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A genetically encoded tool to increase cellular NADH/NAD+ ratio in living cells

Nature Chemical Biology (2023)Cite this article

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Abstract

Impaired redox metabolism is a key contributor to the etiology of many diseases, including primary mitochondrial disorders, cancer, neurodegeneration and aging. However, mechanistic studies of redox imbalance remain challenging due to limited strategies that can perturb redox metabolism in various cellular or organismal backgrounds. Most studies involving impaired redox metabolism have focused on oxidative stress; consequently, less is known about the settings where there is an overabundance of NADH reducing equivalents, termed reductive stress. Here we introduce a soluble transhydrogenase from Escherichia coli (EcSTH) as a novel genetically encoded tool to promote reductive stress in living cells. When expressed in mammalian cells, EcSTH, and a mitochondrially targeted version (mitoEcSTH), robustly elevated the NADH/NAD+ ratio in a compartment-specific manner. Using this tool, we determined that metabolic and transcriptomic signatures of the NADH reductive stress are cellular background specific. Collectively, our novel genetically encoded tool represents an orthogonal strategy to promote reductive stress.

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Fig. 1: Screening of bacterial STHs for their ability to elevate NADH levels in mammalian cells.
Fig. 2: Determination of EcSTH kinetic parameters and visualization of EcSTH activity in live cells using genetically encoded sensors.
Fig. 3: Metabolic features of the NADH reductive stress in HeLa cells.
Fig. 4: RNA-seq of HeLa cells expressing LbNOX, EcSTH and mitoEcSTH.
Fig. 5: The anti-proliferative effect of EcSTH and mitoEcSTH expression is cellular background specific.
Fig. 6: In vivo elevation of the hepatic cytosolic NADH/NAD+ ratio upregulates GDF15 expression.

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Data availability

RNA-seq data presented in this work are available at the Gene Expression Omnibus database under accession number GEO GSE232115. The H. sapiens GRCh38 reference genome was used for mapping RNA-seq reads. Source data are provided with this paper.

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Acknowledgements

We thank members of the Thompson laboratory at MSKCC, H. Sies, M. Clasquin, A. Caudy and A. Rosebrock for discussion and feedback. We thank A. T. Truong (Agilent Technologies), B. Webb (Center for Metabolomics and Mass Spectrometry, Scripps Research) and D. Scott (Sanford Burnham Prebys Discovery Cancer Metabolism Core) for technical support. This work was supported by grants from the National Institutes of Health (R00GM121856, R03AG067301, R35GM142495 and R35GM142495-02S1 to V.C. and R01DK134675 to R.P.G.). The Seahorse XFe96 analyzer in the Saez laboratory (Scripps Research) was supported by 1S10OD16357. This is manuscript number 1072 from The Scintillon Institute.

Author information

Author notes

  1. Mina L. Heacock

    Present address: Calibr, The Scripps Research Institute, La Jolla, CA, USA

  2. Evana N. Abdulaziz

    Present address: Process Development Associate, Amgen, Thousand Oaks, CA, USA

  3. These authors contributed equally: Xingxiu Pan, Mina L. Heacock, Evana N. Abdulaziz.

Authors and Affiliations

  1. Laboratory of Redox Biology and Metabolism, Scintillon Institute, San Diego, CA, USA

    Xingxiu Pan, Mina L. Heacock, Evana N. Abdulaziz, Austin L. Zuckerman, Canglin Yao & Valentin Cracan

  2. Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Sara Violante & Justin R. Cross

  3. Program in Mathematics and Science Education, University of California San Diego, San Diego, CA, USA

    Austin L. Zuckerman

  4. Program in Mathematics and Science Education, San Diego State University, San Diego, USA

    Austin L. Zuckerman

  5. Liver Center, Division of Gastroenterology, Massachusetts General Hospital, Boston, MA, USA

    Nirajan Shrestha & Russell P. Goodman

  6. Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA

    Valentin Cracan

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Contributions

V.C. conceived the study. X.P., M.L.H. and E.N.A. performed all experiments with assistance from A.L.Z. and C.Y. C.Y. performed enzyme kinetics experiments. S.V. and J.R.C. performed LC–MS experiments. X.P. performed imaging experiments and analyzed RNA-seq data. N.S. and R.P.G. performed experiments in mice. V.C., X.P. and M.L.H. wrote the manuscript with input from all the authors. All authors contributed to editing the manuscript and approved the final version.

Corresponding author

Correspondence to Valentin Cracan.

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Competing interests

V.C. is listed as an inventor on a patent application on the therapeutic uses of LbNOX and TPNOX (US patent application US20190017034A1). The remaining authors declare no competing interests.

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Nature Chemical Biology thanks Melanie McReynolds, Yi Yang 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 SoNar imaging of HeLa cells expressing EcSTH and mitoEcSTH.

(a) Left panel is a schematic representation of live cell imaging using genetically encoded biosensor SoNar. The fluorescence intensity of the SoNar channel is represented in pseudocolor. The ratio of fluorescent intensities from excitation at 400 and 488 nm reflects cellular NADH/NAD+ ratio. High F400/488 ratio corresponds to high cellular NADH/NAD+ ratio and vice versa. To the right are widefield images of HeLa cells with lentivirus mediated LUC, LbNOX, EcSTH, mitoEcSTH expression under Dox control transiently expressing SoNar, in basal medium (DMEM without pyruvate, fluorescent vitamins and phenol red with 5 mM glucose, 25 mM HEPES, pH 7.4 and 1% dialyzed FBS), or after addition of 1 μM Ant A, or when cells were switched to the basal medium without glucose but with 10 mM pyruvate. Scale bars: 50 µm. (b-c) Quantification of the time course measurements of the fluorescence ratio (F400/488) for HeLa cells with lentivirus mediated LUC, LbNOX, EcSTH, mitoEcSTH expression under Dox control transiently expressing SoNar for conditions shown in (a). Values are mean ± s.d.; n = 8, 8, 10, 10 in (b), n = 7, 6, 6, 7 in (c) biologically independent samples.

Source data

Extended Data Fig. 2 iNAP1 imaging of HeLa cells expressing EcSTH and mitoEcSTH.

(a) Left panel is a schematic representation of principle of live cell imaging using genetically encoded biosensor iNAP1. The fluorescence intensity of the iNAP1 channel is represented in pseudocolor. The ratio of fluorescent intensities from excitations at 400 and 488 reflects cellular levels of NADPH. High F400/488 ratio corresponds to high NADPH and vice versa. To the right are widefield images of HeLa cells with lentivirus mediated LUC, LbNOX, EcSTH, mitoEcSTH expression under Dox control transiently expressing iNAP1, in basal medium (DMEM without pyruvate, fluorescent vitamins and phenol red with 5 mM glucose, 25 mM HEPES, pH 7.4 and 1% dialyzed FBS), or when cells were switched to the basal medium without glucose but with 10 mM pyruvate. Scale bars: 50 µm. Quantification of the time course measurements of the fluorescence ratio (F400/488) for HeLa cells with lentivirus mediated LUC, LbNOX, EcSTH, mitoEcSTH expression under Dox control transiently expressing iNAP1 in the basal medium followed by replacement with basal medium without glucose but with 10 mM pyruvate (b), after addition of G6PDi (c), after addition of 6-AN (d), or after addition of diamide (e). Values are mean ± s.d.; n = 9, 8, 8, 8 in (b), n = 8, 8, 8, 8 in (c), n = 8, 8, 8, 8 in (d), n = 8, 9, 7, 8 in (e) biologically independent samples.

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Extended Data Fig. 3 Impact of EcSTH and mitoEcSTH expression on oxygen consumption and acidification rates.

Oxygen consumption rate (OCR) (a) and extracellular acidification rate (ECAR) (b) of HeLa cells expressing EcSTH and mitoEcSTH before and after addition of 1 µM antimycin A (ANT) measured in pyruvate free HEPES/DMEM+dFBS media. Values are mean ± s.d.; n = 12, 12, 12, 10 for OCR traces in (a), n = 4 for bar graphs depicting OCR quantification in (a), n = 12, 12, 12, 10 for ECAR tracers in (b), n = 4 for bar graphs depicting ECAR quantification in (b) biologically independent samples. The statistical significance indicated for (a-b) represents a One-Way ANOVA followed by uncorrected Fisher′s least significant difference test.

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Extended Data Fig. 4 Activity of EcSTH and mitoEcSTH in HeLa cells with inhibited ETC or under hypoxia conditions.

The total NADH/NAD+ (a) and NADPH/NADP+ (b) ratios measured in HeLa cells expressing EcSTH and mitoEcSTH three hours after changing to fresh pyruvate-free DMEM+dFBS with 1 μM antimycin A (ANT), 1 μM piericidin A (Pier), or 1 mM pyruvate (PYR). The total NADH/NAD+ (c) and NADPH/NADP+ (d) ratios measured in HeLa cells expressing EcSTH and mitoEcSTH incubated for 24 hours at 5%CO2/1%O2 hypoxia. Values are mean ± s.d.; n = 8, 4, 4, 3, 4, 4, 4, 3, 8, 4, 4, 3 in (a), n = 4 in (b), n = 8 in (c, d) biologically independent samples. The statistical significance indicated for (a-d) represents a One-Way ANOVA followed by uncorrected Fisher′s least significant difference test. NS, no significant difference.

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Extended Data Fig. 5 Metabolic features of LbNOX, EcSTH and mitoEcSTH expression in HeLa cells.

Targeted metabolomics in HeLa cells expressing LbNOX (a), EcSTH (b) and mitoEcSTH (c). LUC expressing HeLa cells were used as a control in (a-c). The statistical significance indicated for (a-c) represents p value cutoff = 0.05, fold change cutoff = 1.5 and Welch t-test (FDR corrected).

Extended Data Fig. 6 Representative redox couples in HeLa cells expressing EcSTH and mitoEcSTH.

Intracellular lactate/pyruvate (a), 2-hydroxybutyrate/2-ketobutyrate (2HB/2KB) (b), 3-hydroxybutyrate/acetoacetate (3HB/AcAc) (c), glycerol 3-phosphate/dihydroxyacetone phosphate (Gro3P/DHAP) (d), C16:1/C16:0 (e) and AMP/ATP (f) ratios measured in HeLa cells expressing EcSTH and mitoEcSTH. In (a-f) values are mean ± s.d.; n = 3 biologically independent samples. Statistically significant differences were calculated by using a Welch ANOVA followed by unpaired t-test. NS, no significant difference.

Source data

Extended Data Fig. 7 Rescue of the anti-proliferative effect of EcSTH expression with extracellular electron acceptors.

Proliferation of HeLa cells with EcSTH and mitoEcSTH expression in DMEM+dFBS when supplemented with exogenous electron acceptors (10 mM pyruvate (PYR), 10 mM oxaloacetate (OAA) or 10 mM α-ketobutyrate (2KB)). Values are mean ± s.d.; n = 3 biologically independent samples.

Source data

Extended Data Fig. 8 Analysis of GO terms linked to NAD(P) redox metabolism.

(a-b) Venn diagrams displaying the similarities and differences of genes from indicated gene sets. Yellow circles include all genes in the GO term ‘oxidation-reduction’ pathways (see Fig. 4e). The salmon circle contains all genes in the GO term ‘cholesterol biosynthetic’ pathways (see Fig. 4e). The light brown circle indicates the NAD(P)ome (genes in humans that encode NAD+ or NADP+ dependent enzymes as defined in27). The up-regulated genes are marked in red and down-regulated genes are marked in blue.

Extended Data Fig. 9 Impact of EcSTH and mitoEcSTH expression on NAD(P)H/NAD(P)+ ratios and proliferation in different cell lines.

Western blot analysis of EcSTH and mitoEcSTH expression in A549 lung adenocarcinoma cells (a), MIA PaCa-2 epithelial tumor tissue of the pancreas (e) and PANC-1 pancreatic duct epithelioid carcinoma (i). (a, e, i) Representative blots are shown. The total NADH/NAD+ ratios in A549 (b), MIA PaCa-2 (f) and PANC-1 (j) cells expressing EcSTH and mitoEcSTH. The total NADPH/NADP+ ratios in A549 (c), MIA PaCa-2 (g) and PANC-1 (k) cells expressing EcSTH and mitoEcSTH. Proliferation of A549 (d), MIA PaCa-2 (h) and PANC-1 (l) cells expressing EcSTH and mitoEcSTH in pyruvate-free DMEM+dFBS. LUC and LbNOX expressing HeLa cells were used as controls in (a-l). Values are mean ± s.d.; n = 8 in (b-c), n = 6 in (f, g, j, k) biologically independent samples. The statistical significance indicated for (b, c, f, g, j, k) represents a Welch ANOVA followed by unpaired t-test. NS, no significant difference. For growth curves (d, h, l) error bars represent mean ± s.d.; n = 3 biologically independent samples.

Source data

Extended Data Fig. 10 Untargeted metabolomics of C2C12 cells expressing EcSTH and mitoEcSTH.

Untargeted metabolomics in C2C12 cells expressing LbNOX (a), EcSTH (b) and mitoEcSTH (c). LUC expressing C2C12 cells were used as a control in (a–c). The statistical significance indicated for (a-c) represents p value cutoff = 0.05, fold change cutoff = 1.5 and Welch t-test (FDR corrected).

Supplementary information

Supplementary Information

Supplementary Figs. 1–17.

Reporting Summary

Supplementary Tables 1–12

Relevant to Methods (Tables 1–5), raw metabolomics peak areas (Tables 6–7) and RNA-seq (Tables 8–12).

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Source Data Extended Data Fig. 9

Uncut western blot for Extended Data Fig. 9a.

Source Data Extended Data Fig. 9

Uncut western blot for Extended Data Fig. 9e.

Source Data Extended Data Fig. 9

Uncut western blot for Extended Data Fig. 9i.

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Pan, X., Heacock, M.L., Abdulaziz, E.N. et al. A genetically encoded tool to increase cellular NADH/NAD+ ratio in living cells. Nat Chem Biol (2023). https://doi.org/10.1038/s41589-023-01460-w

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