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The MARCHF6 E3 ubiquitin ligase acts as an NADPH sensor for the regulation of ferroptosis

Nature Cell Biology volume 24, pages 1239–1251 (2022)Cite this article

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Abstract

Ferroptosis is a unique form of cell death caused by excessive iron-dependent lipid peroxidation. The level of the anabolic reductant NADPH is a biomarker of ferroptosis sensitivity. However, specific regulators that detect cellular NADPH levels, thereby modulating downstream ferroptosis cascades, are largely unknown. We show here that the transmembrane endoplasmic reticulum MARCHF6 E3 ubiquitin ligase recognizes NADPH through its C-terminal regulatory region. This interaction upregulates the E3 ligase activity of MARCHF6, thus downregulating ferroptosis. We also found that MARCHF6 mediates the degradation of the key ferroptosis effectors ACSL4 and p53. Furthermore, inhibiting ferroptosis rescued the growth of MARCHF6-deficient tumours and peri-natal lethality of Marchf6–/– mice. Together, these findings identify MARCHF6 as a previously unknown NADPH sensor in the ubiquitin system and a crucial regulator of ferroptosis.

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Fig. 1: MARCHF6 ablation increases lipid peroxidation.
Fig. 2: MARCHF6 suppresses ferroptosis.
Fig. 3: Ferroptosis inducers stabilize MARCHF6 substrates.
Fig. 4: MARCHF6 regulates NADPH metabolism and vice versa.
Fig. 5: C-terminal region of MARCHF6 regulates the activity of the Ub ligase.
Fig. 6: NADPH binds directly to MRR and increases MARCHF6 activity.
Fig. 7: MARCHF6 promotes degradation of ferroptosis effectors ACSL4 and p53.
Fig. 8: MARCHF6 promotes tumour growth and foetal development.

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Data availability

RNA-seq data in wild-type and MARCHF6-KO HeLa cells were deposited in the Gene Expression Omnibus database under accession number GSE173282. All other data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank A. Varshavsky (CALTECH), J. P. Mahaffey (Edanz), B.-H. Lee (DGIST) and S. T. Baek (POSTECH) for helpful comments on this manuscript and technical assistance. We also thank the present and former members of the Hwang and Lee laboratories for their advice and help. This work was supported by grants from the Korean Government (MSIP) NRF-2020R1A3B2078127 and NRF-2017R1A5A1015366 (C.-S.H.), NRF-2021R1A2C3004006 (Y.L.) and the BK21 Plus program (C.-S.H. and Y.L.).

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Authors and Affiliations

  1. Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea

    Kha The Nguyen, Sang-Hyeon Mun, Jihye Yang, Jongeun Lee, Ok-Hee Seok, Eunjeong Kim, Dasom Kim, Dong-Young Seo, Yoontae Lee & Cheol-Sang Hwang

  2. Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea

    So Young An & Jeong-Yong Suh

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Contributions

K.T.N., J.-Y.S., Y.L. and C.-S.H. designed the study and wrote the manuscript. K.T.N., S.-H.M., J.Y., J.L., D.K., O.-H.S., D.-Y.S., S.Y.A. and E.K. performed the experiments.

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Correspondence to Yoontae Lee or Cheol-Sang Hwang.

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Extended data

Extended Data Fig. 1 MARCHF6 ablation increases lipid ROS production.

a, The ER membrane-embedded MARCHF6 contains 14 transmembrane domains and 8 cytosol-facing regions9. Both the RING domain-containing N-terminal region and the C-terminal element (CTE)-containing C-terminal region face the cytosol10. Here, the C-terminal region is denoted as the MARCHF6-regulatory region (MRR) to reflect its role in the activation of the E3 Ub ligase more explicitly. b, Levels of lipid ROS in HeLa cells treated with control or MARCHF6 siRNAs for 48 h. c, Immunoblotting of MARCHF6 in HeLa cells treated with control or MARCHF6 siRNAs. d, Same as in (c) but with various wild-type and MARCHF6-KO cell lines. In (c–d), images are representative of three independent experiments. e, Levels of lipid ROS in wild-type and MARCHF6-KO HEK293T cells with or without erastin (10 µM) treatment for 16 h. f, Same as in (e) but with or without RSL3 (0.05 µM) treatment for 4 h. g, Same as in (e) with HCT116 cells. h, Same as in (g) but with or without RSL3 (1.5 µM) for 6 h. Panels (b,e–h) show representative histograms of the fluorescent signal ratios of oxidized (FITC) to reduced (PE-TR) C11-BODIPY. Right: the ratio of oxidized to total (oxidized + reduced) C11-BODIPY signals (as indicated by the bar); data are expressed as mean ± SD (n = 3 biologically independent samples); two-tailed t-test in (b) and two-way ANOVA with Tukey’s HSD post hoc test in (e–h). Significant P values are indicated in the figures.

Source data

Extended Data Fig. 2 MARCHF6 ablation decreases NADPH production.

a, Relative viability of MARCHF6-KO HEK293T cells in the presence of mock, DFO (100 µM), Fer-1 (5 µM), Z-VAD (20 µM), or Nec-1 (40 µM) under RSL3 (0.15 µM) treatment for 24 h. Relative cell viability in each group was normalized to the level with DMSO in the control as 100%. Error bars with mean ± SD (n = 3 biologically independent samples); one-way ANOVA with Tukey’s HSD post hoc test. Significant P values are indicated in the figure. b, Functional analysis of the identified DEGs between wild-type and MARCHF6-KO HeLa cells using WikiPathways enrichment. Shown are the top-scoring pathways downregulated in MARCHF6-KO HeLa cells. c, Same as in (b) but shown are the top-scoring pathways upregulated in MARCHF6-KO HeLa cells. d, A heat map indicating normalized gene expression profiles of NADP(H) metabolism-related proteins and key ferroptosis effectors in three independent wild-type and MARCHF6-KO HeLa cells. e, Schematic representation of the NADPH biosynthetic pathway. The downregulated genes in MARCHF6-KO HeLa cells are indicated in red. f,g, Relative NADPH and NADP(H) levels in wild-type and MARCHF6-KO HeLa cells. Data are expressed as mean ± SD (n = 3 biologically independent samples); two-tailed t-test. A significant P value is indicated in the figures. h, Relative NADP(H) levels of HeLa cells treated with mock, erastin, or erastin + Fer-1. i, Same as in (h) but with mock, FAC, or DFO. j, Same as in (h) but with control or two different NADK shRNAs. In (h–j), error bars with mean ± SD (n = 3 biologically independent samples); one-way ANOVA with Tukey’s HSD post hoc test. Significant P values are indicated in the figures.

Source data

Extended Data Fig. 3 NADPH depletion inhibits the degradation of MARCHF6 substrates.

a, CHX-chase experiments of SM in HeLa cells treated with control and two different NADK shRNAs. b–d, CHX-chase experiments of SMha, PLIN2ha, and MARCHF63f in HeLa cells treated with control or G6PD siRNAs for 72 h. e, Same as in (b) but with endogenous SM and siRNA treatment for 48 h. In (a–e), right: quantification data with mean ± SD (n = 3 biologically independent experiments); two-way ANOVA. Significant P values are indicated in the figures. The levels of SMha, PLIN2ha, MARCHF6ha, and SM in G6PD siRNA or NADK#1 shRNA-treated HeLa cells at the beginning of chases were taken as 100%. f, Ub-affinity pull-down assay of SMha from the extracts of SMha-expressing HeLa cells. Cells were treated with control or NADK siRNA in the presence of the proteasome inhibitor MG132 (10 µM) for 12 h before being subjected to Ub-affinity pull-down assays. g, Same as in (f) but with PLIN2ha from the extracts of PLIN2ha-expressing HeLa cells. h, Chemical crosslinking and coimmunoprecipitation of MARCHF6-KO HeLa cells expressing \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{C}}}}9{{{\mathrm{A}}}}}\) with anti-flag, followed by SDS-PAGE and immunoblotting with anti-SM, anti-NADK, and anti-flag. Cells were treated with control or NADK siRNAs for 72 h before being subjected to immunoprecipitation. i, Same as in (h) but with immunoblotting with anti-PLIN2. In (f–i), images are representative of at least three independent experiments.

Source data

Extended Data Fig. 4 MRR of MARCHF6 binds directly to UBE2G2 and UBE2J2.

a, Three-dimensional structure modelling of MARCHF6 predicted by AlphaFold226 was obtained from UniProt (https://www.uniprot.org/uniprot/O60337). RING, MRR, MarA, and MarI are indicated in the model. b, GST pull-down assay of GST-MRR (25 µg) with purified UBE2G2ha (2 µg). c, Same as in (b) but with purified \(\small {{{\mathrm{UBE}}}}2{{{\mathrm{J}}}}2_{{{{\mathrm{ha}}}}}^{{{{\mathrm{TM}}}}}\) (2 µg). d, Coimmunoprecipitations of HeLa extracts (1 mg) expressing empty vector, \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{1-910}\), or \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{1-869}\) together with UBE2G2ha. e, Same as in (d) but with UBE2J2ha. In (b–e), images are representative of three independent experiments.

Source data

Extended Data Fig. 5 MRR is required for the degradation of MARCHF6 substrates.

a, CHX-chase experiments of MARCHF63f and its indicated truncations in HeLa cells. b, Steady-state levels of MARCHF63f and its indicated mutants, and \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{MRR}}}}}\) in HeLa cells. Here, red indicates upregulation of MARCHF63f mutants. c, Schematic illustration of the MRR residues responsible for MARCHF6 self-degradation, which includes a summary of data in (a) and (b). Residues in red altered MARCHF6 self-degradation. d, Steady-state levels of SMha in HeLa cells expressing SMha with empty vector, MARCHF63f, \(\small \small {{{\mathrm{MARF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{MRR}}}}}\), or \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{C}}}}9{{{\mathrm{A}}}}}\). Immunoblotting with anti-ha, anti-flag and anti-tubulin. e, Same as in (d) but with PLIN2ha. In (a,b,d,e), images are representative of three independent experiments. f, Relative mRNA levels of \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{1 - 910}\) and \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{1 - 900}\) (see also Fig. 5g). Error bars with mean ± SD (n = 3 biologically independent samples); two-tailed t-test; ns, not significant.

Source data

Extended Data Fig. 6 MRR strongly promotes MARCHF6 activity.

a, SDS-PAGE of purified MARCHF63f (5 µg) and \(\small {{{\mathrm{MARF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{MRR}}}}}\) (5 µg), UBE2G2 (10 µg), and UBE2J2ΔTM (10 µg), followed by Coomassie brilliant blue staining. b, In vitro ubiquitylation assays of MARCHF63f or \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{MRR}}}}}\) (0.1 µM) with UBE2G2 (1 µM), UBE2J2ΔTM (0.2 µM), myc-Ub (20 µM), UBA1 (0.1 µM), and ATP (2 mM) for 20 min at 30 °C. UBE2J2ΔTM alone produced, to some extent, poly-Ub chains, whereas few poly-Ub chains were observed with UBE2G2 alone. Both MARCHF63f−UBE2J2ΔTM and MARCHF63f−UBE2G2 strongly promoted poly-Ub chain formation with higher overall yields and processivity. The combinations of MARCHF63f−UBE2J2ΔTM and MARCHF63f−UBE2G2 formed poly-Ub chains more strongly and greater processivity than MARCHF63f−UBE2J2ΔTM or MARCHF63f−UBE2G2 alone. However, the overall yield and processivity of poly-Ub chain formation were most dramatically reduced by \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{MRR}}}}}\). c, Diagrams for RINGha only and RINGha-MRR fusion. d, Same as in (b) but with RINGha (0.5 µM) and RINGha-MRR (0.5 µM). The purified linear RINGha-MRR fusion strongly promoted poly-Ub chain formation in the presence of UBE2J2ΔTM and/or UBE2G2, whereas RINGha only produced di- or tri-Ub adducts. e, Same as in (d) but with immunoblotting using the anti-K48 linkage-specific Ub antibody. f, In vitro ubiquitylation assays of RINGha-MRR (0.5 µM) with Ub, Lys-lacking K0-Ub, and the indicated single Lys-containing Ub mutants (each 25 µM), followed by immunoblotting with anti-Ub. g, Scheme for di-Ub formation assay using fluorescent dye-labelled UbR48 with a K48-to-R mutation as a donor and UbAA with C-terminal diglycine (GG) to di-alanine (AA) as an acceptor. h, Di-Ub formation assays of RINGha (0.5 µM) or RINGha-MRR (0.5 µM) upon increasing concentrations of the acceptor UbAA for 15 min at 30 °C. i, Same as in (h) but with linkage-specific tetra-Ubs (each 2 µM)]. j, Same as in (g) but with UbR48 and UbAA-GST (each 10 µM). In (a,b,d,e,f,h,i,j), images represent at least two independent experiments.

Source data

Extended Data Fig. 7 MRR binds specifically to NADPH.

a, 2′,5′-ADP-agarose affinity chromatography with purified RINGha and RINGha-MRR (2 µg). b, Same as in (a) but with purified GST and GST-MRR (2 µg) in the presence of NAD+, NADH, NADP+, NADPH, or ATP (2 mM each). c, ITC analyses of the interaction between GST-MRR and NADPH (top), NADP+ (middle), or NADH (bottom). In (a,b) and (c), images are representative of three and two independent experiments, respectively. d, CHX-chase experiments of SM and PLIN2 in MARCHF6-KO HeLa cells expressing MARCHF63f or \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{L}}}}888{{{\mathrm{A}}}}}\). Prior to CHX pulse, these cells were treated with control or NADK siRNAs for 72 h. Bottom: quantification data with mean ± SD (n = 3 biologically independent experiments); two-way ANOVA. Significant P values are indicated in the figures. The levels of SM or PLIN2 in NADK siRNA-treated MARCHF6-KO cells expressing \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{L}}}}888{{{\mathrm{A}}}}}\) at the beginning of chases were taken as 100%. e, Relative viability of MARCHF6-KO HeLa cells expressing empty vector, MARCHF63f, \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{G}}}}885{{{\mathrm{L}}}}}\), or \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{L}}}}888{{{\mathrm{A}}}}}\) under erastin (10 µM) treatment for 24 h. f, Same as in (e) but with or without RSL3 (0.15 µM). In (e,f), relative cell viability in each group was normalized to its DMSO controls as 100%. Quantification data with mean ± SD (n = 3 biologically independent samples); one-way ANOVA with Tukey’s HSD post hoc test. A significant P value is indicated in the figures; ns, not significant.

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Extended Data Fig. 8 MARCHF6 regulates the expression of ferroptosis effectors.

a, Immunoblotting of ferroptosis effectors SM, ACSL4, p53, SLC7A11, GPX4, and NRF2 in MARCHF6-KO HeLa cells expressing empty vector, MARCHF63f, \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{G}}}}885{{{\mathrm{L}}}}}\), or \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{L}}}}888{{{\mathrm{A}}}}}\). b, CHX chases for 0 and 4 h of the indicated ferroptosis-related proteins in wild-type and MARCHF6-KO HeLa cells. c, Same as in (b) but with A549 cells. In (a–c), images are representative of three independent experiments. d, CHX-chase experiment of ACSL4 in wild-type and MARCHF6-KO HeLa cells. e, Same as in (d) but with p53. In (d,e), bottom: quantification data with mean ± SD (n = 3 biologically independent experiments); two-way ANOVA. Significant P values are indicated in the figures. f–h, Relative mRNA levels of SLC7A11, p53, and GPX4 in wild-type and MARCHF6-KO HeLa or A549 cells, respectively. Error bars with mean ± SD (n = 3 biologically independent samples); two-tailed t-test. Significant P values are indicated in the figures; ns, not significant. i, Chemical crosslinking and coimmunoprecipitation of ACSL4ha from MARCHF6-KO HeLa cells coexpressing MARCHF63f and ACSL4ha. j, Same as in (i) but with p53ha. In (i,j), images are representative of three independent experiments.

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Extended Data Fig. 9 MARCHF6 promotes tumour growth.

a, Cell proliferation assay in wild-type and MARCHF6-KO A549 cells. Cell viability was measured every 24 h using the CellTiter Glo viability kit. Quantification data with mean ± SEM (n = 3 biologically independent samples); two-way ANOVA; ns, not significant. b, In vivo subcutaneous tumour growth curves of wild-type and MARCHF6-KO A549 cells. Tumour volumes were measured every 4 days. Quantification data with mean ± SEM (n = 5 mice); two-way ANOVA. A significant P value is indicated in the figure. c, Images of wild-type and MARCHF6-KO A549 xenograft tumours dissected from mice following the last assessment of tumour sizes. d, Average xenograft weights of the dissected wild-type and MARCHF6-KO A549 tumours. Error bars with mean ± SEM (n = 5 mice); two-tailed t-test. A P value is indicated in the figure. e, Representative images of 4-HNE immunohistochemical and haematoxylin and eosin (H&E) staining of six wild-type and MARCHF6-KO A549 xenograft tumours with indicated treatments. Arrowheads and dark-brown staining indicate the lysed tumour cells and 4-HNE-positive cells, respectively. Scale bars, 50 µM. See also Fig. 8a–d for details.

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Extended Data Fig. 10 Developmental phenotypes of Marchf6–/– mice.

a, Non-Mendelian inheritance ratios from the interbreeding of heterozygous Marchf6+/– mice. The percentages (numbers) of observed Marchf6–/– embryos (E18.5) and pups (P0) are shown in red. b, Perinatal lethality and growth defects of whole-body Marchf6–/– mice. Marchf6–/– mice displayed perinatal lethality and growth defects compared with wild-type Marchf6+/+ mice; scale bar: 1 cm. c, Embryo morphologies of littermates with the indicated genotypes (E18.5). d, Relative body weights of wild-type and Marchf6–/– mice at E18.5. Error bars with mean ± SEM (n = 6 Marchf6+/+ and 8 Marchf6–/–– embryos); two-tailed t-test. A significant P value is indicated in the figure. e, Representative images of H&E staining from livers of three Marchf6+/+ and Marchf6–/– embryos at E18.5. Arrowheads indicate dead cells with damaged membrane, cytoplasm, and nuclei. Scale bars of the images, top: 1 mm (top); middle: 50 µm; bottom: 10 µm. f, Representative images of 4-HNE immunohistochemical and H&E staining of livers from three Marchf6+/+ and Marchf6–/– embryos at E18.5, which were fed normal and vitamin E-enriched diets. Dark-brown staining indicates 4-HNE-positive cells. Scale bar, 50 µM. g, Same as in (f) but with mice intraperitoneally injected with DMSO or Fer-1 (2 mg/kg) daily starting from E9.5. Scale bar, 50 µM. See also Fig. 8e–g for details.

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Supplementary information

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Supplementary Tables 1–7

Supplementary Table 1. DEGs identified in wild-type versus MARCHF6-KO HeLa cells. Supplementary Table 2. Downregulated and upregulated genes in MARCHF6-KO HeLa cells. Supplementary Table 3. Chemical, enzymes, commercial kits and software used in this study. Supplementary Table 4. Antibodies used in this study. Supplementary Table 5. Plasmids used in this study. Supplementary Table 6. Cell lines, mice and bacteria used in this study. Supplementary Table 7. Oligonucleotides used in this study.

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Nguyen, K.T., Mun, SH., Yang, J. et al. The MARCHF6 E3 ubiquitin ligase acts as an NADPH sensor for the regulation of ferroptosis. Nat Cell Biol 24, 1239–1251 (2022). https://doi.org/10.1038/s41556-022-00973-1

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