1. Active site labeling of cysteine cathepsins by a straightforward diazomethylketone probe derived from the N-terminus of human cystatin C.
- Author
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Garenne, Thibaut, Saidi, Ahlame, Gilmore, Brendan F., Niemiec, Elżbieta, Roy, Vincent, Agrofoglio, Luigi A., Kasabova, Mariana, Lecaille, Fabien, and Lalmanach, Gilles
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CATHEPSINS , *KETONES , *CYSTATINS , *PEROXIDASE , *N-terminal residues , *AFFINITY labeling - 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 (k ass = 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. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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