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Sensing of mitochondrial DNA by ZBP1 promotes RIPK3-mediated necroptosis and ferroptosis in response to diquat poisoning

Cell Death & Differentiation (2024)Cite this article

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Abstract

Diquat (DQ) poisoning is a severe medical condition associated with life-threatening implications and multiorgan dysfunction. Despite its clinical significance, the precise underlying mechanism remains inadequately understood. This study elucidates that DQ induces instability in the mitochondrial genome of endothelial cells, resulting in the accumulation of Z-form DNA. This process activates Z-DNA binding protein 1 (ZBP1), which then interacts with receptor-interacting protein kinase 3 (RIPK3), ultimately leading to RIPK3-dependent necroptotic and ferroptotic signaling cascades. Specific deletion of either Zbp1 or Ripk3 in endothelial cells simultaneously inhibits both necroptosis and ferroptosis. This dual inhibition significantly reduces organ damage and lowers mortality rate. Notably, our investigation reveals that RIPK3 has a dual role. It not only phosphorylates MLKL to induce necroptosis but also phosphorylates FSP1 to inhibit its enzymatic activity, promoting ferroptosis. The study further shows that deletion of mixed lineage kinase domain-like (Mlkl) and the augmentation of ferroptosis suppressor protein 1 (FSP1)-dependent non-canonical vitamin K cycling can provide partial protection against DQ-induced organ damage. Combining Mlkl deletion with vitamin K treatment demonstrates a heightened efficacy in ameliorating multiorgan damage and lethality induced by DQ. Taken together, this study identifies ZBP1 as a crucial sensor for DQ-induced mitochondrial Z-form DNA, initiating RIPK3-dependent necroptosis and ferroptosis. These findings suggest that targeting the ZBP1/RIPK3-dependent necroptotic and ferroptotic pathways could be a promising approach for drug interventions aimed at mitigating the adverse consequences of DQ poisoning.

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Fig. 1: ZBP1, RIPK3, and MLKL are crucial components for the development of fatal multiple organ injury induced by DQ.
Fig. 2: Specific deletion of Zbp1, Ripk3, and Mlkl in endothelial cells, rather than hematopoietic cells, provides protection against the fatal multiple organ injury induced by DQ.
Fig. 3: DQ induces mitochondrial damage to release Z-DNA and activate ZBP1.
Fig. 4: The Zα and RHIM domain facilitates the activation of ZBP1 and its interaction with RIPK3 to trigger cell death in response to DQ stimulation.
Fig. 5: ZBP1/RIPK3 mediates necroptosis and ferroptosis induced by DQ.
Fig. 6: DQ-triggered ferroptosis is mediated through the RIPK3/FSP1 axis.
Fig. 7: Vitamin K counteracts DQ induced cell death both in vitro and in vivo through restoring FSP1 activity.
Fig. 8: Schematic model.

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Data availability

The datasets used in the current study are available from the corresponding author (YX) upon reasonable request. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD045977. The single-cell sequencing data generated in this study have been deposited in the National Center for Biotechnology Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE244858).The original western blot data are provided in Supplementary Materials (Original western blots).

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Acknowledgements

We are grateful to Prof. Jiahuai Han (Xiamen University), Prof. Wei Mo (Xiamen University) and Ben Lu (Central South University), for experimental materials. Yanfang Xu was supported by Fujian Research and Training Grants for Young and Middle-aged Leaders in Healthcare (2022QNRCYX-XYF), and Outstanding Young Talents Program of the First Affiliated Hospital of Fujian Medical University (YJCQN-A-XYF2021).

Funding

This work was supported by grants from National Natural Science Foundation of China (U23A20410), Young and Middle-aged Scientific Research Major Project of Fujian Provincial Health Commission (No. 2021ZQNZD004), Joint Funds for the innovation of science and Technology of Fujian province (2021Y9100), Program of the First Affiliated Hospital of Fujian Medical University (YJRC4104), and Fujian Province Finance Project (2020B009).

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  1. These authors contributed equally: Kunmei Lai, Junjie Wang, Siyi Lin.

Authors and Affiliations

  1. Department of Nephrology, Blood Purification Research Center, the First Affiliated Hospital, Fujian Medical University, Fuzhou, 350005, China

    Kunmei Lai, Junjie Wang, Siyi Lin, Zhimin Chen, Keng Ye, Ying Yuan, Yujiao Lin & Yanfang Xu

  2. Research Center for Metabolic Chronic Kidney Disease, the First Affiliated Hospital, Fujian Medical University, Fuzhou, 350005, China

    Kunmei Lai, Junjie Wang, Siyi Lin, Zhimin Chen, Keng Ye, Ying Yuan, Yujiao Lin & Yanfang Xu

  3. Department of Nephrology, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, 350212, China

    Kunmei Lai, Junjie Wang, Siyi Lin, Zhimin Chen, Keng Ye, Ying Yuan, Yujiao Lin & Yanfang Xu

  4. Central Laboratory, the First Affiliated Hospital, Fujian Medical University, Fuzhou, 350005, China

    Kunmei Lai, Huabin Ma & Yanfang Xu

  5. Department of Intensive Care Unit, the First Affiliated Hospital, Fujian Medical University, Fuzhou, 350005, China

    Guo Lin

  6. State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian, 361102, China

    Chuan-Qi Zhong

  7. Laboratory Animal Research Center, Xiamen University, Xiamen, Fujian, 361102, China

    Jianfeng Wu

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Contributions

YX, HM and JW designed research. KL, JW, SL, GL, KY, YY, YJ, JW, HM performed the mice and molecular experiments. ZC analyzed the single-cell RNA sequencing data. CQZ. conducted the MS data. HM and YX wrote the draft manuscript. YX HM and JW supervised the research and performed writing-review and editing. All authors verified the data and approved the final version of the manuscript.

Corresponding authors

Correspondence to Jianfeng Wu, Huabin Ma or Yanfang Xu.

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The animal experiments were accomplished in compliance with ethical standards. All animal experiments were approved by the Laboratory Animal Management and Ethics Committee of Fujian Medical University with approved number IACUC FJMU 2022-0886 and were performed in accordance with the “China Guide for the Protection and Use of Laboratory Animals”.

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Lai, K., Wang, J., Lin, S. et al. Sensing of mitochondrial DNA by ZBP1 promotes RIPK3-mediated necroptosis and ferroptosis in response to diquat poisoning. Cell Death Differ (2024). https://doi.org/10.1038/s41418-024-01279-5

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