An E1‐Catalyzed Chemoenzymatic Strategy to Isopeptide‐N‐Ethylated Deubiquitylase‐Resistant Ubiquitin Probes

Abstract

Triazole‐based deubiquitylase (DUB)‐resistant ubiquitin (Ub) probes have recently emerged as effective tools for the discovery of Ub chain‐specific interactors in proteomic studies, but their structural diversity is limited. A new family of DUB‐resistant Ub probes is reported based on isopeptide‐N‐ethylated dimeric or polymeric Ub chains, which can be efficiently prepared by a one‐pot, ubiquitin‐activating enzyme (E1)‐catalyzed condensation reaction of recombinant Ub precursors to give various homotypic and even branched Ub probes at multi‐milligram scale. Proteomic studies using label‐free quantitative (LFQ) MS indicated that the isopeptide‐N‐ethylated Ub probes may complement the triazole‐based probes in the study of Ub interactome. Our study highlights the utility of modern protein synthetic chemistry to develop structurally and new families of tool molecules needed for proteomic studies.

Development and application of ubiquitin-based chemical probes

“…Such probes usually contain one or more Ub units and an enrichment/purication tag. [33][34][35][36][37][38][39][40] Ub units can be either natural or mimetics that do not impact the interaction with linkage-specic interactors ( Fig. 4a).…”

“…Third, the transesterication of E1 and Ub to form an active E1-Ub thioester can be intercepted by the addition of thiol, leading to the formation of Ub (1-76)thioester in good yields (e.g., 50 mg L À1 ). 36,79,80 Finally, Liu et al found that a small molecule cyanylating reagent (2-nitro-5thiocyanatobenzoic acid) can modify a recombinant protein at its C-terminus via nucleophilic acyl substitution, generating a protein hydrazide if the nucleophile is hydrazine.…”

“…To obtain a Ub conjugate incorporating a closer mimic of the native isopeptide bond, a bifunctional handle was appended onto a substrate protein through aminoethylation reaction with Cys. 36,78 Ligation of the substrate with the Ub-thioester using NCL followed by auxiliary removal led to the formation of the isopeptide bond mimetics. Two different bifunctional handles were developed: one that results in mimetics bearing only one atom different from the native isopeptide bonds, which may provide ideal tools for biochemical and structural studies; 78 and the other producing an N-ethyl isopeptide bond that is stable to DUB and therefore, useful for screening Ub chain binding proteins from cell lysates.…”

“…Two different bifunctional handles were developed: one that results in mimetics bearing only one atom different from the native isopeptide bonds, which may provide ideal tools for biochemical and structural studies; 78 and the other producing an N-ethyl isopeptide bond that is stable to DUB and therefore, useful for screening Ub chain binding proteins from cell lysates. 36 3.3.5 Thiol-ene coupling strategy. Thiol-ene coupling can be used for nonenzymatic synthesis of Ub chains.…”

“…In addition, triazole-free DUB-resistant Ub probes were developed containing N-ethyl isopeptide bonds. 36 These probes were used to prole the interactome of K29 Ubs, which captured some new interactors not identied by the triazole probes. The main limitation of the Ub pull-down probes is that weak interactors cannot be captured.…”