A General Chemical Ligation Approach Towards Isopeptide‐Linked Ubiquitin and Ubiquitin‐Like Assay Reagents

摘要

Thiolysine-mediated chemical ligation has generated fluorescence polarisation assay reagents based on isopeptide-linked ubiquitin-like protein conjugates. These have been used to monitor the activity of ubiquitin(-like) proteases. Thus, it is now possible to generate assay reagents that contain substrate-derived elements around the isopeptide linkage, with no practical limitation. Ubiquitin (Ub) and ubiquitin-like proteins (Ubls) form a family of small and highly conserved post-translational modifiers that become linked to target proteins and thus modulate their function (such as degradation, trafficking and signalling).1 The linkage between a Ub(l) and a target protein most frequently consists of an isopeptide bond between the C-terminal carboxylate of Ub(l) and the ε-amine of a lysine residue. Ub(l) ligation requires the concerted action of enzymes E1, E2 and E3, defined combinations of which provide specificity for the protein target.2 Next to human Ub, 17 Ubls from nine phylogenetic classes have been reported.3 Each has its own discrete conjugation and deconjugation enzymes and has a distinct effect on its cellular target. The best-studied Ubls are Nedd8 and SUMO. For example, neddylation of cullin–RING E3 ligases is required for their enzymatic activity.4 The three human SUMO proteins (SUMO-1, SUMO-2 and SUMO-3) are conjugated to diverse target proteins, thereby often altering their interaction with other proteins through interactions between SUMO and SUMO-binding motifs.5 Specific deconjugating enzymes remove Ub and Ubls from target proteins. By doing so, they achieve three major functions.6 First, as Ub and Ubls are often translated as pro-proteins, they cleave the C termini of Ub and Ubls to generate the mature forms. Secondly, these proteases can reverse Ub(l) signalling functions and recycle free Ub and Ubls. Thirdly, in those cases where chains exist, such as for Ub and SUMO-2 and -3, proteases can perform a chain-editing function. As deregulation of Ub(l) deconjugating activity is linked to the occurrence of a variety of diseases, these are of interest as potential drug targets,7 and consequently, good assay reagents are required to report enzymatic activity and inhibition. Current assay reagents are mainly based on a Ub(l) part connected by a linear peptide bond to a reporter module—either a fluorogenic or latent enzyme that becomes active upon Ub(l) processing.7c In addition, besides lacking the native isopeptide linkage, such reagents cannot be functionalised (beyond the reporter module) to resemble a more physiologically relevant substrate. A previously reported fluorescence anisotropy/fluorescence polarisation (FP) assay reagent for Ub(l) deconjugating enzymes is based on a fluorophore-labelled lysine, or a peptide linked to Ub by an isopeptide bond (Figure 1).8 This reagent has two characteristics that make it well-suited for high-throughput investigations of catalytic action.9 First, it is the only reported assay reagent that incorporates an isopeptide linkage;8 secondly, its physiological relevance (and potentially its affinity for a deconjugating enzyme) can be enhanced by functionalising the assay reagent with substrate-derived elements around the isopeptide linkage.10 FP assay. When a fluorophore, covalently attached to a small molecule (e.g. a small peptide) is excited by polarised light, it will emit predominantly depolarised light. When it is bound to a high molecular weight molecule (e.g. Ub or a Ubl) the emitted light is much less depolarised. By following the change in fluorescence polarisation, the activity can be monitored. P, polarisation. Because of the cumbersome enzymatic preparation required for this type of reagent, it has not become the standard in this field. To overcome the limitations set by enzymatic reactions, we and others recently reported methods for the site- and chemoselective Ub modification of peptides.11 In this approach, isopeptide-linked Ub-conjugates are prepared by native