Lack of NAD(P)+ transhydrogenase activity in patients with primary adrenal insufficiency due to NNT variants.

BACKGROUND: Pathogenic variants in the nicotinamide nucleotide transhydrogenase gene (NNT) are a rare cause of primary adrenal insufficiency (PAI), as well as functional impairment of the gonads. OBJECTIVE: Despite the description of different homozygous and compound heterozygous NNT variants in PAI patients, the extent to which the function and expression of the mature protein are compromised remains to be clarified. DESIGN: The activity and expression of mitochondrial NAD(P)+ transhydrogenase (NNT) were analyzed in blood samples obtained from patients diagnosed with PAI due to genetically confirmed variants of the NNT gene (n = 5), heterozygous carriers as their parents (n = 8), and healthy controls (n = 26). METHODS: NNT activity was assessed by a reverse reaction assay standardized for digitonin-permeabilized peripheral blood mononuclear cells (PBMCs). The enzymatic assay was validated in PBMC samples from a mouse model of NNT absence. Additionally, the PBMC samples were evaluated for NNT expression by western blotting and reverse transcription quantitative polymerase chain reaction and for mitochondrial oxygen consumption. RESULTS: NNT activity was undetectable (<4% of that of healthy controls) in PBMC samples from patients, independent of the pathogenic genetic variant. In patients' parents, NNT activity was approximately half that of the healthy controls. Mature NNT protein expression was lower in patients than in the control groups, while mRNA levels varied widely among genotypes. Moreover, pathogenic NNT variants did not impair mitochondrial bioenergetic function in PBMCs. CONCLUSIONS: The manifestation of PAI in NNT-mutated patients is associated with a complete lack of NNT activity. Evaluation of NNT activity can be useful to characterize disease-causing NNT variants.

Coenzyme Q10 and nicotinamide nucleotide transhydrogenase: Sentinels for mitochondrial hydrogen peroxide signaling.

Mitochondria use hydrogen peroxide (H2O2) as a mitokine for cell communication. H2O2 output for signaling depends on its rate of production and degradation, both of which are strongly affected by the redox state of the coenzyme Q10 (CoQ) pool and NADPH availability. Here, we propose the CoQ pool and nicotinamide nucleotide transhydrogenase (NNT) have evolved to be central modalities for mitochondrial H2O2 signaling. Both factors play opposing yet equally important roles in dictating H2O2 availability because they are connected to one another by two central parameters in bioenergetics: electron supply and Δp. The CoQ pool is the central point of convergence for electrons from various dehydrogenases and the electron transport chain (ETC). The increase in Δp creates a significant amount of protonic backpressure on mitochondria to promote H2O2 genesis through CoQ pool reduction. These same factors also drive the activity of NNT, which uses electrons and the Δp to eliminate H2O2. In this way, electron supply and the magnitude of the Δp manifests as a redox connection between the two sentinels, CoQ and NNT, which serve as opposing yet equally important forces required for budgeting H2O2. Taken together, CoQ and NNT are sentinels linked through mitochondrial bioenergetics to manage H2O2 availability for interorganelle and intercellular redox signaling.

LncRNA NNT-AS1/hsa-miR-485-5p/HSP90 axis in-silico and clinical prospect correlated-to histologic grades-based CRC stratification: A step toward ncRNA Precision.

The oncogenic effects of long non-coding RNA (lncRNA) Nicotinamide Nucleotide Transhydrogenase-antisense RNA1 (NNT-AS1) role in colorectal cancer (CRC) hasn't been sufficiently inspected in relation to the Homo sapiens (hsa)-microRNA (miR)- 485-5p/ heat shock protein 90 (HSP90) axis, clinically. qRT-PCR was performed to detect lncRNA NNT-AS1 and hsa-miR-485-5p expression levels in 60 Egyptian patients' sera. HSP90 serum level was quantified using Enzyme-linked immunosorbent assay (ELISA). The relative expression level of the studied non-coding RNAs as well as the HSP90 ELISA concentration were correlated with patients clinicopathological characteristics and correlated to each other. The axis diagnostic utility in comparison with carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) tumor markers (TMs) was studied by receiver operating characteristic (ROC) curve analysis. The relative lncRNA NNT-AS1 expression level fold change 56.7 (13.5-112) and HSP90 protein ELISA level 6.68 (5.14-8.77) (ng/mL) were elevated, while, for hsa-miR-485-5p 0.0474 (0.0236-0.135) expression fold change was repressed in CRC Egyptian patients' cohort sera, being compared to 28 apparently healthy control subjects. LncRNA NNT-AS1 specificity is 96.4% and a sensitivity of 91.7%, hsa-miR-485-5p showed 96.4% specificity, 90% sensitivity, and for HSP90 89.3%, 70% specificity and sensitivity, respectively. Those specificities and sensitivities were superior to the classical CRC TMs. A significant negative correlation was found between hsa-miR-485-5p with lncRNA NNT-AS1 (r = -0.933) expression fold change or with HSP90 protein blood level (r = -0.997), but, significant positive correlation was there between lncRNA NNT-AS1 and HSP90 (r = 0.927). LncRNA NNT-AS1/hsa-miR-485-5p/HSP90 axis could be a prospect for CRC development as well as diagnosis. Being correlated and related to CRC histologic grades 1-3, therefore, lncRNA NNT-AS1/hsa-miR-485-5p/HSP90 axis (not individually) expression approved clinically and in silico, could aid treatment precision.

IL-1ß-associated NNT acetylation orchestrates iron-sulfur cluster maintenance and cancer immunotherapy resistance.

Interleukin-1ß (IL-1ß) is a key protein in inflammation and contributes to tumor progression. However, the role of IL-1ß in cancer is ambiguous or even contradictory. Here, we found that upon IL-1ß stimulation, nicotinamide nucleotide transhydrogenase (NNT) in cancer cells is acetylated at lysine (K) 1042 (NNT K1042ac) and thereby induces the mitochondrial translocation of p300/CBP-associated factor (PCAF). This acetylation enhances NNT activity by increasing the binding affinity of NNT for NADP+ and therefore boosts NADPH production, which subsequently sustains sufficient iron-sulfur cluster maintenance and protects tumor cells from ferroptosis. Abrogating NNT K1042ac dramatically attenuates IL-1ß-promoted tumor immune evasion and synergizes with PD-1 blockade. In addition, NNT K1042ac is associated with IL-1ß expression and the prognosis of human gastric cancer. Our findings demonstrate a mechanism of IL-1ß-promoted tumor immune evasion, implicating the therapeutic potential of disrupting the link between IL-1ß and tumor cells by inhibiting NNT acetylation.

CRISPR-based DNA methylation editing of NNT rescues the cisplatin resistance of lung cancer cells by reducing autophagy.

Cisplatin is recommended as a first-line chemotherapeutic agent against advanced non-small cell lung cancer (NSCLC), but acquired resistance substantially limits its clinical efficacy. Recently, DNA methylation has been identified as an essential contributor to chemoresistance. However, the precise DNA methylation regulatory mechanism of cisplatin resistance remains unclear. Here, we found that nicotinamide nucleotide transhydrogenase (NNT) was silenced by DNA hypermethylation in cisplatin resistance A549 (A549/DDP) cells. Also, the DNA hypermethylation of NNT was positively correlated to poor prognosis in NSCLC patients. Overexpression of NNT in A549/DDP cells could reduce their cisplatin resistance, and also suppressed their tumor malignancy such as cell proliferation and clone formation. However, NNT enhanced sensitivity of A549/DDP cells to cisplatin had little to do with its function in mediating NADPH and ROS level, but was mainly because NNT could inhibit protective autophagy in A549/DDP cells. Further investigation revealed that NNT could decrease NAD+ level, thereby inactivate SIRT1 and block the autophagy pathway, while re-activation of SIRT1 through NAD+ precursor supplementation could antagonize this effect. In addition, targeted demethylation of NNT CpG island via CRISPR/dCas9-Tet1 system significantly reduced its DNA methylation level and inhibited the autophagy and cisplatin resistance in A549/DDP cells. Thus, our study found a novel chemoresistance target gene NNT, which played important roles in cisplatin resistance of lung cancer cells. Our findings also suggested that CRISPR-based DNA methylation editing of NNT could be a potential therapeutics method in cisplatin resistance of lung cancer.

Nicotinamide Nucleotide Transhydrogenase Is Essential for Adrenal Steroidogenesis: Clinical and In Vitro Lessons.

CONTEXT: Nicotinamide nucleotide transhydrogenase (NNT) acts as an antioxidant defense mechanism. NNT mutations cause familial glucocorticoid deficiency (FGD). How impaired oxidative stress disrupts adrenal steroidogenesis remains poorly understood. OBJECTIVE: To ascertain the role played by NNT in adrenal steroidogenesis. METHODS: The genotype-phenotype association of a novel pathogenic NNT variant was evaluated in a boy with FGD. Under basal and oxidative stress (OS) induced conditions, transient cell cultures of the patient's and controls' wild-type (WT) mononuclear blood cells were used to evaluate antioxidant mechanisms and mitochondrial parameters (reactive oxygen species [ROS] production, reduced glutathione [GSH], and mitochondrial mass). Using CRISPR/Cas9, a stable NNT gene knockdown model was built in H295R adrenocortical carcinoma cells to determine the role played by NNT in mitochondrial parameters and steroidogenesis. NNT immunohistochemistry was assessed in fetal and postnatal human adrenals. RESULTS: The homozygous NNT p.G866D variant segregated with the FGD phenotype. Under basal and OS conditions, p.G866D homozygous mononuclear blood cells exhibited increased ROS production, and decreased GSH levels and mitochondrial mass than WT NNT cells. In line H295R, NNT knocked down cells presented impaired NNT protein expression, increased ROS production, decreased the mitochondrial mass, as well as the size and the density of cholesterol lipid droplets. NNT knockdown affected steroidogenic enzyme expression, impairing cortisol and aldosterone secretion. In human adrenals, NNT is abundantly expressed in the transition fetal zone and in zona fasciculata. CONCLUSION: Together, these studies demonstrate the essential role of NNT in adrenal redox homeostasis and steroidogenesis.

Metabolic differences and differentially expressed genes between C57BL/6J and C57BL/6N mice substrains.

C57BL/6J (B6J) and C57BL/6N (B6N) mice are the most frequently used substrains in C57BL/6 (B6) inbred mice, serving as physiological models for in vivo studies and as background strains to build transgenic mice. However, the differences in metabolic phenotypes between B6J and B6N mice are not coherent, and genotypic differences in metabolically important tissues have not been well studied. The phenotypic differences between B6J and B6N substrains have often been attributed to the role of the nicotinamide nucleotide transhydrogenase (Nnt) gene, whereby B6J has a spontaneous missense mutation of Nnt. Nevertheless, phenotypic differences between the two cannot be explained by Nnt mutations alone, especially in metabolic traits. Therefore, we aimed to investigate the genetic cause of the phenotypic differences between B6J and B6N mice. Determining consistent genetic differences across multiple tissues involved in metabolic traits such as subcutaneous and visceral white adipose tissues, brown adipose tissue, skeletal muscle, liver, hypothalamus, and hippocampus, may help explain phenotypic differences in metabolism between the two substrains. We report candidate genes along with comparative data on body weight, tissue weight, blood components involved in metabolism, and energy balance of B6J and B6N mice. Insulin degrading enzyme, adenylosuccinate synthase 2, and ectonucleotide triphosphate diphosphohydrolase 4 were highly expressed in B6J mice compared with those in B6N mice, and Nnt, WD repeat and FYVE domain containing 1, and dynein light chain Tctex-type 1 were less expressed in B6J mice compared with those in B6N mice in all seven tissues. Considering the extremely wide use of both substrains and their critical importance in generating transgenic and knock-out models, these findings guide future research across several interrelated fields.

Insights on the potential oncogenic impact of long non-coding RNA nicotinamide nucleotide transhydrogenase antisense RNA 1 in different cancer types; integrating pathway(s) and clinical outcome(s) association.

Long non-coding RNAs (lncRNAs) are becoming more prevalent in the cancer field arena, with functional roles in both oncogenic and onco-suppressive pathways. Despite their widespread aberrant expression in a range of human malignancies, the biological activities of the ncRNAs majority are unknown. All showed the involvement of the lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1). Since NNT-AS1 influences cellular proliferation, invasion, migration, apoptosis, and metastasis, this lncRNA appears to be linked to deregulating the normal cellular processes driving malignancy. This was observed in breast cancer (BC), gastric cancer (GC), colorectal cancer (CRC), epithelial ovarian cancer (EOC), and hepatocellular carcinoma (HCC). The current narrative non-systematic review will discuss "the significance of lncRNAs in cancer", as well as "lncRNAs future potential application(s) as diagnostic or predictive biomarkers", therefore, comprising an opportunity as treatment target(s). The review will have a special emphasis on lncRNA NNT-AS1.

Long-Term Follow-Up of Three Family Members with a Novel NNT Pathogenic Variant Causing Primary Adrenal Insufficiency.

Nicotinamide nucleotide transhydrogenase (NNT) deficiency causes primary adrenal insufficiency (PAI) and possibly some extra-adrenal manifestations. A limited number of these patients were previously described. We present the clinical and genetic characteristics of three family members with a biallelic novel pathogenic variant in the NNT gene. The patients were followed until the ages of 21.6, 20.2, and 4.2 years. PAI was diagnosed in the eldest two brothers after an Addisonian crisis and the third was diagnosed at the age of 4.5 months in the asymptomatic stage due to the genetic screening of family members. Whole exome sequencing with a targeted interpretation of variants in genes related to PAI was performed in all the patients. The urinary steroid metabolome was determined by gas chromatography-mass spectrometry in the asymptomatic patient. The three patients, who were homozygous for c.1575dup in the NNT gene, developed isolated glucocorticoid deficiency. The urinary steroid metabolome showed normal excretion of cortisol metabolites. The adolescent patients had slow pubertal progression with low-normal testicular volume, while testicular endocrine function was normal. Bone mineral density was in the range for osteopenia in both grown-up siblings. Echocardiography revealed no structural or functional heart abnormalities. This article is among the first with a comprehensive and chronologically-detailed description of patients with NNT deficiency.

Regulation of immune cell function by nicotinamide nucleotide transhydrogenase.

Redox homeostasis is elemental for the normal physiology of all cell types. Cells use multiple mechanisms to tightly regulate the redox balance. The onset and progression of many metabolic and aging-associated diseases occur due to the dysregulation of redox homeostasis. Thus, it is critical to identify and therapeutically target mechanisms that precipitate abnormalities in redox balance. Reactive oxygen species (ROS) produced within the immune cells regulate homeostasis, hyperimmune and hypoimmune cell responsiveness, apoptosis, immune response to pathogens, and tumor immunity. Immune cells have both cytosolic and organelle-specific redox regulatory systems to maintain appropriate levels of ROS. Nicotinamide nucleotide transhydrogenase (NNT) is an essential mitochondrial redox regulatory protein. Dysregulation of NNT function prevents immune cells from mounting an adequate immune response to pathogens, promotes a chronic inflammatory state associated with aging and metabolic diseases, and initiates conditions related to a dysregulated immune system such as autoimmunity. Although many studies have reported on NNT in different cell types, including cancer cells, relatively few studies have explored NNT in immune cells. This review provides an overview of NNT and focuses on the current knowledge of NNT in the immune cells.

Widespread discrepancy in Nnt genotypes and genetic backgrounds complicates granzyme A and other knockout mouse studies.

Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma-/- mouse studies having informed our understanding of GZMA's physiological function. We show herein that Gzma-/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma-/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J GzmaS211A mouse provided the first insights into GZMA's bioactivity free of background issues, with circulating proteolytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.

Nicotinamide nucleotide transhydrogenase mutation analysis in Chinese patients with thyroid dysgenesis.

Thyroid dysgenesis (TD) accounts for 80% cases of congenital hypothyroidism, which is the most common neonatal disorder. Until now, the gene mutations have been reported associated with TD can only account for 5% cases, suggesting the genetic heterogeneity of the pathology. Nicotinamide nucleotide transhydrogenase (NNT) plays a crucial role in regulating redox homeostasis, patients carrying NNT mutations have been described with a clinical phenotype of hypothyroidism. As TD risk is increased in the context of several syndromes and redox homeostasis is vital for thyroid development and function, NNT might be a candidate gene involved in syndromic TD. Therefore, we performed target sequencing (TS) in 289 TD patients for causative mutations in NNT and conducted functional analysis of the gene mutations. TS and Sanger sequence were used to screen the novel mutations. For functional analysis, we performed western blot, measurement of NADPH/NADPtotal and H2 O2 generation, cell proliferation, and wounding healing assay. As a result, three presumably pathogenic mutations (c.811G > A, p.Ala271Ser; c.2078G > A, p.Arg693His; and c.2581G > A, p.Val861Met) in NNT had been identified. Our results showed the damaging effect of NNT mutations on stability and catalytic activity of proteins and redox balance of cells. In conclusion, our findings provided novel insights into the role of the NNT isotype in thyroid physiopathology and broaden the spectrum of pathogenic genes associated with TD. However, the pathogenic mechanism of NNT in TD is still need to be investigated in further study.

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.

Identification and Characterization of a Novel Soluble Pyridine Nucleotide Transhydrogenase from Streptomyces avermitilis.

Soluble pyridine nucleotide transhydrogenase (STH) transfers hydride between NADH and NADPH to maintain redox balance. In the present study, the sth gene from Gram-positive bacterium Streptomyces avermitilis (SaSTH) was expressed in Escherichia coli, and the recombinant STH protein was purified to homogeneity. Activity assays indicated that SaSTH was able to catalyze transhydrogenase reactions by using NADH or NADPH as reductants and thio-NAD+ as an oxidant. The apparent Km value for NADPH (74.5 µM) was lower than that for NADH (104.0 µM) and the apparent kcat/Km for NADPH (2704.7 mM-1 s-1) was higher than that for NADH (1129.8 mM-1 s-1). SaSTH showed optimal activity at 25 °C and at a pH of 6.2. Heat-inactivation studies revealed that SaSTH remained stable below 55 °C and that approximately 50% activity was preserved at 57 °C for 20 min. Analyses also showed that SaSTH activity was inhibited by divalent ions, particularly Co2+, Ni2+, and Zn2+. In addition, the transhydrogenase activity of SaSTH was inhibited by ATP and strongly stimulated by ADP and AMP. In summary, we characterized a recombinant enzyme exhibiting STH activity from Gram-positive bacteria for the first time. Our findings provide new options for cofactor engineering and industrial biocatalytic processes.

NADPH-to-NADH conversion by mitochondrial transhydrogenase is indispensable for sustaining anaerobic metabolism in Euglena gracilis.

Euglena gracilis produces ATP in the anaerobic mitochondria with concomitant wax ester formation, and NADH is essential for ATP formation and fatty acid synthesis in the mitochondria. This study demonstrated that mitochondrial cofactor conversion by nicotinamide nucleotide transhydrogenase (NNT), converting NADPH/NAD+ to NADP+ /NADH, is indispensable for sustaining anaerobic metabolism. Silencing of NNT genes significantly decreased wax ester production and cellular viability during anaerobiosis but had no such marked effects under aerobic conditions. An analogous phenotype was observed in the silencing of the gene encoding a mitochondrial NADP+ -dependent malic enzyme. These results suggest that the reducing equivalents produced in glycolysis are shuttled to the mitochondria as malate, where cytosolic NAD+ regeneration is coupled with mitochondrial NADPH generation.

Serum long non­coding RNA NNT­AS1 protected by exosome is a potential biomarker and functions as an oncogene via the miR­496/RAP2C axis in colorectal cancer.

Increasing evidence has indicated that long non­coding RNAs (lncRNAs) serve an essential role in carcinogenesis and cancer development. It has been reported that lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT­AS1) serves a crucial role in several types of cancer. However, the clinical significance of circulating NNT­AS1 expression in colorectal cancer (CRC) remains to be elucidated. The current study aimed to investigate the potential role of NNT­AS1 and the clinical significance of its serum expression levels in patients with CRC. The expression of NNT­AS1 was measured in 40 pairs of tumor and adjacent normal tissues from patients with CRC via reverse transcription­quantitative PCR. The serum expression levels of NNT­AS1 were assayed in an independent cohort of healthy controls and patients with CRC. The levels of NNT­AS1 were also compared between paired preoperative and postoperative serum samples. In addition, the presence of exosomal NNT­AS1 in serum was explored. Furthermore, the biological roles of NNT­AS1 were investigated in CRC cells in vitro. The expression of NNT­AS1 was significantly upregulated in tumor tissues compared with adjacent normal tissues (P<0.05). A higher level of NNT­AS1 was associated with an advanced CRC stage. The serum levels of NNT­AS1 were significantly upregulated in patients with CRC compared with healthy subjects (P<0.05). Furthermore, the NNT­AS1 levels were significantly decreased in postoperative samples compared with preoperative samples (P<0.01). In addition, it was also identified that NNT­AS1 was upregulated in CRC exosomes (P<0.01), whereas no significant difference was observed in NNT­AS1 levels between serum and exosomes. Silencing of NNT­AS1 inhibited the proliferation, migration and invasion of CRC cells. It was also identified that NNT­AS1 exerted its effects via regulation of the microRNA­496/Ras­related protein Rap­2c axis. The present study demonstrated that circulating NNT­AS1, which may be protected by exosomes, could be a novel potential biomarker and therapeutic target in CRC.

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.

Silencing long non-coding RNAs nicotinamide nucleotide transhydrogenase antisense RNA 1 inhibited papillary thyroid cancer cell proliferation, migration and invasion and promoted apoptosis via targeting miR-199a-5p.

The increasing incidence of papillary thyroid cancer (PTC) has attracted many researchers to investigate the mechanism underlying PTC progression. This study explored the growth and apoptosis of PTC cells based on an lncRNA regulatory mechanism. The expression of nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) in PTC cell lines and PTC tissues was analyzed by qRT-PCR. The mutual binding site between NNT-AS1 and miR-199a-5p was predicted by starBase and confirmed by dual-luciferase reporter assay. The correlation between NNT-AS1 and miR-199a-5p was shown by Pearson correlation test. The viability, clone formation, migration, invasion and apoptosis of TPC-1 and IHH-4 cells were examined by CCK-8, colony formation, wound-healing, transwell, and flow cytometry assays, respectively. The expressions of Bax, cleaved Caspase-3, Bcl-2, E-Cadherin, N-Cadherin and SNAIL in TPC-1 and IHH-4 cells were determined by Western blot or qRT-PCR. NNT-AS1 expression was upregulated in PTC cells and tissues. In TPC-1 cells, silencing NNT-AS1 inhibited viability, clone formation, migration, and invasion as well as the expressions of N-Cadherin, SNAIL and Bcl-2, but promoted the expressions of E-Cadherin, Bax, and cleaved caspase-3. The effects of NNT-AS1 overexpression on IHH-4 cells were opposite to those of silencing NNT-AS1. In PTC tissues, miR-199a-5p was low-expressed and targeted by NNT-AS1, and it was negatively correlated with NNT-AS1. MiR-199a-5p inhibitor promoted TPC-1 cell progression, but miR-199a-5p mimic inhibited IHH-4 cell progression. NNT-AS1 and miR-199a-5p exerted opposite effects on PTC cells. Silencing NNT-AS1 inhibited PTC cell proliferation, migration and invasion, but promoted apoptosis via upregulation of miR-199a-5p.

Mylk3 null C57BL/6N mice develop cardiomyopathy, whereas Nnt null C57BL/6J mice do not.

The C57BL/6J and C57BL/6N mice have well-documented phenotypic and genotypic differences, including the infamous nicotinamide nucleotide transhydrogenase (Nnt) null mutation in the C57BL/6J substrain, which has been linked to cardiovascular traits in mice and cardiomyopathy in humans. To assess whether Nnt loss alone causes a cardiovascular phenotype, we investigated the C57BL/6N, C57BL/6J mice and a C57BL/6J-BAC transgenic rescuing NNT expression, at 3, 12, and 18 mo. We identified a modest dilated cardiomyopathy in the C57BL/6N mice, absent in the two B6J substrains. Immunofluorescent staining of cardiomyocytes revealed eccentric hypertrophy in these mice, with defects in sarcomere organisation. RNAseq analysis identified differential expression of a number of cardiac remodelling genes commonly associated with cardiac disease segregating with the phenotype. Variant calling from RNAseq data identified a myosin light chain kinase 3 (Mylk3) mutation in C57BL/6N mice, which abolishes MYLK3 protein expression. These results indicate the C57BL/6J Nnt-null mice do not develop cardiomyopathy; however, we identified a null mutation in Mylk3 as a credible cause of the cardiomyopathy phenotype in the C57BL/6N.

NAD(P) transhydrogenase has vital non-mitochondrial functions in malaria parasite transmission.

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form (NADP) are vital for cell function in all organisms and form cofactors to a host of enzymes in catabolic and anabolic processes. NAD(P) transhydrogenases (NTHs) catalyse hydride ion transfer between NAD(H) and NADP(H). Membrane-bound NTH isoforms reside in the cytoplasmic membrane of bacteria, and the inner membrane of mitochondria in metazoans, where they generate NADPH. Here, we show that malaria parasites encode a single membrane-bound NTH that localises to the crystalloid, an organelle required for sporozoite transmission from mosquitos to vertebrates. We demonstrate that NTH has an essential structural role in crystalloid biogenesis, whilst its enzymatic activity is required for sporozoite development. This pinpoints an essential function in sporogony to the activity of a single crystalloid protein. Its additional presence in the apicoplast of sporozoites identifies NTH as a likely supplier of NADPH for this organelle during liver infection. Our findings reveal that Plasmodium species have co-opted NTH to a variety of non-mitochondrial organelles to provide a critical source of NADPH reducing power.