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TFAM is an autophagy receptor that limits inflammation by binding to cytoplasmic mitochondrial DNA

Abstract

When cells are stressed, DNA from energy-producing mitochondria can leak out and drive inflammatory immune responses if not cleared. Cells employ a quality control system called autophagy to specifically degrade damaged components. We discovered that mitochondrial transcription factor A (TFAM)—a protein that binds mitochondrial DNA (mtDNA)—helps to eliminate leaked mtDNA by interacting with the autophagy protein LC3 through an autolysosomal pathway (we term this nucleoid-phagy). TFAM contains a molecular zip code called the LC3 interacting region (LIR) motif that enables this binding. Although mutating TFAM’s LIR motif did not affect its normal mitochondrial functions, more mtDNA accumulated in the cell cytoplasm, activating inflammatory signalling pathways. Thus, TFAM mediates autophagic removal of leaked mtDNA to restrict inflammation. Identifying this mechanism advances understanding of how cells exploit autophagy machinery to selectively target and degrade inflammatory mtDNA. These findings could inform research on diseases involving mitochondrial damage and inflammation.

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Fig. 1: Autophagy is involved in the removal of cytoplasm-exposed mtDNA and TFAM.
Fig. 2: The mtDNA–TFAM complex associates with LC3B in the cytoplasm.
Fig. 3: TFAM interacts with LC3B.
Fig. 4: TFAM mediates the degradation of mtDNA via the autophagy lysosomal pathway.
Fig. 5: TFAM knockdown exacerbates inflammation through the cGAS–STING pathway.
Fig. 6: The LIR motif of TFAM is required for interaction with LC3B and degradation of mtDNA.
Fig. 7: TFAM-mediated nucleoid-phagy attenuates cGAS–STING inflammatory signalling.

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Data availability

The NGS raw data reported in this paper have been deposited in the Genome Sequence Archive49 of the National Genomics Data Center50, China National Center for Bioinformation/Bejing lnstitute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA007049) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human. Source data are provided with this paper. All of the other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank L. Yu (Tsinghua University), H. Zhang (Institute of Biophysics, Chinese Academy of Sciences) and Q. Chen (Nankai University) for constructive suggestions during the preparations of the manuscript. We extend our gratitude to Q. Chen and L. Liu for generously providing the necessary plasmids and cell lines as gifts for this project. Additionally, we thank Y. Li (Cryo-EM Facility of Tsinghua University, Branch of National Protein Science Facility) for section and immunogold labelling. We also acknowledge the Core Facility of the School of Basic Medical Sciences for instrument and equipment support. This project is supported by the National Natural Science Foundation of China (numbers 92254307, 32300629, 82170461 and 32170758), the Guangdong Provincial Education Reform Project (number 01-408-2301057XM), the Key Discipline of Guangzhou Education Bureau (Basic Medicine; grant number: 201851839), open research funds from The Sixth Affiliated Hospital of Guangzhou Medical University at Qingyuan People’s Hospital, the Discipline Construction Project of Guangzhou Medical University (02-410-2206329 and 15001019004006) and the Guangzhou Medical University Research Capacity Improvement Program (02-410-2302292XM).

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Contributions

D.F., H.L., H.Z. and G.Z. designed and supervised the study. D.F., H.L. and C.Zhen wrote the manuscript. H.L., C.Zhen, J.X. and L.Z. performed most of the experiments and revised the manuscript. X.L. and Z.Liu provided the purified protein samples. Z.Luo performed the experiment related to C. elegans. S.Lu performed the NGS experiments and analysis. H.F, S.Lin, H.J., Y.C., J.C., Z.C., K.D., J.S., Z.W., Y.H., T.M., C.Zhou, Z.H., H.H., Q.Z. and P.H. contributed to the manuscript and fruitful discussions.

Corresponding author

Correspondence to Du Feng.

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The authors declare no competing interests.

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Nature Cell Biology thanks Rayk Behrendt, Masaaki Komatsu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The levels of cytosol-released TFAM and mtDNA are associated with autophagy in HeLa cells under oxidative stress.

a, b, SDS-PAGE (a) and statistical analysis on TFAM level (b) in HeLa cells upon different dose of H2O2 for 4 h (b, n = 3 independent experiments). c, HeLa cells overexpressing TFAM stimulated with H2O2, MG132 and Baf A1 (n = 3 independent experiments). d, e, Experimental plan to separate and extract the cytoplasm with light and dense organelles components (d). Validation of optimal NP-40 concentration for permeabilizing the plasma membrane in HeLa cells. (e, n = 3 independent experiments). f, g, Representative images (f) and percentages (g) of extra-mitochondrial TFAM in HeLa cells upon H2O2 and Baf A1. Yellow arrows, TFAM inside mitochondria; Green arrows, extra-mitochondrial TFAM. Scale bar, 10 μm in merged and 1 μm in zoom images. (g, n = 15 cells from 3 independent experiments). h, HeLa cells upon H2O2 and Baf A1. Cytosolic mtDNA was detected by primer ND2 and D-loop using PCR (n = 3). i–k, Representative images (i) and percentages (k) of mtDNA (j) or extra-mitochondrial TFAM (k) in ATG7-deficient HeLa cells with or without VBIT4 upon H2O2. White arrows, extra-mitochondrial TFAM-mtDNA complex. (n = 15 cells from 3 independent experiments in j and k) Scale bar, 10 μm in merged and 1 μm in zoom images. l, m, ATG7-deficient HeLa cells were stimulated with H2O2 and VBIT4. Cytosolic mtDNA was detected by primer ND1 (l, n = 3 independent experiments) or ND4 (m, n = 3 independent experiments) using qPCR. n, o, SDS-PAGE analysis (n) and statistical analysis on TFAM level (o, n = 3 independent experiments) in FIP200 knockdown HeLa cells upon H2O2. p, q, Representative images (p, n = 3 independent experiments) and co-localization analysis (q) of cytosolic TFAM-DsRed colocalizing with FIP200 in HeLa cells upon H2O2. White arrows, co-localization of mtDNA and GFP-quenched RFP-LC3 puncta. Scale bar, 10 μm in merged and 1 μm in zoom images. All magnify scale bar, 1 μm.Two-tailed unpaired Student’s t-test was used. n.s., no significance. Numerical source data, including exact P values and unprocessed blots, are provided in source data.

Source data

Extended Data Fig. 2 The association between cytosol-released TFAM-mtDNA and autophagy in THP1 cells under oxidative stress.

a, b, SDS-PAGE analysis (a) and statistical analysis on TFAM level (b) in THP1 cells stimulated with different dose of H2O2 for 1 h (b, n = 3 independent experiments, compared with 0 mM, from 1 mM to 4 mM: n.s., no significance, *P = 0.0014, ***P = 0.0002, respectively). c–e, SDS-PAGE analysis (c), statistical analysis (d) and TFAM expression detected by RT-qPCRI) in HeLa cells stimulated with H2O2, MG132 and Baf A1.(d,e, n = 3 independent experiments, n.s., no significance, *P = 0.0240, **P = 0.0032). f, The ideal concentration of NP-40 that can permeabilize the plasma membrane is verified in THP1 cells. Lamin B1 (nucleus), VDAC (mitochondria) and CytC (mitochondrial intermembrane space) are the markers for dense organelles, while LAMP1 (lysosome) is the marker of light organelles (n = 3 independent experiments). g, h, Cytosol, organelle, and whole cell lysate (WCL) components from THP1 cells treated with H2O2, MG132 and Baf A1. Samples were verified by immunoblotting (g) and related statistical analysis (h, n = 3 independent experiments, n.s., no significance, ***P = 0.0004, ****P < 0.0001). i, j, Representative images (i) and percentages (j) of mtDNA (PicoGreen) released outside mitochondria (DeepRed-Mito) in THP1 cells treated with H2O2 and Baf A1. Yellow arrows, mtDNA inside mitochondria; Green arrows, mtDNA outside mitochondria. Scale bar, 10 μm in merged and 1 μm in zoom images. All magnify scale bar, 1 μm. n = 20 cells from 3 independent experiments independent experiments. **P = 0.0054, ****P < 0.0001).Two-tailed unpaired Student’s t-test was used. Numerical source data, including exact P values and unprocessed blots, are provided in source data.

Source data

Extended Data Fig. 3 Cytosolic retention of mtDNA and TFAM induced by inflammatory triggers is also related to autophagy.

ac, SDS-PAGE analysis (a), statistical analysis on TFAM level (b, n = 3, ***P = 0.0002, **P = 0.0041) and TFAM expression detected by RT-qPCR (c, n = 3 independent experiments, **P = 0.0039, *P = 0.0241) in THP1 cells stimulated with LPS, ATP and Baf A1. d, e, Cytosol, organelle, and WCL components from in THP1 cells stimulated with LPS, ATP and Baf A1. Samples were verified by immunoblotting (d) and related statistical analysis (e). f–i, Representative images (f,h) and percentages (g,i) of TFAM (anti-TFAM; f,g) and mtDNA (anti-DNA; h,i) released outside mitochondria (DeepRed-Mito) in THP1 cells treated with LPS, ATP and Baf A1. Yellow arrows, TFAM or mtDNA inside mitochondria; Green arrows, TFAM or mtDNA outside mitochondria. Scale bar, 10 μm in merged and 1 μm in zoom images. In g and i, n = 15 cells from 3 independent experiments, **P = 0.0047, ***P = 0.0001, ****P < 0.0001. j, Expression patterns of the cytoplasmic mtDNA and nDNA of THP1 cells in control, H2O2, and LPS + ATP groups. The mean values were used to calculate SEM (error bars) and P values. n.s., no significance. two-tailed unpaired Student’s t-test. Numerical source data, including exact P values and unprocessed blots, are provided in source data.

Source data

Extended Data Fig. 4 Cytosol released mtDNA or HMG-5 colocalizes with autophagosomes in Caenorhabditis elegans under oxidative stress.

a, b, Representative images (a) and numbers (b) of GFP::LGG-1 puncta in hypodermal seam cells in a transgenic C. elegans expressing gfp::lgg-1 stimulated with H2O2. Ctrl n = 18 cells and H2O2 n = 20 cells from 3 independent experiments, ****P < 0.0001. Scale bar, 10 μm in merged and 1 μm in zoom images. c, d, Representative images (c) and numbers (d) of mCherry::LGG-1 puncta in hypodermal seam cells in a transgenic C. elegans expressing mCherry::lgg-1 stimulated with H2O2. Ctrl n = 18 cells and H2O2 n = 18 cells from 3 independent experiments, ****P < 0.0001. Scale bar, 10 μm in merged and 1 μm in zoom images. e, Representative images revealed the co-localization between mtDNA (anti-DNA) and mCherry::HMG-5 in hypodermis cells in a transgenic C. elegans expressing mCherry::hmg-5. Scale bar, 10 μm in merged and 1 μm in zoom images. f, Representative images revealed the co-localization between mtDNA (anti-DNA, green) and mitochondria (DeepRed-mito) in hypodermis cells of C. elegans stimulated with H2O2. White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. Scale bar, 10 μm in merged and 1 μm in zoom images. g, Representative images revealed the co-localization between mCherry::HMG-5 (Red) and mitochondria (MitoTracker, green) in hypodermis cells of C. elegans expressing mCherry::hmg-5 with H2O2. White boxed regions in the panels are enlarged. The white arrows indicate mCherry::HMG-5 outside mitochondria. Scale bar, 10 μm in merged and 1 μm in zoom images. All magnify scale bar, 1 μm.The mean values were used to calculate SEM (error bars) and P values. ***P ≤ 0.001. two-tailed unpaired Student’s t-test. Numerical source data, including exact P values, are provided in source data.

Source data

Extended Data Fig. 5 Cytosol released mtDNA and HMG-5 colocalizes with LC3 in Caenorhabditis elegans under oxidative stress.

a, b, Representative images (a) and co-localization analysis (b) of GFP::LGG-1 colocalizing mCherry::HMG-5 in hypodermal seam cells of C. elegans expressing gfp::lgg-1 and mCherry::hmg-5 stimulated with H2O2. White boxed regions in the panels are enlarged. The white arrows indicate mCherry::HMG-5 in autophagosome. Scale bar, 10 μm in merged and 1 μm in zoom images. c, d, Representative images (c) and co-localization analysis (d) of mtDNA (anti-DNA) and colocalizing mCherry::LGG-1 in hypodermis cells of C. elegans expressing mCherry:: lgg-1 stimulated with H2O2. White boxed regions in the panels are enlarged. The white arrows indicate mtDNA in autophagosome. Scale bar, 10 μm in merged and 1 μm in zoom images. e, f, Representative images verified the location of HMG-5-FLAG between TFAM (anti-TFAM, e) and mitochondria (anti-TOM20, f) in HeLa cells expressing HMG-5-FLAG without any stimuli. Scale bar, 10 μm in merged and 1 μm in zoom images. g, h, Representative images (g) and co-localization analysis (h) of HMG-5 colocalizing with GFP-LC3B marked autophagosome in GFP-LC3B stably-expressed HeLa cells were stimulated with H2O2 and Baf A1. White arrows in control group, the colocalization between HMG-5-FLAG and TFAM; White arrows in treatment group, the colocalization between HMG-5-FLAG/TFAM and GFP-LC3B. Scale bar, 10 μm in merged and 1 μm in zoom images.

Source data

Extended Data Fig. 6 The interaction between LC3B or other LC3 family proteins and TFAM or HMG-5.

a, Co-immunoprecipitation with anti-LC3B to validate the interaction between TFAM and LC3B in WCL of THP1 cells stimulated by H2O2 and Baf A1. IgG was used as negative control. b, Co-immunoprecipitation with anti-GFP to validate the interaction between HMG-5-FLAG and GFP-LC3B in cytoplasmic lysates of GFP-LC3B stably-expressed HeLa cells expressing stimulated by H2O2 and Baf A1. FLAG-Vector was used as negative control. c, d, Recombinant His-TFAM pulled down a series of recombinant LC3 family proteins (c, GST-LC3A, GST-LC3C; d, GST-GABARAP L1, GST-GABARAP L2), but failed to pull down GST-GABARAP (d). Unprocessed blots are provided in source data, n = 3 independent experiments.

Source data

Extended Data Fig. 7 TFAM binds to LC3B, instead of TREX1, to mediate the autophagy-lysosomal degradation of mtDNA.

a, b, Representative images (a) and co-localization analysis (b) of TFAM-mtDNA complex colocalizing with GFP-quenched RFP-LC3B in HeLa cells upon H2O2. White arrows indicate the co-localization between TFAM-mtDNA complex and GFP-quenched RFP-LC3B puncta. Scale bar, 10 μm in merged and 1 μm in zoom images. c, d, Representative images (c) and co-localization analysis (d) of cytosolic TFAM-mtDNA complex colocalizing with autophagosome in GFP-LC3B-stably transfected HeLa cells stimulated with H2O2 and Baf A1. White arrows indicate TFAM-mtDNA complex colocalizing with mitochondria without stress. Yellow arrows indicate the co-localization between TFAM-mtDNA complex and GFP-LC3B-marked autophagosomes outside mitochondria. Scale bar, 10 μm in merged and 1 μm in zoom images. e, Co-immunoprecipitation with anti-FLAG to validate the interaction between TFAM-FLAG and LC3B or TREX1 in WCL of GFP-LC3B-transfected HeLa cells expressing TFAM-FLAG stimulated by H2O2 and Baf A1. FLAG-Vector was used as negative control. f, g, HeLa cells expressing HA-TREX1 stimulated by H2O2. The amount of cytosolic mtDNA was detected by ND1 (f) and ND4 (g) using qPCR. In f and g, n = 3 independent experiments. The mean values were used to calculate SEM (error bars) and P values. n.s., no significance. two-tailed unpaired Student’s t-test. Numerical source data, including exact P values and unprocessed blots, are provided in source data.

Source data

Extended Data Fig. 8 Oxidative stress-induced inflammatory response not mtDNA releasing requires STING.

a, b, SDS-PAGE analysis (a) and statistical analysis on p-IRF3/IRF3 level (b) in HeLa cells stimulated with H2O2 and Baf A1. n = 3 independent experiments, **P = 0.0078, *P = 0.012 for ctrl vs H2O2, *P = 0.0442 for H2O2 vs H2O2+Baf A1. c, d, SDS-PAGE analysis (c) and statistical analysis on p-IRF3/IRF3 level (d) in HeLa cells stimulated with H2O2 and Baf A1. n = 3, *P = 0.0159, **P = 0.0027, ***P = 0.001. e, f, IFNβ expression in HeLa cells stimulated with H2O2 and Baf A1 (e, n = 4 independent experiments, *P = 0.0433, **P = 0.0096, ***P = 0.0004) or THP1 cells stimulated with H2O2 and Baf A1 (f, n = 5 independent experiments, ***P = 0.0007, *P = 0.0157 for ctrl vs Baf A1, *P = 0.0325 for H2O2 vs H2O2+Baf A1). g, IFNβ expression in THP1 cells stimulated with LPS, ATP and Baf A1. n = 3 independent experiments, *P = 0.0221, **P = 0.0055, ****P < 0.0001. h, i, HeLa cells (h) and THP1 cells (i) were examined TFAM knockdown by RNA interference. j, k, Representative images (j) and percentages (k) of mtDNA (PicoGreen) or TFAM(DeepRed-Mito) released outside mitochondria in STING-deficient HeLa cells with or without H2O2. n = 30 cells each group form 3 independent experiments, ****P < 0.0001. Scale bar, 10 μm in merged and 1 μm in zoom images. The mean values were used to calculate SEM (error bars) and P values. n.s., no significance. two-tailed unpaired Student’s t-test. Numerical source data, including exact P values and unprocessed blots, are provided in source data.

Source data

Extended Data Fig. 9 The LIR motif of TFAM-mediated interaction of LC3 is essential for the degradation of mtDNA.

a, The potentially functional LIR motifs in the protein sequence of TFAM were predicted by the iLIR Autophagy Database. b, c, The expressions (b) and locations (c) of different plasmids related to mutation or deletion of the core residue of LIR1 or LIR2 on TFAM are verified. Scale bar, 10 μm. d–h, HeLa cells with TFAM knockdown were rescued by TFAM-FLAG WT, or TFAM-FLAG Δ6 without any stimulus. Samples from these cytosolic lysates were immunoprecipitated by anti-FLAG antibody. Cytosolic TFAM-FLAG-precipitated mtDNA was detected by qPCR with ND1 (d, n = 3 independent experiments),ND2 (e, n = 3 independent experiments),ND4 (f, n = 3 independent experiments), and D-loop (g, n = 3 independent experiments). Nuclear DNA level in WCL detected by primer 18 S was used to standardize. The expression levels of plasmids are verified (h). i, j, TFAM knockdown THP1 cells were rescued by TFAM-FLAG-WT or TFAM-FLAG-Δ6 with LPS and ATP. Cytosolic mtDNA was detected by primer ND1 (i, n = 5 independent experiments) or ND4 (j, n = 5 independent experiments) using qPCR. Nuclear DNA level in WCL detected by primer 18 S was used to standardize. k, l, Flow cytometric analysis (k) and statistical analysis (l) of MMP ΔΨm (TMRM) in HeLa cells expressing TFAM-FLAG-WT or TFAM-FLAG-Δ6 upon H2O2. n = 3 independent experiments. m, n, Flow cytometric analysis (m) and statistical analysis (n) of intracellular ROS levels (DHE) in HeLa cells expressing TFAM-FLAG-WT or TFAM-FLAG-Δ6 upon H2O2. n = 3 independent experiments, ***P = 0.0007. Gating strategy of k and m are indicated in source data. o–q, Representative images (o), co-localization analysis (p) and statistical analysis (q) of mtKeima in HeLa cells expressing TFAM-FLAG WT or TFAM-FLAG Δ6 with H2O2. n = 3 independent experiments. Scale bar,10 μm. The mean values were used to calculate SEM (error bars) and P values. n.s., no significance. two-tailed unpaired Student’s t-test. Numerical source data, including exact P values and unprocessed blots, are provided in source data.

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Extended Data Fig. 10 The key LIR motif of HMG-5 to mediate the interaction of LC3 and functions in Caenorhabditis elegans.

a, Typical LIR sequences were manually aligned with HMG-5. The schematic representation of different plasmids related to deletion of the core residue of LIR1-4 on HMG-5. b, Co-immunoprecipitation with anti-GFP to validate the interaction between LC3B and different mutations of HMG-5-FLAG in WCL of GFP-LC3B-transfected HeLa cells with H2O2 and Baf A1. HMG-5-FLAG WT is used as the positive control. c, d, Irg-1 (c; n = 4 independent experiments, ***P = 0.0009, **P = 0.0024, *P = 0.0391 for HMG-5 WT- H2O2 vs HMG-5 -LIR3 H2O2, *P = 0.0109 for HMG-5 LIR3 ctrl vs HMG-5 -WT ctrl) or ctl-1 (d; n = 4 independent experiments, ***P = 0.0002, **P = 0.0033, *P = 0.0383) gene expression in C. elegans expressing HMG-5-FLAG WT or HMG-5-FLAG Δ3 treated with H2O2. The mean values were used to calculate SEM (error bars) and P values. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; n.s., no significance. two-tailed unpaired Student’s t-test. Numerical source data, including exact P values and unprocessed blots, are provided in source data.

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Source Data Fig. 1

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Source Data Extended Data Fig. 1

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Unprocessed western blots, image and flow cytometry image.

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Liu, H., Zhen, C., Xie, J. et al. TFAM is an autophagy receptor that limits inflammation by binding to cytoplasmic mitochondrial DNA. Nat Cell Biol 26, 878–891 (2024). https://doi.org/10.1038/s41556-024-01419-6

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