Resolving the paradox of ferroptotic cell death: Ferrostatin-1 binds to 15LOX/PEBP1 complex, suppresses generation of peroxidized ETE-PE, and protects against ferroptosis

Tamil S Anthonymuthu¹, Yulia Y Tyurina², Wan-Yang Sun³, Karolina Mikulska-Ruminska⁴, Indira H Shrivastava⁵, Vladimir A Tyurin², Fatma B Cinemre⁶, Haider H Dar², Andrew P VanDemark⁷, Theodore R Holman⁸, Yoel Sadovsky⁹, Brent R Stockwell¹⁰, Rong-Rong He¹¹, Ivet Bahar¹², Hülya Bayır¹³, Valerian E Kagan¹⁴

Affiliations

¹ Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA; Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA, USA; Children's Neuroscience Institute, University of Pittsburgh, Pittsburgh, PA, USA; Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA.
² Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA.
³ Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA; Guangdong Engineering Research Center of Chinese Medicine & Disease Susceptibility, Jinan University, College of Pharmacy, Guangzhou, China.
⁴ Department of Computational and System Biology, University of Pittsburgh, Pittsburgh, PA, USA; Institute of Physics, Faculty of Physics Astronomy and Informatics, Nicolaus Copernicus University in Toruń, Grudziadzka 5, 87-100 Torun, Poland.
⁵ Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Computational and System Biology, University of Pittsburgh, Pittsburgh, PA, USA.
⁶ Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA; Sakarya University School of Medicine, Sakarya, Turkey.
⁷ Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA.
⁸ Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, USA.
⁹ Magee-Womens Research Institute and Departments of OBGYN and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, USA.
¹⁰ Department of Biological Sciences and Department of Chemistry, Columbia University, New York, NY, USA.
¹¹ Guangdong Engineering Research Center of Chinese Medicine & Disease Susceptibility, Jinan University, College of Pharmacy, Guangzhou, China.
¹² Department of Computational and System Biology, University of Pittsburgh, Pittsburgh, PA, USA.
¹³ Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA; Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA, USA; Children's Neuroscience Institute, University of Pittsburgh, Pittsburgh, PA, USA; Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA. Electronic address: bayihx@ccm.upmc.edu.
¹⁴ Children's Neuroscience Institute, University of Pittsburgh, Pittsburgh, PA, USA; Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA; Navigational Redox Lipidomics Group, Institute for Regenerative Medicine, IM Sechenov First Moscow State Medical University, Russian Federation. Electronic address: kagan@pitt.edu.

PMID: 33126055
PMCID: PMC7596334
DOI: 10.1016/j.redox.2020.101744

Resolving the paradox of ferroptotic cell death: Ferrostatin-1 binds to 15LOX/PEBP1 complex, suppresses generation of peroxidized ETE-PE, and protects against ferroptosis

Tamil S Anthonymuthu et al. Redox Biol. 2021 Jan.

. 2021 Jan:38:101744.

doi: 10.1016/j.redox.2020.101744. Epub 2020 Oct 16.

Authors

Affiliations

¹ Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA; Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA, USA; Children's Neuroscience Institute, University of Pittsburgh, Pittsburgh, PA, USA; Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA.
² Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA.
³ Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA; Guangdong Engineering Research Center of Chinese Medicine & Disease Susceptibility, Jinan University, College of Pharmacy, Guangzhou, China.
⁴ Department of Computational and System Biology, University of Pittsburgh, Pittsburgh, PA, USA; Institute of Physics, Faculty of Physics Astronomy and Informatics, Nicolaus Copernicus University in Toruń, Grudziadzka 5, 87-100 Torun, Poland.
⁵ Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Computational and System Biology, University of Pittsburgh, Pittsburgh, PA, USA.
⁶ Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA; Sakarya University School of Medicine, Sakarya, Turkey.
⁷ Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA.
⁸ Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, USA.
⁹ Magee-Womens Research Institute and Departments of OBGYN and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, USA.
¹⁰ Department of Biological Sciences and Department of Chemistry, Columbia University, New York, NY, USA.
¹¹ Guangdong Engineering Research Center of Chinese Medicine & Disease Susceptibility, Jinan University, College of Pharmacy, Guangzhou, China.
¹² Department of Computational and System Biology, University of Pittsburgh, Pittsburgh, PA, USA.
¹³ Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA; Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA, USA; Children's Neuroscience Institute, University of Pittsburgh, Pittsburgh, PA, USA; Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA. Electronic address: bayihx@ccm.upmc.edu.
¹⁴ Children's Neuroscience Institute, University of Pittsburgh, Pittsburgh, PA, USA; Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA; Navigational Redox Lipidomics Group, Institute for Regenerative Medicine, IM Sechenov First Moscow State Medical University, Russian Federation. Electronic address: kagan@pitt.edu.

PMID: 33126055
PMCID: PMC7596334
DOI: 10.1016/j.redox.2020.101744

Abstract

Hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) is a ferroptotic cell death signal. HpETE-PE is produced by the 15-Lipoxygenase (15LOX)/Phosphatidylethanolamine Binding Protein-1 (PEBP1) complex or via an Fe-catalyzed non-enzymatic radical reaction. Ferrostatin-1 (Fer-1), a common ferroptosis inhibitor, is a lipophilic radical scavenger but a poor 15LOX inhibitor arguing against 15LOX having a role in ferroptosis. In the current work, we demonstrate that Fer-1 does not affect 15LOX alone, however, it effectively inhibits HpETE-PE production by the 15LOX/PEBP1 complex. Computational molecular modeling shows that Fer-1 binds to the 15LOX/PEBP1 complex at three sites and could disrupt the catalytically required allosteric motions of the 15LOX/PEBP1 complex. Using nine ferroptosis cell/tissue models, we show that HpETE-PE is produced by the 15LOX/PEBP1 complex and resolve the long-existing Fer-1 anti-ferroptotic paradox.

Keywords: 15-Lipoxygenase; Ferroptosis; Ferrostatin-1; Hydroperoxy-eicosatetraenoyl-phosphatidylethanolamines; Phospholipid peroxidation.

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Figures

**Scheme 1**
Two alternative oxidation mechanisms: enzymatic (15LOX/PEBP1-driven) and/or non-enzymatic (Fe-driven) reactions of phospholipids (PL) (with the allyl hydrogen shown explicitly as PLH) oxidation yielding hydroperoxy-phospholipids (PLOOH) and their inhibition by ferrostatin-1 (Fer-1).

**Fig. 1**
Oxidation of ETE and ETE-PE by 15LOX2 and 15LOX2/PEBP1 complex. a) Effect of Fer-1 on ETE oxidation by 15LOX2 and 15LOX2/PEBP1; b) Oxidation of ETE-PE by 15LOX2 and 15LOX2/PEBP1; c) Inhibition of 15LOX2 and 15LOX2/PEBP1 catalyzed ETE-PE oxidation by Fer-1. The IC50 = 10.1 nM (for 15LOX2/PEBP1; two-way ANOVA with Sidak test).

**Fig. 2**
Fer-1 binds 15LOX2/PEBP1 complex and affects its functional dynamics. a) Fer-1 binding poses (red sticks) cluster in three regions (sites 1–3). b) Histograms of binding population counts (ordinate) and corresponding binding energies (abscissa) for each site. Note the sharp difference between sites 1,3 and 2, where binding occurs to 15LOX2/PEBP1 (red-bars) only. c) Comparison of global dynamics of 15LOX2/PEBP1 complex in absence (red-curve) and presence of Fer-1 (black-curve). d) Final conformers (at100 ns) observed in MD simulations of PEBP1 binding to 15LOX2, with (right) and without (left) Fer-1. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

**Fig. 3**
Inhibitory effect of Fer-1 on free radical and enzymatic lipid peroxidation. a) Matrix plot representing the fraction of PEox (red circles), PCox (grey circles), PSox (green circles) and PIox (blue circles) species among the total PLox generated by Fe/ascorbate (top) and RSL3 (bottom) in MLE cells. The total amount of PLox is specified at the bottom of each plot; b) Dot-plot showing amounts of oxidized PL species inhibited by Fer-1 in RSL3 (y-axis) vs. Fe/ascorbate (x-axis). The amount is the difference between RSL3 or Fe/Asc treated cells in the absence and presence of Fer-1 respectively. Larger dot denotes significant differences between the levels in RSL3-Fer-1 and Fe/Asc-Fer-1, smaller dot denotes the non-significant values. Area in dashed rectangle is enlarged as inset, n = 3; c) Cell death induced by Fe/ascorbate (upper panel) and RSL3 (lower panel) in MLE cells. n = 7; d) total PE oxidation in cells exposed to Fe/ascorbate ± Fer-1, RSL3±Fer-1. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

**Fig. 4**
Proportion of enzymatic and non-enzymatic PL oxidation during ferroptosis. Ratio of changes in free radical induced non-specific PCox products (amount of PCox in oxidation induced condition – amount of PCox in basal condition) to specific enzymatic ETE-PE oxidation products (amount of ETE-PEox in oxidation induced condition – amount of ETE-PEox in basal condition) (primary y-axis) for two systems HBE+ΔwspF and MLE + Fe/ascorbate defines the complete enzymatic and free radical oxidation, respectively. Using these values, the percentage of free radical oxidation (secondary y-axis) was calculated for various ferroptotic conditions. *P < 0.05 vs MLE + Fe/Asc, One way ANOVA.

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References

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Resolving the paradox of ferroptotic cell death: Ferrostatin-1 binds to 15LOX/PEBP1 complex, suppresses generation of peroxidized ETE-PE, and protects against ferroptosis

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Resolving the paradox of ferroptotic cell death: Ferrostatin-1 binds to 15LOX/PEBP1 complex, suppresses generation of peroxidized ETE-PE, and protects against ferroptosis

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