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

Biochemical and Biophysical Research Communications
Thibaut GarenneGilles Lalmanach

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.

References

Jan 20, 2004·Trends in Cell Biology·Amos BaruchMatthew Bogyo
Nov 24, 2005·Biochimica Et Biophysica Acta·Céline PerdereauGilles Lalmanach
Mar 18, 2008·FEBS Letters·Virginie Hervé-GrépinetGilles Lalmanach
May 30, 2008·Frontiers in Bioscience : a Journal and Virtual Library·Vito TurkDusan Turk
Oct 6, 2010·The Journal of Clinical Investigation·Jochen ReiserThomas Reinheckel
Sep 20, 2011·Expert Opinion on Therapeutic Patents·Jac Wijkmans, Jan Gossen
Oct 26, 2011·Biochimica Et Biophysica Acta·Vito TurkDušan Turk
Oct 26, 2013·Nucleic Acids Research·Neil D RawlingsAlex Bateman
Jun 7, 2014·Annual Review of Biochemistry·Laura E Sanman, Matthew Bogyo
Sep 3, 2014·Biological Chemistry·Gilles LalmanachMariana Kasabova

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Citations

Feb 24, 2016·Journal of Immunological Methods·Alex SteimleJulia-Stefanie Frick
Jan 23, 2016·Biochemical and Biophysical Research Communications·Caroline S HughesBrendan F Gilmore
Feb 23, 2020·Free Radical Biology & Medicine·Mylène WartenbergGilles Lalmanach

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