Label-free quantitative analysis of the membrane proteome of Bace1 protease knock-out zebrafish brains

Authors

  • Sebastian Hogl,

    1. German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
    2. Technische Universität München, Neuroproteomics, Munich, Germany
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  • Frauke van Bebber,

    1. German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
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  • Bastian Dislich,

    1. German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
    2. Technische Universität München, Neuroproteomics, Munich, Germany
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  • Peer-Hendrik Kuhn,

    1. German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
    2. Technische Universität München, Neuroproteomics, Munich, Germany
    3. Institute for Advanced Study, Technische Universität München, Garching, Germany
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  • Christian Haass,

    1. German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
    2. Adolf Butenandt Institute, Ludwig-Maximilians University, Munich Biochemistry, Munich, Germany
    3. Munich Center for Systems Neurology (SyNergy), Munich, Germany
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  • Bettina Schmid,

    1. German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
    2. Adolf Butenandt Institute, Ludwig-Maximilians University, Munich Biochemistry, Munich, Germany
    3. Munich Center for Systems Neurology (SyNergy), Munich, Germany
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  • Stefan F. Lichtenthaler

    Corresponding author
    1. Technische Universität München, Neuroproteomics, Munich, Germany
    2. Institute for Advanced Study, Technische Universität München, Garching, Germany
    3. Munich Center for Systems Neurology (SyNergy), Munich, Germany
    • German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
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Correspondence: Dr. Stefan F. Lichtenthaler, German Center for Neurodegenerative Diseases (DZNE), Munich, and Technische Universität München, Max-Lebsche-Platz 30, 81377 Munich, Germany

E-mail: stefan.lichtenthaler@dzne.de

Fax: +49-0-89/7095-8420

Abstract

The aspartyl protease BACE1 cleaves neuregulin 1 and is involved in myelination and is a candidate drug target for Alzheimer's disease, where it acts as the β-secretase cleaving the amyloid precursor protein. However, little is known about other substrates in vivo. Here, we provide a proteomic workflow for BACE1 substrate identification from whole brains, combining filter-aided sample preparation, strong-anion exchange fractionation, and label-free quantification. We used bace1-deficient zebrafish and quantified differences in protein levels between wild-type and bace1 −/− zebrafish brains. Over 4500 proteins were identified with at least two unique peptides and quantified in both wild-type and bace1 −/− zebrafish brains. The majority of zebrafish membrane proteins did not show altered protein levels, indicating that Bace1 has a restricted substrate specificity. Twenty-four membrane proteins accumulated in the bace1 −/− brains and thus represent candidate Bace1 substrates. They include several known BACE1 substrates, such as the zebrafish homologs of amyloid precursor protein and the cell adhesion protein L1, which validate the proteomic workflow. Additionally, several candidate substrates with a function in neurite outgrowth and axon guidance, such as plexin A3 and glypican-1 were identified, pointing to a function of Bace1 in neurodevelopment. Taken together, our study provides the first proteomic analysis of knock-out zebrafish tissue and demonstrates that combining gene knock-out models in zebrafish with quantitative proteomics is a powerful approach to address biomedical questions.

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