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The C99 domain of the amyloid precursor protein is a disordered membrane phase-preferring protein

Ricardo Capone, Ajit Tiwari, Arina Hadziselimovic, Yelena Peskova, James M. Hutchison, Charles R. Sanders, View ORCID ProfileAnne K. Kenworthy
doi: https://doi.org/10.1101/2020.11.25.397893
Ricardo Capone
1Department of Biochemistry, Vanderbilt University, Nashville, TN, 37240 USA
2Center for Structural Biology, Vanderbilt University, Nashville, TN, 37240 USA
3Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, 37232
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Ajit Tiwari
3Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, 37232
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Arina Hadziselimovic
1Department of Biochemistry, Vanderbilt University, Nashville, TN, 37240 USA
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Yelena Peskova
4Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA, 22903 USA
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James M. Hutchison
1Department of Biochemistry, Vanderbilt University, Nashville, TN, 37240 USA
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Charles R. Sanders
1Department of Biochemistry, Vanderbilt University, Nashville, TN, 37240 USA
2Center for Structural Biology, Vanderbilt University, Nashville, TN, 37240 USA
eDepartment of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37232 USA
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Anne K. Kenworthy
3Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, 37232
4Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA, 22903 USA
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  • ORCID record for Anne K. Kenworthy
  • For correspondence: akk7hp@virginia.edu
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Abstract

Processing of the amyloid precursor protein (APP) via the amyloidogenic pathway is associated with the etiology of Alzheimer’s disease. The cleavage of APP by β-secretase to generate the transmembrane 99-residue C-terminal fragment (C99) and subsequent processing of C99 by γ-secretase to yield amyloid-β (Aβ) peptides are essential steps in this pathway. Biochemical evidence suggests amyloidogenic processing of C99 occurs in cholesterol- and sphingolipid-enriched liquid ordered phase membrane raft domains. However, direct evidence that C99 preferentially associates with rafts has remained elusive. Here, we test this idea by quantifying the affinity of C99-GFP for raft domains in cell-derived giant plasma membrane vesicles. We find that C99 is essentially excluded from ordered domains in HeLa cells, SH-SY5Y cells and neurons, instead exhibiting a strong (roughly 90%) affinity for disordered domains. The strong association of C99 with disordered domains occurs independently of its cholesterol binding activity, homodimerization, or the familial Alzheimer disease Arctic mutation. Finally, we confirm previous studies suggesting that C99 is processed in the plasma membrane by α-secretase, in addition to the well-known γ-secretase. These findings suggest that C99 itself lacks an intrinsic affinity for raft domains, implying either that amyloidogenic processing of the protein occurs in disordered regions of the membrane, that processing involves a marginal sub-population of C99 found in rafts, or that as-yet-unidentified protein-protein interactions involving C99 in living cells drive it into rafts to promote its cleavage therein.

Competing Interest Statement

The authors have declared no competing interest.

  • Abbreviations

    Aβ
    amyloid-β
    APP
    amyloid precursor protein
    BACE1
    β-site amyloid precursor protein cleaving enzyme 1
    DRM
    detergent resistant membrane
    GPMV
    giant plasma membrane vesicle
    GUV
    giant unilamellar vesicle
    Ld
    liquid disordered
    Lo
    liquid ordered
    TfR
    transferrin receptor
    TMD
    transmembrane domain
    v
    number of GPMVs measured
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    Posted November 25, 2020.
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    The C99 domain of the amyloid precursor protein is a disordered membrane phase-preferring protein
    Ricardo Capone, Ajit Tiwari, Arina Hadziselimovic, Yelena Peskova, James M. Hutchison, Charles R. Sanders, Anne K. Kenworthy
    bioRxiv 2020.11.25.397893; doi: https://doi.org/10.1101/2020.11.25.397893
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    The C99 domain of the amyloid precursor protein is a disordered membrane phase-preferring protein
    Ricardo Capone, Ajit Tiwari, Arina Hadziselimovic, Yelena Peskova, James M. Hutchison, Charles R. Sanders, Anne K. Kenworthy
    bioRxiv 2020.11.25.397893; doi: https://doi.org/10.1101/2020.11.25.397893

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