RNF149, was identified 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-specific Ub conjugation profiles and identifying previously unknown E3 Ub ligases for potential therapeutic applications.</p><p></p>

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

  • 期刊论文

  • 发表日期：

    2025年1月6日

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  • Siqi Shen,
  • Hang Yin

摘要

The E2 ubiquitin (Ub)-conjugating enzyme primarily determines Ub conjugation as Ub-isopeptide (lysine), Ub-oxyester (serine/threonine) or Ub-thioester (cysteine). However, E2-specific Ub conjugation profiles within cells remain elusive. Here we developed the fusion E2–Ub-R74G profiling (FUSEP) strategy to access E2-specific Ub conjugation profiles in cells with amino acid resolution. The probe-specific leucine-glycine-glycine-glycine-modified Ub remnant enables systematic studies of non-lysine Ub conjugation and provides site-specific information. Multiple E2 enzymes were found to be involved in non-lysine ubiquitination. Profiling with UBE2D3–Ub-R74G probes identified a post-translational modification, tyrosine ubiquitination, in human Cullin-1, a scaffold protein for Cullin-RING E3 Ub ligases. This modification is distinct from lysine ubiquitination. A single-pass membrane-bound E3 ligase, RNF149, was identified 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-specific Ub conjugation profiles and identifying previously unknown E3 Ub ligases for potential therapeutic applications.