Abstract
Several chemical approaches have been applied to develop Ub-based substrates and probes selective toward one or a narrow subset of deubiquitinases (DUBs). Since DUBs are highly specific toward ubiquitin and exhibit low activity toward shorter peptides, it is challenging to design truly selective chemical tools to investigate one DUB in biological samples. Incorporating amino acids other than canonical LRG at the P4–P2 positions in the Ub improves DUB activity and selectivity toward Ub derivatives. Here, we describe the protocol for identifying selective peptide sequences using a hybrid combinatorial substrate library (HyCoSuL) approach that can be introduced in the C-terminal motif of Ub. Furthermore, we describe the synthesis protocol of Ub-based probes and substrates containing unnatural amino acids and the application of Ub-based probes to detect DUBs in cell lysates.
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Acknowledgments
This work was supported by the National Science Center grant 2015/17/N/ST5/03072 (Preludium 9) in Poland (W.R.) and the “TEAM/2017-4/32” project, which is carried out within the TEAM program of the Foundation for Polish Science, co-financed by the European Union under the European Regional Development Fund (M.D.).
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Rut, W., Zmudzinski, M., Drag, M. (2023). Design and Synthesis of Ubiquitin-Based Chemical Tools with Unnatural Amino Acids for Selective Detection of Deubiquitinases. In: Maupin-Furlow, J., Edelmann, M.J. (eds) Deubiquitinases. Methods in Molecular Biology, vol 2591. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2803-4_5
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DOI: https://doi.org/10.1007/978-1-0716-2803-4_5
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