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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.

Mutations in the mitochondrial genome (mtDNA) are ubiquitous in humans and can lead to a broad spectrum of disorders. However, due to the presence of multiple mtDNA molecules in the cell, co-existence of mutant and wild-type mtDNAs (termed heteroplasmy) can mask disease phenotype unless a threshold of mutant molecules is reached. Importantly, the mutant mtDNA level can change across lifespan as mtDNA segregates in an allele- and cell-specific fashion, potentially leading to disease. Segregation of mtDNA is mainly evident in hepatic cells, resulting in an age-dependent increase of mtDNA variants, including non-synonymous potentially deleterious mutations. Here we modeled mtDNA segregation using a well-established heteroplasmic mouse line with mtDNA of NZB/BINJ and C57BL/6N origin on a C57BL/6N nuclear background. This mouse line showed a pronounced age-dependent NZB mtDNA accumulation in the liver, thus leading to enhanced respiration capacity per mtDNA molecule. Remarkably, liver-specific atg7 (autophagy related 7) knockout abolished NZB mtDNA accumulat ion, resulting in close-to-neutral mtDNA segregation through development into adulthood. prkn (parkin RBR E3 ubiquitin protein ligase) knockout also partially prevented NZB mtDNA accumulation in the liver, but to a lesser extent. Hence, we propose that age-related liver mtDNA segregation is a consequence of macroautophagic clearance of the less-fit mtDNA. Considering that NZB/BINJ and C57BL/6N mtDNAs have a level of divergence comparable to that between human Eurasian and African mtDNAs, these findings have potential implications for humans, including the safe use of mitochondrial replacement therapy.Abbreviations: Apob: apolipoprotein B; Atg1: autophagy-related 1; Atg7: autophagy related 7; Atp5a1: ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1; BL6: C57BL/6N mouse strain; BNIP3: BCL2/adenovirus E1B interacting protein 3; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mt-Atp8: mitochondrially encoded ATP synthase 8; MT-CO1: mitochondrially encoded cytochrome c oxidase I; MT-CO2: mitochondrially encoded cytochrome c oxidase II; mt-Co3: mitochondrially encoded cytochrome c oxidase III; mt-Cytb: mitochondrially encoded cytochrome b; mtDNA: mitochondrial DNA; MUL1: mitochondrial ubiquitin ligase activator of NFKB 1; nDNA: nuclear DNA; Ndufa9: NADH:ubiquinone oxireductase subunit A9; NDUFB8: NADH:ubiquinone oxireductase subunit B8; Nnt: nicotinamide nucleotide transhydrogenase; NZB: NZB/BINJ mouse strain; OXPHOS: oxidative phosphorylation; PINK1: PTEN induced putative kinase 1; Polg2: polymerase (DNA directed), gamma 2, accessory subunit; Ppara: peroxisome proliferator activated receptor alpha; Ppia: peptidylprolyl isomerase A; Prkn: parkin RBR E3 ubiquitin protein ligase; P10: post-natal day 10; P21: post-natal day 21; P100: post-natal day 100; qPCR: quantitative polymerase chain reaction; Rpl19: ribosomal protein L19; Rps18: ribosomal protein S18; SD: standard deviation; SEM: standard error of the mean; SDHB: succinate dehydrogenase complex, subunit B, iron sulfur (Ip); SQSTM1: sequestosome 1; Ssbp1: single-stranded DNA binding protein 1; TFAM: transcription factor A, mitochondrial; Tfb1m: transcription factor B1, mitochondrial; Tfb2m: transcription factor B2, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; UQCRC2: ubiquinol cytochrome c reductase core protein 2; WT: wild-type.

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.

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

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.

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

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.

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.

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.

Protein engineering has been used to enhance the activities, selectivities, and stabilities of enzymes. Frequently tradeoffs are observed, where improvements in some features can come at the expense of others. Nature uses modular assembly of active sites for complex, multi-step reactions, and natural "swing arm" mechanisms have evolved to transfer intermediates between active sites. Biomimetic polyethylene glycol (PEG) swing arms modified with NAD(H) have been explored to introduce synthetic swing arms into fused oxidoreductases. Here we report that increasing NAD(H)-PEG swing arms can improve the activity of synthetic formate:malate oxidoreductases as well as the thermal and operational stabilities of the biocatalysts. The modular assembly approach enables the K M values of new enzymes to be predictable, based on the parental enzymes. We describe four unique synthetic transhydrogenases that have no native homologs, and this platform could be easily extended for the predictive design of additional synthetic cofactor-independent transhydrogenases.

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.

Increasing evidence has indicated that long non-coding RNAs (lncRNAs) serve an essential role in carcinogenesis and cancer development. It has been reported that lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) serves a crucial role in several types of cancer. However, the clinical significance of circulating NNT-AS1 expression in colorectal cancer (CRC) remains to be elucidated. The current study aimed to investigate the potential role of NNT-AS1 and the clinical significance of its serum expression levels in patients with CRC. The expression of NNT-AS1 was measured in 40 pairs of tumor and adjacent normal tissues from patients with CRC via reverse transcription-quantitative PCR. The serum expression levels of NNT-AS1 were assayed in an independent cohort of healthy controls and patients with CRC. The levels of NNT-AS1 were also compared between paired preoperative and postoperative serum samples. In addition, the presence of exosomal NNT-AS1 in serum was explored. Furthermore, the biological roles of NNT-AS1 were investigated in CRC cells in vitro. The expression of NNT-AS1 was significantly upregulated in tumor tissues compared with adjacent normal tissues (P

Lactobacillus buchneri and Oenococcus oeni are two unique ethanol-tolerant Gram-positive bacteria species. Genome comparison analyses revealed that L. buchneri and O. oeni possess a pntAB locus that was absent in almost all other lactic acid bacteria (LAB) genomes. Our hypothesis is that the pntAB locus contributes to the ethanol tolerance trait of these two distinct ethanol-tolerant organisms. The pntAB locus, consisting of the pntA and pntB genes, codes for NADP(H) transhydrogenase subunits. This membrane-bound transhydrogenase catalyzes the reduction of NADP+ and is known as an important enzyme in maintaining cellular redox balance. In this study, the transhydrogenase operon from L. buchneri NRRL B-30929 and O. oeni PSU-1 were cloned and analyzed. The LbpntB shared 71.0% identity with the O. oeni (OopntB). The entire pntAB locus was expressed in Lactococcus lactis ssp . lactis IL1403 resulting in an increased tolerance to ethanol (6%), butanol (1.8%) and isopropanol (1.8%) when compared to the control strain. However, the recombinant E. coli cells carrying the entire pntAB locus did not show any improved ethanol tolerance. Independent expression of OopntB and LbpntB in recombinant E. coli BL21(DE3)pLysS host demonstrated higher tolerance to ethanol when compared with a control E. coli BL21(DE3)pLysS strain carrying pET28b vector. Ethanol tolerance comparison of E. coli strains carrying LbpntB and OopntB showed that LbpntB conferred higher ethanol tolerance (4.5%) and resulted in greater biomass, while the OopntB conferred lower ethanol tolerance (4.0%) resulted lower biomass. Therefore, the pntB gene from L. buchneri is a better choice in generating higher ethanol tolerance. This is the first study to uncover the role of pntAB locus on ethanol tolerance.

Corynebacterium glutamicum SNK 118 was metabolically engineered with improved l-arginine titer. Considering the crucial role of NADPH level in l-arginine production, pntAB (membrane-bound transhydrogenase) and ppnK (NAD+ kinase) were co-expressed to increase the intracellular NADPH pool. Expression of pntAB exhibited significant effects on NADPH supply and l-arginine synthesis. Furthermore, argR and farR, encoding arginine repressor ArgR and transcriptional regulator FarR, respectively, were removed from the genome of C. glutamicum. The competitive branch pathway gene ldh was also deleted. Eventually, an engineered C. glutamicum JML07 was obtained for l-arginine production. Fed-batch fermentation in 5-L bioreactor employing strain JML07 allowed production of 67.01 g L−1 l-arginine with productivity of 0.89 g L−1 h−1 and yield of 0.35 g g−1 glucose. This study provides a productive l-arginine fermentation strain and an effective cofactor manipulating strategy for promoting the biosynthesis of NADPH-dependent metabolites.

Nicotinamide nucleotide transhydrogenase (NNT) is involved in decreasing melanogenesis through tyrosinase degradation induced by cellular redox changes. Nicotinamide is a component of coenzymes, such as NAD+, NADH, NADP+, and NADPH, and its levels are modulated by NNT. Vitamin C and polydeoxyribonucleotide (PDRN) are also known to decrease skin pigmentation. We evaluated whether a mixture of nicotinamide, vitamin C, and PDRN (NVP-mix) decreased melanogenesis by modulating mitochondrial oxidative stress and NNT expression in UV-B-irradiated animals and in an in vitro model of melanocytes treated with conditioned media (CM) from UV-B-irradiated keratinocytes. The expression of NNT, GSH/GSSG, and NADPH/NADP+ in UV-B-irradiated animal skin was significantly decreased by UV-B radiation but increased by NVP-mix treatment. The expression of NNT, GSH/GSSG, and NADPH/NADP+ ratios decreased in melanocytes after CM treatment, although they increased after NVP-mix administration. In NNT-silenced melanocytes, the GSH/GSSG and NADPH/NADP+ ratios were further decreased by CM compared with normal melanocytes. NVP-mix decreased melanogenesis signals, such as MC1R, MITF, TYRP1, and TYRP2, and decreased melanosome transfer-related signals, such as RAB32 and RAB27A, in UV-B-irradiated animal skin. NVP-mix also decreased MC1R, MITF, TYRP1, TYRP2, RAB32, and RAB27A in melanocytes treated with CM from UV-irradiated keratinocytes. The expression of MC1R and MITF in melanocytes after CM treatment was unchanged by NNT silencing. However, the expression of TYRP1, TYRP2, RAB32, and RAB27A increased in NNT-silenced melanocytes after CM treatment. NVP-mix also decreased tyrosinase activity and melanin content in UV-B-irradiated animal skin and CM-treated melanocytes. In conclusion, NVP-mix decreased mitochondrial oxidative stress by increasing NNT expression and decreased melanogenesis by decreasing MC1R/MITF, tyrosinase, TYRP1, and TYRP2.

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.

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.

The redox states of NAD and NADP are linked to each other in the mitochondria thanks to the enzyme nicotinamide nucleotide transhydrogenase (NNT) which, by utilizing the mitochondrial membrane potential (m&Delta, &Psi, ), catalyzes the transfer of redox potential between these two coenzymes, reducing one at the expense of the oxidation of the other. In order to define NNT reaction direction in CF cells, NNT activity under different redox states of cell has been investigated. Using spectrophotometric and western blotting techniques, the presence, abundance and activity level of NNT were determined. In parallel, the levels of NADPH and NADH as well as of mitochondrial and cellular ROS were also quantified. CF cells showed a 70% increase in protein expression compared to the Wt sample, however, regarding NNT activity, it was surprisingly lower in CF cells than healthy cells (about 30%). The cellular redox state, together with the low m&Delta, pushes to drive NNT reverse reaction, at the expense of its antioxidant potential, thus consuming NADPH to support NADH production. At the same time, the reduced NNT activity prevents the NADH, produced by the reaction, from causing an explosion of ROS by the damaged respiratory chain, in accordance with the reduced level of mitochondrial ROS in NNT-loss cells. This new information on cellular bioenergetics represents an important building block for further understanding the molecular mechanisms responsible for cellular dysfunction in cystic fibrosis.

Background HIF2a and lipid accumulation play key roles in the progression of clear cell renal cell carcinoma (ccRCC). Tumor cell “slimming” is a new concept in which tumor cells with abnormal lipids efficiently consume lipids to inhibit tumor progression without producing additional ATP. However, their respective regulatory mechanisms are still unclear. The purpose of this study is uncovering the links between these three key elements of ccRCC to elucidate new mechanisms of ccRCC metabolic abnormalities and providing a basis for new drug development for ccRCC. Methods Bioinformatics screening and analyses were performed in ccRCC according to TCGA‐KIRC database. qRT‐PCR, luciferase reporter assay, western blot, chromatin immunoprecipitation (ChIP) assays, and other biological methods were used to explore and verify related pathways. Various cell line models and animal models were used to perform related functional experiments. Results Screening based on sequencing data after HIF2a knockdown and three independent mitochondrial metabolism‐related gene sets showed that nicotinamide nucleotide transhydrogenase (NNT) was a mediator between HIF2a and tumor cells “slimming.” Further research showed that NNT had significant prognostic predictive value and was downregulated in ccRCC. It is regulated by HIF2a and can significantly activate lipid browning‐mediated tumor cell “slimming.” Mechanistic investigations indicated that HIF2a enhanced the expression of miR‐455‐5p via binding to HIF2a‐related response elements in the miR‐455‐5p promoter, which suppresses NNT expression by binding to its 3′ untranslated region. Conclusions Our study revealed a novel mechanism by which HIF2a decreased NNT level through a microRNA that suppressed tumor cell “slimming,” resulting in the progression of ccRCC. This mechanism provides a fresh perspective of lipid accumulation in ccRCC and may help target novel strategies for the treatment of tumors with abnormal lipid metabolism., A model could be suggested in which HIF2a increases miR‐455‐5p expression by binding to HIF2a response elements in the miR‐455‐5p promoter, which suppresses NNT expression by binding to its 3'UTR. Meanwhile, NNT promotes lipid browning to consume lipids, resulting in the suppression of ccRCC progression.

Orientadores: Roger Frigério Castilho, Annelise Francisco Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: A NAD(P)+ transidrogenase mitocondrial, também conhecida como transidrogenase de nucleotídeos de nicotinamida (NNT), é uma proteína integral da membrana mitocondrial interna e catalisa uma reação reversível que consiste na transferência de um hidreto entre NAD(H) e NADP(H) acoplada ao transporte de um próton entre o espaço intermembranar e a matriz mitocondrial. Em condições fisiológicas, a reação da NNT ocorre predominantemente no sentido da redução de NADP+ (i.e., reação direta), fazendo com que essa enzima seja uma importante fonte mitocondrial de NADPH, o qual é utilizado por sistemas antioxidantes da organela e em vias de biossíntese. A supressão da atividade da NNT tem relevância não só como abordagem experimental para avaliação da contribuição desta enzima em diferentes contextos fisiológicos e patológicos, como também consiste em uma possível abordagem terapêutica em condições patológicas específicas. A inibição da atividade NNT pode ser obtida por modificações moleculares como silenciamento de sua expressão, mutações ou com o uso de inibidores químicos. O presente trabalho objetivou avaliar a eficácia de alguns dos principais compostos propostos como inibidores químicos da NNT, diante de possíveis efeitos inespecíficos destes sobre a respiração mitocondrial. Foram avaliados os compostos: 4-cloro-7-nitrobenzofuran (NBD-Cl), N,N?-diciclo-hexilcarbodiimida (DCC), palmitoil-CoA, palmitoil-L-carnitina e rhein. Diferentes concentrações desses compostos foram testadas em mitocôndrias isoladas de fígado de camundongo quanto a (1) capacidade de inibir as reações direta e reversa da NNT em meio contendo detergente, e (2) efeitos sobre a função respiratória de mitocôndrias intactas, estimada pelo consumo de oxigênio estimulado por ADP e pelo estado respiratório não-fosforilante após adição de oligomicina. De forma geral, concentrações destes compostos capazes de gerar uma inibição parcial das reações direta e reversa da NNT em mitocôndrias, ocasionaram um prejuízo significativo na função respiratória mitocondrial. Dentre os compostos testados, o NBD-Cl mostrou uma melhor relação entre a inibição das reações da NNT e menor impacto sobre a função respiratória mitocondrial. Tendo em vista esse resultado, foram realizados testes adicionais com NBD-Cl em cultura primária de astrócitos sem NNT funcional, onde foi feita a contagem de células viáveis, medida da metabolização de MTT (brometo de 3-(4,5-dimetiltiazol-2-yl)-2,5-difeniltetrazolium) e dosagem de ATP após 48 h de exposição dessa cultura a diferentes concentrações de NDB-Cl. Nesses experimentos, as concentrações de NBD-Cl que causaram somente uma inibição parcial das reações direta e reversa da NNT (i.e., 10 e 20 µM) diminuíram significativamente a proliferação e viabilidade de astrócitos, demonstrando haver um efeito tóxico do NBD-Cl. Em outro conjunto de experimentos, observou-se que o NBD-Cl inibiu parcialmente a capacidade de mitocôndrias isoladas de fígado de camundongos com NNT intacta metabolizarem o peroxido orgânico tert-butil hidroperóxido, o que pode ser devido a inibição da NNT, mas também devido a efeitos tóxicos e inibitórios sobre outras proteínas mitocondriais. Os dados aqui apresentados permitem concluir que, embora os compostos testados realmente tenham apresentado efeitos inibitórios sobre as reações direta e reversa da NNT, concentrações efetivas desses compostos causam efeitos indesejáveis significativos sobre a função respiratória mitocondrial e viabilidade celular. Abstract: The mitochondrial NAD(P)+ transhydrogenase, also known as nicotinamide nucleotide transhydrogenase (NNT), is an integral protein of the inner mitochondrial membrane that catalyzes a reversible transfer of a hydride between NAD(H) and NADP(H) coupled to the transport of a proton between the intermembrane space and mitochondrial matrix. In most physiological conditions, the NNT reaction occurs towards NADP+ reduction (i.e., forward reaction); because of that this enzyme is an important mitochondrial source of NADPH, which is used by antioxidant systems in the organelle and in biosynthetic pathways. The suppression of NNT activity is relevant not only as an experimental approach to assess the contribution of this enzyme in different physiological and pathological contexts, but also consists of a possible therapeutic approach in specific pathological conditions. The inhibition of NNT activity can be achieved by molecular modifications such as silencing its expression, mutations or with the use of chemical inhibitors. The present study aimed at evaluating the efficacy of the main chemical compounds proposed as NNT inhibitors in view of their possible effects on other mitochondrial components. The compounds 4-chloro-7-nitrobenzofuran (NBD-Cl), N,N?-dicyclohexylcarbodiimide (DCC), palmitoyl-CoA, palmitoyl-L-carnitine and rhein were evaluated. Different concentrations of these compounds were tested in isolated mitochondria from mouse liver regarding (1) the ability to inhibit the direct and reverse reactions of NNT in detergent-solubilized mitochondria, and (2) effects on respiratory function of intact mitochondria, estimated by ADP-stimulated oxygen consumption and non-phosphorylating respiratory state due to oligomycin addition. In general, concentrations of these compounds capable of generating a partial inhibition of the direct and reverse activities of NNT in mitochondria, significantly impaired mitochondrial respiratory function. Among the compounds assayed, NBD-Cl showed a better relationship between NNT reaction inhibition and lower impact on mitochondrial oxygen consumption. In view of this result, additional experiments were carried out with NBD-Cl in a primary culture of astrocytes devoid of functional NNT, where it was evaluated cell number, metabolism of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and ATP content after 48 h of exposure to different NDB-Cl concentrations. In these experiments, NBD-Cl concentrations able to partially inhibit the direct and reverse NNT reactions (i.e., 10 and 20 µM) significantly decreased the proliferation and viability of astrocytes, demonstrating that there is a toxic effect of NBD-Cl. In another set of experiments, NBD-Cl partially decreased the ability of isolated mouse liver mitochondria with intact NNT to metabolize the organic peroxide tert-butyl hydroperoxide, which might be explained by inhibition of NNT, but also can be due to toxicity and inhibitory effects on other mitochondrial proteins. The data presented here allow us to conclude that, although the tested compounds indeed presented inhibitory effects on the direct and reverse activities of NNT, at effective concentrations they caused significant undesirable effects on mitochondrial respiratory function and cell viability. The mitochondrial NAD(P)+ transhydrogenase, also known as nicotinamide nucleotide transhydrogenase (NNT), is an integral protein of the inner mitochondrial membrane that catalyzes a reversible transfer of a hydride between NAD(H) and NADP(H) coupled to the transport of a proton between the intermembrane space and mitochondrial matrix. In most physiological conditions, the NNT reaction occurs towards NADP+ reduction (i.e., forward reaction); because of that this enzyme is an important mitochondrial source of NADPH, which is used by antioxidant systems in the organelle and in biosynthetic pathways. The suppression of NNT activity is relevant not only as an experimental approach to assess the contribution of this enzyme in different physiological and pathological contexts, but also consists of a possible therapeutic approach in specific pathological conditions. The inhibition of NNT activity can be achieved by molecular modifications such as silencing its expression, mutations or with the use of chemical inhibitors. The present study aimed at evaluating the efficacy of the main chemical compounds proposed as NNT inhibitors in view of their possible effects on other mitochondrial components. The compounds 4-chloro-7-nitrobenzofuran (NBD-Cl), N,N?-dicyclohexylcarbodiimide (DCC), palmitoyl-CoA, palmitoyl-L-carnitine and rhein were evaluated. Different concentrations of these compounds were tested in isolated mitochondria from mouse liver regarding (1) the ability to inhibit the direct and reverse reactions of NNT in detergent-solubilized mitochondria, and (2) effects on respiratory function of intact mitochondria, estimated by ADP-stimulated oxygen consumption and non-phosphorylating respiratory state due to oligomycin addition. In general, concentrations of these compounds capable of generating a partial inhibition of the direct and reverse activities of NNT in mitochondria, significantly impaired mitochondrial respiratory function. Among the compounds assayed, NBD-Cl showed a better relationship between NNT reaction inhibition and lower impact on mitochondrial oxygen consumption. In view of this result, additional experiments were carried out with NBD-Cl in a primary culture of astrocytes devoid of functional NNT, where it was evaluated cell number, metabolism of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and ATP content after 48 h of exposure to different NDB-Cl concentrations. In these experiments, NBD-Cl concentrations able to partially inhibit the direct and reverse NNT reactions (i.e., 10 and 20 µM) significantly decreased the proliferation and viability of astrocytes, demonstrating that there is a toxic effect of NBD-Cl. In another set of experiments, NBD-Cl partially decreased the ability of isolated mouse liver mitochondria with intact NNT to metabolize the organic peroxide tert-butyl hydroperoxide, which might be explained by inhibition of NNT, but also can be due to toxicity and inhibitory effects on other mitochondrial proteins. The data presented here allow us to conclude that, although the tested compounds indeed presented inhibitory effects on the direct and reverse activities of NNT, at effective concentrations they caused significant undesirable effects on mitochondrial respiratory function and cell viability. Mestrado Fisiopatologia Médica Mestra em Ciências

Nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) is a long noncoding RNA (lncRNA) that has been shown to be overexpressed in hepatocellular carcinoma (HCC). However, the molecular mechanism involving NNT-AS1 in HCC remains to be extensively investigated. The activation of TGF-β signaling inhibits tumor-infiltrating lymphocytes (TILs) and results in tumor immune evasion. We thus planned to explore the mechanism by which NNT-AS1 activates the TGF-β signaling pathway and inhibits TILs in HCC. High levels of NNT-AS1 were detected in HCC tissues by both RNAscope and real-time quantitative PCR (RT-qPCR) assays. The levels of proteins involved in TGF-β signaling and those of CD4 T lymphocytes were quantified by immunohistochemistry (IHC). HCC cell lines (HepG2 and Huh7) were used to explore the effects of NNT-AS1 on TGF-β signaling activation. In these analyses, RNAscope detection demonstrated that NNT-AS1 levels were significantly increased in HCC cancer tissues ( P = 0.0001 ). In addition, the elevated NNT-AS1 levels in cancer tissue were further confirmed by RT-qPCR analysis of HCC cancer tissues ( n = 64 ) and normal tissues ( n = 26 ) ( P = 0.0003 ). Importantly, the overall survival time of HCC patients who exhibited higher levels of NNT-AS1 expression was significantly shorter than that of HCC patients who had lower levels of NNT-AS1 expression ( P = 0.0402 ). Further mechanistic investigation indicated that NNT-AS1 inhibition significantly decreased the levels of TGF-β, TGFBR1, and SMAD5 in HCC cells. In HCC tissues, IHC detection showed that relatively high NNT-AS1 levels were associated with a reduction in infiltrated CD4 lymphocyte numbers. In conclusion, this research identifies a novel mechanism by which NNT-AS1 impairs CD4 T cell infiltration via activation of the TGF-β signaling pathway in HCC.

H

Human lung tumors engage in substantial mitochondrial metabolism. Here, Ward et al. demonstrate that nicotinamide nucleotide transhydrogenase (NNT) supports oxidative metabolism in NSCLC by mitigating Fe-S cluster oxidation., Human lung tumors exhibit robust and complex mitochondrial metabolism, likely precipitated by the highly oxygenated nature of pulmonary tissue. As ROS generation is a byproduct of this metabolism, reducing power in the form of nicotinamide adenine dinucleotide phosphate (NADPH) is required to mitigate oxidative stress in response to this heightened mitochondrial activity. Nicotinamide nucleotide transhydrogenase (NNT) is known to sustain mitochondrial antioxidant capacity through the generation of NADPH; however, its function in non-small cell lung cancer (NSCLC) has not been established. We found that NNT expression significantly enhances tumor formation and aggressiveness in mouse models of lung tumor initiation and progression. We further show that NNT loss elicits mitochondrial dysfunction independent of substantial increases in oxidative stress, but rather marked by the diminished activities of proteins dependent on resident iron-sulfur clusters. These defects were associated with both NADPH availability and ROS accumulation, suggesting that NNT serves a specific role in mitigating the oxidation of these critical protein cofactors., Graphical Abstract

Nucleotide nicotinamide transhydrogenase contributes to non-small cell lung carcinoma., In this study, Ward et al. (https://doi.org/10.1084/jem.20191689) provide exciting evidence that nucleotide nicotinamide transhydrogenase (NNT), a mitochondrial matrix–located enzyme harnessing the proton gradient to generate NADPH using NADH, markedly contributes to non-small cell lung carcinoma (NSCLC), which is abrogated in the murine C57BL/6J background, a strain known to be deficient in NNT.

Compensatory changes in energy expenditure occur in response to positive and negative energy balance, but the underlying mechanism remains unclear. Under low energy demand, the mitochondrial electron transport system is particularly sensitive to added energy supply (i.e. reductive stress), which exponentially increases the rate of H(2)O(2) (JH(2)O(2)) production. H(2)O(2) is reduced to H(2)O by electrons supplied by NADPH. NADP(+) is reduced back to NADPH by activation of mitochondrial membrane potential–dependent nicotinamide nucleotide transhydrogenase (NNT). The coupling of reductive stress-induced JH(2)O(2) production to NNT-linked redox buffering circuits provides a potential means of integrating energy balance with energy expenditure. To test this hypothesis, energy supply was manipulated by varying flux rate through β-oxidation in muscle mitochondria minus/plus pharmacological or genetic inhibition of redox buffering circuits. Here we show during both non-ADP– and low-ADP–stimulated respiration that accelerating flux through β-oxidation generates a corresponding increase in mitochondrial JH(2)O(2) production, that the majority (∼70–80%) of H(2)O(2) produced is reduced to H(2)O by electrons drawn from redox buffering circuits supplied by NADPH, and that the rate of electron flux through redox buffering circuits is directly linked to changes in oxygen consumption mediated by NNT. These findings provide evidence that redox reactions within β-oxidation and the electron transport system serve as a barometer of substrate flux relative to demand, continuously adjusting JH(2)O(2) production and, in turn, the rate at which energy is expended via NNT-mediated proton conductance. This variable flux through redox circuits provides a potential compensatory mechanism for fine-tuning energy expenditure to energy balance in real time.

In heart failure, a functional block of complex I of the respiratory chain provokes superoxide generation, which is transformed to H2O2 by dismutation. The Krebs cycle produces NADH, which delivers electrons to complex I, and NADPH for H2O2 elimination via isocitrate dehydrogenase and nicotinamide nucleotide transhydrogenase (NNT). At high NADH levels, α-ketoglutarate dehydrogenase (α-KGDH) is a major source of superoxide in skeletal muscle mitochondria with low NNT activity. Here, we analyzed how α-KGDH and NNT control H2O2 emission in cardiac mitochondria. In cardiac mitochondria from NNT-competent BL/6N mice, H2O2 emission is equally low with pyruvate/malate (P/M) or α-ketoglutarate (α-KG) as substrates. Complex I inhibition with rotenone increases H2O2 emission from P/M, but not α-KG respiring mitochondria, which is potentiated by depleting H2O2-eliminating capacity. Conversely, in NNT-deficient BL/6J mitochondria, H2O2 emission is higher with α-KG than with P/M as substrate, and further potentiated by complex I blockade. Prior depletion of H2O2-eliminating capacity increases H2O2 emission from P/M, but not α-KG respiring mitochondria. In cardiac myocytes, downregulation of α-KGDH activity impaired dynamic mitochondrial redox adaptation during workload transitions, without increasing H2O2 emission. In conclusion, NADH from α-KGDH selectively shuttles to NNT for NADPH formation rather than to complex I of the respiratory chain for ATP production. Therefore, α-KGDH plays a key role for H2O2 elimination, but is not a relevant source of superoxide in heart. In heart failure, α-KGDH/NNT-dependent NADPH formation ameliorates oxidative stress imposed by complex I blockade. Downregulation of α-KGDH may, therefore, predispose to oxidative stress in heart failure.

2-Pyrone-4,6-dicarboxylic acid (PDC) is a pseudoaromatic dicarboxylic acid and is a promising biobased building block chemical that can be used to make diverse polyesters with novel functionalities. In this study, Escherichia coli was metabolically engineered to produce PDC from glucose. First, an efficient biosynthetic pathway for PDC production from glucose was suggested by in silico metabolic flux simulation. This best pathway employs a single-step biosynthetic route to protocatechuic acid (PCA), a metabolic precursor for PDC biosynthesis. On the basis of the selected PDC biosynthetic pathway, a shikimate dehydrogenase (encoded by aroE)-deficient E. coli strain was engineered by introducing heterologous genes of different microbial origin encoding enzymes responsible for converting 3-dehydroshikimate (DHS) to PDC, which allowed de novo biosynthesis of PDC from glucose. Next, production of PDC was further improved by applying stepwise rational metabolic engineering strategies. These include elimination of feedback inhibition on 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (encoded by aroG) by overexpressing a feedback-resistant variant, enhancement of the precursor phosphoenolpyruvate supply by changing the native promoter of the ppsA gene with the strong trc promoter, and reducing accumulation of the major byproduct DHS by overexpression of a DHS importer (encoded by shiA). Furthermore, cofactor (NADP+/NADPH) utilization was manipulated through genetic modifications of the E. coli soluble pyridine nucleotide transhydrogenase (encoded by sthA), and the resultant impact on PDC production was investigated. Fed-batch fermentation of the final engineered E. coli strain allowed production of 16.72 g/L of PDC from glucose with the yield and productivity of 0.201 g/g and 0.172 g/L/h, respectively, representing the highest PDC production performance indices reported to date.

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., Graphical abstract fx1

Background/Aims: Increasing evidence demonstrates that long non-coding RNAs (lncRNAs) play critical regulatory roles in cancers, including osteosarcoma. A previous study showed that Nicotinamide Nucleotide Transhydrogenase-antisense RNA1 (NNT-AS1) was aberrantly expressed in several types of cancer. However, the potential biological roles and regulatory mechanisms of NNT-AS1 in osteosarcoma progression remain unknown. Methods: Quantitative RT-PCR was performed to examine the expression of NNT-AS1 in human tissues and cells. The biological functions of NNT-AS1 were determined by CCK-8, colony formation, Flow cytometry and Transwell assays in vitro. A mouse xenograft model was performed to investigate the effect of NNT-AS1 on tumor growth in vivo. Results: In this study, we found the expression of NNT-AS1 was significantly increased in tumor tissues compared to adjacent normal tissues. Furthermore, upregulated NNT-AS1 expression predicted poor prognosis and was an independent and significant risk factor for osteosarcoma patient survival. Further experiments revealed that NNT-AS1 knockdown significantly inhibited cell proliferation by inducing cell cycle arrest and promoting apoptosis in osteosarcoma cells. Moreover, NNT-AS1 silencing suppressed cell migration and invasion in vitro. In a tumor xenograft model, knockdown of NNT-AS1 suppressed tumor growth of OS-732 cells in vivo. Conclusions: Taken together, these findings indicate that NNT-AS1 functions as an oncogene in osteosarcoma and could be a novel diagnostic and therapeutic target for osteosarcoma.

Background The six-carbon circular non-proteinogenic compound l-pipecolic acid is an important chiral drug intermediate with many applications in the pharmaceutical industry. In the present study, we developed a metabolically engineered strain of Escherichia coli for the overproduction of l-pipecolic acid from glucose. Results The metabolic pathway from l-lysine to l-pipecolic acid was constructed initially by introducing lysine cyclodeaminase (LCD). Next, l-lysine metabolic flux from glucose was amplified by the plasmid-based overexpression of dapA, lysC, and lysA under the control of the strong trc promoter to increase the biosynthetic pool of the precursor l-lysine. Additionally, since the catalytic efficiency of the key enzyme LCD is limited by the cofactor NAD+, the intracellular pyridine nucleotide concentration was rebalanced by expressing the pntAB gene encoding the transhydrogenase, which elevated the proportion of LCD with bound NAD+ and enhanced l-pipecolic acid production significantly. Further, optimization of Fe2+ and surfactant in the fermentation process resulted in 5.33 g/L l-pipecolic acid, with a yield of 0.13 g/g of glucose via fed-batch cultivation. Conclusions We expanded the metabolic pathway for the synthesis of the chiral pharmaceutical intermediate l-pipecolic acid in E. coli. Using the engineered E. coli, a fast and efficient fermentative production of l-pipecolic acid was achieved. This strategy could be applied to the biosynthesis of other commercially and industrially important chiral compounds containing piperidine rings. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0666-0) contains supplementary material, which is available to authorized users.

C57BL/6J and C57BL/6N inbred mice are widely, and often interchangeably, used for stem cell research; yet, these substrains harbor discrete genetic differences that can cause phenotypic disparities. In this issue of Stem Cell Reports, Morales-Hernández et al. identify one particular difference—disruption of Nicotinamide Nucleotide Transhydrogenase (Nnt)—that increases reactive oxygen exposure and impairs hematopoietic progenitor cell function in C57BL/6J, as compared to C57BL/6N, mice. : C57BL/6J and C57BL/6N inbred mice are widely, and often interchangeably, used for stem cell research; yet, these substrains harbor discrete genetic differences that can cause phenotypic disparities. In this issue of Stem Cell Reports, Morales-Hernández et al. identify one particular difference—disruption of Nicotinamide Nucleotide Transhydrogenase (Nnt)—that increases reactive oxygen exposure and impairs hematopoietic progenitor cell function in C57BL/6J, as compared to C57BL/6N, mice.

The enzyme nicotinamide nucleotide transhydrogenase (NNT) transfers hydride from NADH to NADP+ coupled to H+ translocation across the inner mitochondrial membrane. In a recent study, Kampjut and Sazanov reveal that the bifunctional NNT mechanism rules the NAD(P)+/NAD(P)H interconversion ratio, which in turn regulates antioxidant defense and sirtuin actions.

The atherosclerosis prone LDL receptor knockout mice (Ldlr−/−, C57BL/6J background) carry a deletion of the NADP(H)-transhydrogenase gene (Nnt) encoding the mitochondrial enzyme that catalyzes NADPH synthesis. Here we hypothesize that both increased NADPH consumption (due to increased steroidogenesis) and decreased NADPH generation (due to Nnt deficiency) in Ldlr−/− mice contribute to establish a macrophage oxidative stress and increase atherosclerosis development. Thus, we compared peritoneal macrophages and liver mitochondria from three C57BL/6J mice lines: Ldlr and Nnt double mutant, single Nnt mutant and wild-type. We found increased oxidants production in both mitochondria and macrophages according to a gradient: double mutant > single mutant > wild-type. We also observed a parallel up-regulation of mitochondrial biogenesis (PGC1a, TFAM and respiratory complexes levels) and inflammatory (iNOS, IL6 and IL1b) markers in single and double mutant macrophages. When exposed to modified LDL, the single and double mutant cells exhibited significant increases in lipid accumulation leading to foam cell formation, the hallmark of atherosclerosis. Nnt deficiency cells showed up-regulation of CD36 and down-regulation of ABCA1 transporters what may explain lipid accumulation in macrophages. Finally, Nnt wild-type bone marrow transplantation into LDLr−/− mice resulted in reduced diet-induced atherosclerosis. Therefore, Nnt plays a critical role in the maintenance of macrophage redox, inflammatory and cholesterol homeostasis, which is relevant for delaying the atherogenesis process.

The mitochondrial, inner-membrane-associated, reversible NADPH

Redox cofactors play an important role in biosynthetic and catabolic reactions and the transfer of energy for the cell. Therefore, studying the relationship between cofactor perturbation and metabolism is a useful approach to improve the yield of target products. To study RNA accumulation and metabolism when intracellular cofactor balance was impaired, the water-forming NADH oxidase (NoxE) from Lactococcus lactis and membrane-bound transhydrogenase (PntAB) from Escherichia coli were expressed in Candidatropicalis no. 121, respectively. Expression of noxE significantly decreased the intracellular NADH/NAD+ ratio, but the NADPH/NADP+ ratio did not differ significantly. PntAB increased the intracellular NADH pool, while the NADPH/NADP+ ratio decreased. The perturbation of the cofactors caused a large redistribution of metabolic fluxes. The biomass and RNA content decreased by 11.0% and 10.6% in pAUR-noxE strain, respectively, while the RNA content increased by 5.5% and the biomass showed no signification difference in pAUR-pntAB strain. Expression of noxE and pntAB led to decreases and increases in the ATP concentration and yield of RNA, respectively, which also indicated that ATP plays an important role in the RNA biosynthesis.

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 &gt, 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.

Perinatal hypoxia is a critical complication during delivery and is mostly studied in animal models of postnatal hypoxic-ischemic brain injury. We here studied the effects of postnatal hypoxic-ischemic brain injury in two different sub-strains of C57BL/6 mice, i.e. C57BL/6J and C57BL/6N mice. These two sub-strains show different metabolic properties, for instance an impaired glucose tolerance in C57BL/6J mice. Genetically, this was linked to differences in their nicotinamide nucleotide transhydrogenase (Nnt) genes: In C57BL/6J mice, exons 7–11 of the Nnt gene are deleted, resulting in the absence of functional Nnt protein. The mitochondrial Nnt-protein is one of several enzymes that catalyses the generation of NADPH, which in turn is important for the elimination of reactive oxygen species (ROS). As ROS is thought to contribute to the pathophysiology of hypoxia-ischemia, the lack of Nnt might indirectly increase ROS levels and therefore result in increased brain damage. We therefore hypothesize that lesion score and lesion size will increase in C57BL/6J mice as compared to C57BL/6N mice. Surprisingly, the results showed exactly the opposite: C57BL/6J mice showed a decrease in lesion score and size, associated with a reduced number of apoptotic cells and activated microglia. In contrast, the number of cells with ROS-induced DNA modifications (detected by 8OHdG) was higher in C57BL/6J than C57BL/6N mice. In conclusion, C57BL/6J mice showed reduced ischemic consequences after postnatal hypoxic-ischemic brain injury compared to C57BL/6N mice, with the exception of the amount of ROS-induced DNA-damage. These differences might relate to the lack of Nnt, but also to a modified metabolic setting (cardiovascular parameters, oxygen and glucose metabolism, immune function) in C57BL/6J mice.

Acetobacter ghanensis LMG 23848(T) and Acetobacter senegalensis 108B are acetic acid bacteria that originate from a spontaneous cocoa bean heap fermentation process and that have been characterised as strains with interesting functionalities through metabolic and kinetic studies. As there is currently little genetic information available for these species, whole-genome sequencing of A. ghanensis LMG 23848(T) and A. senegalensis 108B and subsequent data analysis was performed. This approach not only revealed characteristics such as the metabolic potential and genomic architecture, but also allowed to indicate the genetic adaptations related to the cocoa bean fermentation process. Indeed, evidence was found that both species possessed the genetic ability to be involved in citrate assimilation and displayed adaptations in their respiratory chain that might improve their competitiveness during the cocoa bean fermentation process. In contrast, other properties such as the dependence on glycerol or mannitol and lactate as energy sources or a less efficient acid stress response may explain their low competitiveness. The presence of a gene coding for a proton-translocating transhydrogenase in A. ghanensis LMG 23848(T) and the genes involved in two aromatic compound degradation pathways in A. senegalensis 108B indicate that these strains have an extended functionality compared to Acetobacter species isolated from other ecosystems.

Nicotinamide-nucleotide-transhydrogenase (Nnt) is a mitochondrial protein. It is altered and functionally lacking in the C57BL/6J sub-strain. This leads to the generation of more radical oxygen species than in the C57BL/6N sub-strain. During studies on the effect of Nnt in perinatal hypoxia the cerebral vasculature was investigated in postnatal day 9 mice using post mortem arterial filling with silicone rubber compounds. Surprisingly, the tiny vessels were no longer uniformly filled and a bleb-like pattern occurred in both sub-strains. Furthermore, considerably more bleb-like spots were observed in the C57Bl/6J sub-strain than in the C57Bl/6N sub-strain. These blebs might be the result of feathery vessels bursting. It remains unclear how the mechanisms in the used strains differ. Nnt might influence the vascular structure or its development and mechanisms and should be investigated further.

Endothelial dysfunction is a critical, initiating step in the development of hypertension (HTN) and mitochondrial reactive oxygen species (ROS) are important contributors to endothelial dysfunction. Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in the nicotinamide nucleotide transhydrogenase (Nnt) gene that are associated with endothelial dysfunction and increased risk for HTN. NNT is emerging as an important enzyme that regulates mitochondrial NADPH levels and mitochondrial redox balance by supporting the thiol dependent peroxidase systems in the mitochondria. We have previously shown that the absence of NNT in C57Bl/6J animals promotes a more severe hypertensive phenotype through reductions in •NO and endothelial dependent vessel dilation. However, the impact of NNT on human endothelial cell function remains unclear. We utilized NNT directed shRNA in human aortic endothelial cells to test the hypothesis that NNT critically regulates mitochondrial redox balance and endothelial function in response to angiotensin II (Ang II). We demonstrate that NNT expression and activity are elevated in response to the mitochondrial dysfunction and oxidative stress associated with Ang II treatment. Knockdown of NNT led to a significant elevation of mitochondrial ROS production and impaired glutathione peroxidase and glutathione reductase activities associated with a reduction in the NADPH/NADP+ ratio. Loss of NNT also promoted mitochondrial dysfunction, disruption of the mitochondrial membrane potential, and impaired ATP production in response to Ang II. Finally, we observed that, while the loss of NNT augmented eNOS phosphorylation at Ser1177, neither eNOS activity nor nitric oxide production were similarly increased. The results from these studies clearly demonstrate that NNT is critical for the maintenance of mitochondrial redox balance and mitochondrial function. Loss of NNT and disruption of redox balance leads to oxidative stress that compromises eNOS activity that could have a profound effect on the endothelium dependent regulation of vascular tone., Graphical abstract Image 1

The effects of asbestos fibers on human immune cells have not been well documented. We have developed a continuously exposed cell line model using the human T-lymphotropic virus 1 (HTLV-1)-immortalized human T cell line MT-2. Sublines continuously exposed to chrysotile (CH) or crocidolite (CR) showed acquired resistance to asbestos-induced apoptosis following transient and high-dose re-exposure with fibers. These sublines in addition to other immune cells such as natural killer cells or cytotoxic T lymphocytes exposed to asbestos showed a reduction in anti-tumor immunity. In this study, the expression of genes and molecules related to antioxidative stress was examined. Furthermore, complexes related to oxidative phosphorylation were investigated since the production of reactive oxygen species (ROS) is important when considering the effects of asbestos in carcinogenesis and the mechanisms involved in resistance to asbestos-induced apoptosis. In sublines continuously exposed to CH or CR, the expression of thioredoxin decreased. Interestingly, nicotinamide nucleotide transhydrogenase (NNT) expression was markedly enhanced. Thus, knockdown of NNT was then performed. Although the knockdown clones did not show any changes in proliferation or occurrence of apoptosis, these clones showed recovery of ROS production with returning NADPH/NADP+ ratio that increased with decreased production of ROS in continuously exposed sublines. These results indicated that NNT is a key factor in preventing ROS-induced cytotoxicity in T cells continuously exposed to asbestos. Considering that these sublines showed a reduction in anti-tumor immunity, modification of NNT may contribute to recovery of the anti-tumor effects in asbestos-exposed T cells. Keywords: Asbestos, T cell, Continuous exposure, Oxidative phosphorylation, nicotinamide nucleotide transhydrogenase (NNT)

The non-conventional d-xylose metabolism called the Dahms pathway which only requires the expression of at least three enzymes to produce pyruvate and glycolaldehyde has been previously engineered in Escherichia coli. Strains that rely on this pathway exhibit lower growth rates which were initially attributed to the perturbed redox homeostasis as evidenced by the lower intracellular NADPH concentrations during exponential growth phase. NADPH-regenerating systems were then tested to restore the redox homeostasis. The membrane-bound pyridine nucleotide transhydrogenase, PntAB, was overexpressed and resulted to a significant increase in biomass and glycolic acid titer and yield. Furthermore, expression of PntAB in an optimized glycolic acid-producing strain improved the growth and product titer significantly. This work demonstrated that compensating for the NADPH demand can be achieved by overexpression of PntAB in E. coli strains assimilating d-xylose through the Dahms pathway. Consequently, increase in biomass accumulation and product concentration was also observed.

Unraveling the mechanisms of microbial adaptive evolution following genetic or environmental challenges is of fundamental interest in biological science and engineering. When the challenge is the loss of a metabolic enzyme, adaptive responses can also shed significant insight into metabolic robustness, regulation, and areas of kinetic limitation. In this study, whole-genome sequencing and highresolution C-13-metabolic flux analysis were performed on 10 adaptively evolved pgi knockouts of Escherichia coli. Pgi catalyzes the first reaction in glycolysis, and its loss results in major physiological and carbon catabolism pathway changes, including an 80% reduction in growth rate. Following adaptive laboratory evolution (ALE), the knockouts increase their growth rate by up to 3.6-fold. Through combined genomic-fluxomic analysis, we characterized the mutations and resulting metabolic fluxes that enabled this fitness recovery. Large increases in pyridine cofactor transhydrogenase flux, correcting imbalanced production of NADPH and NADH, were enabled by direct mutations to the transhydrogenase genes sthA and pntAB. The phosphotransferase system component crr was also found to be frequently mutated, which corresponded to elevated flux from pyruvate to phosphoenolpyruvate. The overall energy metabolism was found to be strikingly robust, and what have been previously described as latently activated Entner-Doudoroff and glyoxylate shunt pathways are shown here to represent no real increases in absolute flux relative to the wild type. These results indicate that the dominant mechanism of adaptation was to relieve the rate-limiting steps in cofactor metabolism and substrate uptake and to modulate global transcriptional regulation from stress response to catabolism.

Orientador: Helena Coutinho Franco de Oliveira Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A transidrogenase de nucleotídeo de nicotinamida mitocondrial (NNT) é a principal enzima geradora de NA(D)PH em mitocôndrias, o qual é utilizado para detoxificação de peróxido de hidrogênio mitocondrial. Estudos prévios mostraram que a mutação nessa enzima está associada a alterações do estado redox de mitocôndrias, redução de secreção de insulina e aumento de obesidade induzida por dieta em animais C57BL/6J provenientes do Jackson Laboratory (B6-J) quando comparados a linhagens sem esta mutação (por exemplo, C57BL/6JUnib e C57BL/6N). Neste trabalho, investigamos o metabolismo glicídico e lipídico visando entender o papel da NNT na suscetibilidade ao diabetes e à obesidade. Para tanto, estudamos sub-linhagens C57BL6 isogênicas independentes, com e sem mutação da NNT, B6-J e B6-UNI, respectivamente, assim como linhagens congênicas (com >99% de homogeneidade de background genético) com e sem a mutação da NNT (NNT-/- vs NNT+/+). Estes estudos foram realizados tanto com dieta normal (padrão) quanto sob dieta hiperlipídica (high fat ¿ HF, 20 semanas). No primeiro estudo avaliamos o efeito da NNT sobre a homeostase glicêmica. Observamos que, em relação aos não mutantes (B6-UNI), os animais deficientes de NNT (B6-J) apresentaram intolerância à glicose, tanto por deficiência de secreção de insulina quanto por resistência periférica ao hormônio. Nas ilhotas pancreáticas, além da redução de secreção de insulina estimulada por glicose, houve redução de autofluorescência de NAD(P)H e aumento de produção de H2O2. Verificamos também que os animais deficientes de NNT apresentaram resistência hepática à insulina determinada pelo teste de conversão de piruvato à glicose e pela redução da sinalização hepática da insulina (fosforilação da AKT). Todos estes achados foram observados tanto em dieta normal como em HF. No entanto, em linhagens congênicas, demonstramos que os animais deficientes de NNT (NNT-/-) também eram intolerantes à glicose, porém com a função secretória de insulina preservada, tanto em dieta normal quanto em HF. A intolerância à glicose nos animais NNT-/- é explicada pela maior resistência periférica à insulina, de modo global (teste de tolerância à insulina) e hepática (conversão de piruvato a glicose e fosforilação da AKT). Baseado em estudos prévios mostrando anormalidades redox em mitocôndrias hepáticas com deficiência de NNT, nossa hipótese é de que estas anormalidades são responsáveis pela resistência hepática à insulina. Assim, tratamos os animais por um mês com o antioxidante TEMPOL, o que de fato reverteu a intolerância à glicose causada pela deficiência de NNT. Assim, concluímos que a NNT é importante para sinalização hepática de insulina, mas não altera a secreção de insulina. Provavelmente outras diferenças genéticas entre as linhagens B6-J e B6-UNI são responsáveis pelo comprometimento da secreção de insulina estimulada por glicose nos animais C57BL/6J. No segundo estudo avaliamos o papel da NNT sobre o metabolismo lipídico. Observamos que os animais B6-J deficientes em NNT apresentam aumento de adiposidade corporal e de triglicérides (TG) hepáticos, aumento de leptinemia e redução de metabolismo corporal (respirometria). Estes achados foram obtidos tanto em dieta normal quanto em dieta HF. O aumento de gordura hepática pode ser explicado por aumento de retenção de lípides da dieta. Quando investigamos as linhagens congênicas, observamos que os animais NNT-/- só permanecem mais obesos quando em dieta hiperlipídica, enquanto a esteatose hepática está presente tanto em dieta normal quanto em HF. Os mecanismos responsáveis pela esteatose hepática estão associados à redução de secreção hepática de VLDL-TG. Assim, propomos que a disfunção mitocondrial prejudica a sinalização da insulina, sendo a resistência à insulina determinante do acúmulo lipídico hepático, principalmente por comprometer a secreção de VLDL. Em síntese, a deficiência da NNT é determinante da adiposidade induzida por dieta, mas não explica diferenças nos depósitos adiposos em dieta normal observadas nas linhagens C57BL6 independentes. O aumento da adiposidade induzido por dieta é explicado por redução do metabolismo corporal e a esteatose hepática pela resistência à insulina nos animais deficientes de NNT Abstract: Mitochondrial nicotinamide nucleotide transhydrogenase (NNT) is the main source of NA(D)PH in mitochondria, which is used for detoxification of mitochondrial hydrogen peroxide. Previous studies have shown that the mutation in this enzyme is associated with changes in the redox state of mitochondria, reduction of insulin secretion and increase of diet-induced obesity in C57BL/6J mice from the Jackson Laboratory (B6-J) when compared to strains without this mutation (e.g., C57BL/6JUnib). In this work, we investigate the glucose and lipid metabolism in order to understand the role of NNT in the susceptibility to diabetes and obesity. For this, we studied independent isogenic C57BL6 sub line, with and without mutation of NNT, B6-J and B6-UNI, respectively, as well as congenic lineages (with >99% genetic background homogeneity) with and without the NNT mutation (NNT-/- vs. NNT+/+). These studies were performed with either normal (standard) diet or high fat (HF, 20 weeks) diet. In the first study we evaluated the effect of NNT on glycemic homeostasis. We observed that in relation to the non-mutants (B6-UNI), NNT deficient animals (B6-J) presented glucose intolerance due to both insulin secretion deficiency and peripheral resistance to the hormone. In the pancreatic islets, in addition to the reduction of insulin secretion stimulated by glucose, there was a reduction of autofluorescence of NAD(P)H and increase of H2O2 production. NNT-deficient animals had hepatic insulin resistance as determined by the pyruvate-to-glucose test and reduced hepatic insulin signaling (pAKT). All these findings were observed in both normal and HF diet fed mice. However, in congenic strains, we demonstrated that NNT deficient animals (NNT-/-) were also glucose intolerant, but with secretory insulin function preserved, both in normal and HF diet. Glucose intolerance in NNT-/- animals is explained by increased peripheral insulin resistance, overall (insulin tolerance test) and in liver (conversion of pyruvate to glucose and phosphorylation of AKT). Based on previous studies showing redox abnormalities in hepatic mitochondria with NNT deficiency, our hypothesis is that these abnormalities are responsible for hepatic insulin resistance. Thus, we treated the animals for a month with the antioxidant TEMPOL, which in fact reversed the glucose intolerance caused by NNT deficiency. Thus, we conclude that NNT is important for hepatic insulin signaling, but does not alter insulin secretion. Probably other genetic differences between the B6-J and B6-UNI strains besides NNT mutation are responsible for the impairment of glucose-stimulated insulin secretion in C57BL/6J animals. In the second study we evaluated the role of NNT on lipid metabolism. We observed that NNT-deficient B6-J mice present increased body adiposity and hepatic triglycerides (TG), increased leptinemia and reduced body metabolism (respirometry). These findings were obtained both in the normal diet and in the HF diet. Increased liver fat can be explained by increased dietary lipid retention. When we investigated the congenic lines, we observed that the NNT-/- animals only remain more obese when on a hyperlipidic diet, while hepatic steatosis is present in both normal and HF diet. The mechanisms responsible for hepatic steatosis are associated with reduced hepatic secretion of VLDL-TG. Thus, we propose that mitochondrial dysfunction impairs insulin signaling, and insulin resistance is determinant of hepatic lipid accumulation, mainly because it compromises the secretion of VLDL. In summary, NNT deficiency is determinant of diet-induced adiposity but does not explain differences in fat deposits observed in independent C57BL6 strains under normal dietary. The increase in diet-induced adiposity is explained by reduced body metabolism and hepaticsteatosis by insulin resistance in NNT-deficient animals Doutorado Fisiologia Doutora em Biologia Funcional e Molecular FAPESP 2014/02819-0 CNPQ CAPES

Understanding how microbes adapt to changing conditions is fundamental to biological science and engineering. For example, adaptation is a key driver of antimicrobial resistance, and adaptive laboratory evolution has become a key tool in biotechnology. Here, we present a comprehensive genetic and fluxomic analysis of 10 adaptively evolved Escherichia coli phosphoglucose isomerase (pgi) knockout strains. The loss of PGI, a key enzyme in glycolysis, results in massive redirection of carbon catabolic flux and reduction in growth rate. Adaptive evolution results in a 3.6-fold increase in growth rate, enabled by key mutations and metabolic flux rewiring. These include global transcriptional regulators, cofactor transhydrogenases, and the phosphotransferase system component crr. Overcoming key bottlenecks, rather than a broad metabolic response, is the dominant mechanism of adaptation.

SummaryMitochondrial reactive oxygen species (ROS) play a central role in most aging-related diseases. ROS are produced at the respiratory chain that demands NADH for electron transport and are eliminated by enzymes that require NADPH. The nicotinamide nucleotide transhydrogenase (Nnt) is considered a key antioxidative enzyme based on its ability to regenerate NADPH from NADH. Here, we show that pathological metabolic demand reverses the direction of the Nnt, consuming NADPH to support NADH and ATP production, but at the cost of NADPH-linked antioxidative capacity. In heart, reverse-mode Nnt is the dominant source for ROS during pressure overload. Due to a mutation of the Nnt gene, the inbred mouse strain C57BL/6J is protected from oxidative stress, heart failure, and death, making its use in cardiovascular research problematic. Targeting Nnt-mediated ROS with the tetrapeptide SS-31 rescued mortality in pressure overload-induced heart failure and could therefore have therapeutic potential in patients with this syndrome.

The membrane protein transhydrogenase in animal mitochondria and bacteria couples reduction of NADP+ by NADH to proton translocation. Recent X-ray data on Thermus thermophilus transhydrogenase indicate a significant difference in the orientations of the two dIII components of the enzyme dimer (Leung et al., 2015). The character of the orientation change, and a review of information on the kinetics and thermodynamics of transhydrogenase, indicate that dIII swivelling might assist in the control of proton gating by the redox state of bound NADP+/NADPH during enzyme turnover.