Synthetic and semi-synthetic strategies to study ubiquitin signaling

Abstract

The post-translational modification ubiquitin can be attached to the ɛ-amino group of lysine residues or to a protein's N-terminus as a mono ubiquitin moiety. Via its seven intrinsic lysine residues and its N-terminus, it can also form ubiquitin chains on substrates in many possible ways. To study ubiquitin signals, many synthetic and semi-synthetic routes have been developed for generation of ubiquitin-derived tools and conjugates. The strength of these methods lies in their ability to introduce chemo-selective ligation handles at sites that currently cannot be enzymatically modified. Here, we review the different synthetic and semi-synthetic methods available for ubiquitin conjugate synthesis and their contribution to how they have helped investigating conformational diversity of diubiquitin signals. Next, we discuss how these methods help understanding the ubiquitin conjugation-deconjugation system by recent advances in ubiquitin ligase probes and diubiquitin-based DUB probes. Lastly, we discuss how these methods help studying post-translational modification of ubiquitin itself.

Profiling DUBs and Ubl-specific proteases with activity-based probes

“…In addition, ABPs can be applied to determine the active fraction of a recombinantly expressed and purified enzyme or to study the effect of specific enzyme modifications or mutations with respect to the enzyme's activity and its substrate specificities (Mevissen et al, 2013. ABPs are also very useful tools for gaining insight into the structural characteristics of an enzyme, where an enzyme-ABP complex mimics a certain state of the reaction between the enzyme and its substrate (Basters et al, 2017;van Tilburg, Elhebieshy, & Ovaa, 2016). Also, by designing and testing different structural variants of an ABP (Flierman et al, 2016;Mulder, El Oualid, ter Beek, & Ovaa, 2014), one can identify preferences of a given enzyme for certain structural features (Bekes et al, 2015(Bekes et al, , 2016Mevissen et al, 2016).…”

Enhanced Delivery of Synthetic Labelled Ubiquitin into Live Cells by Using Next‐Generation Ub–TAT Conjugates

“…Ub can also form polymeric chains (PolyUb) through the attachment of one Ub unit to the N‐terminal methionine residue or one of the seven lysine side chains of another Ub unit . Although ubiquitination has been extensively studied by use of various synthetic in vitro tools, there is an increasing need for in vivo tools to allow study of ubiquitin conjugation in live cells . Therefore, the generation of methods that deliver Ub into live cells is key to furthering our understanding of ubiquitination in cells in real time.…”

Evaluating enzyme activities and structures of DUBs

“…The Ub field has benefited greatly from the past efforts of biochemists and chemical biologists who have reconstituted Ub signals in vitro and generated tools for capturing or measuring DUB activities, many of which can be readily produced or are commercially available (Ekkebus, Flierman, Geurink, & Ovaa, 2014;van Tilburg, Elhebieshy, & Ovaa, 2016). Critically, the nature of the DUB in question and the type of information sought must be considered before choosing a suitable substrate, as each comes with advantages and disadvantages.…”

“…Obtaining sitespecifically ubiquitinated substrate is not an easy feat. Various strategies exist currently to chemically ubiquitinate an unnatural amino acid target introduced into a protein backbone either through orthogonal genetic coding or total peptide synthesis (Gopinath, Ohayon, Nawatha, & Brik, 2016;van Tilburg et al, 2016). The resulting ubiquitinated substrate can be used biochemically to assess DUB activity (Bavikar et al, 2011) or structurally to understand enzyme-substrate interactions (Morgan et al, 2016).…”