A Novel Photoaffinity-Based Probe for Selective Detection of Cathepsin L Active Form

Authors

  • Ana Torkar,

    1. Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana (Slovenia)
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  • Dr. Sarah Bregant,

    1. CEA, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO) iBiTec-S, 91191 Gif-sur-Yvette (France)
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  • Dr. Laurent Devel,

    1. CEA, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO) iBiTec-S, 91191 Gif-sur-Yvette (France)
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  • Dr. Marko Novinec,

    1. Faculty of Chemistry and Chemical Technology, University of Ljubljana, Cesta v Mestni log 88a, 1000 Ljubljana (Slovenia)
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  • Dr. Brigita Lenarčič,

    1. Faculty of Chemistry and Chemical Technology, University of Ljubljana, Cesta v Mestni log 88a, 1000 Ljubljana (Slovenia)
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  • Dr. Tamara Lah,

    1. Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana (Slovenia)
    2. Faculty of Chemistry and Chemical Technology, University of Ljubljana, Cesta v Mestni log 88a, 1000 Ljubljana (Slovenia)
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  • Dr. Vincent Dive

    Corresponding author
    1. CEA, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO) iBiTec-S, 91191 Gif-sur-Yvette (France)
    • CEA, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO) iBiTec-S, 91191 Gif-sur-Yvette (France)
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Abstract

Detecting the active forms of proteases by using activity-based probes in complex proteomes has become an intensively investigated field of research over the past years because many pathogenic conditions involve alterations in protease activities. The detection of lysosomal cysteine proteases, the cathepsins, has mostly relied on the use of probes that incorporate reactive electrophilic moieties to modify a cysteine in the active site covalently. Here we report the first example of an activity-based probe that targets the cathepsins and incorporates a photoactivatable benzophenone group for covalent labelling. When tested on a set of five cathepsins (B, K, L, S and V), this probe selectively labelled the active site of cathepsin L. Furthermore, when tested on crude cell extracts, the probe specifically detected cathepsin L quantities as low as a few picomoles. This study suggests that photoaffinity labelling is a promising approach for developing highly selective and useful cathepsin L probes. In particular, this probe might allow the detection of small amounts of the secreted active cathepsin L form in the cellular microenvironment in vitro and ex vivo.

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