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Mitochondrial NADP+ is essential for proline biosynthesis during cell growth

Abstract

Nicotinamide adenine dinucleotide phosphate (NADP+) is vital to produce NADPH, a principal supplier of reducing power for biosynthesis of macromolecules and protection against oxidative stress. NADPH exists in separate pools, in both the cytosol and mitochondria; however, the cellular functions of mitochondrial NADPH are incompletely described. Here, we find that decreasing mitochondrial NADP(H) levels through depletion of NAD kinase 2 (NADK2), an enzyme responsible for production of mitochondrial NADP+, renders cells uniquely proline auxotrophic. Cells with NADK2 deletion fail to synthesize proline, due to mitochondrial NADPH deficiency. We uncover the requirement of mitochondrial NADPH and NADK2 activity for the generation of the pyrroline-5-carboxylate metabolite intermediate as the bottleneck step in the proline biosynthesis pathway. Notably, after NADK2 deletion, proline is required to support nucleotide and protein synthesis, making proline essential for the growth and proliferation of NADK2-deficient cells. Thus, we highlight proline auxotrophy in mammalian cells and discover that mitochondrial NADPH is essential to enable proline biosynthesis.

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Fig. 1: Loss of NADK2 renders cells dependent on proline for cell proliferation.
Fig. 2: NADK2 is required for anchorage-independent cell growth.
Fig. 3: NADK2 is required for the maintenance of proline levels.
Fig. 4: In vivo infusions with 13C isotopes reveal that proline synthesis occurs mainly in the pancreas.
Fig. 5: NADK2 activity is required for glutamine-dependent proline biosynthesis.
Fig. 6: Proline levels do not alter the NADK2-dependent regulation of mitochondrial respiration.
Fig. 7: Proline becomes limiting for nucleotide synthesis in NADK2-deficient cells.
Fig. 8: NADK2 loss inhibits protein synthesis and triggers activation of the GCN2–eIF2α–ATF4 pathway.

Data availability

Supplementary Information including Supplementary Tables 13 and a figure exemplifying the gating strategy for Extended Data Fig. 6 are provided with this paper. All other data are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank C. Llamas and P. Mishra for guidance with Seahorse assays; C. Yang for assistance with xenograft experiments; L. Zacharias, M. Martin and T. Mathews for mass spectrometry assistance; and N. Loof, T. Shih and the Moody Foundation Flow Cytometry Facility for flow cytometry assistance. The research was supported by the Cancer Prevention and Research Institute of Texas (CPRIT; RR190087). G.H. is a CPRIT Scholar. R.J.D. is an HHMI Program investigator, the Robert L. Moody, Sr. Faculty Scholar at University of Texas Southwestern Medical Center and Joel B. Steinberg, M.D. Chair in Paediatrics. J.A.P. III is supported in part by Vanderbilt Center for Undiagnosed Diseases (VCUD; 5U01HG007674).

Author information

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Authors

Contributions

D.H.T. and R.K. performed and analysed experiments. H.R. performed immunoblots, cell proliferation assays and analysed data. M.H.S., D.B., H.S.V. and F.C. performed LC–MS/MS analyses. A.S. performed in vivo infusion experiments. J.A.P. III provided patient-derived fibroblasts. R.J.D. provided intellectual contribution, discussed ideas and data and reviewed the manuscript. G.H. conceived and directed the project, designed the experiments, analysed the data and wrote the manuscript. G.H. and D.H.T. prepared the manuscript.

Corresponding author

Correspondence to Gerta Hoxhaj.

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Competing interests

R.J.D. is an advisor for Agios Pharmaceuticals and Vida Ventures. All other authors declare no competing interests.

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Peer review information Nature Metabolism thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: George Caputa.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 NADK2-deficient cells require proline for cell proliferation.

a, A549 and K562 ∆NADK2 cells stably reconstituted with either empty vector (Vec) or NADK2 were injected subcutaneously into athymic nude mice. Tumour growth curves are shown as average of all tumours from the data shown in Fig. 1c,d. Data are presented as mean ± SEM from 7 tumors (A549) or 3 or 4 tumours (K562). *P < 0.05 for pairwise comparisons calculated using a one-sided Student’s t-test. b, Immunoblots and cell proliferation of wild-type or single cell-derived knockout cells of NADK in HEK-293E cells grown in DMEM with 10% serum. Proliferation rate was assessed 72 h post-plating and normalized to Day 0. c, Relative proliferation rate was assessed in three consecutive days from wild-type or isogenic ∆NADK2 cells stably expressing either empty vector, NADK2 or NADK. Immunoblots for NADK, NADK2 and β-actin are shown. d, Relative proliferation rate as in (b) from isogenic ∆NADK2 HeLa cells stably expressing either empty vector or NADK2. Cells were grown in 10% dialyzed serum or supplemented with oleate (0.1 mM). e, Relative proliferation rate as in (b) from wild-type or ∆NADK2 HeLa cells. Cells were grown in 10% dialyzed serum for 72 hours or supplemented with NAC (5 mM), nucleosides (inosine 0.1 mg/ml uridine, 0.1 mg/ml), non-essential amino acid (NEAA) mixture (1X), pyruvate (10 mM) or aspartate (10 mM). f, Relative proliferation rate as in (b) from isogenic ∆NADK2 K562 cells stably expressing either empty vector or NADK2 and supplemented with the indicated individual non-essential amino acids at concentration present in human plasma-like medium (HPLM). g, Relative proliferation rate as in (b) from isogenic ∆NADK2 HEK-293E cells stably expressing either empty vector or NADK2, and supplemented with the indicated individual non-essential amino acids at 10X concentration present in human plasma-like medium (HPLM). Related to Fig. 1f. h, Relative proliferation rate as in (b) from HCT116 cells with stable shRNA-mediated knockdown of NADK2 grown in the presence or absence of proline (2 mM). i, Normalized peak areas of mitochondrial NAD + (M + 4), NADP + (M + 4) and NADPH (M + 4) from labeling with 13C3-15N-nicotinamide are shown. ∆NADK2 HEK293E cells stably expressing HA-Mito were stably reconstituted with either empty vector or NADK2. HEK-293E cells expressing (Myc-Mito) were used as a negative control (1-Ctrl). HA-tagged mitochondria were isolated from equal amount of protein for each condition and metabolites were analyzed by targeted LC-MS/MS. Data are presented as the mean ± SD (b-i) from n = 4 of biologically independent samples for (b), n = 3 for (c,g,h,i), n = 12 for (d), n = 3–9 for (e), n = 3–5 for (f) and are representative of at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 for comparisons calculated using a two-sided Student’s t-test for (b) and with one-way ANOVA test and Tukey’s post-hoc test for (c-i).

Source data

Extended Data Fig. 2 NADK2-deficient tumors display reduced proline levels.

a,b, Immunoblots and peak areas of proline and other amino acid are shown from A549 (a) and K562 (b) xenograft tumours that are NADK2-deficient (blue) or that express NADK2 from tumors in Fig. 1c,d. Each condition represents four tumors, and levels of NADK2 are assessed by immunoblotting and shown below the graphs. Data are presented as the mean ± SD from n = 4 of biologically independent samples. **P < 0.01 for comparisons was calculated using a two-sided Student’s t-test.

Source data

Extended Data Fig. 3 Glutamine-dependent proline synthesis is dependent on NADK2 (Data supporting Fig. 5).

a, Schematic of 13C5-glutamine labelling. b, Fractional abundance (%) of glutamine (M + 5) from experiment presented in Fig. 5b. c, Fractional abundance (%) of glutamine (M + 5) from experiment presented in Fig. 5c. d, Fractional abundance (%) of glutamine (M + 5), glutamate (M + 5) and proline (M + 5) from isogenic ∆NADK2 A549 cells stably expressing either empty vector (-) or NADK2 cDNA and labeled for 3 hours with 13C5-glutamine. Wildtype counterparts of each cell line are shown as controls. e, Fractional abundance (%) of glutamine (M + 5), glutamate (M + 5) and proline (M + 5) from isogenic ∆NADK2 K562 cells stably expressing either empty vector (-) or NADK2 cDNA and labeled for 3 hours with 13C5-glutamine. Wildtype counterparts of each cell line are shown as controls. f, Immunoblots from HCT116 cells stably expressing a control shRNA (Ctrl), NADK2 shRNA, or two shRNAs against P5CS. g, Fractional abundance (%) of glutamine (M + 5), glutamate (M + 5) and proline (M + 5) from HCT116 cells stably expressing a control shRNA (Ctrl) or two shRNAs against P5CS. Immunoblots are shown in (f). h, Fractional abundance (%) of glutamine (M + 5), glutamate (M + 5) and proline (M + 5) from HCT116 cells stably expressing a control shRNA (Ctrl) or an shRNA against NADK2. Immunoblots are shown in (f). Data are presented as the mean ± SD from n = 4 of biologically independent samples for (b-e) and n = 3 or 4 for (g,h) and are representative of at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 for comparisons was calculated using a two-sided Student’s t-test for (h) and with one-way ANOVA test and Tukey’s post-hoc test for (b-e,g).

Source data

Extended Data Fig. 4 NADK2 regulated proline synthesis for cell growth. Data supporting Fig. 5.

a, Immunoblots of NADK2 and proline biosynthesis genes shown for wild-type or isogenic ∆NADK2 HEK293E cells stably expressing either empty vector or NADK2 grown in DMEM supplemented with 10% serum in the presence or absence of proline (0.2 mM). Data are representative of at least two independent experiments. b, Immunoblots as in (a) for wild-type or isogenic ∆NADK2 A549 cells stably expressing either empty vector or NADK2 grown in DMEM supplemented with 10% serum in the presence or absence of proline (0.2 mM). Data are representative of at least two independent experiments. c, Immunoblot and fractional abundance (%) of glutamine (M + 5) from experiment presented in Fig. 2d. d, Schematic showing the sequence conservation of D161 among NADK2 orthologs across different species. e, Fractional abundance (%) of glutamine (M + 5) from experiment presented in Fig. 5e. f, Fractional abundance (%) of glutamine (M + 5) from experiment presented in Fig. 5f. g, Relative proliferation rate as in (Fig. 5g), but from isogenic ∆NADK2 HEK293E cells stably expressing either empty vector or NADK2. Cells were grown in 10% dialyzed serum for 72 hours or supplemented with ornithine (0.5 mM), dimethyl α-ketoglutarate (0.25 mM), or with ornithine (0.5 mM) and dimethyl α-ketoglutarate (0.25 mM). h, Normalized peak areas of ornithine (M + 7) from 3 hours labeling with 13C5-15N2-ornithine are shown from experiment presented in Fig. 5i. Data are presented as the mean ± SD from n = 4 of biologically independent samples for (c), n = 3 for (e-h) and are representative of at least two independent experiments. *P < 0.05, **P < 0.01 for comparisons calculated using a two-sided Student’s t-test for (c,h) and with one-way ANOVA test and Tukey’s post-hoc test for (e-g).

Source data

Extended Data Fig. 5 Proline does not affect the NADK2-dependent regulation of ROS levels.

a, Relative proliferation in ∆NADK2 HeLa cells cultured in DMEM with 10% dialyzed serum for 72 hours in the presence or absence of proline (2 mM) or PRODH inhibitor L-THFA (5 mM). Data is normalized to cells grown in the presence of proline. b, Relative proliferation as in (a) in HeLa ∆NADK2 cells reconstituted with NADK2. c, Schematic of putative functions of proline axis mediating ATP generation or defense against ROS. d,e, Mean fluorescence intensity for CellRox Green staining (ROS) in ∆NADK2 HeLa (d) or HEK-293E (e) cells stably expressing either empty vector or NADK2 cultured in 10% dialyzed serum in the presence or absence of proline (2 mM). f,g, GSH, GSSG and GSH/GSSG ratio quantified from ∆NADK2 HeLa (d) or HEK-293E (e) cells cultured in 10% dialyzed serum in the presence or absence of proline (2 mM). h,i, Extracellular Acidification Rate (ECAR) for ∆NADK2 HeLa (e) or HEK-293E (f) cells stably expressing either empty vector or NADK2 cultured in the presence or absence of proline (2 mM). Wildtype counterparts are shown (Supporting Fig. 6). Data are presented as the mean ± SD from n = 3 of biologically independent samples for (a,b), n = 4 for (d,e,f,g), n = 11 or 12 for (h), and n = 9–11 for (i), and are representative of at least two independent experiments. **P < 0.01, ***P < 0.001 for comparisons calculated using a two-sided Student’s t-test for (f,g) and with one-way ANOVA test and Tukey’s post-hoc test for (a,b,d,e,h,i).

Source data

Extended Data Fig. 6 NADK2 loss results in impairment of cell cycle progression.

a, Cell cycle profiles (histograms) at indicated time points after release from G1 phase arrest induced by double-thymidine block, from ∆NADK2 HeLa cells stably expressing either empty vector or WT NADK2, cultured in the presence or absence of proline (0.2 mM) as indicated. Data are analyzed using the FlowJo’s Watson Pragmatic cell cycle platform algorithm and are representative of at least two independent experiments performed in duplicates or tripicates. b, Cell cycle profiles from asynchronous ∆NADK2 HeLa cells stably expressing either empty vector or WT NADK2, cultured in the presence or absence of proline (0.2 mM) as indicated. Biological duplicates are shown. Data are analyzed using the FlowJo’s Watson Pragmatic cell cycle platform algorithm and are representative of at least two independent experiments performed in duplicates or triplicates.

Source data

Extended Data Fig. 7 NADK2 regulates nucleotide synthesis (Data supporting Fig. 7).

a, Schematic of 13C6-glucose labelling. b, Schematic of 3-13C-serine labeling. c, Fractional abundance (%) of Serine (M + 1), 10-Formyl-THF (M + 1), and dTMP (M + 1) from isogenic ∆NADK2 HEK-293E cells stably expressing either empty vector or NADK2 cDNA and labeled for 6 hours with 3-13C-serine. d, Relative incorporation of radiolabel from 14C-glycine, 14C-aspartate, or 3H-uridine (6 hours labelling) from isogenic ∆NADK2 HEK-293E cells stably expressing either empty vector (-) or NADK2 cDNA grown in the presence or absence of proline (0.2 mM). e, Relative incorporation of radiolabel from 3H-uridine (6 hours labelling) from isogenic ∆NADK2 HeLa, or K562 cells stably expressing either empty vector or NADK2 cDNA grown in the presence or absence of proline (0.2 mM). Performed in parallel with experiment shown in Fig. 7c (HeLa) and Fig. 7d (K562). Data are presented as the mean ± SD from n = 3–4 of biologically independent samples (c,d,e) and are representative of at least two independent experiments. **P < 0.01, ***P < 0.001 for comparisons calculated using a two-sided Student’s t-test for (c) and with one-way ANOVA test and Tukey’s post-hoc test for (d,e). f, Immunoblots of pentose pathway enzymes (TKT, TALDO, PGD, G6PD) and nucleotide biosynthesis genes (PPAT, GART) shown for isogenic ΔNADK2 HeLa, K562 or HEK-293E cells stably expressing either empty vector or NADK2, grown in DMEM supplemented with 10% serum in the presence or absence of proline (0.2 mM) for 48 hours. Data are representative of two independent experiments.

Source data

Extended Data Fig. 8 Proline availability regulates transcript levels of multiple nucleotide biosynthesis genes. (Data supporting Fig. 7).

a,b, Transcript levels are shown for the indicated nucleotide biosynthesis genes from isogenic ΔNADK2 HeLa (a), or K562 (b) cells stably expressing either empty vector or NADK2 grown in DMEM supplemented with 10% serum in the presence or absence of proline (0.2 mM). Data are presented as the mean ± SEM from =3 of biologically independent samples that were each measured in technical triplicates. *P < 0.05, **P < 0.01 for comparisons calculated using one-way ANOVA test and Tukey’s post-hoc test.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–3 and gating strategy for Extended Data Fig. 6

Reporting Summary

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Tran, D.H., Kesavan, R., Rion, H. et al. Mitochondrial NADP+ is essential for proline biosynthesis during cell growth. Nat Metab 3, 571–585 (2021). https://doi.org/10.1038/s42255-021-00374-y

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