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.

Structural Insights Into the Initiation and Elongation of Ubiquitination by Ubr1

“…Ubc2 was first chemically modified with the bifunctional adaptor molecule 1 36 at the catalytic Cys to form molecule 2. Subsequently, the S-acetamidomethyl (Acm) group on molecule 2 was removed to expose a β-mercaptoethylamine group for NCL with a Ub thioester (Ub-MesNa) 36 , generating Ubc2~Ub carrying an additional thiol group (molecule 3). Finally, a stable intermediate mimic was obtained by the creation of a disulfide bond between the thiol groups of molecule 3 and a Ub-Degron carrying the K48C point mutation (Fig.…”

“…2a inset). Ubc2 was first chemically modified with the bifunctional adaptor molecule 1 36 at the catalytic Cys to form molecule 2.…”

Use of a Removable Backbone Modification Strategy to Prevent Aspartimide Formation in the Synthesis of Asp Lactam Cyclic Peptides†

“…Asp‐based lactam cyclic peptide A‐183 and cyclo [Asp9,Lys13] KIIIA7‐14 lactam cyclic peptides have been successfully synthesized using an RBM strategy, wherein a 2‐hydroxy‐4‐methoxy‐ 5‐nitrobenzaldehyde group is appended to the residue immediately before Asp(OTbe), to prevent aspartimide formation. [ 32‐36 ] This RBM strategy may constitute a simple, efficient, and general method for the synthesis of Asp‐based lactam cyclic peptides, which we established to be inaccessible by direct Fmoc SPPS.…”

One-Pot, Photocontrolled Enzymatic Assembly of the Structure-Defined Heterotypic Polyubiquitin Chain

“…Polyubiquitination is a post-translational modification that plays essential roles in the precise and dynamic regulation of various cellular processes, including protein quality control and intracellular signaling. − In a canonical polyubiquitin chain with a single linkage type, each ubiquitin unit is conjugated via any of eight amino groups, seven lysine residues (K6, K11, K27, K29, K33, K48, and K63), and N-terminal methionine (M1), resulting in eight kinds of divergences with respect to the linkage type. The structural diversity in the linkage type and length − dictates interactions with the reader proteins underlying the diverse signals mediated by polyubiquitin chains. − Recently, the heterotypic polyubiquitin chain, which includes multiple linkage types in a single ubiquitin chain, has attracted much attention because of its further potential diversity in the structure . Considering a heterotypic chain with only two linkage types, the number of possible combinations reaches 28.…”