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Article Dans Une Revue Biochimie Année : 2019

Imaging of extracellular cathepsin S activity by a selective near infrared fluorescence substrate-based probe

Résumé

We designed a near-infrared fluorescent substrate-based probe (SBP), termed MG101, for monitoring extracellular cathepsin S (CatS) activity. We conceived a fused peptide hairpin loop-structure, combining a CatS recognition domain, an electrostatic zipper (with complementary charges of a polyanionic (D-Glu)5 segment and a polycationic (D-Arg)5 motif, as well as a N and C terminal Förster resonance energy transfer pair (donor: AlexaFluor680; quencher: BHQ3) to facilitate activity-dependent imaging. MG101 showed excellent stability since no fluorescence release corresponding to a self-dequenching was observed in the presence of either 2 M NaCl or after incubation at a broad range of pH (2.2-8.2). Cathepsins B, D, G, H, and K, neutrophil elastase and proteinase 3 did not cleave MG101, while CatS, and to a lesser extent CatL, hydrolysed MG101 at pH 5.5. However MG101 was fully selective for CatS at pH 7.4 (kcat/Km = 140,000 M-1 s-1) and sensitive to low concentration of CatS (<1 nM). The selectivity of MG101 was successfully endorsed ex vivo, as it was hydrolysed in cell lysates derived from wild-type but not knockout CatS murine spleen. Furthermore, application of the SBP probe with confocal microscopy confirmed the secretion of active CatS from THP-1 macrophages, which could be abrogated by pharmacological CatS inhibitors. Taken together, present data highlight MG101 as a novel near-infrared fluorescent SBP for the visualization of extracellular active CatS from macrophages and other cell types.

Dates et versions

hal-03677252 , version 1 (24-05-2022)

Identifiants

Citer

Mylène Wartenberg, Ahlame Saidi, Mathieu Galibert, Alix Joulin-Giet, Julien Burlaud-Gaillard, et al.. Imaging of extracellular cathepsin S activity by a selective near infrared fluorescence substrate-based probe. Biochimie, 2019, 166, pp.84-93. ⟨10.1016/j.biochi.2019.03.013⟩. ⟨hal-03677252⟩
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