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A Targeted Protease Substrate for a Quantitative Determination of Protease Activities in the Endolysosomal Pathway

Authors

  • Rainer Fischer Dr.,

    1. Eberhard Karls University Tübingen, Department of Molecular Biology, Interfaculty Institute for Cell Biology, Auf der Morgenstelle 15, 72076 Tübingen, Germany, Fax: (+49) 7071-295891
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    • These authors contributed equally to this work

  • Daniel Bächle Dr.,

    1. Eberhard Karls University Tübingen, Medical and Natural Science Research Center, Ob dem Himmelreich 7, 72074 Tübingen, Germany
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    • These authors contributed equally to this work

  • Mariola Fotin-Mleczek Dr.,

    1. Eberhard Karls University Tübingen, Department of Molecular Biology, Interfaculty Institute for Cell Biology, Auf der Morgenstelle 15, 72076 Tübingen, Germany, Fax: (+49) 7071-295891
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  • Günther Jung Prof. Dr.,

    1. Eberhard Karls University Tübingen, Institute for Organic Chemistry, Auf der Morgenstelle 18, 72076 Tübingen, Germany
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  • Hubert Kalbacher Dr.,

    1. Eberhard Karls University Tübingen, Medical and Natural Science Research Center, Ob dem Himmelreich 7, 72074 Tübingen, Germany
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  • Roland Brock Priv.-Doz. Dr.

    1. Eberhard Karls University Tübingen, Department of Molecular Biology, Interfaculty Institute for Cell Biology, Auf der Morgenstelle 15, 72076 Tübingen, Germany, Fax: (+49) 7071-295891
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Abstract

Inside the cell, proteases act in concert in the degradation of proteins and peptides. In order to understand the significance of an individual proteolytic activity within an ensemble of proteases, protocols and probes are required that enable a quantitative determination of the contribution of a protease to the break-down of a given substrate. Here we present a fluorescence resonance energy transfer-based probe and protocols for a quantitative determination of proteolytic activities inside the endolysosomal compartment. A peptide substrate that is readily cleaved by different cathepsins is flanked by fluorescein and tetramethylrhodamine-labeled lysine residues. Efficient endolysosomal targeting of the substrate is achieved by N-terminal elongation with the cell-penetrating peptide nona-arginine. The proteasome inhibitor lactacystin has a small, but significant effect on the break-down of the substrate, thus demonstrating that only a minor fraction of the peptide reaches the cytoplasm in its intact form. Nona-arginine therefore constitutes a highly efficient low-molecular-weight moiety for targeting the endolysosomal compartment.

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