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Characterization of the binding of cytosolic phospholipase A2 alpha and NOX2 NADPH oxidase in mouse macrophages

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Abstract

Background

Previous studies have demonstrated that cytosolic phospholipase A2α (cPLA2α) is required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Moreover, upon stimulation, cPLA2α translocates to the plasma membranes by binding to the assembled oxidase, forming a complex between its C2 domain and the PX domain of the cytosolic oxidase factor, p47phox in human phagocytes. Intravenous administration of antisense against cPLA2α that significantly inhibited its expression in mouse peritoneal neutrophils and macrophages also inhibited superoxide production, in contrast to cPLA2α knockout mice that showed normal superoxide production. The present study aimed to determine whether there is a binding between cPLA2α-C2 domain and p47phox-PX in mouse macrophages, to further support the role of cPLA2α in oxidase regulation also in mouse phagocytes.

Methods and results

A significant binding of mouse GST-p47phox-PX domain fusion protein and cPLA2α in stimulated mouse phagocyte membranes was demonstrated by pull-down experiments, although lower than that detected by the human p47phox-PX domain. Substituting the amino acids Phe98, Asn99, and Gly100 to Cys98, Ser99, and Thr100 in the mouse p47phox-PX domain (present in the human p47phox-PX domain) caused strong binding that was similar to that detected by the human p47phox-PX domain

Conclusions

The binding between cPLA2α-C2 and p47phox-PX domains exists in mouse macrophages and is not unique to human phagocytes. The binding between the two proteins is lower in the mice, probably due to the absence of amino acids Cys98, Ser 99, and Thr100in the p47phox-PX domain that facilitate the binding to cPLA2α.

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Acknowledgements

This research was supported by a donation from Miss Dorothy Polayes, USA entitled: the role of cPLA2α in oxidative stress and the pathogenesis of inflammation.

Funding

This research was supported by a donation from Miss Dorothy Polayes, USA entitled: the role of cPLA2α in oxidative stress and the pathogenesis of inflammation.

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Authors and Affiliations

  1. Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, 84105, Beer Sheva, Israel

    Yulia Solomonov, Nurit Hadad & Rachel Levy

Authors
  1. Yulia Solomonov
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  2. Nurit Hadad
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  3. Rachel Levy
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Contributions

YS designed and performed the experiments and analyzed data. NH helped in methodology. RL guided the study and wrote the manuscript. All authors read the manuscript and approved its publication.

Corresponding author

Correspondence to Rachel Levy.

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The authors declare that they have no conflict of interest.

Ethical approval

The study was approved by the institutional Human Research Committee of the Soroka University Medical Center (No. 0370-16-SOR) and by the Ben-Gurion University of the Negev committee for ethical care and use of animals in experiments, Authorization No. IL-23-05-2017.

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All authors approved the manuscript and agreed to publish it in Molecular Biology Reports.

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Solomonov, Y., Hadad, N. & Levy, R. Characterization of the binding of cytosolic phospholipase A2 alpha and NOX2 NADPH oxidase in mouse macrophages. Mol Biol Rep 49, 3511–3518 (2022). https://doi.org/10.1007/s11033-022-07191-w

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