Facile Synthesis of Native and Protease‐Resistant Ubiquitylated Peptides

Abstract

The reversible post-translational modification (PTM) of eukaryotic proteins by ubiquitin (Ub) regulates key cellular processes including protein degradation and gene transcription. Studies of the mechanistic roles for protein ubiquitylation require quantities of homogenously modified substrates that are typically inaccessible from natural sources or by enzymatic ubiquitylation in vitro. Therefore, we developed a facile and scalable methodology for site-specific chemical ubiquitylation. Our semisynthetic strategy utilized a temporary ligation auxiliary, 2-(aminooxy)ethanethiol, to direct ubiquitylation at specific lysines in peptide substrates. Mild reductive removal of the auxiliary after ligation yielded ubiquitylated peptides with the native isopeptide linkage. Alternatively, retention of the ligation auxiliary yielded protease-resistant analogues of ubiquitylated peptides. Importantly, our strategy was fully compatible with protein sulfhydryl groups, as demonstrated by the synthesis of peptides modified by the human small ubiquitin-related modifier 3 (SUMO-3) protein.

Allosteric Activation of Ubiquitin-Specific Proteases by β-Propeller Proteins UAF1 and WDR20

“…To validate these results, we used a Bio-Layer Interferometry-based direct binding assay to compare the binding of a substrate mimetic to a USP12 active site mutant (C48S) with or without the two activators. In these assays, we replaced Ub-AMC with a Ub-peptide conjugate, Ub~(AKA), which contains a natural Ub-lysine iso-peptide linkage imitating the physiological substrates of USP12 (Weller et al, 2014). In agreement with our enzyme kinetics data, our binding studies revealed negligible changes in the substrate binding affinity of USP12 induced by UAF1 or WDR20 (Figure 6A and S6).…”

Chemical Methods for Protein Ubiquitination

“…Recently, Chatterjee et al reported an auxiliary-mediated ubiquitination method employing 2-mercaptoethoxyl group as the auxiliary [16]. The 2-mercaptoethoxyl auxiliary was introduced to the α-amine of Gly76 which had been attached to the lysine side chain during SPPS.…”

Peeling away the layers of ubiquitin signaling complexities with synthetic ubiquitin–protein conjugates

“…Chief among those that afford native isopeptide linkages are native chemical ligation (NCL) and expressed chemical ligation (EPL). Both NCL and EPL utilize temporary auxiliaries to facilitate chemical ligation with a Ub C-terminal thioester (Figure 2A) [16-18]. The auxiliaries are typically removed under reducing conditions to furnish a native linkage.…”