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.

RIP3 targets pyruvate dehydrogenase complex to increase aerobic respiration in TNF-induced necroptosis

Abstract

Receptor-interacting protein kinase 3 (RIP3)-regulated production of reactive oxygen species (ROS) positively feeds back on tumour necrosis factor (TNF)-induced necroptosis, a type of programmed necrosis. Glutamine catabolism is known to contribute to RIP3-mediated ROS induction, but the major contributor is unknown. Here, we show that RIP3 activates the pyruvate dehydrogenase complex (PDC, also known as PDH), the rate-limiting enzyme linking glycolysis to aerobic respiration, by directly phosphorylating the PDC E3 subunit (PDC-E3) on T135. Upon activation, PDC enhances aerobic respiration and subsequent mitochondrial ROS production. Unexpectedly, mixed-lineage kinase domain-like (MLKL) is also required for the induction of aerobic respiration, and we further show that it is required for RIP3 translocation to meet mitochondria-localized PDC. Our data uncover a regulation mechanism of PDC activity, show that PDC activation by RIP3 is most likely the major mechanism activated by TNF to increase aerobic respiration and its by-product ROS, and suggest that RIP3-dependent induction of aerobic respiration contributes to pathologies related to oxidative stress.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: TNF induces an increase in aerobic respiration in a RIP3-dependent manner.
Fig. 2: Induction of OCR is not due to the side effect of zVAD.
Fig. 3: TNF-induced increase in aerobic respiration is responsible for ROS induction in necroptosis.
Fig. 4: PDC is positively involved in TNF-induced necroptosis.
Fig. 5: RIP3 targets PDC to upregulate aerobic respiration.
Fig. 6: RIP3 activates PDC by phosphorylating PDC-E3 on T135.
Fig. 7: MLKL is required for PDC activation.

References

  1. 1.

    Laster, S. M., Wood, J. G. & Gooding, L. R. Tumor necrosis factor can induce both apoptic and necrotic forms of cell lysis. J. Immunol. 141, 2629–2634 (1988).

    CAS  PubMed  Google Scholar 

  2. 2.

    Kerr, J. F., Wyllie, A. H. & Currie, A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257 (1972).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Zhang, D. W. et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 325, 332–336 (2009).

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    He, S. et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-α. Cell 137, 1100–1111 (2009).

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Cho, Y. S. et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137, 1112–1123 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Wu, X. N. et al. Distinct roles of RIP1-RIP3 hetero- and RIP3-RIP3 homo-interaction in mediating necroptosis. Cell. Death Differ. 21, 1709–1720 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Orozco, S. et al. RIPK1 both positively and negatively regulates RIPK3 oligomerization and necroptosis. Cell. Death Differ. 21, 1511–1521 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Chen, W. et al. Ppm1b negatively regulates necroptosis through dephosphorylating Rip3. Nat. Cell. Biol. 17, 434–444 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148, 213–227 (2012).

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Zhao, J. et al. Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proc. Natl. Acad. Sci. USA 109, 5322–5327 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Wang, H. et al. Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol. Cell. 54, 133–146 (2014).

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Chen, X. et al. Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. Cell. Res. 24, 105–121 (2014).

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Dondelinger, Y. et al. MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell. Rep. 7, 971–981 (2014).

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Cai, Z. et al. Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat. Cell. Biol. 16, 55–65 (2014).

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Goossens, V. et al. Redox regulation of TNF signaling. BioFactors 10, 145–156 (1999).

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Schulze-Osthoff, K. et al. Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J. Biol. Chem. 267, 5317–5323 (1992).

    CAS  PubMed  Google Scholar 

  17. 17.

    Schulze-Osthoff, K., Beyaert, R., Vandevoorde, V., Haegeman, G. & Fiers, W. Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. EMBO J. 12, 3095–3104 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Tait, S. W. et al. Widespread mitochondrial depletion via mitophagy does not compromise necroptosis. Cell. Rep. 5, 878–885 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Schenk, B. & Fulda, S. Reactive oxygen species regulate Smac mimetic/TNFα-induced necroptotic signaling and cell death. Oncogene 34, 5796–5806 (2015).

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Zhang, Y. et al. RIP1 autophosphorylation is promoted by mitochondrial ROS and is essential for RIP3 recruitment into necrosome. Nat. Commun. 8, 14329 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Kim, Y. S., Morgan, M. J., Choksi, S. & Liu, Z. G. TNF-induced activation of the Nox1 NADPH oxidase and its role in the induction of necrotic cell death. Mol. Cell. 26, 675–687 (2007).

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Turrens, J. F., Freeman, B. A., Levitt, J. G. & Crapo, J. D. The effect of hyperoxia on superoxide production by lung submitochondrial particles. Arch. Biochem. Biophys. 217, 401–410 (1982).

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Turrens, J. F. Mitochondrial formation of reactive oxygen species. J. Physiol. 552, 335–344 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Jastroch, M., Divakaruni, A. S., Mookerjee, S., Treberg, J. R. & Brand, M. D. Mitochondrial proton and electron leaks. Essays Biochem. 47, 53–67 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Harris, R. A., Bowker-Kinley, M. M., Huang, B. & Wu, P. Regulation of the activity of the pyruvate dehydrogenase complex. Adv. Enzym. Reg. 42, 249–259 (2002).

    CAS  Article  Google Scholar 

  26. 26.

    Roche, T. E. et al. Distinct regulatory properties of pyruvate dehydrogenase kinase and phosphatase isoforms. Prog. Nucleic Acid. Res. Mol. Biol. 70, 33–75 (2001).

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Kaplon, J. et al. A key role for mitochondrial gatekeeper pyruvate dehydrogenase in oncogene-induced senescence. Nature 498, 109–112 (2013).

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Hitosugi, T. et al. Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism. Mol. Cell. 44, 864–877 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Temkin, V., Huang, Q., Liu, H., Osada, H. & Pope, R. M. Inhibition of ADP/ATP exchange in receptor-interacting protein-mediated necrosis. Mol. Cell. Biol. 26, 2215–2225 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Hanson, G. T. et al. Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J. Biol. Chem. 279, 13044–13053 (2004).

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Narendra, D., Tanaka, A., Suen, D. F. & Youle, R. J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J. Cell. Biol. 183, 795–803 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Festjens, N. et al. Butylated hydroxyanisole is more than a reactive oxygen species scavenger. Cell. Death Differ. 13, 166–169 (2006).

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Goossens, V., Grooten, J. & Fiers, W. The oxidative metabolism of glutamine. A modulator of reactive oxygen intermediate-mediated cytotoxicity of tumor necrosis factor in L929 fibrosarcoma cells. J. Biol. Chem. 271, 192–196 (1996).

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Zachar, Z. et al. Non-redox-active lipoate derivates disrupt cancer cell mitochondrial metabolism and are potent anticancer agents in vivo. J. Mol. Med. 89, 1137–1148 (2011).

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    El Sayed, S. M. et al. Safety and outcome of treatment of metastatic melanoma using 3-bromopyruvate: a concise literature review and case study. Chin. J. Cancer 33, 356–364 (2014).

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Deck, L. M. et al. Selective inhibitors of human lactate dehydrogenases and lactate dehydrogenase from the malarial parasite Plasmodium falciparum. J. Med. Chem. 41, 3879–3887 (1998).

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Hiromasa, Y., Fujisawa, T., Aso, Y. & Roche, T. E. Organization of the cores of the mammalian pyruvate dehydrogenase complex formed by E2 and E2 plus the E3-binding protein and their capacities to bind the E1 and E3 components. J. Biol. Chem. 279, 6921–6933 (2004).

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Schell, J. C. et al. A role for the mitochondrial pyruvate carrier as a repressor of the Warburg effect and colon cancer cell growth. Mol. Cell. 56, 400–413 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Thapa, R. J. et al. Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases. Proc. Natl. Acad. Sci. USA 110, E3109–3118 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Zhong, C. Q. et al. Quantitative phosphoproteomic analysis of RIP3-dependent protein phosphorylation in the course of TNF-induced necroptosis. Proteomics 14, 713–724 (2014).

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Grassian, A. R., Metallo, C. M., Coloff, J. L., Stephanopoulos, G. & Brugge, J. S. Erk regulation of pyruvate dehydrogenase flux through PDK4 modulates cell proliferation. Genes. Dev. 25, 1716–1733 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Rodriguez, D. A. et al. Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis. Cell. Death Differ. 23, 76–88 (2016).

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Boveris, A. & Chance, B. The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem. J. 134, 707–716 (1973).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Hennet, T., Richter, C. & Peterhans, E. Tumour necrosis factor-alpha induces superoxide anion generation in mitochondria of L929 cells. Biochem. J. 289, 587–592 (1993).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Joplin, R. et al. Subcellular localization of pyruvate dehydrogenase dihydrolipoamide acetyltransferase in human intrahepatic biliary epithelial cells. J. Pathol. 176, 381–390 (1995).

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Turrens, J. F. & Boveris, A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem. J. 191, 421–427 (1980).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Starkov, A. A. et al. Mitochondrial alpha-ketoglutarate dehydrogenase complex generates reactive oxygen species. J. Neurosci. 24, 7779–7788 (2004).

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Nakagawa, T. et al. Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature 434, 652–658 (2005).

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Linkermann, A. et al. Two independent pathways of regulated necrosis mediate ischemia–reperfusion injury. Proc. Natl. Acad. Sci. USA 110, 12024–12029 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Baines, C. P. et al. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434, 658–662 (2005).

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Malatesha, G., Singh, N. K., Bharija, A., Rehani, B. & Goel, A. Comparison of arterial and venous pH, bicarbonate, PCO2 and PO2 in initial emergency department assessment. Emerg. Med. J. 24, 569–571 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Lin, J. et al. A role of RIP3-mediated macrophage necrosis in atherosclerosis development. Cell. Rep. 3, 200–210 (2013).

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    Kim, J. W., Tchernyshyov, I., Semenza, G. L. & Dang, C. V. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell. Metab. 3, 177–185 (2006).

    Article  PubMed  Google Scholar 

  54. 54.

    Moriwaki, K., Bertin, J., Gough, P. J., Orlowski, G. M. & Chan, F. K. Differential roles of RIPK1 and RIPK3 in TNF-induced necroptosis and chemotherapeutic agent-induced cell death. Cell. Death Dis. 6, e1636 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Chen, X. et al. Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. Cell. Res. 24, 105–121 (2014).

    CAS  Article  PubMed  Google Scholar 

  56. 56.

    Zhang, D. W. et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 325, 332–336 (2009).

    CAS  Article  PubMed  Google Scholar 

  57. 57.

    Lin, J. et al. A role of RIP3-mediated macrophage necrosis in atherosclerosis development. Cell. Rep. 3, 200–210 (2013).

    CAS  Article  PubMed  Google Scholar 

  58. 58.

    Yoshida, S. et al. Molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis. Proc. Natl. Acad. Sci. USA 110, E1604–1612 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Hanson, G. T. et al. Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J. Biol. Chem. 279, 13044–13053 (2004).

    CAS  Article  PubMed  Google Scholar 

  60. 60.

    Waypa, G. B. et al. Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells. Circ. Res. 106, 526–535 (2010).

    CAS  Article  PubMed  Google Scholar 

  61. 61.

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Frezza, C. et al. Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Nature 477, 225–228 (2011).

    CAS  Article  PubMed  Google Scholar 

  65. 65.

    Wu, X. et al. Investigation of receptor interacting protein (RIP3)-dependent protein phosphorylation by quantitative phosphoproteomics. Mol. Cell. Proteom. 11, 1640–1651 (2012).

    Article  Google Scholar 

  66. 66.

    Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M. & Zhuang, X. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat. Methods 8, 1027–1036 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  67. 67.

    French, J. B. et al. Spatial colocalization and functional link of purinosomes with mitochondria. Science 351, 733–737 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Narendra, D., Tanaka, A., Suen, D. F. & Youle, R. J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J. Cell. Biol. 183, 795–803 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Wieckowski, M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J. & Pinton, P. Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nat. Protoc. 4, 1582–1590 (2009).

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Natural Science Foundation of China (91429301), the National Basic Research Program of China (973 Program; 2015CB553800 and 2014CB541804), the National Natural Science Foundation of China (31420103910, 31330047 and 81788104), the 111 Project (B12001), the National Science Foundation of China for Fostering Talents in Basic Research (J1310027) and the Open Research Fund of State Key Laboratory of Cellular Stress Biology, Xiamen University.

Author information

Affiliations

Authors

Contributions

Z.Y. and J.H. conceived and designed the experiments. Z.Y., Y.W., Y.Z. and X.C. performed the experiments. X.H. and C.-Q.Z. performed the GC-MS and MS experiments and analysed the obtained results. H.N., Y.L. and J.W. helped to prepare cell lines for the study. S.Z. and D.Z. provided technical support. Z.Y. and J.H. interpreted the data and wrote the paper.

Corresponding author

Correspondence to Jiahuai Han.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures, Legends, Table Legends.

Life Sciences Reporting Summary

Supplementary Table 1

Statistics source data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yang, Z., Wang, Y., Zhang, Y. et al. RIP3 targets pyruvate dehydrogenase complex to increase aerobic respiration in TNF-induced necroptosis. Nat Cell Biol 20, 186–197 (2018). https://doi.org/10.1038/s41556-017-0022-y

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