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MESH1 is a cytosolic NADPH phosphatase that regulates ferroptosis

Nature Metabolism volume 2, pages 270–277 (2020)Cite this article

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Abstract

Critical to the bacterial stringent response is the rapid relocation of resources from proliferation toward stress survival through the respective accumulation and degradation of (p)ppGpp by RelA and SpoT homologues. While mammalian genomes encode MESH1, a homologue of the bacterial (p)ppGpp hydrolase SpoT, neither (p)ppGpp nor its synthetase has been identified in mammalian cells. Here, we show that human MESH1 is an efficient cytosolic NADPH phosphatase that facilitates ferroptosis. Visualization of the MESH1–NADPH crystal structure revealed a bona fide affinity for the NADPH substrate. Ferroptosis-inducing erastin or cystine deprivation elevates MESH1, whose overexpression depletes NADPH and sensitizes cells to ferroptosis, whereas MESH1 depletion promotes ferroptosis survival by sustaining the levels of NADPH and GSH and by reducing lipid peroxidation. The ferroptotic protection by MESH1 depletion is ablated by suppression of the cytosolic NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. Collectively, these data shed light on the importance of cytosolic NADPH levels and their regulation under ferroptosis-inducing conditions in mammalian cells.

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Fig. 1: MESH1 is a mammalian NADPH phosphatase.
Fig. 2: Structure of the hMESH1 (D66K)–NADPH complex.
Fig. 3: MESH1 regulates cellular NADP(H) levels and ferroptosis.
Fig. 4: The ferroptosis resistance phenotypes are caused by the loss of the cytosolic NADPH phosphatase activity of MESH1.

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Data availability

The data that support the findings of this study are available from the corresponding authors upon request. The coordinates of the hMesh1 D66K–NADPH complex have been deposited in the Protein Data Bank under accession code 5VXA. The RNA-seq data have been deposited into NCBI GEO with accession number GSE114282. Source data for Figs. 1, 3 and 4 are presented with the paper.

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Acknowledgements

This work was supported in part by DOD grants (grant nos. W81XWH-17-1-0143, W81XWH-15-1-0486 and W81XWH-19-1-0842 to J.-T.C.), the Emerson Collective (to J.-T.C.), NIH grants no. R01GM124062 and R21AI149205 (to J.-T.C. and P.Z.) and no. R01GM115355 (to P.Z.), the Duke Bridge Fund, the Duke Cancer Institute (DCI) Discovery fund and the DCI Pilot fund. X-ray diffraction data were collected at the Southeast Regional Collaborative Access Team (SER-CAT) 22-BM beamline at the Advanced Photon Source, Argonne National Laboratory, supported by the US Department of Energy, Office of Science and the Office of Basic Energy Sciences under Contract no. W-31-109-Eng-38. We appreciate the great suggestions and help from the X. Shen laboratory.

Author information

Author notes

  1. These authors contributed equally: Chien-Kuang Cornelia Ding, Joshua Rose, Tianai Sun.

Authors and Affiliations

  1. Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA

    Chien-Kuang Cornelia Ding, Tianai Sun, Jianli Wu, Po-Han Chen, Chao-Chieh Lin, Hana Lee & Jen-Tsan Chi

  2. Duke Center for Genomic and Computational Biology, Duke University, Durham, NC, USA

    Chien-Kuang Cornelia Ding, Tianai Sun, Jianli Wu, Po-Han Chen, Chao-Chieh Lin, Wen-Hsuan Yang, Hana Lee, Emily Xu, Sarah Tian, Jadesola Akinwuntan & Jen-Tsan Chi

  3. Department of Biochemistry, Duke University Medical Center, Durham, NC, USA

    Joshua Rose, Wen-Hsuan Yang, Jinshi Zhao, Ziqiang Guan & Pei Zhou

  4. Department of Biomedical Engineering, Duke University, Durham, NC, USA

    Kai-Yuan Chen

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Contributions

The experimental strategy was conceived by J.-T.C. and P.Z. and further developed by C.-K.C.D. and J.R. X-ray crystallography was conducted by J.R. and analysed by J.R. and P.Z. Enzymatic assays were performed and analysed by J.R., J.Z. and P.Z. Mass spectrometry measurements were carried out by Z.G. Measurements of the enzymatic activity and NADP(H) concentration in the cell lysates were carried out by C.-K.C.D., J.W. and J.R. Cell culture and transcriptome profiling experiments were carried out by C.-K.C.D. and K.-Y.C. with assistance from J.W., T.S., P.-H.C., E.X., S.T., H.L. and J.A. C.-C.L. contributed to the NADPH sensor and phenotype experiments. Lipid peroxidation measurements were carried out by W.-H.Y. C.-K.C.D., J.R., J.-T.C. and P.Z. wrote the manuscript with input from all co-authors.

Corresponding authors

Correspondence to Pei Zhou or Jen-Tsan Chi.

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Extended data

Extended Data Fig. 1 MESH1 contributes to the NADPH phosphatase activity in cells.

NADPH phosphatase activity of HEK-293T was estimated by incubating NADPH with cell lysates with different MESH1 status; phosphate release over time was measured by the malachite green assay and normalized by the total lysate protein (μmol/min/mg total lysate protein). a, Significantly decreased cellular NADPH phosphatase activity by silencing MESH1 in HEK-293T cells. b, Increased cellular NADPH phosphatase activity with overexpression of WT MESH1, but not with overexpression of enzymatically deficient MESH1 (MESH1-E65A) in HEK-293T cells. Black dots represent siNT or empty vector samples, blue dots represent siMESH1 samples, red dots represent MESH1-WT samples, and grey dots represent MESH1-E65A samples. Error bars represent s.d. (n=3 biologically independent samples per condition). Statistical analysis: one way ANOVA with Tukey’s HSD post-hoc, **P<0.01, N.S., not significant. Source data are provided in the Source Data File.

Source data

Extended Data Fig. 2 MESH1 asymmetric unit, omit map, and Zn2+ anomalous phasing.

a, Crystallographic asymmetric unit is shown with two protomers of hMESH1 in the ribbon diagram and NADPH molecules in the stick model. b, 2mFo-DFc omit maps for the ADP (left and middle) and nicotinamide riboside moieties (right) of the NADPH molecules contoured to 0.8σ in purple mesh. c, The active site of each protomer is shown along with density from Zn2+ anomalous scattering contoured to 6σ in black mesh.

Extended Data Fig. 3 Up-regulation of MESH1 protein and its regulation of NADP(H) and cell survival during ferroptosis.

a, b, The levels of MESH1 protein were determined by Western blots of MESH1 and β-tubulin protein in RCC4 cells treated with (a) DMSO vs. erastin (3 µM; 12 h) or (b) 200 or 0 µM for 20 h. All the representative western blots were repeated for 3 biologically independent times with similar results. c, Overexpression of WT MESH1, but not the MESH1-E65A mutant, significantly reduced the intracellular NADP(H) content in RCC4 cells. d, Overexpression of MESH1-WT, but not MESH1-E65A, sensitized the cell death to erastin treatment in RCC4 cells. Black dots represent empty vector samples, red dots represent MESH1-WT samples, and grey dots represent MESH1-E65A samples. e, MESH1 silencing restored the intracellular NAPDH level decreased by erastin treatment. HEK-293T cells transfected with the NADPH fluorescent biosensor, iNAP1, and siNT or two independent siMESH1 RNAs were treated with 20 µM of erastin. The ratio of fluorescence excited at 420 nm and 485 nm indicated the cellular NADPH level. The fluorescence ratios of individual treatments were normalized to that of the control condition. (n=6 biologically independent samples). f, The viability of RCC4 cells transfected with siNT (black) or two independent siMESH1 (blue) and treated with indicated concentrations of erastin for one day. Each dot represents one sample; there are three biologically independent samples (n=3) in each group unless otherwise specified. Error bars indicate s.d. Statistical analysis: one-way ANOVA with Tukey’s HSD post-hoc test, *P<0.05, **P<0.01. Source data are provided in the Source Data File.

Source data

Extended Data Fig. 4 MESH1 depletion conferred resistance to ferroptosis in multiple assays.

a–c, Erastin-induced cell death is rescued by MESH1 silencing in RCC4 cells using multiple cell death and viability assays: a, Cytotox-Flour for measuring protease release in the media due to cell death; b, Crystal violet staining for attached viable cells; and c, CellTiter-Glo for intracellular ATP and viability. Error bars indicate s.d. (n=3 biologically independent samples per condition). Statistical analysis: one-way ANOVA with Tukey’s HSD post-hoc test, *P<0.05, **P<0.01. Source data are provided in the Source Data File.

Source data

Extended Data Fig. 5 MESH1 depletion increased resistance to ferroptosis in multiple cell lines.

a-h. Erastin-induced ferroptotic cell death of indicated cell lines was rescued by MESH1 silencing as accessed by CellTiter-Glo: a, HEK-293T cells exposed to erastin 2.5 μM for one day; b, lung adenocarcinoma cell line H1975 with erastin 10 μM for two days; c, breast cancer cell line MDA-MB-231 with erastin 2.5 μM for one day; d, prostate cancer cell line PC3 with erastin 10 μM for one day; e, fibrosarcoma cell line HT1080 with erastin 2.5 μM for one day; f, Ewing’s sarcoma cell line A673 with erastin 2.5 μM for one day; g, pancreatic carcinoma cell line PANC1 with erastin 2.5 μM for one day, and h, clear cell renal carcinoma cell line 786-O with erastin 5 μM for one day. Black dots represent siNT samples, blue dots represent siMESH1 samples. Error bars indicate s.d. (n=3 biologically independent samples per condition). Statistical analysis: one-way ANOVA with Tukey’s HSD post-hoc test, *P<0.05, **P<0.01. Source data are provided in the Source Data File.

Source data

Extended Data Fig. 6 MESH1-depletion increased the ratio of reduced glutathione over oxidized glutathione.

a–d, The level of indicated metabolites in RCC4 transfected with non-targeting (NT) siRNA or MESH1-targeting siRNA were quantified by mass spectrometry and normalized by DNA content, a, cysteine; b, oxidized glutathione (GSSG); c, reduced glutathione (GSH) (effect size 1.746, p=0.0491); and d, normalized intensity ratio of GSH/GSSG. n=5 biologically independent samples per condition. Each box plot was defined by a lower line (at the 25th percentile), a central line (at the centre), and a higher line (at the 75th percentile). The whisker boundaries marked the minima and the maxima of the data. Statistical analysis: two-tail student’s t-test, *P<0.05. Source data are provided in the Source Data File.

Source data

Extended Data Fig. 7 MESH1 silenced cells were re-sensitized to erastin-induced cell death upon NADK silencing.

Percentage of cell viability of HEK-293T cells after treatment with erastin 10 μM for one day, showing simultaneously silencing MESH1 with NADK, but not NADK2, restored sensitivity to erastin-induced ferroptosis. Black dots represent siNT samples, blue dots represent siMESH1 samples. Error bars indicate s.d. (for siNT and siMESH1 n=6 biologically independent samples per condition; for other genetic conditions n=3 biologically independent samples). Statistical analysis: one-way ANOVA with Tukey’s HSD post-hoc test *P<0.05, **P<0.01. Source data are provided in the Source Data File.

Source data

Extended Data Fig. 8 Relative expression levels of genes involved in the NADPH and glutathione metabolisms.

Three plates of RCC4 cells were sequenced and the relative expression levels of the indicated genes were calculated by RPKM (Reads per kilo base per million mapped reads). Error bars indicate s.d. (n=3 biologically independent samples per condition).

Supplementary information

Supplementary Information

Supplementary Table 1

Reporting Summary

Supplementary Data 1

Chemical structure data.

Source data

Source Data Fig. 1

Unprocessed western blots and/or gels for Fig. 1g,h.

Source Data Fig. 1

Raw data and statistics for Fig. 1c,g.

Source Data Fig. 3

Unprocessed western blots and/or gels for Fig. 3c.

Source Data Fig. 3

Raw data and statistics for Fig. 3b,c,e–i.

Source Data Fig. 4

Unprocessed western blots and/or gels for Fig. 4a.

Source Data Fig. 4

Raw data and statistics for Fig. 4a–d.

Source Data Extended Data Fig. 1

Unprocessed western blots and/or gels for Fig. 1a,b.

Source Data Extended Data Fig. 1

Raw data and statistics for Extended Data Fig. 1a,b.

Source Data Extended Data Fig. 3

Unprocessed western blots and/or gels for Fig. 3a–d.

Source Data Extended Data Fig. 3

Raw data and statistics for Extended Data Fig. 3c–f.

Source Data Extended Data Fig. 4

Raw data and statistics for Extended Data Fig. 4a–c.

Source Data Extended Data Fig. 5

Raw data and statistics for Extended Data Fig. 5a–h.

Source Data Extended Data Fig. 6

Raw data and statistics for Extended Data Fig. 6a–d.

Source Data Extended Data Fig. 7

Raw data and statistics for Extended Data Fig. 7.

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Ding, CK.C., Rose, J., Sun, T. et al. MESH1 is a cytosolic NADPH phosphatase that regulates ferroptosis. Nat Metab 2, 270–277 (2020). https://doi.org/10.1038/s42255-020-0181-1

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