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Mitochondrial NADPH fuels mitochondrial fatty acid synthesis and lipoylation to power oxidative metabolism

Nature Cell Biology volume 27, pages 790–800 (2025)Cite this article

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An Author Correction to this article was published on 22 May 2025

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Abstract

Nicotinamide adenine dinucleotide phosphate (NADPH) is a vital electron donor essential for macromolecular biosynthesis and protection against oxidative stress. Although NADPH is compartmentalized within the cytosol and mitochondria, the specific functions of mitochondrial NADPH remain largely unexplored. Here we demonstrate that NAD+ kinase 2 (NADK2), the principal enzyme responsible for mitochondrial NADPH production, is critical for maintaining protein lipoylation, a conserved lipid modification necessary for the optimal activity of multiple mitochondrial enzyme complexes, including the pyruvate dehydrogenase complex. The mitochondrial fatty acid synthesis (mtFAS) pathway utilizes NADPH for generating protein-bound acyl groups, including lipoic acid. By developing a mass-spectrometry-based method to assess mammalian mtFAS, we reveal that NADK2 is crucial for mtFAS activity. NADK2 deficiency impairs mtFAS-associated processes, leading to reduced cellular respiration and mitochondrial translation. Our findings support a model in which mitochondrial NADPH fuels the mtFAS pathway, thereby sustaining protein lipoylation and mitochondrial oxidative metabolism.

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Fig. 1: NADK2 loss impairs pyruvate oxidation.
Fig. 2: NADK2 is required for protein lipoylation.
Fig. 3: Mitochondrial NADPH, but not ROS, or proline, is required for protein lipoylation.
Fig. 4: Mitochondrial NADPH sustains mtFAS.
Fig. 5: NADK2 is crucial for cellular respiration and mitochondrial translation.

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Data availability

The raw metabolomics data are deposited in the Mendeley Repository at https://data.mendeley.com/datasets/x4tkj57mb5/1. The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE60 partner repository with the dataset identifier PXD060981. All other data are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

The code used to generate the gene coessentiality networks is available via Zenodo at https://doi.org/10.5281/zenodo.14902791 (ref. 61).

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Acknowledgements

We thank Y. Yang (East China University of Science and Technology) for providing iNAP plasmids34 and Y. Zhang for insightful discussions. We also thank the UTSW Quantitative Light Microscopy Core, a shared resource of the Harold C. Simmons Cancer Center, supported in part by NCI (1P30 CA142543-01), the UT Southwestern Proteomics Core and the US Department of Agriculture, Agriculture Research Service that supports D.K.A. The research is supported by the NIH (grant no. R01GM143236 to G.H., grant no. 3R01GM143236-02S1 (equipment grant) to G.H.), Welch foundation (grant no. I-2067-20240404 to G.H), CPRIT (grant no. RP240035 to G.H.) and USDA National Institute of Food and Agriculture Award (grant no. 2021-67013-33778 to D.K.A.). G.H. is a recipient of the Pew-Stewart Scholar, CPRIT Scholar (CPRIT; RR190087), ACS Scholar awards (RSG-22-177-01-TBE) and V Scholar (V2021-019). D.K. is supported by a CPRIT training grant (grant no. RP210041 to D.K.) and C.M. by an NIH F30 fellowship (grant no. DK137407 to C.M.).

Author information

Authors and Affiliations

  1. Children’s Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Dohun Kim, Rushendhiran Kesavan, Kevin Ryu, Trishna Dey, Cameron Menezes, Prakash P. Praharaj, Bookyung Ko, Mona H. Soflaee, Harrison J. Tom, Harrison Brown, Hieu S. Vu, Prashant Mishra, Feng Cai & Gerta Hoxhaj

  2. Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Austin Marckx

  3. Donald Danforth Plant Science Center, St. Louis, MO, USA

    Stewart Morley & Doug K. Allen

  4. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Shih-Chia Tso & Chad A. Brautigam

  5. Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Chad A. Brautigam

  6. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Andrew Lemoff

  7. Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Marcel Mettlen

  8. United States Department of Agriculture, Agriculture Research Service, St. Louis, MO, USA

    Doug K. Allen

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Contributions

D.K. performed and analysed most experiments. R.K., T.D., C.M., P.P.P., M.H.S., K.R., H.B., H.J.T. and P.M. conducted experiments and analysed and discussed data. S.M. and D.K.A. contributed to the mtFAS analysis, A.M. to the computational work, A.L. to the post-translational modification analysis, R.K. and M.M. to the microscopy, S.T. and C.A.B. to the ITC and B.K., F.C. and H.S.V. to mass spectrometry. D.K. and G.H. conceptualized the study and wrote the manuscript, and G.H. directed the study. All authors reviewed the manuscript.

Corresponding author

Correspondence to Gerta Hoxhaj.

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Nature Cell Biology thanks Yihui Shen, Huiyong Yin 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 NADK2 supports pyruvate oxidation and tumor growth.

(a) Fractional abundance (%) of the indicated TCA cycle metabolites from isogenic ∆NADK2 A375 cells expressing either empty vector or NADK2 and labeled with [13C3]-pyruvate for 30 min in 0.2 mM proline. (n = 4 biological replicates). (b) As in (a), but the experiments were performed in isogenic ∆NADK2 A549 cells expressing either empty vector or NADK2 and labeled with [13C3]-pyruvate in the presence of 0.2 mM proline for 15, 30, or 60 min. (n = 4 biological replicates). Related to Fig. 1d. (c) As in (b), but the experiments were performed in isogenic ∆NADK2 HeLa cells expressing either empty vector or NADK2. (n = 4 biological replicates). Related to Fig. 1e. (d) As in (b), but the cells were labeled with [13C3]-pyruvate for 1 h. The peak area was normalized to protein abundance. (n = 4 biological replicates). (e) As in (d), but the experiments were performed in isogenic ∆NADK2 HeLa cells expressing either empty vector or NADK2. (n = 4 biological replicates). (f) Relative citrate (M + 2)/ pyruvate (M + 2) ratio from (d). (n = 4 biological replicates). (g) Relative citrate (M + 2)/ pyruvate (M + 2) ratio from (e). (n = 4 biological replicates). (h) Schematic of A375 tumor xenograft. Related to Fig. 1l, m. (i) Tumor growth from isogenic ∆NADK2 A375 cells expressing either empty vector or NADK2. (n = 8 biologically independent animals). Related to Fig. 1l, m. (j) The size of tumors derived from isogenic ∆NADK2 A375 cells expressing either empty vector or NADK2 at the end of the experiment. (n = 8 biologically independent tumors).Related to Fig. 1l, m. (k) Normalized proline abundance from tumors derived from isogenic ∆NADK2 A375 cells expressing either empty vector or NADK2. (n = 4 biologically independent tumors). Data are presented as the mean ± standard deviation (a-g, j, and k) or mean ± s.e.m. (i). *P < 0.05, **P < 0.01, and ***P < 0.001 were calculated using a two-tailed Student’s t-test (a-c, and i-k) or one-way ANOVA with Tukey’s post hoc test (d-g).

Source data

Extended Data Fig. 2 Impact of NADK2 on redox ratios and the TCA cycle.

(a) The relative levels of NAD+ and NADH and NAD+/NADH ratio in mitochondria isolated from isogenic ∆NADK2 HeLa cells expressing either empty vector or NADK2. The cells were cultured in the presence of 0.2 mM proline. (n = 4 biologically independent replicates). (b) The relative levels of NAD+ and NADH and NAD+/NADH ratio in mitochondria isolated from isogenic ∆NADK2 A549 cells expressing either empty vector or NADK2. The cells were cultured in the presence of 0.2 mM proline. (n = 4 biologically independent replicates). (c) Coomassie staining of immunoprecipitated DLAT-HA from isogenic ∆NADK2 HeLa cells expressing either empty vector or NADK2 and transfected with DLAT-HA. The cells were cultured in the presence of 0.2 mM proline. The bands were cut out and subjected to mass spectrometry for detecting lipoylated peptides. (d) The abundance of lipoylated DLAT as described in (c). (e) Schematic of [13C5]-glutamine tracing. (f) The normalized peak area (labeled and total pools) of glutamine in isogenic ΔNADK2 HeLa cells expressing either empty vector or NADK2 labeled with [13C5]-glutamine for 1 h. The peak areas were normalized to the protein amount assessed with a BCA assay. (n = 4 biological replicates). (g) As in (f), but the normalized peak area (labeled and total pools) of glutamate are shown. (n = 4 biological replicates). (h) As in (f), but the normalized peak area (labeled and total pools) of alpha-ketoglutarate are shown. (n = 4 biological replicates). (i) As in (f), but the normalized peak area (labeled and total pools) of succinyl-CoA are shown. (n = 4 biological replicates). (j) The relative OGDH activity from mitochondrial extracts of isogenic ΔNADK2 HeLa cells expressing either empty vector or NADK2, which were grown in DMEM containing 10% FBS and 0.2 mM proline. (n = 3 biological replicates). Data are presented as the mean ± standard deviation (a,b,f-j). *P < 0.05, **P < 0.01, and ***P < 0.001 were calculated using a two-tailed Student’s t-test (a,b,f-j).

Source data

Extended Data Fig. 3 Impact of ROS, proline, and lipids on protein lipoylation.

(a) Immunoblots showing DLAT lipoylation, DLAT, NADK2, and vinculin levels from isogenic ΔNADK2 A549 cells expressing either empty vector or NADK2. Cells were grown in the presence of 0.2 mM proline and treated with the indicated antioxidants for 48 h. GSHee; glutathione ethyl ester (5 mM), NAC; N-acetyl cysteine (1 mM), and Trolox (5 μM). (b) Immunoblotting was done as in (a), but cells were grown in the presence or absence of 0.2 mM proline. (c) Immunoblotting was done as in (a), but cells were grown in the presence of 0.2 mM proline and treated with octanoic acid (50 μM), lipoic acid (50 μM), palmitic acid (100 μM), oleic acid (100 μM), low-density lipoprotein (LDL, 50 μg/ml), or high-density lipoprotein (HDL, 50 μg/ml) for 48 h. (d) Immunoblotting was done as in (a) but in ΔNADK2 A549 cells expressing either empty vector or NADK2. (e) Immunoblotting was done as in (a), but from WT or isogenic ΔNADK2 A549 cells expressing empty vector, NADK2, D161A NADK2, MTS-NADK, or cytosolic NADK. D161A NADK2: kinase-dead NADK2; MTS: Mitochondrial Targeting Sequence. (f) The abundance of intracellular lipoic acid from isogenic ∆NADK2 HeLa or A549 cells expressing either empty vector or NADK2 treated with either vehicle or lipoic acid (50 μM) for 48 h assessed via LC/MS. The peak areas were normalized to the protein amount. (n = 4 biologically independent replicates). Data are presented as the mean ± standard deviation. **P < 0.01 was calculated using a two-tailed Student’s t-test. (g) Immunoblots showing ACP, NADK2, and vinculin levels from WT or ∆NADK2 HeLa cells.

Source data

Extended Data Fig. 4 The synthesis and detection of acyl-ACP standards via LC/MS.

(a) Schematic illustrating workflow for synthesizing acyl-DSL standard for acyl-ACP measurement. (b) The mass spectrometry parameters of various acyl-DSLs. (c-j) The chromatogram of synthesized standards (black) and biological HeLa WT (blue) for Apo-ACP (c), Holo-ACP (d), C2-ACP (e), C4-ACP (f), C6-ACP (g), C8-ACP (h), C10-ACP (i), C12-ACP (j) run on LC/MS.

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Extended Data Fig. 5 Impact of NADK2 and mtFAS on mitochondrial translation.

(a) Coomassie staining of purified ACP-FLAG from WT and ΔMECR HeLa cells, which was then subjected to Asp-N peptidase treatment for assessment of acyl-ACP species via LC/MS. (b) Representative immunofluorescence images for ACP-FLAG (green) detected with anti-FLAG antibody MitoTracker Red (red), and nuclei (blue) stained with Hoechst for WT and ΔNADK2 HeLa cells cultured in the presence of 0.2 mM proline. Scale bars, 10 µm. (c) The NADK2 coessentiality network showing the top 6 enrichment gene clusters. (d) The MECR coessentiality network showing the top 6 enrichment gene clusters. (e) Volcano plot illustrating the log2 fold change of mitoribosome-related proteins identified in ACP immunoprecipitates from wild-type (WT), ΔNADK2, and ΔMECR HeLa cells. Proteins belonging to the mitochondrial ribosomal protein large subunit (MRPL) or small subunit (MRPS), as well as other interactors, are indicated and color-coded. (f) Schematic illustrating the workflow of click chemistry-based mitochondrial translation assay.

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Kim, D., Kesavan, R., Ryu, K. et al. Mitochondrial NADPH fuels mitochondrial fatty acid synthesis and lipoylation to power oxidative metabolism. Nat Cell Biol 27, 790–800 (2025). https://doi.org/10.1038/s41556-025-01655-4

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