Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio

Abstract

A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD(+)) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD(+)/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD(+)/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state.

Figure 1. H₂O-forming NADH oxidase from L. brevis (LbNOX

(A) Reaction catalyzed by LbNOX. (B) UV-visible spectrum of purified LbNOX. Protein (83 µM FAD active sites) in oxidized form (solid line) and after addition of excess of sodium dithionite, reduced form (dashed line). Inset: SDS-PAGE of purified LbNOX. (C) Simultaneous measurement of NADH and oxygen consumption by LbNOX. NADH and LbNOX were added as indicated by arrows. (D) Dependence of the specific activity of recombinant LbNOX on the concentration of NADH and NADPH. Reported values for V_max, k_cat and K_M for NADH represent the mean ± S.D. from n=4 independent experiments. (E) Crystal structure of the catalytic dimer of LbNOX. Each of the two-fold symmetry related monomers (cyan and green ribbons) contain bound FAD, shown here in sphere (CPK) representation. Details of the catalytic center on the si-face of FAD and of the substrate selectivity loop are shown in fig. S3A–C.

Figure 2. Expression and activity of LbNOX in human cells

(A) Western blot of LbNOX and mitoLbNOX in HeLa cells after 24-hour induction with water or doxycycline (300 ng/ml). Representative gel from one of three independent experiments. (B) Subcellular localization of LbNOX and mitoLbNOX in HeLa cells determined by cell fractionation. LRPPRC is a mitochondrial marker and Actin is a cytosolic marker. Representative gel from one of three independent experiments. (C) Subcellular localization of LbNOX and mitoLbNOX in HeLa cells determined using fluorescence microscopy. Tomm20 is a marker of mitochondria. (D) Effect of LbNOX and mitoLbNOX expression in HeLa cells on basal, piericidin-resistant and antimycin-resistant oxygen consumption measured with a XF24 extracellular flux analyzer. Mean ± S.E., n=3 independent experiments.

Figure 3. Effect of LbNOX and mitoLbNOX on NAD⁺/NADH ratios, metabolic fluxes, PDH phosphorylation and gluconeogenesis

(A–C) Effect of LbNOX and mitoLbNOX expression in HeLa cells on (A) cytoplasmic NADH concentrations determined with fluorescence microscopy using SoNar expressing cells (n=7), (B) intracellular and secreted lactate/pyruvate ratio determined by LC-MS (n=4), and (C) intracellular NAD⁺/NADH ratios determined by HPLC (n=4). Student’s t-test. ns P > 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Mean ± S.E. (D) Effect of LbNOX and mitoLbNOX expression in HeLa cells on release rate of pyruvate, aspartate and succinate, determined by comparing concentrations in spent versus fresh media. Student’s t-test. ns P > 0.05, ** P < 0.01, *** P < 0.001. Mean ± S.E., n=3 replicates from one experiment. (E) Effect of LbNOX and mitoLbNOX expression in HeLa cells on PDH phosphorylation. Representative gel from one of three independent experiments. (F) Effect of adenoviral transduction of GFP, LbNOX or mitoLbNOX on primary rat hepatocyte gluconeogenesis in DMEM containing no glucose, no glutamine and no pyruvate using either no substrate, 5mM pyruvate, or 5mM lactate. One-way ANOVA followed by Tukey’s multiple comparisons test. ns P > 0.05, * P < 0.05, ** P < 0.01, **** P < 0.0001. Mean ± S.E., n=3 (no substrate, pyruvate) or n=7 (lactate) independent experiments. (G) Effect of LbNOX and mitoLbNOX on secreted β-hydroxybutyrate/acetoacetate ratio in rat hepatocytes performing gluconeogenesis from lactate as a substrate. Metabolite levels determined using LC-MS. One-way ANOVA followed by Tukey’s multiple comparisons test. ns P > 0.05, * P < 0.05, ** P < 0.01. Mean ± S.E., n=10 independent experiments.

Figure 4. NAD⁺ recycling rescues proliferation in cells with impaired ETC

(A) Effect of pyruvate, oxaloacetate, lactate and malate addition on proliferation of HeLa Tet3G Luciferase cells in the presence of 200 µM uridine and in the presence or absence of 1 µM piericidin. Mean ± S.E., n=5 independent experiments. (B) Effect of LbNOX and mitoLbNOX expression in HeLa cells on inhibition of cell proliferation by 1 µM piericidin, 1 µM antimycin, 10 µg/ml chloramphenicol and 30 ng/ml ethidium bromide in the presence of 200 µM uridine. Mean ± S.E., n=3 independent experiments.