Inhibition of nicotinamide nucleotide transhydrogenase in rat liver submitochondrial particles by dicyclohexylcarbodi-imide and butanedione.
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ABSTRACT:
SUBMITTER: Moody AJ
PROVIDER: S-EPMC1154171 | biostudies-other | 1983 Mar
REPOSITORIES: biostudies-other
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Project description:Nicotinamide nucleotide transhydrogenase (TH) is an enzyme complex in animal mitochondria and bacteria that utilizes the electrochemical proton gradient across membranes to drive the production of NADPH. The enzyme plays an important role in maintaining the redox balance of cells with implications in aging and a number of human diseases. TH exists as a homodimer with each protomer containing a proton-translocating transmembrane domain and two soluble nucleotide binding domains that mediate hydride transfer between NAD(H) and NADP(H). The three-domain architecture of TH is conserved across species but polypeptide composition differs substantially. The complex domain coupling mechanism of TH is not fully understood despite extensive biochemical and structural characterizations. Herein the progress is reviewed, focusing mainly on structural findings from 3D crystallization of isolated soluble domains and more recently of the transmembrane domain and the holo-enzyme from Thermus thermophilus. A structural perspective and impeding challenges in further elucidating the mechanism of TH are discussed.
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Project description:Using targeted exome sequencing, we identified mutations in NNT, an antioxidant defense gene, in individuals with familial glucocorticoid deficiency. In mice with Nnt loss, higher levels of adrenocortical cell apoptosis and impaired glucocorticoid production were observed. NNT knockdown in a human adrenocortical cell line resulted in impaired redox potential and increased reactive oxygen species (ROS) levels. Our results suggest that NNT may have a role in ROS detoxification in human adrenal glands.
| S-EPMC3386896 | biostudies-literature
Project description:Adrenocortical carcinoma (ACC) is an aggressive malignancy with poor response to chemotherapy. In this study, we evaluated a potential new treatment target for ACC, focusing on the mitochondrial reduced form of NAD phosphate (NADPH) generator nicotinamide nucleotide transhydrogenase (NNT). NNT has a central role within mitochondrial antioxidant pathways, protecting cells from oxidative stress. Inactivating human NNT mutations result in congenital adrenal insufficiency. We hypothesized that NNT silencing in ACC cells will induce toxic levels of oxidative stress. To explore this, we transiently knocked down NNT in NCI-H295R ACC cells. As predicted, this manipulation increased intracellular levels of oxidative stress; this resulted in a pronounced suppression of cell proliferation and higher apoptotic rates, as well as sensitization of cells to chemically induced oxidative stress. Steroidogenesis was paradoxically stimulated by NNT loss, as demonstrated by mass spectrometry-based steroid profiling. Next, we generated a stable NNT knockdown model in the same cell line to investigate the longer lasting effects of NNT silencing. After long-term culture, cells adapted metabolically to chronic NNT knockdown, restoring their redox balance and resilience to oxidative stress, although their proliferation remained suppressed. This was associated with higher rates of oxygen consumption. The molecular pathways underpinning these responses were explored in detail by RNA sequencing and nontargeted metabolome analysis, revealing major alterations in nucleotide synthesis, protein folding, and polyamine metabolism. This study provides preclinical evidence of the therapeutic merit of antioxidant targeting in ACC as well as illuminating the long-term adaptive response of cells to oxidative stress.
| S-EPMC6093335 | biostudies-literature
Project description:RNA-seq samples used to support a study in to the understanding of antioxidant targeting in adrenocortical carcinoma
2019-02-01 | GSE106873 | GEO
Project description:To study the physiological roles of NADH and NADPH homeostasis in cancer, we studied the effect of NNT knockdown on physiology of SK-Hep1 cells. NNT knockdown cells show limited abilities to maintain NAD+ and NADPH levels and have reduced proliferation and tumorigenicity. There is an increased dependence of energy production on oxidative phosphorylation. Studies with stable isotope tracers have revealed that under the new steady-state metabolic condition, the fluxes of TCA and glycolysis decrease while that of reductive carboxylation increases. Increased [α-ketoglutarate]/[succinate] ratio in NNT-deficient cells results in decrease in HIF-1α level and expression of HIF-1α regulated genes. Reduction in NADPH level leads to repression of HDAC1 activity and an increase in p53 acetylation. These findings suggest that NNT is essential to homeostasis of NADH and NADPH pools, anomalies of which affect HIF-1α- and HDAC1-dependent pathways, and hence retrograde response of mitochondria.
| S-EPMC5426036 | biostudies-literature
Project description:From the foregoing considerations, the energy-linked transhydrogenase reaction emerges as a powerful and flexible element in the network of redox and energy interrelationships that integrate mitochondrial and cytosolic metabolism. Its thermodynamic features make it possible for the reaction to respond readily to challenges, either on the side of NADPH utilization or on the side of energy depletion. Yet, the kinetic features are designed to prevent a wasteful input of energy when other sources of reducing equivalents to NADP are available, or to deplete the redox potential of NADPH in other than emergency conditions. By virtue of these characteristics, the energy-linked transhydrogenase can act as an effective buffer system, guarding against an excessive depletion of NADPH, preventing uncontrolled changes in key metabolites associated with NADP-dependent enzymes and calling on the supply of reducing equivalents from NAD-linked substrates only under conditions of high demand for NADPH. At the same time, it can provide an emergency protection against a depletion of energy, especially in situations of anoxia where a supply of reducing equivalents through NADP-linked substrates can be maintained. The flexibility of this design makes it possible that the functions of the energy-linked transhydrogenase vary from one tissue to another and are readily adjustable to different metabolic conditions.
| S-EPMC1135030 | biostudies-other
Project description:1. The effects of dicyclohexylcarbodi-imide, oligomycin A and aurovertin on enzyme systems related to respiratory-chain phosphorylation were compared. Dicyclohexylcarbodi-imide and oligomycin A have very similar functional effects, giving 50% inhibition of ATP-utilizing and ATP-generating systems at concentrations below 0.8nmole/mg. of submitochondrial-particle protein. Aurovertin is a more potent inhibitor of ATP synthesis, giving 50% inhibition at 0.2nmole/mg. of protein. However, aurovertin is a less potent inhibitor of ATP-utilizing systems: the ATP-driven energy-linked nicotinamide nucleotide transhydrogenase is 50% inhibited at 3.0nmoles/mg. of protein and the ATP-driven reduction of NAD(+) by succinate is 50% inhibited at 0.95nmole/mg. of protein. 2. With EDTA-particles (prepared by subjecting mitochondria to ultrasonic radiation at pH9 in the presence of 2mm-EDTA) the maximum stimulation of the ATP-driven partial reactions is effected by similar concentrations of oligomycin A and dicylcohexylcarbodi-imide, but the latter is less effective. The stimulatory effects of suboptimum concentrations of dicyclohexylcarbodi-imide and oligomycin A are additive. Aurovertin does not stimulate these reactions or interfere with the stimulation by the other inhibitors. 3. Dicyclohexylcarbodi-imide and oligomycin A stimulate the aerobic energy-linked nicotinamide nucleotide transhydrogenase of EDTA-particles, but the optimum concentration is higher than that required for the ATP-driven partial reactions. Aurovertin has no effect on this reaction. 4. The site of action of dicyclohexylcarbodi-imide is in CF(0), the mitochondrial fraction that confers oligomycin sensitivity on F(1) mitochondrial adenosine triphosphatase.
| S-EPMC1198830 | biostudies-other
Project description:Membrane bound nicotinamide nucleotide transhydrogenase (TH) catalyses the hydride transfer from NADH to NADP+. Under physiological conditions, this reaction is endergonic and must be energized by the pmf, coupled to transmembrane proton transport. Recent structures of transhydrogenase holoenzymes suggest new mechanistic details, how the long-distance coupling between hydride transfer in the peripheral nucleotide binding sites and the membrane-localized proton transfer occurs that now must be tested experimentally. Here, we provide protocols for the efficient expression and purification of the Escherichia coli transhydrogenase and its reconstitution into liposomes, alone or together with the Escherichia coli F1F0 ATP synthase. We show that E. coli transhydrogenase is a reversible enzyme that can also work as a NADPH-driven proton pump. In liposomes containing both enzymes, NADPH driven H+-transport by TH is sufficient to instantly fuel ATP synthesis, which adds TH to the pool of pmf generating enzymes. If the same liposomes are energized with ATP, NADPH production by TH is stimulated > sixfold both by a pH gradient or a membrane potential. The presented protocols and results reinforce the tight coupling between hydride transfer in the peripheral nucleotide binding sites and transmembrane proton transport and provide powerful tools to investigate their coupling mechanism.
| S-EPMC8551311 | biostudies-literature
Project description:Overcoming oxidative stress is a critical step for tumor growth and metastasis, however the underlying mechanisms in gastric cancer remain unclear. In this study, we found that overexpression of nicotinamide nucleotide transhydrogenase (NNT) was associated with shorter overall and disease free survival in gastric cancer. The NNT is considered a key antioxidative enzyme based on its ability to regenerate NADPH from NADH. Knockdown of NNT caused significantly NADPH reduction, induced high levels of ROS and significant cell apoptosis under oxidative stress conditions such as glucose deprival and anoikis. In vivo experiments showed that NNT promoted tumor growth, lung metastasis and peritoneal dissemination of gastric cancer. Moreover, intratumoral injection of NNT siRNA significantly suppressed gastric tumor growth in patient-derived xenograft (PDX) models. Overall, our study highlights the crucial functional roles of NNT in redox regulation and tumor progression and thus raises an important therapeutic hypothesis in gastric cancer.
| S-EPMC6079569 | biostudies-literature
Project description:Circulating fatty acids (FAs) increase with obesity and can drive mitochondrial damage and inflammation. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial protein that positively regulates nicotinamide adenine dinucleotide phosphate (NADPH), a key mediator of energy transduction and redox homeostasis. The role that NNT-regulated bioenergetics play in the inflammatory response of immune cells in obesity is untested. Our objective was to determine how free fatty acids (FFAs) regulate inflammation through impacts on mitochondria and redox homeostasis of peripheral blood mononuclear cells (PBMCs). PBMCs from lean subjects were activated with a T cell-specific stimulus in the presence or absence of generally pro-inflammatory palmitate and/or non-inflammatory oleate. Palmitate decreased immune cell expression of NNT, NADPH, and anti-oxidant glutathione, but increased reactive oxygen and proinflammatory Th17 cytokines. Oleate had no effect on these outcomes. Genetic inhibition of NNT recapitulated the effects of palmitate. PBMCs from obese (BMI >30) compared to lean subjects had lower NNT and glutathione expression, and higher Th17 cytokine expression, none of which were changed by exogenous palmitate. Our data identify NNT as a palmitate-regulated rheostat of redox balance that regulates immune cell function in obesity and suggest that dietary or therapeutic strategies aimed at increasing NNT expression may restore redox balance to ameliorate obesity-associated inflammation.
| S-EPMC6406569 | biostudies-literature