Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis

Abstract

Enigmatic lipid peroxidation products have been claimed as the proximate executioners of ferroptosis—a specialized death program triggered by insufficiency of glutathione peroxidase 4 (GPX4). Using quantitative redox lipidomics, reverse genetics, bioinformatics and systems biology, we discovered that ferroptosis involves a highly organized oxygenation center, wherein oxidation in endoplasmic-reticulum-associated compartments occurs on only one class of phospholipids (phosphatidylethanolamines (PEs)) and is specific toward two fatty acyls—arachidonoyl (AA) and adrenoyl (AdA). Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 (ACSL4) acts as a specific antiferroptotic rescue pathway. Lipoxygenase (LOX) generates doubly and triply-oxygenated (15-hydroperoxy)-diacylated PE species, which act as death signals, and tocopherols and tocotrienols (vitamin E) suppress LOX and protect against ferroptosis, suggesting a homeostatic physiological role for vitamin E. This oxidative PE death pathway may also represent a target for drug discovery.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Oxygenation of esterified AA contributes to RSL3-induced ferroptosis in WT and Acsl4 KO Pfa1 cells.
Figure 2: Effects of exogenous AA on RSL3-triggered ferroptosis.
Figure 3: Screening of phospholipids and their oxidation products identifies ferroptosis death signals.
Figure 4: Oxygenated PE species identified in ferroptotic Gpx4 KO cells and kidney of Gpx4 KO mice.
Figure 5: Labeling with AA-d8 unravels pathways leading to oxygenated diacylated PE ferroptotic signals.
Figure 6: A 15-LOX phospholipid oxidation product, 15-hydroperoxy-SAPE, triggers ferroptosis in WT and Acsl4 KO Pfa1 cells.

Accession codes

Accessions

Protein Data Bank

References

  1. 1

    Allocati, N., Masulli, M., Di Ilio, C. & De Laurenzi, V. Die for the community: an overview of programmed cell death in bacteria. Cell Death Dis. 6, e1609 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Byrne, J.M. et al. Redox cycling of Fe(II) and Fe(III) in magnetite by Fe-metabolizing bacteria. Science 347, 1473–1476 (2015).

    Article  CAS  Google Scholar 

  3. 3

    Dixon, S.J. & Stockwell, B.R. The role of iron and reactive oxygen species in cell death. Nat. Chem. Biol. 10, 9–17 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Dixon, S.J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060–1072 (2012).

    CAS  Article  Google Scholar 

  5. 5

    Yang, W.S. & Stockwell, B.R. Ferroptosis: death by lipid peroxidation. Trends Cell Biol. 26, 165–176 (2016).

    Article  CAS  Google Scholar 

  6. 6

    Yang, W.S. & Stockwell, B.R. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem. Biol. 15, 234–245 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Imai, H. & Nakagawa, Y. Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) in mammalian cells. Free Radic. Biol. Med. 34, 145–169 (2003).

    Article  CAS  Google Scholar 

  8. 8

    Yamanaka, K. et al. A novel fluorescent probe with high sensitivity and selective detection of lipid hydroperoxides in cells. RSC Advances 2, 7894–7900 (2012).

    Article  CAS  Google Scholar 

  9. 9

    Drummen, G.P., van Liebergen, L.C., Op den Kamp, J.A. & Post, J.A. C11-BODIPY581/591, an oxidation-sensitive fluorescent lipid peroxidation probe: (micro)spectroscopic characterization and validation of methodology. Free Radic. Biol. Med. 33, 473–490 (2002).

    Article  CAS  Google Scholar 

  10. 10

    Li, B. & Pratt, D.A. Methods for determining the efficacy of radical-trapping antioxidants. Free Radic. Biol. Med. 82, 187–202 (2015).

    Article  CAS  Google Scholar 

  11. 11

    Küch, E.M. et al. Differentially localized acyl-CoA synthetase 4 isoenzymes mediate the metabolic channeling of fatty acids towards phosphatidylinositol. Biochim. Biophys. Acta 1841, 227–239 (2014).

    Article  CAS  Google Scholar 

  12. 12

    Golej, D.L. et al. Long-chain acyl-CoA synthetase 4 modulates prostaglandin E release from human arterial smooth muscle cells. J. Lipid Res. 52, 782–793 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Doll, S. et al. ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. Nat. Chem. Biol. http://dx.doi.org/10.1038/nchembio.2239 (2016).

  14. 14

    McIntyre, T.M., Prescott, S.M. & Stafforini, D.M. The emerging roles of PAF acetylhydrolase. J. Lipid Res. 50 (Suppl.): S255–S259 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Soh, N. et al. Swallow-tailed perylene derivative: a new tool for fluorescent imaging of lipid hydroperoxides. Org. Biomol. Chem. 5, 3762–3768 (2007).

    Article  CAS  Google Scholar 

  16. 16

    Dixon, S.J. et al. Human haploid cell genetics reveals roles for lipid metabolism genes in nonapoptotic cell death. ACS Chem. Biol. 10, 1604–1609 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Seiler, A. et al. Glutathione peroxidase 4 senses and translates oxidative stress into 12/15-lipoxygenase dependent- and AIF-mediated cell death. Cell Metab. 8, 237–248 (2008).

    Article  CAS  Google Scholar 

  18. 18

    Friedmann Angeli, J.P. et al. Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat. Cell Biol. 16, 1180–1191 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Askari, B. et al. Rosiglitazone inhibits acyl-CoA synthetase activity and fatty acid partitioning to diacylglycerol and triacylglycerol via a peroxisome proliferator-activated receptor-γ-independent mechanism in human arterial smooth muscle cells and macrophages. Diabetes 56, 1143–1152 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    O'Donnell, V.B. & Murphy, R.C. New families of bioactive oxidized phospholipids generated by immune cells: identification and signaling actions. Blood 120, 1985–1992 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Xiao, Y. & Guengerich, F.P. Metabolomic analysis and identification of a role for the orphan human cytochrome P450 2W1 in selective oxidation of lysophospholipids. J. Lipid Res. 53, 1610–1617 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Khanna, S. et al. Molecular basis of vitamin E action: tocotrienol modulates 12-lipoxygenase, a key mediator of glutamate-induced neurodegeneration. J. Biol. Chem. 278, 43508–43515 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Arai, H., Nagao, A., Terao, J., Suzuki, T. & Takama, K. Effect of D-α-tocopherol analogues on lipoxygenase-dependent peroxidation of phospholipid–bile salt micelles. Lipids 30, 135–140 (1995).

    Article  CAS  Google Scholar 

  24. 24

    Dennis, E.A. Diversity of group types, regulation, and function of phospholipase A2. J. Biol. Chem. 269, 13057–13060 (1994).

    CAS  PubMed  Google Scholar 

  25. 25

    van den Brink-van der Laan, E., Killian, J.A. & de Kruijff, B. Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile. Biochim. Biophys. Acta 1666, 275–288 (2004).

    Article  CAS  Google Scholar 

  26. 26

    Lee, A.G. How lipids affect the activities of integral membrane proteins. Biochim. Biophys. Acta 1666, 62–87 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Toppo, S., Flohé, L., Ursini, F., Vanin, S. & Maiorino, M. Catalytic mechanisms and specificities of glutathione peroxidases: variations of a basic scheme. Biochim. Biophys. Acta 1790, 1486–1500 (2009).

    Article  CAS  Google Scholar 

  28. 28

    Uderhardt, S. et al. 12/15-lipoxygenase orchestrates the clearance of apoptotic cells and maintains immunologic tolerance. Immunity 36, 834–846 (2012).

    Article  CAS  Google Scholar 

  29. 29

    Orrenius, S. & Zhivotovsky, B. Cardiolipin oxidation sets cytochrome c free. Nat. Chem. Biol. 1, 188–189 (2005).

    Article  CAS  Google Scholar 

  30. 30

    Kagan, V.E. et al. Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors. Nat. Chem. Biol. 1, 223–232 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Massey, K.A. & Nicolaou, A. Lipidomics of polyunsaturated-fatty-acid-derived oxygenated metabolites. Biochem. Soc. Trans. 39, 1240–1246 (2011).

    Article  CAS  Google Scholar 

  32. 32

    Kuhn, H., Banthiya, S. & van Leyen, K. Mammalian lipoxygenases and their biological relevance. Biochim. Biophys. Acta 1851, 308–330 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Schroeder, F. Regulation of aminophospholipid asymmetry in murine fibroblast plasma membranes by choline and ethanolamine analogues. Biochim. Biophys. Acta 599, 254–270 (1980).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Sessions, A. & Horwitz, A.F. Myoblast aminophospholipid asymmetry differs from that of fibroblasts. FEBS Lett. 134, 75–78 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Garreta, A. et al. Structure and interaction with phospholipids of a prokaryotic lipoxygenase from Pseudomonas aeruginosa. FASEB J. 27, 4811–4821 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Suardíaz, R. et al. Understanding the mechanism of the hydrogen abstraction from arachidonic acid catalyzed by the human enzyme 15-lipoxygenase-2. A quantum mechanics/molecular mechanics free energy simulation. J. Chem. Theory Comput. 12, 2079–2090 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Noguchi, N. et al. The specificity of lipoxygenase-catalyzed lipid peroxidation and the effects of radical-scavenging antioxidants. Biol. Chem. 383, 619–626 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Carlson, B.A. et al. Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration. Redox Biol. 9, 22–31 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Chen, L., Hambright, W.S., Na, R. & Ran, Q. Ablation of the ferroptosis inhibitor glutathione peroxidase 4 in neurons results in rapid motor neuron degeneration and paralysis. J. Biol. Chem. 290, 28097–28106 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Matsushita, M. et al. T cell lipid peroxidation induces ferroptosis and prevents immunity to infection. J. Exp. Med. 212, 555–568 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Wortmann, M. et al. Combined deficiency in glutathione peroxidase 4 and vitamin E causes multiorgan thrombus formation and early death in mice. Circ. Res. 113, 408–417 (2013).

    Article  CAS  Google Scholar 

  42. 42

    Sen, C.K., Khanna, S., Roy, S. & Packer, L. Molecular basis of vitamin E action. Tocotrienol potently inhibits glutamate-induced pp60c-Src kinase activation and death of HT4 neuronal cells. J. Biol. Chem. 275, 13049–13055 (2000).

    Article  CAS  Google Scholar 

  43. 43

    Yang, W.S. et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317–331 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Telmer, C.A. et al. Rapid, specific, no-wash, far-red fluorogen activation in subcellular compartments by targeted fluorogen activating proteins. ACS Chem. Biol. 10, 1239–1246 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Szent-Gyorgyi, C. et al. Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat. Biotechnol. 26, 235–240 (2008).

    Article  CAS  Google Scholar 

  46. 46

    Tardi, P.G., Mukherjee, J.J. & Choy, P.C. The quantitation of long-chain acyl-CoA in mammalian tissue. Lipids 27, 65–67 (1992).

    Article  CAS  Google Scholar 

  47. 47

    Minkler, P.E., Kerner, J., Ingalls, S.T. & Hoppel, C.L. Novel isolation procedure for short-, medium-, and long-chain acyl–coenzyme A esters from tissue. Anal. Biochem. 376, 275–276 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Sun, D., Cree, M.G. & Wolfe, R.R. Quantification of the concentration and 13C tracer enrichment of long-chain fatty acyl–coenzyme A in muscle by liquid chromatography/mass spectrometry. Anal. Biochem. 349, 87–95 (2006).

    Article  CAS  Google Scholar 

  49. 49

    Folch, J., Lees, M. & Sloane Stanley, G.H. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509 (1957).

    CAS  Google Scholar 

  50. 50

    Miller, T.M. et al. Rapid, simultaneous quantitation of mono and dioxygenated metabolites of arachidonic acid in human CSF and rat brain. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877, 3991–4000 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Tejero, J. et al. Peroxidase activation of cytoglobin by anionic phospholipids: mechanisms and consequences. Biochim. Biophys. Acta 1861, 391–401 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Kobe, M.J., Neau, D.B., Mitchell, C.E., Bartlett, S.G. & Newcomer, M.E. The structure of human 15-lipoxygenase-2 with a substrate mimic. J. Biol. Chem. 289, 8562–8569 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Marrink, S.J., Risselada, H.J., Yefimov, S., Tieleman, D.P. & de Vries, A.H. The MARTINI force field: coarse grained model for biomolecular simulations. J. Phys. Chem. B 111, 7812–7824 (2007).

    Article  CAS  Google Scholar 

  54. 54

    Van Der Spoel, D. et al. GROMACS: fast, flexible, and free. J. Comput. Chem. 26, 1701–1718 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Yang, K. et al. Dynamic simulations on the arachidonic acid metabolic network. PLoS Comput. Biol. 3, e55 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Gupta, S., Maurya, M.R., Stephens, D.L., Dennis, E.A. & Subramaniam, S. An integrated model of eicosanoid metabolism and signaling based on lipidomics flux analysis. Biophys. J. 96, 4542–4551 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Hoops, S. et al. COPASI—a COmplex PAthway SImulator. Bioinformatics 22, 3067–3074 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Ruzicka (Thermo Fisher Scientific) for help in obtaining MS3 spectra of PE oxidation products using tribrid Fusion Lumos. Supported by the US National Institutes of Health (P01HL114453 to R.K.M., U19AI068021 to J.G., NS076511 to V.E.K., NS061817 to H.B., P41GM103712 to I.B. and ES020693 to Y.Y.T.), the Human Frontier Science Program (HFSP-RGP0013/2014), and the Deutsche Forschungsgemeinschaft (CO 291/2-3 and CO 291/5-1) to M.C.

Author information

Affiliations

Authors

Contributions

V.E.K., M.C. and H.B. formulated the idea, designed the study and wrote the manuscript. G.M. and J.P.F.A. performed cell experiments. Y.Y.T. and F.Q. performed MS lipid analysis, interpreted data. C.S. and S.W. performed cell imaging experiments. T.A., V.A.T. and A.A.A. performed model systems experiments. D.M. and J.K.-S. performed computational modeling. B.L. and I.B. performed network analysis. S.D., H.H.D., J.J., V.B.R., A.A.K., B.P. and Q.Y. participated in cell or animal experiments. J.G., R.K.M. and B.R.S. participated in formulating the idea and writing the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Valerian E Kagan or Marcus Conrad or Hülya Bayır.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–5 and Supplementary Figures 1–18. (PDF 9201 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kagan, V., Mao, G., Qu, F. et al. Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis. Nat Chem Biol 13, 81–90 (2017). https://doi.org/10.1038/nchembio.2238

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing