Active site labeling of cysteine cathepsins by a straightforward diazomethylketone probe derived from the N-terminus of human cystatin C

Thibaut Garenne, Ahlame Saidi, Brendan F. Gilmore, Elzbieta Niemiec, Vincent Roy, Luigi A Agrofoglio, Mariana Kasabova, Fabian Lecalle, Gilles Lalmanach

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)250-254
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume460
Issue number2
Early online date13 Mar 2015
DOIs
Publication statusPublished - 01 May 2015

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