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bioRxiv · 10.64898/2026.06.17.732910

Revealing interactions between glutathione peroxidase 4 and phosphoinositides

Walters, S. H.·Park, B.·Labrecque, C. L.·Musayev, F. N.·Van Lehn, R. C.·Fuglestad, B.
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Abstract

Glutathione peroxidase 4 (GPx4) is the primary enzyme reducing lipid hydroperoxides, preventing membrane oxidative damage and protecting against ferroptosis. GPx4 is known to engage with lipid headgroups through electrostatic interactions, positioning the substrate for reduction. This work reveals and characterizes binding of highly anionic phosphoinositides (PIP lipids) by GPx4. PIPs are vital lipids in human cells and are central to many signaling processes, particularly in cytosolic facing membranes. Lipid overlay assays confirm interactions between GPx4 and phosphorylated PIPs, comparable to known anionic lipid binders. Protein NMR describes the interaction between GPx4 and PIPs within micelles. The greatest resonance shifting occurs with trisphosphorylated PIP, suggesting that higher anionic charge leads to greater binding, a known driver of GPx4 substrate recognition. Preferred anionic interactions were also confirmed with titration and crystallographic structure analysis of inositol phosphate 4 (IP4). A headgroup-binding site on GPx4 is revealed to be proximal to the cationic membrane interaction site. In conjunction with molecular simulations, these results show that PIP lipid interactions allow full engagement of GPx4 with the membrane and positions the headgroup to allow the lipid tail to interact with the catalytic site. Understanding whether GPx4 preferentially interacts with PIPs will allow better understanding of the protective function of this essential enzyme and a mechanism that may protect essential lipid signaling pathways from oxidative damage. SignificanceThis study allows a deeper structural and mechanistic understanding of GPx4, the primary enzyme that reduces lipid hydroperoxides and prevents ferroptosis. Gaining an understanding of phosphoinositide binding to GPx4 reveals a mechanism for potential preservation of these important signaling molecules and for ferroptosis protection. Observation of a specific binding site for headgroup engagement reveals a plausible lipid interaction mode and functional mechanism of this important cytoprotective enzyme.

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Walters, S. H., Park, B., Labrecque, C. L., Musayev, F. N., Van Lehn, R. C., Fuglestad, B.. 2026-06-21. Revealing interactions between glutathione peroxidase 4 and phosphoinositides. https://doi.org/10.64898/2026.06.17.732910

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