Source Of NADPH Research Articles

Buffy coat removal to mitigate reporting false normal G6PD phenotype results in patients undergoing rasburicase therapy for tumor lysis syndrome

Abstract Glucose-6-phosphate dehydrogenase (G6PD) is the primary source of NADPH in red blood cells (RBCs). NADPH reduces reactive oxygen species (ROS) thereby protecting RBCs from oxidative stress-mediated hemolytic anemia (HA). Individuals with partial or full G6PD deficiency are therefore susceptible to acute HA provoked by medications that generate high ROS loads in vivo, e.g., rasburicase. Rasburicase is an enzyme-based therapeutic used to degrade uric acid (UA) in patients with tumor lysis syndrome (TLS); however, catalysis of UA generates H2O2, a potent ROS, which poses a risk of HA in individuals who are deficient in G6PD activity. Screening G6PD activity prior to rasburicase administration is clinically indicated in ethnic groups with high rates of G6PD deficiency, although reports of HA in rasburicase-treated patients have been reported across low prevalence ethnic groups as well. TLS occurs most commonly during the treatment of highly proliferative neoplasms, such as leukemia and lymphoma. Since WBCs contain significant G6PD activity, an elevated WBC count in these patients can cause the false assurance of low HA risk due to a falsely normal G6PD activity. We sought to establish and evaluate a novel method to mitigate WBC contamination of G6PD measurements in patients undergoing rasburicase therapy. In this study, we first developed and validated an innovative method (“piercing method”) to minimize the interference of WBCs in G6PD measurements. The piercing method demonstrated acceptable accuracy (slope: 1.008; bias: -6.22%) and precision (1.4% CV between 9.9 to 20.7 U/g Hb) and was effective at eliminating a broad range of WBC types and concentrations from whole blood samples (N = 85, tested range = 0.31 – 413.05 K/μL, 93% reduction in WBCs). We compared G6PD activity in rasburicase-treated patients (N = 39) with varying WBC counts before and after buffy coat removal using the piercing method. Average percent change in G6PD activity (post-pierce – pre-pierce) between patients with and without elevated WBCs was substantial (45% versus 12%). In comparing G6PD activity in patients before and after the initiation of rasburicase therapy, there is no evidence that in vivo-generated ROS from rasburicase affect G6PD measurement in vitro in an assay that relies on monitoring absorbance of NADPH (22.2 U/g Hb versus 22.9 U/g Hb, p=0.721). Results demonstrate how WBCs increase risk for false elevation in G6PD activity in patients being considered for rasburicase therapy. At greatest risk are patients with partial G6PD deficiency, e.g., heterozygous females, where G6PD from WBCs could mask deficiency. Literature describing testing of G6PD in specimens with elevated WBCs is lacking. This study shows that the piercing method provides accurate G6PD results in samples with elevated WBCs from patients at risk of TLS, who might develop HA after starting rasburicase therapy.

Strategies to Maintain Redox Homeostasis in Yeast Cells with Impaired Fermentation-Dependent NADPH Generation.

Redox homeostasis is the balance between oxidation and reduction reactions. Its maintenance depends on glutathione, including its reduced and oxidized form, GSH/GSSG, which is the main intracellular redox buffer, but also on the nicotinamide adenine dinucleotide phosphate, including its reduced and oxidized form, NADPH/NADP+. Under conditions that enable yeast cells to undergo fermentative metabolism, the main source of NADPH is the pentose phosphate pathway. The lack of enzymes responsible for the production of NADPH has a significant impact on yeast cells. However, cells may compensate in different ways for impairments in NADPH synthesis, and the choice of compensation strategy has several consequences for cell functioning. The present study of this issue was based on isogenic mutants: Δzwf1, Δgnd1, Δald6, and the wild strain, as well as a comprehensive panel of molecular analyses such as the level of gene expression, protein content, and enzyme activity. The obtained results indicate that yeast cells compensate for the lack of enzymes responsible for the production of cytosolic NADPH by changing the content of selected proteins and/or their enzymatic activity. In turn, the cellular strategy used to compensate for them may affect cellular efficiency, and thus, the ability to grow or sensitivity to environmental acidification.

The Role of Glucose-6-phosphate Dehydrogenase in the Wine Yeast Hanseniaspora uvarum.

Hanseniaspora uvarum is the predominant yeast species in the majority of wine fermentations, which has only recently become amenable to directed genetic manipulation. The genetics and metabolism of H. uvarum have been poorly studied as compared to other yeasts of biotechnological importance. This work describes the construction and characterization of homozygous deletion mutants in the HuZWF1 gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), which provides the entrance into the oxidative part of the pentose phosphate pathway (PPP) and serves as a major source of NADPH for anabolic reactions and oxidative stress response. Huzwf1 deletion mutants grow more slowly on glucose medium than wild-type and are hypersensitive both to hydrogen peroxide and potassium bisulfite, indicating that G6PDH activity is required to cope with these stresses. The mutant also requires methionine for growth. Enzyme activity can be restored by the expression of heterologous G6PDH genes from other yeasts and humans under the control of a strong endogenous promoter. These findings provide the basis for a better adaptation of H. uvarum to conditions used in wine fermentations, as well as its use for other biotechnological purposes and as an expression organism for studying G6PDH functions in patients with hemolytic anemia.

Apollo-NADP+ reveals in vivo adaptation of NADPH/NADP+ metabolism in electrically activated pancreatic β cells.

Several genetically encoded sensors have been developed to study live cell NADPH/NADP+ dynamics, but their use has been predominantly in vitro. Here, we developed an in vivo assay using the Apollo-NADP+ sensor and microfluidic devices to measure endogenous NADPH/NADP+ dynamics in the pancreatic β cells of live zebrafish embryos. Flux through the pentose phosphate pathway, the main source of NADPH in many cell types, has been reported to be low in β cells. Thus, it is unclear how these cells compensate to meet NADPH demands. Using our assay, we show that pyruvate cycling is the main source of NADP+ reduction in β cells, with contributions from folate cycling after acute electrical activation. INS1E β cells also showed a stress-induced increase in folate cycling and further suggested that this cycling requires both increased glycolytic intermediates and cytosolic NAD+. Overall, we show in vivo application of the Apollo-NADP+ sensor and reveal that β cells are capable of adapting NADPH/NADP+ redox during stress.

Fuels for ROS signaling in plant immunity.

Reactive oxygen species (ROS) signaling has an important role in plant innate immune responses and is primarily mediated by NADPH oxidase, also known as respiratory burst oxidase homologs (RBOHs) in plants. NADPH serves as a fuel for RBOHs and limits the rate or amount of ROS production. Molecular regulation of RBOHs has been extensively studied; however, the source of NADPH for RBOHs has received little attention. Here, we review ROS signaling and the regulation of RBOHs in the plant immune system with a focus on NADPH regulation to achieve ROS homeostasis. We propose an idea to regulate the levels of NADPH as part of a new strategy to control ROS signaling and the corresponding downstream defense responses.

Role of NADP+-dependent isocitrate dehydrogenase from muscle tissue of Rana sylvatica in ROS defense during freeze-tolerance

The wood frog, Rana sylvatica, employs freeze tolerance as a winter survival strategy in seasonally cold environments. At subzero temperatures, up to 65–70% of total body water can freeze in extracellular spaces, halting vital functions (breathing, heartbeat) and causing ischemia that, in turn, can have numerous consequences including the generation of damaging reactive oxygen species (ROS). NADPH serves as a key donor of reductive power for most ROS detoxifying enzymes and can be generated by several metabolic pathways. One source of NADPH reducing power is the NADP-dependent isocitrate dehydrogenase (IDH) reaction. The present study evaluated the properties and regulation of IDH from skeletal muscle of R. sylvatica when frogs were exposed to stress conditions: freezing, dehydration or anoxia. Purified IDH exhibited higher affinity for isocitrate under all stress conditions as compared to controls, suggesting that the enzyme is primed to synthesize NADPH relative to the control state. Immunoblotting showed reduced serine and threonine phosphorylation of muscle IDH from frozen frogs and decreased serine phosphorylation on IDH from dehydrated frogs relative to control and anoxic states, demonstrating a reversible phosphorylation regulatory mechanism for IDH activity during freezing stress. Taken together, these results suggest activation and maintenance of IDH activity despite hypometabolic conditions. This initiation in activity of IDH during freezing may play a role in antioxidant defense by contributing to maintenance of the NADPH pool under stress conditions.

Knocking out central metabolism genes to identify new targets and alternating substrates to improve lipid synthesis in Y. lipolytica.

Introduction: Systematic gene knockout studies may offer us novel insights on cell metabolism and physiology. Specifically, the lipid accumulation mechanism at the molecular or cellular level is yet to be determined in the oleaginous yeast Y. lipolytica. Methods: Herein, we established ten engineered strains with the knockout of important genes involving in central carbon metabolism, NADPH generation, and fatty acid biosynthetic pathways. Results: Our result showed that NADPH sources for lipogenesis include the OxPP pathway, POM cycle, and a trans-mitochondrial isocitrate-α-oxoglutarate NADPH shuttle in Y. lipolytica. Moreover, we found that knockout of mitochondrial NAD+ isocitrate dehydrogenase IDH2 and overexpression of cytosolic NADP+ isocitrate dehydrogenase IDP2 could facilitate lipid synthesis. Besides, we also demonstrated that acetate is a more favorable carbon source for lipid synthesis when glycolysis step is impaired, indicating the evolutionary robustness of Y. lipolytica. Discussion: This systematic investigation of gene deletions and overexpression across various lipogenic pathways would help us better understand lipogenesis and engineer yeast factories to upgrade the lipid biomanufacturing platform.

Glucose feeds the tricarboxylic acid cycle via excreted ethanol in fermenting yeast.

Ethanol and lactate are typical waste products of glucose fermentation. In mammals, glucose is catabolized by glycolysis into circulating lactate, which is broadly used throughout the body as a carbohydrate fuel. Individual cells can both uptake and excrete lactate, uncoupling glycolysis from glucose oxidation. Here we show that similar uncoupling occurs in budding yeast batch cultures of Saccharomyces cerevisiae and Issatchenkia orientalis. Even in fermenting S. cerevisiae that is net releasing ethanol, media 13C-ethanol rapidly enters and is oxidized to acetaldehyde and acetyl-CoA. This is evident in exogenous ethanol being a major source of both cytosolic and mitochondrial acetyl units. 2H-tracing reveals that ethanol is also a major source of both NADH and NADPH high-energy electrons, and this role is augmented under oxidative stress conditions. Thus, uncoupling of glycolysis from the oxidation of glucose-derived carbon via rapidly reversible reactions is a conserved feature of eukaryotic metabolism.

Allosteric role of a structural NADP+ molecule in glucose-6-phosphate dehydrogenase activity

Human glucose-6-phosphate dehydrogenase (G6PD) is the main cellular source of NADPH, and thus plays a key role in maintaining reduced glutathione to protect cells from oxidative stress disorders such as hemolytic anemia. G6PD is a multimeric enzyme that uses the cofactors β-D-glucose 6-phosphate (G6P) and "catalytic" NADP+ (NADP+c), as well as a "structural" NADP+ (NADP+s) located ∼25 Å from the active site, to generate NADPH. While X-ray crystallographic and biochemical studies have revealed a role for NADP+s in maintaining the catalytic activity by stabilizing the multimeric G6PD conformation, other potential roles for NADP+s have not been evaluated. Here, we determined the high resolution cryo-electron microscopy structures of human wild-type G6PD in the absence of bound ligands and a catalytic G6PD-D200N mutant bound to NADP+c and NADP+s in the absence or presence of G6P. A comparison of these structures, together with previously reported structures, reveals that the unliganded human G6PD forms a mixture of dimers and tetramers with similar overall folds, and binding of NADP+s induces a structural ordering of a C-terminal extension region and allosterically regulates G6P binding and catalysis. These studies have implications for understanding G6PD deficiencies and for therapy of G6PD-mediated disorders.

Abstract 3038: IDH1 facilitates melanoma cell survival under metabolic stress

Abstract Introduction: Novel immunotherapies and targeted therapies have generated remarkable responses for patients with melanoma relative to historic treatments, but these responses are only seen in about 50% of patients. This suggests that treatment resistance is an ongoing challenge for a significant number of patients with melanoma, leaving an urgent need for improved therapeutic strategies. In our previous studies, we demonstrated that oxidative stress has an important role in drug resistance, and that wild type isocitrate dehydrogenase 1 (IDH1) is a major source of cytosolic NADPH that maintains redox homeostasis of cells under hypoxic and metabolic stress. This critical enzyme has not been thoroughly evaluated in melanoma. Herein, we explored the expression and function of IDH1 in human melanoma and the role of IDH1 in regulating melanoma metabolism. Methods: We evaluated IDH1 expression in primary and metastatic melanoma using The Cancer Genome Atlas (TCGA). We performed IDH1 knockdown by siRNA oligos, and evaluated cell viability by Trypan blue and PicoGreen assays under normal and nutrient-deprived conditions. Cellular reactive oxygen species (ROS) levels were determined by the DCFDA method. Metastatic activity of these cells was measured by transwell migration assays. In order to determine the impact of IDH1 in cellular metabolism, metabolomics profiling was performed by using LC-MS. These experiments were performed in A375 and SK-Mel 28 cell lines. Results: Analysis of TCGA data from melanoma samples showed IDH1 is highly overexpressed in primary and metastatic melanoma, and higher levels are associated with decreased progression-free survival in patients. Further, we validated that IDH1 is overexpressed in tumors by comparing normal skin tissue versus tumor samples by IHC and protein expression arrays. Silencing IDH1 impaired cell proliferation and migration (vs. control) in a nutrient-deprived microenvironment, but this effect was not seen in nutrient abundance. Metabolomics revealed that inhibiting IDH1 significantly decreased NADPH, α-ketoglutarate (αKG), and GSH levels with a corresponding increase in ROS levels and an impairment of mitochondrial function. In addition, silencing IDH1 sensitized melanoma cells to temozolomide (TMZ), a DNA-alkylating agent, as indicated by a decrease in relative cell survival compared with controls. Conclusion: IDH1 plays a critical role in tumorigenesis and chemoresistance in melanoma. Our data show that IDH1 inhibition sensitizes melanoma cells to TMZ therapy. Our study suggests that IDH1 is a potential target in melanoma as a monotherapy or in combination with existing chemotherapies. Citation Format: Mehrdad Zarei, Ali Vaziri-Gohar, Jonathan Hue, Omid Hajihassani, Erryk Katayama, Hallie Graor, Jordan Winter, Luke Rothermel. IDH1 facilitates melanoma cell survival under metabolic stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3038.

Thioredoxin 1 regulates the pentose phosphate pathway via ATM phosphorylation after experimental subarachnoid hemorrhage in rats

Subarachnoid hemorrhage (SAH), a type of hemorrhagic stroke, is a neurological emergency with high morbidity and mortality. Early brain injury (EBI) after SAH is the leading cause of poor prognosis in SAH patients. TRX system is a NADPH-dependent antioxidant system which is composed of thioredoxin reductase (TRXR), thioredoxin (TRX). The pentose phosphate pathway (PPP), a pathway through which glucose can be metabolized, is a major source of NADPH. Thioredoxin 1 (TRX1) is a member of thioredoxin system mainly located in cytoplasm. Serine/threonine kinases ataxia telangiectasia mutated (ATM) is an important oxidative stress receptor, and TRX1 can regulate ATM phosphorylation and then affect the activity of PPP key enzyme glucose 6-phosphate dehydrogenase (G6PD). However, whether TRX1 is involved in the regulation of PPP pathway after subarachnoid hemorrhage remains unclear. The results showed that after SAH, the level of TRX1 and phosphor-ATM decreased while the level of TRXR1 increased. G6PD protein level remained unchanged but the activity decreased, and the NADPH contents decreased. Overexpression of TRX1 by lentivirus upregulates the level of phosphor-ATM, G6PD activity and NADPH content. TRX1 overexpression improved short-term and long-term neurobehavioral outcomes and alleviated neuronal impairment in rats. Nissl staining showed that upregulation of TRX1 reduced cortical neuron injury. Our study shows that TRX1 participates in the PPP pathway by regulating phosphorylation ATM, which is accomplished by affecting G6PD activity. TRX1 may be an important target for EBI intervention after SAH.

Role of Cytosolic Malic Enzyme in Oleaginicity of High-Lipid-Producing Fungal Strain Mucor circinelloides WJ11.

Mucor circinelloides, an oleaginous filamentous fungus, is gaining popularity due to its ability to synthesize significant amounts of lipids containing γ-linolenic acid (GLA) that have important health benefits. Malic enzyme (ME), which serves as the main source of NADPH in some fungi, has been found to regulate lipid accumulation in oleaginous fungi. In the present study, the role of two cytosolic ME genes, cmalA and cmalB, in the lipid accumulation of the M. circinelloides high-lipid-producing strain WJ11, was evaluated. Strains overexpressing cmalA and cmalB showed a 9.8- and 6.4-fold rise in specific ME activity, respectively, and an elevation of the lipid content by 23.2% and 5.8%, respectively, suggesting that these genes are involved in lipid biosynthesis. Due to increased lipid accumulation, overall GLA content in biomass was observed to be elevated by 11.42% and 16.85% in cmalA and cmalB overexpressing strains, respectively. Our study gives an important insight into different studies exploring the role of the cmalA gene, while we have for the first time investigated the role of the cmalB gene in the M. circinelloides WJ11 strain.

MTOR inhibition downregulates glucose-6-phosphate dehydrogenase and induces ROS-dependent death in T-cell acute lymphoblastic leukemia cells

mTOR activation is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL) and is associated with resistance to glucocorticoid (GC)-based chemotherapy. We previously showed that altering redox homeostasis primes T-ALL cells to GC-induced apoptosis. Here we investigated the connection between the mTOR pathway and redox homeostasis using pharmacological inhibitors and gene silencing.In vitro studies performed on T-ALL cell lines and CG-resistant patient-derived T-ALL xenograft (PDX) cells showed that the mTOR inhibitor everolimus increased reactive oxygen species (ROS) levels, augmented lipid peroxidation, and activated the ROS-controlled transcription factor NRF2. These effects were accompanied by a decrease in the levels of NADPH and of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP), which is a major source of cytosolic NADPH needed for maintaining the cellular ROS-scavenging capacity. The mTOR inhibitor everolimus induced mitochondrial inner membrane depolarization and dose-dependent apoptosis of T-ALL cells, but did not kill normal T-cells. Importantly, the combination of everolimus and the GC dexamethasone had a synergistic effect on killing T-ALL cells. The effects of mTOR inhibition were blunted by ROS scavengers and phenocopied by siRNA-mediated G6PD silencing. In vivo studies of NOD/SCID mice inoculated with refractory T-ALL PDX demonstrated that everolimus overcame dexamethasone resistance in conditions of high tumor burden that mimicked the clinical setting of acute leukemia.These findings provide insight into the crosstalk between mTOR and ROS homeostasis in T-ALL cells and furnish mechanistic evidence to support the combination of glucocorticoids with mTOR inhibitors as a therapeutic avenue for treating refractory T-ALL.

The Non-phosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase GapN Is a Potential New Drug Target in Streptococcus pyogenes.

The strict human pathogen Streptococcus pyogenes causes infections of varying severity, ranging from self-limiting suppurative infections to life-threatening diseases like necrotizing fasciitis or streptococcal toxic shock syndrome. Here, we show that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase GapN is an essential enzyme for S. pyogenes. GapN converts glyceraldehyde 3-phosphate into 3-phosphoglycerate coupled to the reduction of NADP to NADPH. The knock-down of gapN by antisense peptide nucleic acids (asPNA) significantly reduces viable bacterial counts of S. pyogenes laboratory and macrolide-resistant clinical strains in vitro. As S. pyogenes lacks the oxidative part of the pentose phosphate pathway, GapN appears to be the major NADPH source for the bacterium. Accordingly, other streptococci that carry a complete pentose phosphate pathway are not prone to asPNA-based gapN knock-down. Determination of the crystal structure of the S. pyogenes GapN apo-enzyme revealed an unusual cis-peptide in proximity to the catalytic binding site. Furthermore, using a structural modeling approach, we correctly predicted competitive inhibition of S. pyogenes GapN by erythrose 4-phosphate, indicating that our structural model can be used for in silico screening of specific GapN inhibitors. In conclusion, the data provided here reveal that GapN is a potential target for antimicrobial substances that selectively kill S. pyogenes and other streptococci that lack the oxidative part of the pentose phosphate pathway.

Role of mitochondrial inorganic polyphosphate in maintaining mammalian bioenergetics and redox balance, by the regulation of the pentose phosphate pathway

The PPP occurs parallel to glycolysis in the cytosol, and it is the major cellular source of NADPH and Ribose-5-phosphate (R5P). While NADPH plays a critical role in maintaining cellular antioxidant defenses, R5P serves as a substrate for nucleotide synthesis, thereby governing redox homeostasis and anabolic biosynthesis. Dysregulated PPP has been reported in neurodegenerative disorders as a component of the bioenergetic dysregulation and redox imbalance which are present in these pathologies and known to induce elevated oxidative stress and ultimately increased apoptosis.

Dichloroacetate reactivates pyruvate-supported peroxide removal by liver mitochondria and prevents NAFLD aggravation in NAD(P)+ transhydrogenase-null mice consuming a high-fat diet

The mechanisms by which a high-fat diet (HFD) promotes non-alcoholic fatty liver disease (NAFLD) appear to involve liver mitochondrial dysfunction and redox imbalance. The functional loss of the enzyme NAD(P)+ transhydrogenase, a main source of mitochondrial NADPH, results in impaired mitochondrial peroxide removal, pyruvate dehydrogenase inhibition by phosphorylation, and progression of NAFLD in HFD-fed mice. The present study aimed to investigate whether pharmacological reactivation of pyruvate dehydrogenase by dichloroacetate attenuates the mitochondrial redox dysfunction and the development of NAFLD in NAD(P)+ transhydrogenase-null (Nnt−/−) mice fed an HFD (60% of total calories from fat). For this purpose, Nnt−/− mice and their congenic controls (Nnt+/+) were fed chow or an HFD for 20 weeks and received sodium dichloroacetate or NaCl in the final 12 weeks via drinking water. The results showed that HFD reduced the ability of isolated liver mitochondria from Nnt−/− mice to remove peroxide, which was prevented by the dichloroacetate treatment. HFD-fed mice of both Nnt genotypes exhibited increased body and liver mass, as well as a higher content of hepatic triglycerides, but dichloroacetate treatment attenuated these abnormalities only in Nnt−/− mice. Notably, dichloroacetate treatment decreased liver pyruvate dehydrogenase phosphorylation levels and prevented the aggravation of NAFLD in HFD-fed Nnt−/− mice. Conversely, dichloroacetate treatment elicited moderate hepatocyte ballooning in chow-fed mice, suggesting potentially toxic effects. We conclude that the protection against HFD-induced NAFLD by dichloroacetate is associated with its role in reactivating pyruvate dehydrogenase and reestablishing the pyruvate-supported liver mitochondrial capacity to handle peroxide in Nnt−/− mice.

The genome of a nonphotosynthetic diatom provides insights into the metabolic shift to heterotrophy and constraints on the loss of photosynthesis.

Summary Although most of the tens of thousands of diatom species are photoautotrophs, a small number of heterotrophic species no longer photosynthesize. We sequenced the genome of a nonphotosynthetic diatom, Nitzschia Nitz4, to determine how carbon metabolism was altered in the wake of this trophic shift. Nitzschia Nitz4 has retained its plastid and plastid genome, but changes associated with the transition to heterotrophy were cellular‐wide and included losses of photosynthesis‐related genes from the nuclear and plastid genomes, elimination of isoprenoid biosynthesis in the plastid, and remodeling of mitochondrial glycolysis to maximize adenosine triphosphte (ATP) yield. The genome contains a β‐ketoadipate pathway that may allow Nitzschia Nitz4 to metabolize lignin‐derived compounds.Diatom plastids lack an oxidative pentose phosphate pathway (oPPP), leaving photosynthesis as the primary source of NADPH to support essential biosynthetic pathways in the plastid and, by extension, limiting available sources of NADPH in nonphotosynthetic plastids.The genome revealed similarities between nonphotosynthetic diatoms and apicomplexan parasites for provisioning NADPH in their plastids and highlighted the ancestral absence of a plastid oPPP as a potentially important constraint on loss of photosynthesis, a hypothesis supported by the higher frequency of transitions to parasitism or heterotrophy in lineages that have a plastid oPPP.

Mitochondrial NAD(P)+ Transhydrogenase: From Molecular Features to Physiology and Disease.

Significance: Proton-translocating NAD(P)+ transhydrogenase, also known as nicotinamide nucleotide transhydrogenase (NNT), catalyzes a reversible reaction coupling the protonmotive force across the inner mitochondrial membrane and hydride (H-, a proton plus two electrons) transfer between the mitochondrial pools of NAD(H) and NADP(H). The forward NNT reaction is a source of NADPH in the mitochondrial matrix, fueling antioxidant and biosynthetic pathways with reductive potential. Despite the greater emphasis given to the net forward reaction, the reverse NNT reaction that oxidizes NADPH also occurs in physiological and pathological conditions. Recent Advances: NNT (dys)function has been linked to various metabolic pathways and disease phenotypes. Most of these findings have been based on spontaneous loss-of-function Nnt mutations found in the C57BL/6J mouse strain (NntC57BL/6J mutation) and disease-causing Nnt mutations in humans. The present review focuses on recent advances based on the mouse NntC57BL/6J mutation. Critical Issues: Most studies associating NNT function with disease phenotypes have been based on comparisons between different strains of inbred mice (with or without the NntC57BL/6J mutation), which creates uncertainties over the actual contribution of NNT in the context of other potential genetic modifiers. Future Directions: Future research might contribute to understanding the role of NNT in pathological conditions and elucidate how NNT regulates physiological signaling through its forward and reverse reactions. The importance of NNT in redox balance and tumor cell proliferation makes it a potential target of new therapeutic strategies for oxidative-stress-mediated diseases and cancer. Antioxid. Redox Signal. 36, 864-884.

NADPH supply and the contribution of NAD(P)+ transhydrogenase (NNT) to H2O2 balance in skeletal muscle mitochondria

H2O2 is endogenously generated and its removal in the matrix of skeletal muscle mitochondria (SMM) is dependent on NADPH likely provided by NAD(P)+ transhydrogenase (NNT) and isocitrate dehydrogenase (IDH2). Importantly, NNT activity is linked to mitochondrial protonmotive force. Here, we demonstrate the presence of NNT function in detergent-solubilized and intact functional SMM isolated from rats and wild type (Nnt+/+) mice, but not in SMM from congenic mice carrying a mutated NNT gene (Nnt−/-). Further comparisons between SMM from both Nnt mouse genotypes revealed that the NADPH supplied by NNT supports up to 600 pmol/mg/min of H2O2 removal under selected conditions. Surprisingly, SMM from Nnt−/- mice removed exogenous H2O2 at wild-type levels and exhibited a maintained or even decreased net emission of endogenous H2O2 when substrates that support Krebs cycle reactions were present (e.g., pyruvate plus malate or palmitoylcarnitine plus malate). These results may be explained by a compensation for the lack of NNT, since the total activities of concurrent NADP+-reducing enzymes (IDH2, malic enzymes and glutamate dehydrogenase) were ~70% elevated in Nnt−/- mice. Importantly, respiratory rates were similar between SMM from both Nnt genotypes despite differing NNT contributions to H2O2 removal and their implications for an evolving concept in the literature are discussed. We concluded that NNT is capable of meaningfully sustaining NADPH-dependent H2O2 removal in intact SMM. Nonetheless, if the available substrates favor non-NNT sources of NADPH, the H2O2 removal by SMM is maintained in Nnt−/- mice SMM.

Impact of activation of neotrehalosadiamine/kanosamine biosynthetic pathway on the metabolism of Bacillus subtilis.

The pentose phosphate (PP) pathway is one of the major sources of cellular NADPH. A B. subtilis zwf mutant that lacks glucose-6-phosphate dehydrogenase (the enzyme that catalyzes the first step of the PP pathway) showed inoculum-dose-dependent growth. This growth defect was suppressed by glcP disruption, which causes the upregulation of an autoinducer neotrehalosadiamine (NTD)/kanosamine biosynthetic pathway. A metabolome analysis showed that the stimulation of NTD/kanosamine biosynthesis caused significant accumulation of TCA cycle intermediates and NADPH. Because the major malic enzyme YtsJ concomitantly generates NADPH through malate-to-pyruvate conversion, de novo NTD/kanosamine biosynthesis can result in an increase in the intracellular NADPH pool via the accumulation of malate. In fact, a zwf mutant grew in malate-supplemented medium. Artificial induction of glcP in the zwf mutant caused a reduction in the intracellular NADPH pool. Moreover, the correlation between the expression level of the NTD/kanosamine biosynthesis operon ntdABC and the intracellular NADPH pool was confirmed. Our results suggest that NTD/kanosamine has the potential to modulate the carbon-energy metabolism through an autoinduction mechanism.ImportanceAutoinducers enable bacteria to sense cell density and to coordinate collective behavior. NTD/kanosamine is an autoinducer produced by B. subtilis and several close relatives, although its physiological function remains unknown. The most important finding of this study was the significance of de novo NTD/kanosamine biosynthesis in the modulation of the central carbon metabolism in B. subtilis We showed that NTD/kanosamine biosynthesis caused an increase in the NADPH pool via the accumulation of TCA cycle intermediates. These results suggest a possible role for NTD/kanosamine in the carbon-energy metabolism. As Bacillus species are widely used for the industrial production of various useful enzymes and compounds, the NTD/kanosamine biosynthetic pathway might be utilized to control metabolic pathways in these industrial strains.