Mitochondrial NADP(H) generation is essential for proline biosynthesis

Abstract

The coenzyme nicotinamide adenine dinucleotide phosphate (NADP⁺) and its reduced form (NADPH) regulate reductive metabolism in a subcellularly compartmentalized manner. Mitochondrial NADP(H) production depends on the phosphorylation of NAD(H) by NAD kinase 2 (NADK2). Deletion of NADK2 in human cell lines did not alter mitochondrial folate pathway activity, tricarboxylic acid cycle activity, or mitochondrial oxidative stress, but rather led to impaired cell proliferation in minimal medium. This growth defect was rescued by proline supplementation. NADK2-mediated mitochondrial NADP(H) generation was required for the reduction of glutamate and hence proline biosynthesis. Furthermore, mitochondrial NADP(H) availability determined the production of collagen proteins by cells of mesenchymal lineage. Thus, a primary function of the mitochondrial NADP(H) pool is to support proline biosynthesis for use in cytosolic protein synthesis.

Fig. 1.. NADK2 is required to maintain the mitochondrial NADP(H) pool.

(A) DLD1 cells expressing HA-tagged OMP25 protein (DLD1-OMP25HA) were engineered to express control guide RNA (sgCtrl) or two independent guide RNA sequences targeting NADK2 (sgNADK2-1 and sgNADK2-2), and were subjected to Western blot of whole cell or anti-HA immunopurified mitochondria (Mito-IP). (B and C) Colorimetric enzyme-based measurement of total NADP(H) abundance in (B) whole cell or (C) immunopurified mitochondria of DLD1-OMP25HA cells with sgCtrl, sgNADK2-1, or sgNADK2-2, cultured in DMEM/F12 medium. (D) Western blot analysis of JJ012 (mutant IDH1) and CS1 (mutant IDH2) cells with sgCtrl, sgNADK2-1, or sgNADK2-2. (E and F) 2HG abundance measured by gas chromatography-mass spectrometry (GC-MS) in (E) JJ012 and (F) CS1 cells with sgCtrl, sgNADK2-1, or sgNADK2-2. (G) 2HG abundance measured by GC-MS in xenograft tumors formed by CS1 cells with sgCtrl or sgNADK2-2. Error bars in (B) represent mean+SD, n=6; in (C), (E) and (F) represent mean+SD, n=3; in (G) represent mean±SD, n=10. In (C), one-way ANOVA was performed with matched measures. In (F), one-way ANOVA was performed. In (G), two-sided t-test was performed with Welch’s correction. ***P<0.001.

Fig. 2.. Mitochondrial NADP(H) depletion does not have significant effects on folate pathway, TCA cycle activity, or measures of oxidative stress.

(A) Scheme of the tracing strategy, adapted from (9, 10). Catabolism of [2,3,3-²H3]serine in the mitochondrial or cytosolic folate pathway produces singly or doubly deuterated thymidine triphosphate (TTP M+1 or TTP M+2), respectively. (B) Western blot of DLD1 cells with sgCtrl, sgNADK2-1, sgNADK2-2, sgMTHFD2, or sgSHMT2. (C) Isotopologue distribution of TTP measured by liquid chromatography-mass spectrometry (LC-MS) in DLD1 cells denoted in (B), cultured in [2,3,3-²H3]serine-containing medium for 8 hours. (D to G) Isotopologue distribution of the indicated metabolites measured by GC-MS in DLD1 cells with sgCtrl, sgNADK2-1, or sgNADK2-2, cultured in [U-¹³C]glutamine-containing medium for 6 hours. (H) Cellular ROS measured by CM-H₂DCFDA in the indicated DLD1 cells, mock treated or treated with 150 μM H₂O₂ for 4 hours. (I) DLD1 cells expressing Mito-Orp1-roGFP2 and the indicated guide RNA were treated with vehicle (DMSO) or 100 μM MitoPQ for 24 hours. Oxidation status was expressed as percentage of maximal oxidation which was determined by treating cells with 5 mM H₂O₂ for 5 min before harvest. (J) Western blot analysis of whole cell or immunopurified mitochondria of DLD1-OMP25HA cells expressing the indicated guide RNA (K) Western blot of the indicated DLD1 cells mock treated or treated with 500 μM H₂O₂ for 6 hours. SE, short exposure. LE, long exposure. (L) Ferroptosis sensitivity of the indicated DLD1 cells, measured as percentage cell death upon mock, Erastin (5 μM) or RSL3 (0.5 μM) treatment for 24 hours. All error bars in this figure represent mean+SD, n=3.

Fig. 3.. Mitochondrial NADP(H) depletion results in proline auxotrophy.

(A and B) Cell proliferation measured as cell number fold change (Day 4/Day 0) of T47D cells with sgCtrl, sgNADK2-1, or sgNADK2-2, cultured in the indicated medium and supplementation. LA, lipoic acid. Pyr, pyruvate. Cu, cupric sulfate. Zn, zinc sulfate. B12, vitamin B12. A, alanine. D, aspartate. N, asparagine. E, glutamate. P, proline. All supplements are added at the concentrations present in DMEM/F12. (C) Proline abundance measured by GC-MS in the indicated T47D cells cultured in DMEM. (D to F) (D) Western blot, (E) proline abundance measured by GC-MS, and (F) cell proliferation of DMEM-cultured T47D cells with sgCtrl or sgNADK2-2 and ectopically expressing vector or NADK2 cDNA resistant to sgNADK2-2 mediated CRISPR-Cas9 genome editing. (G to I) (G) Western blot, (H) proline abundance measured by GC-MS, and (I) cell proliferation of DMEM-cultured T47D cells with sgCtrl, sgNADK2-1, or sgNADK2-2 and ectopically expressing vector or the POS5 cDNA. All error bars in this figure represent mean+SD, n=3. In (A), (B), (C), (H) and (I), one-way ANOVA was performed. In (E) and (F), two-sided t-test was performed with Welch’s correction. **P<0.01; ***P<0.001; n.s., P>0.05.

Fig. 4.. The mitochondrial NADP(H) pool is required to support proline biosynthesis and collagen production.

(A) Heatmap representing changes of metabolite levels measured by GC-MS in T47D cells with sgCtrl, sgNADK2-1, or sgNADK2-2 cultured in DMEM for 48 hours. The average of 3 biological replicates is shown. For each metabolite, values of sgNADK2-1 and sgNADK2-2 cells are shown as log₂ (fold change) relative to the value of sgCtrl cells. (B) Changes of metabolite levels measured by GC-MS in DMEM/F12 medium used to culture T47D cells with sgCtrl, sgNADK2-1, or sgNADK2-2 for 48 hours. (C and D) (C) proline and (D) glutamate data from (B) re-plotted as normalized values to sgCtrl cells. (E) Proline abundance measured by GC-MS in xenograft tumors formed by CS1 cells with sgCtrl or sgNADK2-2. (F) Scheme of proline biosynthesis pathway in the mitochondria. (G to J) Relative total level and isotopologue distribution of the indicated metabolites measured by LC-MS in MEFs with sgCtrl, sgNadk2-1, or sgNadk2-2, cultured in DMEM containing [U-¹³C]glutamine for 8 hours. (K) Western blot of the indicated MEFs, cultured in DMEM or DMEM supplemented with 300 μM proline. (L) Scheme of ECM extraction and collagen staining in cells and under conditions described in (M). (M) Secreted collagen levels quantified by picro sirius red staining in extracellular matrix (ECM) derived from MEFs with sgCtrl, sgNadk2-1, or sgNadk2-2, cultured for 48 hours in DMEM or DMEM supplemented with 300 μM proline, in the presence of 50 μM ascorbate. (N) Pearson correlation of NADK2 mRNA level and forced vital capacity (FVC) before bronchodilator (pre-BD) as percentage of what was predicted for each patient. Data from GSE32537. (O) Pearson correlation of NADK2 mRNA level and diffusing capacity for carbon monoxide (DLCO) as percentage of what was predicted for each patient. Data from GSE32537. Error bars in (E) represent mean±SD, n=10. All other error bars in this figure represent mean+SD, n=3. In (B to D), one-way ANOVA was performed. In (E) and (M), two-sided t-test was performed with Welch’s correction. *P<0.05; ** P<0.01; ***P<0.001.