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Mitophagy mediated by BNIP3 and NIX protects against ferroptosis by downregulating mitochondrial reactive oxygen species

Cell Death & Differentiation (2024)Cite this article

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Abstract

Mitophagy plays an important role in the maintenance of mitochondrial homeostasis and can be categorized into two types: ubiquitin-mediated and receptor-mediated pathways. During receptor-mediated mitophagy, mitophagy receptors facilitate mitophagy by tethering the isolation membrane to mitochondria. Although at least five outer mitochondrial membrane proteins have been identified as mitophagy receptors, their individual contribution and interrelationship remain unclear. Here, we show that HeLa cells lacking BNIP3 and NIX, two of the five receptors, exhibit a complete loss of mitophagy in various conditions. Conversely, cells deficient in the other three receptors show normal mitophagy. Using BNIP3/NIX double knockout (DKO) cells as a model, we reveal that mitophagy deficiency elevates mitochondrial reactive oxygen species (mtROS), which leads to activation of the Nrf2 antioxidant pathway. Notably, BNIP3/NIX DKO cells are highly sensitive to ferroptosis when Nrf2-driven antioxidant enzymes are compromised. Moreover, the sensitivity of BNIP3/NIX DKO cells is fully rescued upon the introduction of wild-type BNIP3 and NIX, but not the mutant forms incapable of facilitating mitophagy. Consequently, our results demonstrate that BNIP3 and NIX-mediated mitophagy plays a role in regulating mtROS levels and protects cells from ferroptosis.

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Fig. 1: BNIP3 and NIX redundantly contribute to various types of mitophagy in HeLa cells.
Fig. 2: Mitophagy mediated by BNIP3 and NIX plays a crucial role in maintaining mitochondrial function.
Fig. 3: The Nrf2-antioxidant pathway compensates for the loss of mitophagy.
Fig. 4: Mitophagy suppresses ferroptosis induced by compromising antioxidant enzymes.
Fig. 5: Schemes of responses to several oxidative stresses in WT and mitophagy-deficient cells.

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Data availability

All relevant data are available from the authors. Original immunoblot data are provided in the Supplemental Material.

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Acknowledgements

We thank Feng Zhang (Broad Institute of MIT and Harvard) and Eric Campeau (University of Massachusetts Medical School) for providing plasmids and CCRF (Niigata University) for supporting FACS analysis.

Funding

This work was supported in part by the Japan Society for the Promotion of Science KAKENHI grants (16KK0162 and 22K07207 to SY and 19H05712, 18H04858, and 18H04691, to TK), AMED grant (JP21gm6110013, JP23ek0109647 and JP23gm1710006 to TK), and the Takeda Science Foundation (to SY).

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Authors and Affiliations

  1. Department of Cellular Physiology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, 950-8510, Japan

    Shun-ichi Yamashita, Keiichi Inoue, Kentaro Furukawa, Tomoyuki Fukuda & Tomotake Kanki

  2. Center for Cancer Immunotherapy and Immunobiology, Kyoto University Graduate School of Medicine, Kyoto, 606-8501, Japan

    Yuki Sugiura, Yuta Matsuoka & Rae Maeda

  3. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, USA

    David C. Chan

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  1. Shun-ichi Yamashita
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  2. Yuki Sugiura
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Contributions

SY, YS, and TK designed the experiments. SY generated receptor KO cell lines using the CRIPSR/Cas9 genome editing system. SY performed all cellular analyses. YS, YM, and RM performed the metabolomic analysis. SY, KI, KF, TF, DCC, and TK wrote the manuscript.

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Correspondence to Shun-ichi Yamashita or Tomotake Kanki.

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Yamashita, Si., Sugiura, Y., Matsuoka, Y. et al. Mitophagy mediated by BNIP3 and NIX protects against ferroptosis by downregulating mitochondrial reactive oxygen species. Cell Death Differ (2024). https://doi.org/10.1038/s41418-024-01280-y

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