title: Screening ubiquitin specific protease activities using chemically synthesized ubiquitin and ubiquitinated peptides.
abstract: 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.
authors.affiliation: Pôle d'Expertise Biotechnologie, Chimie & Biologie, Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, France.
authors.forename: Marine
authors.initials: M
authors.lastname: Bacchi
authors.affiliation: Pôle d'Expertise Biotechnologie, Chimie & Biologie, Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, France.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Fould
authors.affiliation: Genepep S.A., 12 Rue du Fer à Cheval, 34430 Saint-Jean-de-Védas, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Jullian
authors.affiliation: Genepep S.A., 12 Rue du Fer à Cheval, 34430 Saint-Jean-de-Védas, France.
authors.forename: Aude
authors.initials: A
authors.lastname: Kreiter
authors.affiliation: Genepep S.A., 12 Rue du Fer à Cheval, 34430 Saint-Jean-de-Védas, France.
authors.forename: Amélie
authors.initials: A
authors.lastname: Maurras
authors.affiliation: Pôle d'Expertise Biotechnologie, Chimie & Biologie, Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Nosjean
authors.affiliation: Genepep S.A., 12 Rue du Fer à Cheval, 34430 Saint-Jean-de-Védas, France.
authors.forename: Thibault
authors.initials: T
authors.lastname: Coursindel
authors.affiliation: Genepep S.A., 12 Rue du Fer à Cheval, 34430 Saint-Jean-de-Védas, France.
authors.forename: Karine
authors.initials: K
authors.lastname: Puget
authors.affiliation: Pôle d'Expertise Biotechnologie, Chimie & Biologie, Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Ferry
authors.affiliation: Pôle d'Expertise Biotechnologie, Chimie & Biologie, Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, France. Electronic address: jean.boutin@servier.com.
authors.forename: Jean A
authors.initials: JA
authors.lastname: Boutin
mesh_terms: D002850:Chromatography, Gel; D002942:Circular Dichroism; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D064570:Ubiquitin-Specific Proteases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D010446:Peptide Fragments; D025801:Ubiquitin; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases; D064570:Ubiquitin-Specific Proteases; D008239:Lysine
keywords: Chemical synthesis; Deubiquitinase assay; HPLC; Ubiquitinated peptides