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  • br Synthesis of ubiquitin and

    2020-08-04


    Synthesis of ubiquitin and Ubl reagents The development of the first ubiquitin-based assay reagents relied on transpeptidation reactions. Often trypsin is used in these reactions, which removes the last two glycine residues of ubiquitin under native conditions [29]. During the cleavage reaction, an intermediate ester is formed with the active-site serine residue. This intermediate ester can undergo a transpeptidation reaction resulting in a new peptide bond with an amine nucleophile of choice, if a large amount of nucleophile is present. The fluorogenic substrate Ub-AMC has initially been synthesized using this method [30]. Another method uses intein chemistry for a more convenient preparation of reactive ubiquitin thioester intermediates [31]. These intermediates can be chemically converted to introduce a reactive group in the synthesis of ubiquitin-based active site-directed probes. Intein chemistry is generally preferred over transpeptidation methods as it is more generally applicable [32, 33, 34]. Advances in the total chemical synthesis of ubiquitin have enabled the efficient synthesis of new and improved ubiquitin-based reagents. Using an optimized linear synthesis, ubiquitin can now be easily obtained in high yield and purity [35]. Using this synthetic methodology, ubiquitin can be functionalized with any reactive group, dye or label that is compatible with standard peptide synthesis procedures at any specific position.
    Active-site directed probes and their applications The discovery and study of DUBs and Ubl deconjugating BVT 2733 has been greatly accelerated by the development of activity-based probes (ABPs). These ubiquitin-based probes are suicide substrates that specifically react with the active site cysteine nucleophile of DUBs in an activity-based manner. Their covalent nature can be used to visualize and purify an entire family of active proteins simultaneously. ABPs contain three essential elements (Figure 2A). The targeting element confers specificity for the desired enzyme targets. In the case of DUB probes, the targeting element is ubiquitin. Secondly, the reactive group or warhead, which reacts with the active site of the target enzyme after the targeting element has bound. Finally, a recognition or retrieval element is incorporated to allow for the selective retrieval or visualization of the enzyme-ABP complex [34]. The first of these ubiquitin ABPs was ubiquitin-nitrile, which allowed the labeling of a proteasome-bound cysteine dependent DUB [36]. Many improvements to probe design and synthesis have been reported since [32, 33, 34, 37••]. For instance, these developments led to the discovery that OTU class enzymes act as deubiquitylases [38]. Last year, our lab reported the discovery of a novel warhead for use in activity-based profiling and purification of active deubiquitylating enzymes []. The functionality of this warhead was nicely confirmed and complemented in work reported by Sommer et al. []. The introduction of a chemically inert terminal alkyne moiety at the C-terminus of ubiquitin (Ub-Prg) renders it highly reactive toward deubiquitylating enzymes. It was shown that this novel Ub-Prg probe reacts with all classes of cysteine DUBs. An advantage of this alkyne-based probe is that it is very reactive toward DUBs but does not react with unrelated Cys-proteases under the same conditions. Further reviews on general ABP chemistry can be found elsewhere [40].