Screening ubiquitin specific protease activities using chemically synthesized ubiquitin and ubiquitinated peptides

•Chemically synthesized ubiquitinated peptides permit the study of the catalytic activities of deubiquitinases.•We synthesized, peptides derived from ubiquitinated lysines at the surface of mdm2, and characterized them biophysically.•We used a series of 15 deubiquitinase enzymes to test the use and the practicability of this approach.•The method could be adapted to any peptide susceptible to ubiquitination.•Variations in length and in sequence would permit the study of the Deubiquitinase/peptide recognition rules.

Ubiquitin, a 76 amino acid protein, is a key component that contributes to cellular protein homeostasis. The specificity of this modification is due to a series of enzymes: ligases, attaching the ubiquitin to a lysine, and deubiquitinases, which remove it. More than a hundred of such proteins are implicated in the regulation of protein turnover. Their specificities are only partially understood. We chemically synthesized ubiquitin, attached it to lysines belonging to the protein sequences known to be ubiquitinated. We chose the model protein “murine double minute 2” (mdm2), a ubiquitin ligase, itself ubiquitinated and deubiquitinated. We folded the ubiquitinated peptides and checked their tridimensional conformation. We assessed the use of these substrates with a series of fifteen deubiquitinases to show the potentiality of such an enzymological technique. By manipulating the sequence of the peptide on which ubiquitin is attached, we were able to detect differences in the enzyme/substrate recognition, and to determine that these differences are deubiquitinase-dependent. This approach could be used to understand the substrate/protein relationship between the protagonists of this reaction. The methodology could be customized for a given substrate and used to advance our understanding of the key amino acids responsible for the deubiquitinase specificities.

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