title: Active site labeling of cysteine cathepsins by a straightforward diazomethylketone probe derived from the N-terminus of human cystatin C.
abstract: We designed a straightforward biotinylated probe using the N-terminal substrate-like region of the inhibitory site of human cystatin C as a scaffold, linked to the thiol-specific reagent diazomethylketone group as a covalent warhead (i.e. Biot-(PEG)₂-Ahx-LeuValGly-DMK). The irreversible activity-based probe bound readily to cysteine cathepsins B, L, S and K. Moreover affinity labeling is sensitive since active cathepsins were detected in the nM range using an ExtrAvidin-peroxidase conjugate for disclosure. Biot-(PEG)₂-Ahx-LeuValGly-DMK allowed a slightly more pronounced labeling for cathepsin S with a compelling second-order rate constant for association (kass = 2,320,000 M(-1) s(-1)). Labeling of the active site is dose-dependent as observed using 6-cyclohexylamine-4-piperazinyl-1,3,5-triazine-2-carbonitrile, as competitive inhibitor of cathepsins. Finally we showed that Biot-(PEG)₂-Ahx-LeuValGly-DMK may be a simple and convenient tool to label secreted and intracellular active cathepsins using a myelomonocytic cell line (THP-1 cells) as model.
authors.affiliation: INSERM UMR 1100 «Pathologies Pulmonaires: Protéolyse et Aérosolthérapie», Equipe «Mécanismes Protéolytiques dans l'Inflammation»/Centre d'Etude des Pathologies Respiratoires (CEPR), Université François Rabelais, Faculté de Médecine, Tours, France.
authors.forename: Thibaut
authors.initials: T
authors.lastname: Garenne
authors.affiliation: INSERM UMR 1100 «Pathologies Pulmonaires: Protéolyse et Aérosolthérapie», Equipe «Mécanismes Protéolytiques dans l'Inflammation»/Centre d'Etude des Pathologies Respiratoires (CEPR), Université François Rabelais, Faculté de Médecine, Tours, France.
authors.forename: Ahlame
authors.initials: A
authors.lastname: Saidi
authors.affiliation: Queen's University Belfast, School of Pharmacy, McClay Research Centre, Belfast, United Kingdom.
authors.forename: Brendan F
authors.initials: BF
authors.lastname: Gilmore
authors.affiliation: Université d'Orléans, CNRS UMR 7311, Institut de Chimie Organique et Analytique (ICOA), Orléans, France.
authors.forename: Elżbieta
authors.initials: E
authors.lastname: Niemiec
authors.affiliation: Université d'Orléans, CNRS UMR 7311, Institut de Chimie Organique et Analytique (ICOA), Orléans, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Roy
authors.affiliation: Université d'Orléans, CNRS UMR 7311, Institut de Chimie Organique et Analytique (ICOA), Orléans, France.
authors.forename: Luigi A
authors.initials: LA
authors.lastname: Agrofoglio
authors.affiliation: INSERM UMR 1100 «Pathologies Pulmonaires: Protéolyse et Aérosolthérapie», Equipe «Mécanismes Protéolytiques dans l'Inflammation»/Centre d'Etude des Pathologies Respiratoires (CEPR), Université François Rabelais, Faculté de Médecine, Tours, France.
authors.forename: Mariana
authors.initials: M
authors.lastname: Kasabova
authors.affiliation: INSERM UMR 1100 «Pathologies Pulmonaires: Protéolyse et Aérosolthérapie», Equipe «Mécanismes Protéolytiques dans l'Inflammation»/Centre d'Etude des Pathologies Respiratoires (CEPR), Université François Rabelais, Faculté de Médecine, Tours, France.
authors.forename: Fabien
authors.initials: F
authors.lastname: Lecaille
authors.affiliation: INSERM UMR 1100 «Pathologies Pulmonaires: Protéolyse et Aérosolthérapie», Equipe «Mécanismes Protéolytiques dans l'Inflammation»/Centre d'Etude des Pathologies Respiratoires (CEPR), Université François Rabelais, Faculté de Médecine, Tours, France. Electronic address: gilles.lalmanach@univ-tours.fr.
authors.forename: Gilles
authors.initials: G
authors.lastname: Lalmanach
mesh_terms: D000345:Affinity Labels; D020134:Catalytic Domain; D002403:Cathepsins / Q000737:chemistry*; D002460:Cell Line; D055316:Cystatin C / Q000737:chemistry*; D003978:Diazomethane / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D015335:Molecular Probes
chemical_list: D000345:Affinity Labels; D055316:Cystatin C; D015335:Molecular Probes; C015795:diazomethyl ketones; D003978:Diazomethane; D002403:Cathepsins
keywords: Activity-based probe; Affinity labeling; Cathepsin; Cystatin; Cysteine protease