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Phospholipase iPLA2β averts ferroptosis by eliminating a redox lipid death signal

Nature Chemical Biology volume 17, pages 465–476 (2021)Cite this article

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Abstract

Ferroptosis, triggered by discoordination of iron, thiols and lipids, leads to the accumulation of 15-hydroperoxy (Hp)-arachidonoyl-phosphatidylethanolamine (15-HpETE-PE), generated by complexes of 15-lipoxygenase (15-LOX) and a scaffold protein, phosphatidylethanolamine (PE)-binding protein (PEBP)1. As the Ca2+-independent phospholipase A2β (iPLA2β, PLA2G6 or PNPLA9 gene) can preferentially hydrolyze peroxidized phospholipids, it may eliminate the ferroptotic 15-HpETE-PE death signal. Here, we demonstrate that by hydrolyzing 15-HpETE-PE, iPLA2β averts ferroptosis, whereas its genetic or pharmacological inactivation sensitizes cells to ferroptosis. Given that PLA2G6 mutations relate to neurodegeneration, we examined fibroblasts from a patient with a Parkinson’s disease (PD)-associated mutation (fPDR747W) and found selectively decreased 15-HpETE-PE-hydrolyzing activity, 15-HpETE-PE accumulation and elevated sensitivity to ferroptosis. CRISPR-Cas9-engineered Pnpla9R748W/R748W mice exhibited progressive parkinsonian motor deficits and 15-HpETE-PE accumulation. Elevated 15-HpETE-PE levels were also detected in midbrains of rotenone-infused parkinsonian rats and α-synuclein-mutant SncaA53T mice, with decreased iPLA2β expression and a PD-relevant phenotype. Thus, iPLA2β is a new ferroptosis regulator, and its mutations may be implicated in PD pathogenesis.

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Fig. 1: Effect of a point mutation on iPLA2β function.
Fig. 2: Computational modeling of the interaction of the iPLA2β dimer with membrane-embedded PLs.
Fig. 3: Lipidomics of H109 and fPDR747W cells.
Fig. 4: iPLA2β-deficient cells are sensitive to RSL3-induced ferroptosis.
Fig. 5: Homozygous Pnpla9R748W/R748W mutation leads to a parkinsonian phenotype and ferroptotic oxygenated PE accumulation in the midbrain of mice.
Fig. 6: Accumulation of ferroptotic cell death signals in the substantia nigra of rats exposed to rotenone.

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Data availability

Data generated during the study and included in this article are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the NIH (HL114453, AI156924, AI156923, CA165065, CA243142, AI145406, NS076511, NS061817, P41GM103712, R21NS094854, PA30DA035778 and P01DK096990), the Natural Science Foundation of China (81873209, 81622050, 81903821), the National Science Centre, Poland (grant 2019/35/D/ST4/02203), 111 Project of Chinese MoE (B13038), the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01Y036), GDUPS (2019) and the March of Dimes Prematurity Research Center at the University of Pennsylvania. We are grateful to J. Guererro-Santoro for technical assistance with the PNPLA9 KO BeWo cells.

Author information

Authors and Affiliations

  1. Department of Environmental and Occupational Health and Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA

    Wan-Yang Sun, Vladimir A. Tyurin, Indira H. Shrivastava, Tamil S. Anthonymuthu, Gao-Wei Mao, Haider H. Dar, Alexandr A. Kapralov, Andrew A. Amoscato, Hülya Bayır, Yulia Y. Tyurina & Valerian E. Kagan

  2. Guangdong Engineering Research Center of Chinese Medicine & Disease Susceptibility, Jinan University, Guangzhou, China

    Wan-Yang Sun, Yu-Jia Zhai, Ming-Hai Pan, Hai-Biao Gong, Dan-Hua Lu, Jie Sun, Wen-Jun Duan & Rong-Rong He

  3. International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chinese Ministry of Education (MoE), College of Pharmacy, Jinan University, Guangzhou, China

    Wan-Yang Sun, Yu-Jia Zhai, Ming-Hai Pan, Hai-Biao Gong, Dan-Hua Lu, Jie Sun, Wen-Jun Duan & Rong-Rong He

  4. Institute of Physics, Faculty of Physics, Astronomy and Informatics, Nicolaus Copernicus University in Toruń, Toruń, Poland

    Karolina Mikulska-Ruminska

  5. Department of Computational and Systems Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA

    Karolina Mikulska-Ruminska, Indira H. Shrivastava & Ivet Bahar

  6. Department of Critical Care Medicine, Safar Center for Resuscitation Research, Children’s Neuroscience Institute, Children’s Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, PA, USA

    Tamil S. Anthonymuthu & Hülya Bayır

  7. Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, USA

    Sergey Korolev & Ian Miller

  8. UCL Queen Square Institute of Neurology, Department of Clinical and Movement Neurosciences, University College London, Queen Square, London, UK

    Andrey Y. Abramov & Plamena R. Angelova

  9. Magee-Womens Research Institute and Department of OBGYN, University of Pittsburgh, Pittsburgh, PA, USA

    Ofer Beharier & Yoel Sadovsky

  10. Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA

    Teresa G. Hastings & Timothy J. Greenamyre

  11. Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA

    Charleen T. Chu

  12. Department of Radiation Oncology, University of Pittsburgh, Pittsburgh, PA, USA

    Valerian E. Kagan

  13. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA

    Valerian E. Kagan

  14. Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA

    Valerian E. Kagan

  15. Institute of Regenerative Medicine, IM Sechenov Moscow State Medical University, Moscow, Russia

    Valerian E. Kagan

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  1. Wan-Yang Sun
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  2. Vladimir A. Tyurin
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Contributions

V.E.K., R.-R.H., H.B., Y.Y.T. and Y.S. conceived the study. Y.-J.Z., O.B., H.H.D. and A.A.K. performed experiments with cells. S.K. and I.M. cloned and purified the WT and mutant iPLA2β protein. V.E.K., H.B., C.T.C., T.G.H., R.-R.H. and W.-J.D. designed in vivo experiments; W.-Y.S., M.-H.P., D.-H.L., Y.-J.Z. and J.S. performed in vivo experiments. V.E.K., Y.S., Y.Y.T., H.B., A.Y.A., P.R.A. and R.-R.H. designed in vitro experiments; W.-Y.S. and V.A.T. performed in vitro experiments. W.-Y.S., V.A.T., A.A.A., T.S.A. and H.-B.G. performed MS measurements and analyzed data. W.-Y.S., Y.Y.T. and V.A.T. discussed and interpreted MS results. K.M.-R. and I.H.S. performed computational modeling and analyzed data. I.B. supervised computational studies and interpreted the results. W.-Y.S., I.B., Y.S., C.T.C., I.H.S. and H.B. participated in writing the manuscript. Y.Y.T. and V.E.K. wrote the manuscript.

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Correspondence to Yulia Y. Tyurina, Rong-Rong He or Valerian E. Kagan.

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Extended data

Extended Data Fig. 1 Purification of iPLA2β and analysis of 15-HpETE and its hydrolysis products.

a, Western blot of purified recombinant WT and R693W mutant short variant CHO. The Short variant lacks a 54 amino acid (396-450) insert between ankyrin repeats and catalytic domain. The numbering is adjusted accordingly, such that R747W in Human Long iPLA2β is R693W in the CHO construct. Representative figure from 3 experiments b, Time-courses of ETE (upper panel) and 15-HpETE (lower panel) formation in reactions catalyzed by mutant R747W (red circles) or WT iPLA2β (black circles). 1-SA-2-ETE-PE and 1-SA-2-15-HpETE-PE were used as substrates. Data are means±s.d., N=3 independent experiments, *p=0.0381, ****p<0.0001 for 1-SA-2-ETE-PE data and *p=0.0189, **p=0.0044, ***P=0.0003 for 1-SA-2-15-HpETE-PE data, WT iPLA2β vs iPLA2βR747W at respective time points, two-way ANOVA (Sidak’s post-hoc test). We employed highly purified (>99%) substrates (1-SA-2-ETE-PE and 1-SA-2-15-HpETE-PE) thus free ETE and free 15-HpETE were undetectable in the samples with no enzyme added. c, Detection and identification of 15-HpETE, a hydrolysis product of 1-SA-2-15-HpETE-PE. Base peak chromatogram of molecular ions with m/z 335.2224 corresponding to 1-SA-2-15-HpETE (left insert). MS2 fragmentation pattern of molecular ions with m/z 335.2224 corresponding to 15-HpETE (right panel). Respective structure and fragments formed during MS2 analysis (right insert). d, Effect of substrate concentration on the velocity of iPLA2β WT (left panel) and iPLA2bR474W (right panel) catalyzed reaction. 1-SA-2-ETE-PE (blue circles), 1-SA-2-15-HETE-PE (light red circles) and 1-SA-2-15-HpETE-PE (dark red circles) were used as substrates. Data are presented as 1-SA-2-OH-PE μM/min, Data are means + s.d, N = 4 independent experiments for 1-SA-2-15HpETE-PE, 1-SA-2-15HETE-PE data and 3 for 1-SA-2-ETE-PE data.

Source data

Extended Data Fig. 2 Membrane composition and atomic structure used in MD simulations.

a, Structural formulas of phospholipids used in simulations. b, Spatial distribution in the lipid bilayer. The lipid composition has been deduced from our lipidomics data (see Methods) Here, SAPE is ETE-PE, and SOOH is 15-HpETE-PE. ETE-PE and 15-HpETE-PE were included at the levels of 22% and 4%, respectively. See legend at the bottom for the complete composition. The same composition and spatial distribution have been replicated to construct a larger membrane (100 ×100 Å2 and 250 ×250 Å2 surface area and 50 Å depth/thickness) in a simulation box with a height of 125 Å enclosing the dimeric enzyme bound to the membrane and explicit water molecules.

Extended Data Fig. 3 Comparison of conformational Flexibility of Acyl Chains.

a, Hydroperoxy-group in 15-HpETE-PE comes into close proximity of the membrane surface. Conformations reached in 100 ns MD simulations are shown for two 15-HpETE-PE molecules (left and middle) and one for ETE-PE (right). The distances between terminal amino group N-atom and the peroxidized C15-atom of the sn-2-acyl chain in 15-HpETE-PE and in ETE-PE (using the non-peroxidized equivalent C15-atom) are shown. A total of eight simulations were carried out, four with WT and four with mutant R747W iPLA2β. This distance was < 5 Å in at least two 15-HpETE-PE out of the total of 20 per MD snapshot. No such a short distance was observed for ETE-PE. Results for the two sets of four runs were very similar, showing that the behavior of the fatty acid chains is intrinsic to the oxidized fatty acids, irrespective of the iPLA2β. Similar behavior was also observed in the simulations of the membrane alone (in the absence of enzyme). The results in panel (a) are in qualitative agreement with the Nuclear Overhauser Effects (NOE) data obtained by Greenberg et al., 2007. The authors observed signals for irradiated –N(CH3)3 protons of the choline group and the terminal aldehydic group of the truncated sn-2-oxidized fatty acid chain in oxidized lipids, consistent with ‘whisker’ model. However, no signals were observed for non-oxidized lipids, as these were embedded within the hydrophobic bilayer. Observation of a signal in NOE experiments, corresponds to a distance of < 5 Å. b, Probability distribution of the position of the peroxidized/non-peroxidized carbon (C15, sn-2) for 15-HpETE-PE and ETE-PE along the z-axis of the lipid bilayer. The interface between the two lipid monolayers (red dotted line in the inset) serves as a reference point (z=0, shown in the inset) for the distances of C15 carbon atom from the center of the bilayer. Six random lipids for both, 15-HpETE-PE and ETE-PE, were chosen for the analysis. Carbon index (C15) refers to that shown on the chemical structure in Supplementary Fig. 5a. Shaded boxes denote the approximate range of distances at which maxima occur in the histograms. The C15 ETE-PE (orange box) remains embedded at ~ 5-6 Å, the C15 of 15-HpETE-PE moves closer to the membrane surface at 12-13 Å (blue box). The positions of the head groups at the membrane surface are indicated by grey box. The probability distributions show that the C15 atom (‘peroxidized’) of 1-SA-2-15-HpETE-PE adopts two distinct positions, one close to the membrane surface (blue box) the other buried near the central part of the lipid bilayer (z=0).

Extended Data Fig. 4 Ability of the peroxidized group in 1-SA-2-HpETE-PE to come into close proximity of the protein surface.

a, Probability distribution of the distance between the peroxidized carbon (C15, sn-2) of 1-SA-2-15-HpETE-PE or non-peroxidized carbon (C15, sn-2) of ETE-PE and: (i) protein surface (left panel) and (ii) catalytic site (right panel). For protein surface the closest atom of the lipid was taken into account whereas the reference point for catalytic site was computed based on the closest atom of the lipid to the center of mass of the highly conserved catalytic residue S465 and D598. Carbon index (C15) refers to that shown on the chemical structure in Supplementary Fig. 5a. Arrows denote the maxima in the histograms for C15 (which contains the OOH group, shown in panel b) in 1-SA-2-15-HpETE-PE (blue) and C15 in ETE-PE (orange). The analysis contains results from the second half of three MD trajectories for a WT iPLA2β dimer structure (50–100 ns period of time). b, A snapshot from MD simulations of iPLA2β dimer CAT domains with residues making close contacts with 1-SA-2-15-HpETE-PE and 1-SA-2-ETE-PE highlighted in space filling representation (green). The 1-SA-2-15-HpETE-PE (red-blue-cyan balls) and ETE-PE (grey surface) in the membrane are shown.

Extended Data Fig. 5 Comparison of the intrinsic dynamics of 1-SA-2-15-HpETE-PE and1-SA-2-ETE-PE.

a, Chemical structures. Carbon atom indices are indicated in green for sn-2 and orange for sn-1. Pink boxes highlight the sn-2 chain which is different in the two lipids. Blue arrow points to the peroxidized carbon C15 in 1-SA-2-15-HpETE-PE. b, Order parameters computed from three sets of independent MD runs. Computationally predicted deuterium order parameters (SCD) for sn-1 (upper curves) and sn-2 (lower curves) chains, based on 3 MD runs performed for ETE-PE (thick lines) and three for 1-SA-2-15-HpETE-PE (thin lines). Blue arrow indicates position of C15. In general, the order parameter S=3/2<cos2α>- 1/2 varies in the range [−0.5, 1]; the two limits corresponding to complete order (parallel alignment of the probed bond with respect to the magnetic field, with the angular difference being α=0) and antiparallel orientation (α=90o). SCD=0 for fully disordered states (<cos2α>= 1/3). The fully disordered state SCD=0 is shown by the dotted line. Red arrow shows the position of the oxidized carbon in 1-SA-2-15-HpETE-PE, where a slight increase in order is induced upon oxidation.). Both chains display low order parameters, with the sn-2 chain being more disordered in general than sn-1, except for the terminal C-atom of sn-1 reflecting higher flexibility at the chain terminals. The results are shown for an ensemble of chains in each case, which exhibit highly reproducible patterns, also consistent with previous computational and experimental data. Note that the portion of 1-SA-2-15-HpETE-PE sn-2 near the peroxidation site (C15) exhibit relatively higher ordering, whereas the remaining portions show a mixed behavior. While these differences are small, they are reproducible in independent runs, lending support to the robustness of simulation data. The computed order parameters values are in a good agreement with previously, reported computational and experimental values. Furthermore, while the published results on the effects of peroxidation on lipids dynamics are somewhat contradictory, our computational observations of the peroxidized sn-2 acyl chains for 1-SA-2-15HpETE-PE are in general agreement with those computed for other peroxidized lipids using the same tools.

Extended Data Fig. 6 Homology modeling details of dimer iPLA2β model.

a, Swiss model results for homology modeling which includes global and local quality estimate values, sequence identity and coverage compared to the protein template (PDB code: 6aun) and sequence alignment. Red boxes on the sequence alignment denote regions which was not solved in X-ray structure (6aun) and was modeled using Swiss model server that is Y95-R115, L129-N146, R405-K408, V652-A670. Black box highlight region M1-A80 which was not present in the ANK repeats fragment. b, I-tasser results for homolog modeling of M1-A300 fragment of iPLA2β structure. Cyan ribbon diagram denote iPLA2β dimer solved in X-ray (6aun), red elements of the structures were modeled using Swiss Model server (shown in the panel a, red boxes). M1-A300 fragment of ANK repeat is shown in yellow ribbon diagram and alignment on the crystal structure. Estimated accuracy obtained by I-tasser server for M1-A300 model is also shown.

Extended Data Fig. 7 Time evolution and histograms of interfacial contacts between iPLA2b residues and 15-HpETE-PE molecules observed in MD simulations.

a, Results from 4 × 2 runs (labeled MD1-4) conducted for the WT and R747W mutants are displayed. Residues making contacts are listed along the ordinate, and the time evolution of contacts (atom-atom interactions closer than 3.5 Å with any 15-HpETE-PE atom) is shown in in each case. Colored regions indicate the contacts made by chain A (cyan) and B (dark red). Note that most of the contacts are persistent once formed. b, Histograms of contacts. Residues making the largest number of contacts (counts based on snapshots collected every 50 picoseconds, summed over all runs) are listed, along with the corresponding counts for chains A (top) and B (bottom).

Extended Data Fig. 8 iPLA2β-deficient cells are more sensitive to RSL3-induced ferroptosis compared to WT cells.

a, Total PLA2 activity in H109 and fPDR747W cells in the absence and in the presence of (S)-BEL. Cell supernatants were incubated with 1-SA-2-ETE-PE (left panel) or 1-SA-2-15-HpETE-PE (right panel) for 30 min at 37 °C. Activity is presented as 1-SA-2-OH-PE, pmol/min/mg protein. The background levels of 1-SA-2-OH-PE in H109 and fPDR747W cell supernatants were low and estimated as 2.76 + 0.19 and 2.74 + 0.75 pmols per sample vs 70.5 + 8.5 and 100.7 + 10.5 pmols per sample accumulated in H109 and fPDR747W cell supernatants during incubation in the absence of S-BEL. Data are means ± s.d., **p = 0.0014 for H109 cells, **p = 0.0073 for fPDR743w cells, ****p < 0.0001, N = 3 biologically independent experiments, one-way ANOVA (Tukey post hoc test). b, Heat map showing the content of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol (PI) molecular species in H109 and fPDR747W cells. dPE-diacyl species, pPE-plasmalogens, dPC-diacyl PC species, pPC-plasmalogen species of PC. Data presented as pmol/nmol of total phospholipids, N = 3 biologically independent experiments. c, RSL3-induced accumulation of oxygenated PE (PEox, left), PC (PCox, upper right), PS (PSox, middle right) and PI (PIox, lower right) molecular species in H109 and fPDR747W fibroblasts. Cells were exposed to RSL3 (25 nM) for 14 hrs. Data presented as pmol/mmol of total phospholipids, N = 3 biologically independent experiments. d, RSL3 - induced ferroptosis in WT and iPLA2β KD SHSY5Y cells. Cell were treated with RSL3 (2 μM) for 18 hrs in the absence or in the presence of Fer-1 (0.4 μM). Inset: representative blot of iPLA2β. Data are means±s.d., ****p < 0.0001 vs WT, N = 3 biologically independent experiments, two-way ANOVA (Sidak post-hoc test). e, RSL3-induced ferroptosis in WT and PNPLA9 KO BeWo cells. Cells were incubated with RSL3 (100 nM) for 12 hrs in the absence or in the presence of Fer-1 (0.4 μM). Inset: Typical western blot of iPLA2β obtained from BeWo WT and PNPLA9KO cells. Ferroptosis quantified by LDH release. Data are means±s.d., ****p < 0.0001 vs WT, N = 3 biologically independent experiments, two-way ANOVA (Sidak post-hoc test). f, Content of 1-SA-2-15-HpETE-PE in WT and PNPLA9 KO BeWo cells. Data are means ± s.d., ***p = 0.0002, ****p < 0.0001 vs WT, N = 3 biologically independent experiments, two-way ANOVA (Sidak post-hoc test).

Source data

Extended Data Fig. 9 LPCAT3 KD protects mouse embryonic cells from RSL3 induced death.

a, Representative immunoblots and quantification of LPCAT3 in cells treated with non-targeted siRNA (si-NT) or LPCAT3 siRNA (si-LPCAT3). LPCAT3 levels were quantified from three biological replicates and normalized to actin. Data represent mean ± s.d., *p = 0.0004 vs si-NT, unpaired two-tailed t-test. b, si-NT or LPCAT3 KD cells were exposed to RSL3 (100 nM) and cell death was monitored after 20 hrs by PI staining using flow cytometry. Data are mean ± s.d., N = 3 biologically independent experiments; ****p < 0.0001 vs si-NT control, ##p = 0.0081 vs.si-LPCAT3 control, $$p = 0.0078 vs si-NT/RSL3, one-way ANOVA. c, Quantitative LC/MS-based assessments of lyso-PE (1-SA-2-OH-PE, left) and lyso-PC (1-SA-2OH-PC, right) in MEF cells. Data are mean ± s.d., N = 3 biologically independent experiments, ***p = 0.0008, ****p < 0.0001 vs si-NT, unpaired two-tailed student’s t-test. d, The contents of oxygenated PE (1-SA-2-HpETE-PE, left) and PC (1-SA-2-15-HpETE-PC, right) in MEF cells. Cells were exposed to RSL3 (100 nM) for 20 hrs. Data are mean ± s.d., N = 3 biologically independent experiments, *p = 0.0282, ****p < 0.0001 vs si-NT control, one-way ANOVA, (Tukey’s post-hoc test).

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Extended Data Fig. 10 Content of PE, oxygenated PE in midbrain of rotenone exposed rats and 8-months old WT and A53T mice.

a, Content of PE in substantia nigra of control rats (treated with vehicle, DMSO) and Parkinsonian rats (treated with rotenone for 14 days at a dose of 3 mg/kg/day). Data are presented as pmol/nmol of total phospholipids, N = 6 biologically independent animals. d-diacyl species; p-alkenyl (plasmalogen) species. b, Quantification of oxygenated PE species in substantia nigra of control rats (treated with vehicle, DMSO) and Parkinsonian rats (treated with rotenone for 14 days at the dose of 3 mg/kg/day). Data presented as pmol/mmol of total phospholipids, N = 6 biologically independent animals. c, iPLA2β protein expression in midbrain of 8-months old WT and A53T mice. Inset: Typical western blot of iPLA2β. Data are means ± SD, ****p < 0.0001, N = 5 biologically independent animals, unpaired two-tailed Student’s t-test. d, Content of oxygenated PE species in midbrains of WT and A53T mice. Data are presented as pmol/mmol of total phospholipids. e, Content of 15-HpETE-PE in midbrains of WT and A53T mice. Data are presented as pmol of 1-SA-2-15-HpETE-PE per mmol of total phospholipids, **p = 0.0010 WT vs A53T mice, unpaired two-tailed Student’s t-test. N = 6 biologically independent animals. f, Content of PE in midbrains of WT and A53T mice. Data are presented as pmol/nmol of total phospholipids, N = 5 biologically independent animals. d-diacyl species; p-alkenyl (plasmalogen) species.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–6, Tables 1–3 and uncut gel for Supplementary Fig. 4f

Reporting Summary

Supplementary Video 1

HpETE-PE (shown in space-filling representation) was observed in MD simulations to undergo rapid conformational changes, enabling its lateral diffusion in the membrane and occasional movements toward the surface to expose the peroxidized head. The movie displays a trajectory of 50 ns, with frames recorded every 250 ps. The lipid molecules are color coded as in Supplementary Fig. 2. Additional runs are displayed for HpETE-PE molecules, both resulting (within 50 ns) in the exposure of the peroxidation site of the AA-acyl chain to the membrane surface.

Supplementary Video 2

HpETE-PE (shown in space-filling representation) was observed in MD simulations to undergo rapid conformational changes, enabling its lateral diffusion in the membrane and occasional movements toward the surface to expose the peroxidized head. The movie displays a trajectory of 50 ns, with frames recorded every 250 ps. The lipid molecules are color coded as in Supplementary Fig. 2. Additional runs are displayed for HpETE-PE molecules, both resulting (within 50 ns) in the exposure of the peroxidation site of the AA-acyl chain to the membrane surface.

Supplementary Video 3

HpETE-PE (shown in space-filling representation) was observed in MD simulations to undergo rapid conformational changes, enabling its lateral diffusion in the membrane and occasional movements toward the surface to expose the peroxidized head. The movie displays a trajectory of 50 ns, with frames recorded every 250 ps. The lipid molecules are color coded as in Supplementary Fig. 2. Additional runs are displayed for HpETE-PE molecules, both resulting (within 50 ns) in the exposure of the peroxidation site of the AA-acyl chain to the membrane surface.

Supplementary Video 4

Pole test of representative 7-month-old WT and Pnpla9R748W mice.

Supplementary Video 5

Rotarod test of representative 7-month-old WT and Pnpla9R748W mice.

Source data

Source Data Fig. 1

Unprocessed western blot.

Source Data Fig. 4

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Source Data Fig. 5

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Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 8

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Source Data Extended Data Fig. 9

Unprocessed western blots.

Source Data Extended Data Fig. 10

Unprocessed western blots.

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Sun, WY., Tyurin, V.A., Mikulska-Ruminska, K. et al. Phospholipase iPLA2β averts ferroptosis by eliminating a redox lipid death signal. Nat Chem Biol 17, 465–476 (2021). https://doi.org/10.1038/s41589-020-00734-x

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