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S-Acylation Regulates the Membrane Association and Activity of Calpain-5

Jozsef Gal, Vimala Bondada, Charles B. Mashburn, David W. Rodgers, Dorothy E. Croall, James W. Geddes
doi: https://doi.org/10.1101/2022.01.25.477766
Jozsef Gal
1Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, Kentucky 40536
2Department of Neuroscience, University of Kentucky, Lexington, Kentucky 40536
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  • For correspondence: jgeddes@uky.edu jgal2@uky.edu
Vimala Bondada
1Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, Kentucky 40536
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Charles B. Mashburn
1Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, Kentucky 40536
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David W. Rodgers
3Department of Molecular and Cellular Biochemistry and Center for Structural Biology, University of Kentucky, Lexington, Kentucky 40536
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Dorothy E. Croall
4Department of Molecular and Biomedical Sciences, University of Maine, Orono, Maine 04469
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James W. Geddes
1Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, Kentucky 40536
2Department of Neuroscience, University of Kentucky, Lexington, Kentucky 40536
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  • For correspondence: jgeddes@uky.edu jgal2@uky.edu
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Abstract

Calpain-5 (CAPN5) is a member of the calpain family of calcium-activated neutral thiol proteases. CAPN5 is partly membrane associated, despite its lack of a transmembrane domain. Unlike classical calpains, CAPN5 contains a C-terminal C2 domain. C2 domains often have affinity to lipids, mediating membrane association. We recently reported that the C2 domain of CAPN5 was essential for its membrane association and the activation of its autolytic activity. However, despite the removal of the C2 domain by autolysis, the N-terminal fragment of CAPN5 remained membrane associated. S-acylation, also referred to as S-palmitoylation, is a reversible post-translational lipid modification of cysteine residues that promotes membrane association of soluble proteins. In the present study several S-acylated cysteine residues were identified in CAPN5 with the acyl-PEG exchange method. Data reported here demonstrate that CAPN5 is S-acylated on up to three cysteine residues including Cys-4 and Cys-512, and likely Cys-507. The D589N mutation in a potential calcium binding loop within the C2 domain interfered with the S-acylation of CAPN5, likely preventing initial membrane association. Mutating specific cysteine residues of CAPN5 interfered with both its membrane association and the activation of CAPN5 autolysis. Taken together, our results suggest that the S-acylation of CAPN5 is critical for its membrane localization which appears to favor its enzymatic activity.

Competing Interest Statement

The authors have declared no competing interest.

  • Abbreviations used

    3×FLAG
    tag peptide with the sequence Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr-Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
    3×HA
    tag peptide with the sequence Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Gly-Ser-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Gly-Ser-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala
    ABE
    acyl-biotin exchange
    ADNIV
    Autosomal dominant neovascular inflammatory vitreoretinopathy
    APEx
    acyl-PEG exchange
    APT
    acyl-protein thioesterase
    CBSW
    calpain-type β-sandwich domain
    PEG
    polyethylene glycol
    DAPI
    4’,6-diamidino-2-phenylindole
    SD
    standard deviation
    FBS
    fetal bovine serum
    PEF
    penta-EF hand domain
    PAT
    protein S-acyltransferase
    PC
    protease core domain
    WT
    wild-type
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    Posted January 26, 2022.
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    S-Acylation Regulates the Membrane Association and Activity of Calpain-5
    Jozsef Gal, Vimala Bondada, Charles B. Mashburn, David W. Rodgers, Dorothy E. Croall, James W. Geddes
    bioRxiv 2022.01.25.477766; doi: https://doi.org/10.1101/2022.01.25.477766
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    S-Acylation Regulates the Membrane Association and Activity of Calpain-5
    Jozsef Gal, Vimala Bondada, Charles B. Mashburn, David W. Rodgers, Dorothy E. Croall, James W. Geddes
    bioRxiv 2022.01.25.477766; doi: https://doi.org/10.1101/2022.01.25.477766

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