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Design of human granzyme B variants resistant to serpin B9

Authors

  • Valeria Losasso,

    1. Computational Biophysics, German Research School for Simulation Sciences, Jülich D-52425, Germany
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  • Sonja Schiffer,

    1. Institute for Applied Medical Engineering, Dept. of Experimental Medicine and Immunotherapy, Aachen D-52074, Germany
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  • Stefan Barth,

    1. Institute for Applied Medical Engineering, Dept. of Experimental Medicine and Immunotherapy, Aachen D-52074, Germany
    2. Fraunhofer Institute for Molecular Biology and Applied Ecology, Department of Pharmaceutical Product Development, Aachen D-52074, Germany
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  • Paolo Carloni

    Corresponding author
    1. Computational Biophysics, German Research School for Simulation Sciences, Jülich D-52425, Germany
    2. Institute for Advanced Simulation, Forschungszentrum Jülich, Jülich D-52425, Germany
    • Computational Biophysics, German Research School for Simulation Sciences, Wilhelm-Johnen-Straße, Jülich 52425, Germany
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Abstract

Human granzyme B (hGB) is a serine protease involved in immune-mediated apoptosis. Its cytotoxicity makes it potentially applicable in cancer therapy. However, the effectiveness of hGB can be hampered by the cytosolic expression of a natural protein inhibitor, human Serpin B9 (hSB9). Here, we used computational approaches to identify hGB mutations that can affect its binding to hSB9 without significantly decreasing its catalytic efficiency. Alanine-scanning calculations allowed us to identify residues of hGB important for the interaction with hSB9. Some variants were selected, and molecular dynamic simulations on the mutated hGB in complex with hSB9 in aqueous solution were carried out to investigate the effect of these variants on the stability of the complex. The R28K, R201A, and R201K mutants significantly destabilized the interaction of the protein with hSB9. Consistently, all of these variants also retained their activity in the presence of the Serpin B9 inhibitor in subsequent in vitro assays of wild-type and mutated hGB. In particular, the activity of R201K hGB with and without Serpin B9 is very similar to that of the wild-type protein. Hence, R201K hGB emerges as a promising species for antitumoral therapy applications. Proteins 2012. © 2012 Wiley Periodicals, Inc.

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