Nicotinamide Adenine Dinucleotide Synthesis Research Articles

Protein-protein interactions of the nicotinamide/nicotinate mononucleotide adenylyltransferase of Leishmania braziliensis.

BACKGROUND Nicotinamide adenine dinucleotide (NAD) plays a central role in energy metabolism and integrates cellular metabolism with signalling and gene expression. NAD biosynthesis depends on the enzyme nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT; EC: 2.7.7.1/18), in which converge the de novo and salvage pathways.OBJECTIVE The purpose of this study was to analyse the protein-protein interactions (PPI) of NMNAT of Leishmania braziliensis (LbNMNAT) in promastigotes.METHODS Transgenic lines of L. braziliensis promastigotes were established by transfection with the pSP72αneoαLbNMNAT-GFP vector. Soluble protein extracts were prepared, co-immunoprecipitation assays were performed, and the co-immunoprecipitates were analysed by mass spectrometry. Furthermore, bioinformatics tools such as network analysis were applied to generate a PPI network.FINDINGS Proteins involved in protein folding, redox homeostasis, and translation were found to interact with the LbNMNAT protein. The PPI network indicated enzymes of the nicotinate and nicotinamide metabolic routes, as well as RNA-binding proteins, the latter being the point of convergence between our experimental and computational results.MAIN CONCLUSION We constructed a model of PPI of LbNMNAT and showed its association with proteins involved in various functions such as protein folding, redox homeostasis, translation, and NAD synthesis.

NAD Metabolism in Cancer Therapeutics.

Cancer cells have a unique energy metabolism for sustaining rapid proliferation. The preference for anaerobic glycolysis under normal oxygen conditions is a unique trait of cancer metabolism and is designated as the Warburg effect. Enhanced glycolysis also supports the generation of nucleotides, amino acids, lipids, and folic acid as the building blocks for cancer cell division. Nicotinamide adenine dinucleotide (NAD) is a co-enzyme that mediates redox reactions in a number of metabolic pathways, including glycolysis. Increased NAD levels enhance glycolysis and fuel cancer cells. In fact, nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme for NAD synthesis in mammalian cells, is frequently amplified in several cancer cells. In addition, Nampt-specific inhibitors significantly deplete NAD levels and subsequently suppress cancer cell proliferation through inhibition of energy production pathways, such as glycolysis, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. NAD also serves as a substrate for poly(ADP-ribose) polymerase (PARP), sirtuin, and NAD gylycohydrolase (CD38 and CD157); thus, NAD regulates DNA repair, gene expression, and stress response through these enzymes. Thus, NAD metabolism is implicated in cancer pathogenesis beyond energy metabolism and considered a promising therapeutic target for cancer treatment. In this review, we present recent findings with respect to NAD metabolism and cancer pathogenesis. We also discuss the current and future perspectives regarding the therapeutics that target NAD metabolic pathways.

P7C3-A20 neuroprotection is independent of Wallerian degeneration in primary neuronal culture.

The antiapoptotic, neuroprotective compound P7C3-A20 reduces neurological deficits when administered to murine in-vivo models of traumatic brain injury. P7C3-A20 is thought to exert its activity through small-molecule activation of the enzyme nicotinamide phosphoribosyltransferase. This enzyme converts nicotinamide to nicotinamide mononucleotide, the precursor to nicotinamide adenine dinucleotide synthesis. Alterations to this bioenergetic pathway have been shown to induce Wallerian degeneration (WD) of the distal neurite following injury. This study aimed to establish whether P7C3-A20, through induction of nicotinamide phosphoribosyltransferase activity, would affect the rate of WD. The model systems used were dissociated primary cortical neurons, dissociated superior cervical ganglion neurons and superior cervical ganglion explants. P7C3-A20 failed to show any protection against WD induced by neurite transection or vincristine administration. Furthermore, there was a concentration-dependent neurotoxicity. These findings are important in understanding the mechanism by which P7C3-A20 mediates its effects – a key step before moving to human clinical trials.

NAD-Mediated Metabolic Reprogramming Epigenetically Regulates Gene Expression to Promote Preadipocyte Differentiation

During cell differentiation, intracellular energetic pathways are drastically reorganized. Although this alteration had been considered as a passive process to adapt to environmental change during differentiation, recent studies suggested that the metabolic alteration rather actively regulates the differentiation process. It is known that the dynamic metabolic reprogramming occurs during differentiation of preadipocytes, however its implication in adipogenesis is unknown. In this study, we employed mass spectrometry-based metabolomics and investigated the metabolic change during differentiation of 3T3-L1 preadipocytes. We found that the level of nicotinamide adenine dinucleotide (NAD), a cofactor mediating redox reaction and protein modification, was significantly increased during the differentiation. Consistently Nampt, a rate-limiting enzyme of NAD synthesis, was also upregulated in the early phase of adipogenesis. Further, we found that pharmacological or genetic inhibition of Nampt reduced NAD synthesis and repressed the metabolic reprogramming during adipogenesis. Notably, inhibition of Nampt blocked the gene expression of Pparg and impeded the preadipocyte differentiation. The effect of Nampt inhibition was cancelled by the supplementation of nicotinamide mononucleotide, a precursor of NAD. Our metabolomic analysis also revealed that TCA cycle intermediate, α-ketoglutarate (αKG) was upregulated during the differentiation and Nampt inhibition disturbed the rise of αKG. Interestingly, αKG is known as a cofactor of the demethylation of histones. The ChIP assay revealed that αKG mediated the demethylation of H3K9me3 on Pparg promoter region and promote the gene expression of Pparg during adipogenesis. This process was inhibited by Nampt inhibitor treatment, and was reversed by the supplementation of αKG. Altogether, our data indicated that Nampt-mediated NAD synthesis is necessary for differentiation of preadipocyte. Disclosure K. Okabe: None. I. Usui: None. A. Nawaz: Research Support; Spouse/Partner; Kobayashi Foundation, Kobayashi Foundation, Kobayashi Foundation. S. Fujisaka: Research Support; Self; Mochida Pharmaceutical Co., Ltd., Eli Lilly and Company, MSD K.K., Toyama First Bank. T. Kado: None. Y. Igarashi: None. K. Yagi: None. K. Tobe: Research Support; Self; Bristol-Myers Squibb Company, Takeda Pharmaceutical Company, Teijin Pharma Limited, Japan Diabetes Society, Eli Lilly and Company, MSD K.K., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Pfizer Inc., Astellas Parma Inc, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Tsumura & Co., Ono Pharmaceutical Co., Ltd., Novartis Pharma K.K., Sumitomo Dainippon Pharma Co. T. Nakagawa: None.

Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age-associated insulin resistance.

SummaryNicotinamide adenine dinucleotide (NAD) is an important cofactor that regulates various biological processes, including metabolism and gene expression. As a coenzyme, NAD controls mitochondrial respiration through enzymes of the tricarboxylic acid (TCA) cycle, β‐oxidation, and oxidative phosphorylation and also serves as a substrate for posttranslational protein modifications, such as deacetylation and ADP‐ribosylation by sirtuins and poly(ADP‐ribose) polymerase (PARP), respectively. Many studies have demonstrated that NAD levels decrease with aging and that these declines cause various aging‐associated diseases. In contrast, activation of NAD metabolism prevents declines in NAD levels during aging. In particular, dietary supplementation with NAD precursors has been associated with protection against age‐associated insulin resistance. However, it remains unclear which NAD synthesis pathway is important and/or efficient at increasing NAD levels in vivo. In this study, Nmnat3 overexpression in mice efficiently increased NAD levels in various tissues and prevented aging‐related declines in NAD levels. We also demonstrated that Nmnat3‐overexpressing (Nmnat3 Tg) mice were protected against diet‐induced and aging‐associated insulin resistance. Moreover, in skeletal muscles of Nmnat3 Tg mice, TCA cycle activity was significantly enhanced, and the energy source for oxidative phosphorylation was shifted toward fatty acid oxidation. Furthermore, reactive oxygen species (ROS) generation was significantly suppressed in aged Nmnat3 Tg mice. Interestingly, we also found that concentrations of the NAD analog nicotinamide guanine dinucleotide (NGD) were dramatically increased in Nmnat3 Tg mice. These results suggest that Nmnat3 overexpression improves metabolic health and that Nmnat3 is an attractive therapeutic target for metabolic disorders that are caused by aging.

Identification of the Nicotinamide Salvage Pathway as a New Toxification Route for Antimetabolites

Interest in the modulation of nicotinamide adenine dinucleotide (NAD) metabolome is gaining great momentum because of its therapeutic potential indifferent human disorders. Suppression of nicotinamide salvage by nicotinamide phosphoribosyl transferase (NAMPT) inhibitors, however, gave inconclusive results in neoplastic patients because several metabolic routes circumvent the enzymatic block converging directly on nicotinamide mononucleotide adenylyl transferases (NMNATs) for NAD synthesis. Unfortunately, NMNAT inhibitors have not been identified. Here, we report the identification of Vacor as a substrate metabolized by theconsecutive action of NAMPT and NMNAT2 into the NAD analog Vacor adenine dinucleotide (VAD).This leads to inhibition of both enzymes, as wellas NAD-dependent dehydrogenases, thereby causing unprecedented rapid NAD depletion, glycolytic block, energy failure, and necrotic death of NMNAT2-proficient cancer cells. Conversely, lack of NMNAT2 expression confers complete resistance to Vacor. Remarkably, Vacor prompts VAD formation and growth suppression in NMNAT2-positive neuroblastoma and melanoma xenografts. Our data show the first evidence of harnessing the entire nicotinamide salvage pathway for antimetabolic strategies.

336 Increased Nicotinamide Phosphoribosyltransferase and Cystathionine B-Synthase in Renal Oncocytomas, Renal Transitional Cell Carcinoma, and Renal Cell Carcinoma

Renal cell carcinoma (RCC) and transitional cell carcinoma (TCC) are the two most common renal malignancies, while renal oncocytomas are common benign renal neoplasms. Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the rate-limiting step in nicotinamide adenine dinucleotide synthesis, while cystathionine B-synthase (CBS) catalyzes H2S synthesis. Both Nampt and CBS show concomitant increased expression in some tumors, with their expression positively correlating with higher tumor grade. We employed tissue microarray studies to analyze Nampt and CBS protein levels in benign renal cortex, RCC, TCC, and renal oncocytoma tissue samples. For Nampt, 29 benign, 49 TCCs, 94 RCCs, and six oncocytomas were examined. For CBS, 11 benign, 53 TCCs, 101 RCCs, and six oncocytomas were examined. The expression of both proteins was increased in TCC, RCC, and oncocytomas, compared to benign renal tissue. In RCC, the expression of both enzymes increased with increasing Fuhrman grade. To our knowledge, this is the first demonstration that Nampt and CBS are increased in renal RCC, TCC, and oncocytomas, and also increased with increasing RCC grade. Both enzymes promote tumor growth, survival, and angiogenesis in several malignancies. Increased expression of either enzyme in any oncocytoma type has not been previously reported. While preliminary, our data indicate that increased in Nampt and CBS protein expression, and possibly changes in H2S metabolism may have a role in benign and malignant renal neoplasia.

Conversion of PRPS Hexamer to Monomer by AMPK-Mediated Phosphorylation Inhibits Nucleotide Synthesis in Response to Energy Stress.

Tumors override energy stress to grow. However, how nucleotide synthesis is regulated under energy stress is unclear. We demonstrate here that glucose deprivation or hypoxia results in the AMPK-mediated phosphorylation of phosphoribosyl pyrophosphate synthetase 1 (PRPS1) S180 and PRPS2 S183, leading to conversion of PRPS hexamers to monomers and thereby inhibiting PRPS1/2 activity, nucleotide synthesis, and nicotinamide adenine dinucleotide (NAD) production. Knock-in of nonphosphorylatable PRPS1/2 mutants, which have uninhibited activity, in brain tumor cells under energy stress exhausts cellular ATP and NADPH and increases reactive oxygen species levels, thereby promoting cell apoptosis. The expression of those mutants inhibits brain tumor formation and enhances the inhibitory effect of the glycolysis inhibitor 2-deoxy-d-glucose on tumor growth. Our findings highlight the significance of recalibrating tumor cell metabolism by fine-tuning nucleotide and NAD synthesis in tumor growth.Significance: Our findings elucidate an instrumental function of AMPK in direct regulation of nucleic acid and NAD synthesis in tumor cells in response to energy stress. AMPK phosphorylates PRPS1/2, converts PRPS1/2 hexamers to monomers, and inhibits PRPS1/2 activity and subsequent nucleotide and NAD synthesis to maintain tumor cell growth and survival. Cancer Discov; 8(1); 94-107. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1.

NAD Deficiency, Congenital Malformations, and Niacin Supplementation

BackgroundCongenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients.MethodsWe used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 system.ResultsVariants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects.ConclusionsDisruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.)

Abstract 1942: The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, OT-82, exhibits in vitro and in vivo efficacy against patient-derived xenograft models of high-risk acute lymphoblastic leukemia

Abstract Cancer cells are highly dependent on nicotinamide phosphoribosyltransferase (NAMPT) for the biosynthesis of nicotinamide adenine dinucleotide (NAD). Besides its role in energy metabolism, NAMPT influences the activity of NAD-dependent enzymes, including poly (ADP-ribose) polymerase-1 (PARP-1) and sirtuins, and thereby regulates cellular survival and stress response. Disruption of NAD synthesis through NAMPT inhibition represents a potential therapeutic strategy for treating cancer. The aim of this study was to evaluate the efficacy of the novel NAMPT inhibitor OT-82, initially isolated for its selective toxicity against a panel of adult leukemia cell lines, in a diverse panel of leukemia cell lines in vitro and pediatric acute lymphoblastic leukemia (ALL) patient-derived xenografts (PDXs) in vivo, and to identify potential biomarkers predictive of OT-82 response. OT-82 demonstrated low nanomolar IC50 values (0.9 – 3.4 nM) in 3 ALL and 3 acute myeloid leukemia cell lines as determined by resazurin reduction assays. In vivo efficacy of OT-82 (40 mg/kg x 3 days x 3 weeks, p.o.) was evaluated as a single agent against pediatric ALL PDXs, including B-cell precursor ALL (n = 3), Philadelphia chromosome (Ph) positive ALL (n = 2), Ph-like ALL (n = 2), T-cell ALL (n = 3), and early T-cell precursor ALL (n = 3) in immune deficient (NSG) mice. Response to treatment was assessed by time to event or stringent objective response criteria modeled after the clinical setting. OT-82 was well tolerated, significantly increased event-free survival (EFS) relative to control mice in 11/13 ALL PDXs, and elicited objective responses in 11/13 (85%) PDXs [3 Partial Responses, 4 Complete Responses (CRs) and 4 Maintained CRs (MCRs)]. Analysis of basal protein expression revealed elevated levels of poly (ADP-ribosyl)ated (PARylated) PARP-1 in 4/5 responders versus 0/2 non-responders. In vitro studies examining various chemotherapeutic agents used for childhood leukemia showed synergy between cytarabine (AraC) and OT-82 in an ALL cell line. In an OT-82 sensitive Mixed Lineage Leukemia (MLL) PDX, treatment with AraC (25 mg/kg x 5 days x 2 weeks, i.p.) and OT-82 (40 mg/kg x 3 days x 2 weeks, p.o.) significantly increased EFS compared to OT-82 (P<0.0001) or AraC (P<0.0001) alone. Moreover, the OT-82/AraC combination elicited an MCR compared to OT-82 (CR) and AraC (Progressive Disease) alone. The results herein demonstrate significant activity of OT-82 against leukemia cell lines and a range of pediatric ALL subtypes in vivo, and the potential for PARylated PARP-1 expression as a biomarker for predicting OT-82 response. Moreover, the enhanced activity of OT-82 in combination with AraC over single agent therapy further suggests NAMPT inhibition as an attractive strategy for treating high-risk pediatric ALL. Supported by U01CA199000 from the NCI. Citation Format: Kathryn Evans, Tara Pritchard, Michelle J. Henderson, Klaartje Somers, Mawar Karsa, Leanna Cheung, Raymond Yung, Stephen W. Erickson, Lioubov Korotchkina, Olga Chernova, Andrei Gudkov, Malcolm A. Smith, Richard B. Lock. The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, OT-82, exhibits in vitro and in vivo efficacy against patient-derived xenograft models of high-risk acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1942. doi:10.1158/1538-7445.AM2017-1942

Diversity of the auxotrophic requirements in natural isolates of Escherichia coli.

Isolates of Escherichia coli, except Shigella, are generally prototrophic; they do not require any growth factors to grow in mineral medium. However, a nicotinic acid requirement is common among B2 phylogroup STc95 O18 E. coli clone strains. Nicotinic acid is a precursor of nicotinamide adenine dinucleotide (NAD), an essential molecule that plays central role in cellular metabolism. The defect in NAD synthesis of these strains is due to alterations in de novo biosynthesis pathway nadB gene. Here, by studying growth on minimal medium with glycolytic (glucose) or gluconeogenic (pyruvate or succinate) substrates as the carbon supply in a large panel of E. coli natural isolates representative of the species diversity, we identify an absolute nicotinic acid requirement in non-STc95 strains due in one case to a nadA inactivation. The growth on glucose medium of some extraintestinal pathogenic E. coli strains belonging to various non-O18 B2 phylogroup STc95 clones is restored either by aspartate or nicotinate, demonstrating that the nicotinic acid requirement can also be due to an intracellular aspartate depletion. The auxotrophic requirements depend on the carbon source available in the environment. Moreover, some strains prototrophic in glucose medium become auxotrophic in succinate medium, and conversely, some strainsauxotrophic in glucose medium become prototrophic in succinate medium. Finally, a partial depletion of intracellular aspartate can be observed in some prototrophic strains belonging to various phylogroups. The observed more or less significant depletion according to isolates may be due to differences in tricarboxylic acid cycle enzyme activities. These metabolic defects could be involved in the adaptation of E. coli to its various niches.

Amino-acid sensing and degrading pathways in immune regulation

Indoleamine 2,3-dioxygenases (IDOs) − belonging in the heme dioxygenase family and degrading tryptophan − are responsible for the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). As such, they are expressed by a variety of invertebrate and vertebrate species. In mammals, IDO1 has remarkably evolved to expand its functions, so to become a prominent homeostatic regulator, capable of modulating infection and immunity in multiple ways, including local tryptophan deprivation, production of biologically active tryptophan catabolites, and non-enzymatic cell-signaling activity. Much like IDO1, arginase 1 (Arg1) is an immunoregulatory enzyme that catalyzes the degradation of arginine. Here, we discuss the possible role of amino-acid degradation as related to the evolution of the immune systems and how the functions of those enzymes are linked by an entwined pathway selected by phylogenesis to meet the newly arising needs imposed by an evolving environment.

Salidroside protects PC12 cells from Aβ1‑40‑induced cytotoxicity by regulating the nicotinamide phosphoribosyltransferase signaling pathway.

Alzheimer's disease (AD) is the most common type of senile dementia, which often develops in elderly or presenile individuals. As one of the pathological features of AD, amyloid β-protein (Aβ) causes energy dysmetabolism, thereby inducing cellular damage and apoptosis. Salidroside is the main active component of the traditional Chinese medicine Rhodiola. Previous studies have demonstrated that salidroside exerts a regulatory role in energy metabolism. However, the role and the mechanism of action of salidroside in AD remain unclear. Therefore, the present study used Aβ1–40 to induce damage in PC12 cells, thereby establishing a cell model of AD. In addition, salidroside treatment was performed to investigate the protective effect of salidroside and the underlying mechanisms. Aβ1-40-induced neuronal toxicity reduced cell viability and caused cellular damage. As a result, the expression level of nicotinamide phosphoribosyltransferase (NAMPT) decreased, the synthesis of nicotinamide adenine dinucleotide (NAD+; an energy metabolism-associated coenzyme) became insufficient, and the NAD+/nicotinamide adenine dinucleotide hydride ratio was reduced. Administration of salidroside alleviated Aβ-induced cell damage and increased the expression level of the key protein NAMPT and the synthesis of NAD+. The results of the present study demonstrate that salidroside exerts a protective effect on Aβ1-40-damaged PC12 cells. The underlying mechanism may be associated with the regulation of energy metabolism that relies predominantly on the NAMPT signaling pathway.

EWS-FLI1 confers exquisite sensitivity to NAMPT inhibition in Ewing sarcoma cells

Ewing sarcoma (EwS) is the second most common bone cancer in children and adolescents with a high metastatic potential. EwS development is driven by a specific chromosomal translocation resulting in the generation of a chimeric EWS-ETS transcription factor, most frequently EWS-FLI1.Nicotinamide adenine dinucleotide (NAD) is a key metabolite of energy metabolism involved in cellular redox reactions, DNA repair, and in the maintenance of genomic stability. This study describes targeting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD synthesis, by FK866 in EwS cells. Here we report that blocking NAMPT leads to exhaustive NAD depletion in EwS cells, followed by a metabolic collapse and cell death. Using conditional EWS-FLI1 knockdown by doxycycline-inducible shRNA revealed that EWS-FLI1 depletion significantly reduces the sensitivity of EwS cells to NAMPT inhibition. Consistent with this finding, a comparison of 7 EwS cell lines of different genotypes with 5 Non-EwS cell lines and mesenchymal stem cells revealed significantly higher FK866 sensitivity of EWS-ETS positive EwS cells, with IC50 values mostly below 1nM.Taken together, our data reveal evidence of an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of EwS cells and suggest NAMPT inhibition as a potential new treatment approach for Ewing sarcoma.

Targeting a metabolic pathway to fight the flu

Our antiviral arsenal to fight influenza viruses is limited and we need novel anti-flu drugs. Recently, cellular drug targets came into focus and omics analysis were instrumental to suggest candidate factors. In this issue of The FEBS Journal, Kainov and colleagues used transcriptome data to investigate virus-induced changes in tryptophan metabolism that may serve as immunomodulatory approach against viruses.

Aspects of Tryptophan and Nicotinamide Adenine Dinucleotide in Immunity: A New Twist in an Old Tale.

Increasing evidence underscores the interesting ability of tryptophan to regulate immune responses. However, the exact mechanisms of tryptophan’s immune regulation remain to be determined. Tryptophan catabolism via the kynurenine pathway is known to play an important role in tryptophan’s involvement in immune responses. Interestingly, quinolinic acid, which is a neurotoxic catabolite of the kynurenine pathway, is the major pathway for the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). Recent studies have shown that NAD+, a natural coenzyme found in all living cells, regulates immune responses and creates homeostasis via a novel signaling pathway. More importantly, the immunoregulatory properties of NAD+ are strongly related to the overexpression of tryptophan hydroxylase 1 (Tph1). This review provides recent knowledge of tryptophan and NAD+ and their specific and intriguing roles in the immune system. Furthermore, it focuses on the mechanisms by which tryptophan regulates NAD+ synthesis as well as innate and adaptive immune responses.

Synthesis of a series of NAD+ analogues, potential inhibitors of PARP 1, using ADP conjugates functionalized at the terminal phosphate group

A convenient approach has been proposed to the synthesis of nicotinamide adenine dinucleotide (NAD+) mimetics, which comprise morpholino analogues of nucleosides. The approach is based on the use of ADP conjugates containing an amino group, which is tethered to the terminal phosphate through the aliphatic linker by the phosphodiester bond. We have synthesized four series of the NAD+ mimetics, which differ in the type of the modified nucleoside (2-aminomethylmorpholine (Mor) or 2-aminomethyl-4-carboxymethylmorpholine (MorGly) derivatives), in the linker length, and in the manner of the nucleoside attachment to the ADP derivative. We have studied the efficiency of NAD+ mimetics in the inhibition of the auto-poly(ADP-ribosyl)ation by the PARP 1 enzyme. The linker length, the mode of the attachment of the morpholino nucleoside analogue, and the nature of the heterocyclic base of the modified nucleoside were shown to inf luence the inhibition efficiency.

Stable isotope labeling by essential nutrients in cell culture (SILEC) for accurate measurement of nicotinamide adenine dinucleotide metabolism.

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are conserved metabolic cofactors that mediate reduction-oxidation (redox) reactions throughout all domains of life. The diversity of synthetic routes and cellular processes involving the transfer of reducing equivalents to and from these cofactors makes the accurate quantitation and metabolic tracing of NAD(H) and NADP(H) of broad interest. However, current analytical techniques typically rely on standard curves that do not incorporate confounding effects of the sample matrix. We utilized the essential requirement of niacin and tryptophan for NAD synthesis in mammalian cells to devise a stable isotope labeling by essential nutrients in cell culture (SILEC) method for efficient labeling of intracellular NAD(H) and NADP(H) pools. Coupling this approach with detection by liquid chromatography-high resolution mass spectrometry (LC-HRMS), we demonstrate the utility of incorporating a [13C315N1]-nicotinamide moiety into a library of NAD-derived metabolites for use as internal standards in matrixed samples. Using a two-label system incorporating [13C315N1]-nicotinamide and [13C11]-tryptophan, we quantify the relative contribution of salvage and de novo NAD synthesis, respectively, in S. cerevisiae and HepG2 human hepatocellular carcinoma cells under basal conditions. As a further proof-of-principle, we demonstrate an improvement in the linear range for quantification of NAD and apply this method to analysis of NAD(H) in mouse liver. This method demonstrates the generalizability of SILEC, and provides a simple method for generating a library of stable isotope labeled internal standards for quantifying and tracing the metabolism of cellular and tissue NAD(H) and NADP(H).

Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration.

NAD availability is limiting during liver regeneration, and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. (Hepatology 2017;65:616-630).

Essential role of Bordetella NadC in a quinolinate salvage pathway for NAD biosynthesis.

Nicotinamide adenine dinucleotide (NAD) is produced via de novo biosynthesis pathways and by salvage or recycling routes. The classical Bordetella bacterial species are known to be auxotrophic for nicotinamide or nicotinic acid. This study confirmed that Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis have the recycling/salvage pathway genes pncA and pncB, for use of nicotinamide or nicotinic acid, respectively, for NAD synthesis. Although these Bordetellae lack the nadA and nadB genes needed for de novo NAD biosynthesis, remarkably, they have one de novo pathway gene, nadC, encoding quinolinate phosphoribosyltransferase. Genomic analyses of taxonomically related Bordetella and Achromobacter species also indicated the presence of an 'orphan' nadC and the absence of nadA and nadB. When supplied as the sole NAD precursor, quinolinate promoted B. bronchiseptica growth, and the ability to use it required nadC. Co-expression of Bordetella nadC with the nadB and nadA genes of Paraburkholderia phytofirmans allowed B. bronchiseptica to grow in the absence of supplied pyridines, indicative of de novo NAD synthesis and functional confirmation of Bordetella NadC activity. Expression of nadC in B. bronchiseptica was influenced by nicotinic acid and by a NadQ family transcriptional repressor, indicating that these organisms prioritize their use of pyridines for NAD biosynthesis.