Abstract
Epitope-tagged active-site-directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small-molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor-intensive intein-based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active-site-directed probes and their application to activity-based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull-down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in-gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull-down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells.
Highlights
Ubiquitin (Ub) is a 76 amino acid protein regulator of a wide variety of cellular processes, including proteolysis by the proteasome, regulation of cell division, transcription regulation, and DNA repair
Current activity-based probes that target deubiquitinating enzymes (DUBs) are based on the Ub sequence and are equipped with a reactive C-terminal warhead, for example, vinyl methyl ester (VME), and an N-terminal epitope tag (Figure 1 A), and they react with the active site cysteine residue that is present in most DUBs,[3] as shown in Figure 1 B
We based the synthetic route towards novel UbVME probes on the linear solid-supported total synthesis of Ub that we reported recently,[12] which makes use of four pseudoproline building blocks and two dimethoxybenzyl (Dmb) dipeptides to prevent folding and/or aggregation of the growing peptide chain onresin
Summary
Ubiquitin (Ub) is a 76 amino acid protein regulator of a wide variety of cellular processes, including proteolysis by the proteasome, regulation of cell division, transcription regulation, and DNA repair. Ub modifies protein substrates by forming an isopeptide bond between the C-terminal carboxylate of Ub and the e-amino moiety of a lysine side chain or the N terminus of the target protein or Ub itself.[1] The conjugation of Ub is brought about by the consecutive action of Ub ligases from three classes, while Ub conjugates can be disassembled by any of ~ 100 currently known deubiquitinating enzymes (DUBs) that are encoded in the human genome.
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