Nicotinamide Adenine Dinucleotide Kinase Research Articles

G-protein-coupled estrogen receptor 1 promotes peritoneal metastasis of gastric cancer through nicotinamide adenine dinucleotide kinase 1-mediated redox modulation.

Adipose tissue is the second most important site of estrogen production, where androgens are converted into estrogen by aromatase. While gastric cancer patients often develop adipocyte-rich peritoneal metastasis, the underlying mechanism remains unclear. In this study, we identified the G-protein-coupled estrogen receptor (GPER1) as a promoter of gastric cancer peritoneal metastasis. Functional invitro studies revealed that β-Estradiol (E2) or the GPER1 agonist G1 inhibited anoikis in gastric cancer cells. Additionally, genetic overexpression or knockout of GPER1 significantly inhibited or enhanced gastric cancer cell anoikis invitro and peritoneal metastasis invivo, respectively. Mechanically, GPER1 knockout disrupted the NADPH pool and increased reactive oxygen species (ROS) generation. Conversely, overexpression of GPER1 had the opposite effects. GPER1 suppressed nicotinamide adenine dinucleotide kinase 1(NADK1) ubiquitination and promoted its phosphorylation, which were responsible for the elevated expression of NADK1 at protein levels and activity, respectively. Moreover, genetic inhibition of NADK1 disrupted NADPH and redox homeostasis, leading to high levels of ROS and significant anoikis, which inhibited lung and peritoneal metastasis in cell-based xenograft models. In summary, our study suggests that inhibiting GPER1-mediated NADK1 activity and its ubiquitination may be a promising therapeutic strategy for peritoneal metastasis of gastric cancer.

Biochemical and metabolomics analyses reveal the mechanisms underlying ascorbic acid and chitosan coating mediated energy homeostasis in postharvest papaya fruit

Papaya is a climacteric fruit that undergoes rapid ripening and quality deterioration during postharvest storage, resulting in significant economic losses. This study employed biochemical techniques and targeted metabolomics to investigate the impact of exogenous AsA + CTS application on the energy metabolism regulation of papaya fruit during postharvest storage. We found that AsA + CTS treatment significantly increased the levels of key metabolic compounds and enzymes, such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and the energy charge, as well as the succinic acid content and the activities of succinic dehydrogenase (SDH), cytochrome c oxidase (CCO), H+-ATPase, and Ca2+-ATPase. Moreover, AsA + CTS coating augmented the nicotinamide adenine dinucleotide kinase (NADK) activity and increased the NADH and NADPH concentrations. Regarding sugar metabolism, it increased the activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase and raised d-glucose-6-phosphate levels. These findings suggest that AsA + CTS coating application can mitigate the metabolic deterioration and sustain a primary metabolism homeostasis in papaya fruit by enhancing the tricarboxylic acid (TCA) cycle and pentose phosphate pathway (PPP), thereby preserving their quality attributes during postharvest storage.

Acidic electrolyzed water treatment delays the senescence of ‘Lingwu long’ jujube fruit during cold storage by regulating energy and respiratory metabolism

Jujube fruit may rapidly undergo maturity and senescence during storage, seriously affecting its commercialization. The study aimed to evaluate the role of acidic electrolyzed water (AEW) on energy and respiratory metabolism, and senescence in jujube fruit during cold storage. The results indicated that AEW improved ATP content and energy charge via increasing succinate dehydrogenase, cytochrome C oxidase, H+-ATPase, and Ca2+-ATPase activities and delayed jujube fruit senescence. The treatment was found to improve the activities of nicotinamide adenine dinucleotide kinase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase, and the content of NADPH, and reduce oxidative stress. Transcriptome analysis suggested that AEW regulated the expression of the genes associated with glycolysis, citrate cycle, pentose phosphate pathway, oxidative phosphorylation, ROS metabolism, transcription factors, phenylpropane biosynthesis, cell wall metabolism, and plant hormone signal transduction. Therefore, we concluded that AEW may delay the senescence of postharvest jujube fruit by regulating expression of the relevant genes and metabolic pathways.

Phenyllactic acid maintains the storage quality of ‘Zaosu’ pears by regulating respiration and energy metabolism

Respiration and energy metabolism are intricately linked to the quality and senescence of fruit after harvest. This investigation was carried out to examine alternations in quality parameters, respiration, and energy metabolism of ‘Zaosu’ pears subsequent to phenyllactic acid immersion. Results demonstrated that phenyllactic acid treatment led to a reduction in L* , a* , b* values, ΔE, and mass loss. Additionally, it maintained ascorbic acid, reducing sugar, soluble sugar, soluble solids, and chlorophyll contents; delayed the decrease in flesh firmness and titratable acidity of pears. The activities and gene expressions of glucose-6-phosphate dehydrogenase, glucose phosphate isomerase, hexokinase, nicotinamide adenine dinucleotide kinase, cytochrome C oxidase, and succinate dehydrogenasepears in pears were enhanced by phenyllactic acid. Moreover, phenyllactic acid promoted PbPK, PbAOX, PbH+-ATPase, and PbCa2+-ATPase expressions, while inhibiting malate dehydrogenase activity and the expressions of PbPFK and PbMDH in pears. Phenyllactic acid also led to a decrease in the levels of nicotinamide adenine dinucleotide hydrogen and nicotinamide adenine dinucleotide, while causing an increase in the levels of nicotinamide adenine dinucleotide phosphate hydrogen and nicotinamide adenine dinucleotide phosphate within the fruit. Collectively, postharvest phenyllactic acid treatment can effectively preserve the storage quality of ‘Zaosu’ pears by regulating the activity and gene expression of the key enzymes involved in respiration and energy metabolism.

Acidic electrolyzed oxidizing water delayed the breakdown occurrence in pulp of fresh longan by regulating the metabolisms of respiratory and energy

The efficacy of acidic electrolyzed oxidizing water (AEOW) treatment on the respiratory and energy metabolism during storage of harvested longan and its relationship with pulp breakdown were investigated. Results confirmed that the AEOW treatment exerted a significant inhibitory effect on longan pulp breakdown, with 15.5 % lower pulp breakdown index than the control at 6 d. Compared to the control fruit, the AEOW treatment displayed lower activities of polyphenol oxidase (PPO), ascorbic acid oxidase (AAO), alternate oxidase (AOX), cytochrome C oxidase (CCO), succinate dehydrogenase (SDH), pulp phosphohexose isomerase (PGI), and higher levels of nicotinamide adenine dinucleotide kinase (NADK), glucose-6-phosphate dehydrogenase (G-6-PDH) and glucose-6-phosphate dehydrogenase (6-PGDH). Furthermore, the AEOW treatment increased the amount of NADPH and NADP while decreasing that of NADH and NAD. In terms of energy metabolism, the AEOW treatment maintained higher levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), energy charge, H+-ATPase, Ca2+-ATPase, and Mg2+-ATPase, but lower content of adenosine monophosphate (AMP). Therefore, the AEOW treatment inhibited respiratory metabolism by decreasing the terminal oxidase activities, tricarboxylic acid cycle (TCA cycle) and glycolytic pathway (EMP), and increasing the pentose phosphate pathway (PPP), which helped to maintain a higher energy status and further led to attenuation of longan pulp breakdown.

The role of respiratory metabolism in chilling injury development of Chinese olive fruit during cold storage

Chilling injury (CI) and browning occurrence are pivotal issues during the cold storage of postharvest Chinese olive fruit. This study evaluates the effects of non-chilling temperature (8 °C) and chilling temperature (2 °C) on CI development, browning occurrence, and respiratory metabolism in postharvest Chinese olives. Compared to storage at 8 °C, storage at 2 °C significantly induced CI, accompanied by higher indices of CI and browning, as well as a higher fruit respiratory rate. Storage at 2 °C increased the activities of phosphohexose isomerase (PGI), succinate dehydrogenase (SDH), ascorbic acid oxidase (AAO), and cytochrome C oxidase (CCO) by 55.1%, 11.1%, 26.4%, and 98.8%, respectively, on day 120. On the other hand, storage at 2 °C reduced the activities of NAD kinase (NADK), 6-phosphogluconate dehydrogenase (6-PGDH) + glucose-6-phosphate dehydrogenase (G-6-PDH) by 95.6% and 6.6%, respectively, on day 120. The contents of nicotinamide adenine dinucleotide (NAD) and its reduced form of NAD (NADH) increased by 10.0% and 22.8%, respectively, in fruit stored at 2 °C on day 120. Conversely, the contents of nicotinamide adenine dinucleotide phosphate (NADP) and its reduced form (NADPH) decreased by 58.3% and 64.4%, respectively, in fruit stored 2 °C on day 120. These findings indicate that CI development and browning occurrence in Chinese olives induced by storage at 2 °C were associated with a higher respiratory rate, increased activities of PGI, SDH, AAO, and CCO, higher contents of NAD(H), reduced activities of 6-PGDH + G-6-PDH, and lower contents of NADP(H).

Metabolically distinct roles of NAD synthetase and NAD kinase define the essentiality of NAD and NADP in Mycobacterium tuberculosis.

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated derivative (NADP) are essential cofactors that participate in hundreds of biochemical reactions and have emerged as therapeutic targets in cancer, metabolic disorders, neurodegenerative diseases, and infections, including tuberculosis. The biological basis for the essentiality of NAD(P) in most settings, however, remains experimentally unexplained. Here, we report that inactivation of the terminal enzyme of NAD synthesis, NAD synthetase (NadE), elicits markedly different metabolic and microbiologic effects than those of the terminal enzyme of NADP biosynthesis, NAD kinase (PpnK), in Mycobacterium tuberculosis (Mtb). Inactivation of NadE led to parallel reductions of both NAD and NADP pools and Mtb viability, while inactivation of PpnK selectively depleted NADP pools but only arrested growth. Inactivation of each enzyme was accompanied by metabolic changes that were specific for the affected enzyme and associated microbiological phenotype. Bacteriostatic levels of NAD depletion caused a compensatory remodeling of NAD-dependent metabolic pathways in the absence of an impact on NADH/NAD ratios, while bactericidal levels of NAD depletion resulted in a disruption of NADH/NAD ratios and inhibition of oxygen respiration. These findings reveal a previously unrecognized physiologic specificity associated with the essentiality of two evolutionarily ubiquitous cofactors. IMPORTANCE The current course for cure of Mycobacterium tuberculosis (Mtb)-the etiologic agent of tuberculosis (TB)-infections is lengthy and requires multiple antibiotics. The development of shorter, simpler treatment regimens is, therefore, critical to the goal of eradicating TB. NadE, an enzyme required for the synthesis of the ubiquitous cofactor NAD, is essential for survival of Mtb and regarded as a promising drug target. However, the basis of this essentiality was not clear due to its role in the synthesis of both NAD and NADP. Here, we resolve this ambiguity through a combination of gene silencing and metabolomics. We specifically show that NADP deficiency is bacteriostatic, while NAD deficiency is bactericidal due to its role in Mtb's respiratory capacity. These results argue for a prioritization of NAD biosynthesis inhibitors in anti-TB drug development.

Alternaria alternata stimulates blackhead disease development of ‘Korla’ fragrant pear (Pyrus bretschneideri Rehd) by regulating energy status and respiratory metabolism

Blackhead disease of ‘Korla’ fragrant pear (Pyrus bretschneideri Rehd), caused by the fungus Alternaria alternata (A. alternata), limits postharvest pear fruit storage and transportation. Herein, we explored the effects of A. alternata infection on respiratory metabolism and energy status of pear fruit, and their correlations with disease development. Compared with control fruit, A. alternata-inoculated fruit displayed a higher respiration rate and disease index. Infection increased the gene expression and enzyme activities of phosphohexose isomerase (PGI), alternative oxidase (AOX), succinate dehydrogenase (SDH) and cytochrome C oxidase (CCO) in pear fruit, and decreased those of 6-phosphogluconate dehydrogenase (6PGDH) and glucose-6-phosphate dehydrogenase (G6PDH) in the late storage period. Infection reduced nicotinamide adenine dinucleotide kinase (NADK) activity and expression level in fruit, inhibited NADP accumulation, and increased NAD, NADH, and NADPH contents in the early storage period. Infection also lowered adenosine triphosphate (ATP), adenosine diphosphate (ADP) and energy charge (EC) levels as well as ATPase activity, and enhanced adenosine monophosphate (AMP) content and PbAtpB expression levels, resulting in decreased fruit energy status. All these results suggest that disease development may be connected to the enhancement of the Embden-Meyerhof-Parnas (EMP) pathway, the tricarboxylic acid (TCA) cycle, the cytochrome pathway (CCP), the alternative pathway (AP), the increased then decrease in the pentose phosphate pathway (PPP), and reduced energy status in pear fruit.

A prognostic NAD+ metabolism-related gene signature for predicting response to immune checkpoint inhibitor in glioma.

Nicotinamide adenine dinucleotide (NAD+) metabolism is involved in a series of cancer pathogenesis processes, and is considered a promising therapeutic target for cancer treatment. However, a comprehensive analysis of NAD+ metabolism events on immune regulation and cancer survival has not yet been conducted. Here, we constructed a prognostic NAD+ metabolism-related gene signature (NMRGS) associated with immune checkpoint inhibitor (ICI) efficacy in glioma. 40 NAD+ metabolism-related genes (NMRGs) were obtained from the Reactome database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Glioma cases with transcriptome data and clinical information were obtained from Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA). NMRGS was constructed based on the calculated risk score using univariate analysis, Kaplan-Meier analysis, multivariate Cox regression, and nomogram. This NMRGS was verified in training (CGGA693) and validation (TCGA and CGGA325) cohorts. The immune characteristics, mutation profile, and response to ICI therapy were subsequently analyzed for different NMRGS subgroups. Six NAD+ metabolism-related genes, including CD38, nicotinamide adenine dinucleotide kinase (NADK), nicotinate phosphoribosyltransferase (NAPRT), nicotinamide/nicotinic acid mononucleotide adenylyltransferase 3 (NMNAT3), poly(ADP-Ribose) polymerase family member 6 (PARP6), and poly(ADP-Ribose) polymerase family member 9 (PARP9), were ultimately used to construct a comprehensive risk model for glioma patients. Patients in the NMRGS-high group showed a poorer survival outcome than those in the NMRGS-low group. The area under curve (AUC) indicated that NMRGS has good potential in glioma prognostic prediction. A nomogram with improved accuracy was established based on independent prognostic factors (NMRGS score, 1p19q codeletion status, and WHO grade). Furthermore, patients in the NMRGS-high group showed a more immunosuppressive microenvironment, higher tumor mutation burden (TMB), higher human leucocyte antigen (HLA) expression and a more therapeutic response to ICI therapy. This study constructed a prognostic NAD+ metabolism-related signature associated with the immune landscape in glioma, which can be used for guiding individualized ICI therapy.

Nicotinamide adenine dinucleotide kinase promotes lymph node metastasis of NSCLC via activating ID1 expression through BMP pathway.

Metastasis is a significant cause of high mortality in lung cancer. Lymph node (LN) metastasis is the most common metastatic pathway in non-small cell lung cancer and the most crucial factor affecting the prognosis of NSCLC. Nevertheless, the molecular mechanism underlying metastasis is unknown. We demonstrated that higher NADK expression suggests worsened survival prognosis, and NADK expression positively correlates with the lymph node metastasis rate and TNM and AJCC stages in NSCLC patients. Moreover, patients with LN metastasis show higher NADK expression than those without LN metastasis. NADK can promote NSCLC progression by enhancing the migration, invasion, lymph node metastasis and growth of NSCLC cells. Mechanistically, NADK inhibits the ubiquitination and degradation of BMPR1A by interacting with Smurf1, further activating the BMPs signalling pathway and promoting ID1 transcription. In conclusion, NADK may be a potential diagnostic indicator and a novel therapeutic target for metastatic NSCLC.

(-)-Epigallocatechin Gallate is a Noncompetitive Inhibitor of NAD Kinase.

Nicotinamide adenine dinucleotide kinase (NADK) controls the intracellular NADPH content and provides reducing power for the synthesis of macromolecules and anti-ROS. Moreover, NADK is considered to be a synthetic lethal gene for KRAS mutations. To discover NADK-targeted probes, a high-throughput screening assay was established and optimized with a Z factor of 0.71. The natural product (-)-epigallocatechin gallate (EGCG) was found to be a noncompetitive inhibitor of NADK with K i = 3.28 ± 0.32 μΜ. The direct binding of EGCG to NADK was determined by several biophysical methods, including NMR spectroscopy, surface plasmon resonance (SPR) assay, and hydrogen-deuterium exchange mass spectrometry (HDX-MS). The SPR assay showed a K d of 1.78 ± 1.15 μΜ. The HDX-MS experiment showed that EGCG was bound at the non-substrate-binding sites of NADK. Besides, binding mode prediction and derivative activity analysis revealed a potential structure-activity relationship between EGCG and NADK. Furthermore, EGCG can specifically inhibit the proliferation of KRAS-mutated lung cancer cell lines without affecting KRAS wild-type lung cancer cell lines.

Application of melatonin delays lignification in postharvest water bamboo shoots in association with energy metabolism

Lignification occurs in some vegetables and fruit after harvest and lowers their quality and commercial value. This study investigated the effects of melatonin (MT) on the energy metabolism of postharvest water bamboo shoots (WBS) and their relationship with lignification. We found that lignin accumulation could result in insufficient energy in postharvest WBS. The ATP content of the melatonin treatment group was 1.7 times higher than that of the control. Melatonin slowed down the decrease in the energy status by activating succinic dehydrogenase (SDH), cytochrome c oxidase (CCO), H+-ATPase, Mg2+-ATPase, and Ca2+-ATPase, as well as the transcription levels of their corresponding genes in WBS. Moreover, melatonin treatment increased nicotinamide adenine dinucleotide phosphate (NADP) and nicotinamide adenine dinucleotide phosphate reduced form (NADPH) content by promoting nicotinamide adenine dinucleotide kinase (NADK) activity as well as by maintaining lower nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide reduced form (NADH) levels during storage. The correlation study revealed a negative relationship between lignin accumulation and energy metabolism in postharvest WBS. These findings supported the notion that the application of MT is a viable and effective method for alleviating lignification and supplying adequate energy to harvested WBS.

Structure-based design, synthesis and biological evaluation of a NAD+ analogue targeting Pseudomonas aeruginosa NAD kinase.

Multidrug resistance is a major public health problem that requires the urgent development of new antibiotics and therefore the identification of novel bacterial targets. The activity of nicotinamide adenine dinucleotide kinase, NADK, is essential in all bacteria tested so far, including many human pathogens that display antibiotic resistance leading to the failure of current treatments. Inhibiting NADK is therefore a promising and innovative antibacterial strategy since there is currently no drug on the market targeting this enzyme. Through a fragment-based drug design approach, we have recently developed a NAD+ -competitive inhibitor of NADKs, which displayed in vivo activity against Staphylococcus aureus. Here, we show that this compound, a di-adenosine derivative, is inactive against the NADK enzyme from the Gram-negative bacteria Pseudomonas aeruginosa (PaNADK). This lack of activity can be explained by the crystal structure of PaNADK, which was determined in complex with NADP+ in this study. Structural analysis led us to design and synthesize a benzamide adenine dinucleoside analogue, active against PaNADK. This novel compound efficiently inhibited PaNADK enzymatic activity in vitro with a Ki of 4.6 μm. Moreover, this compound reduced P. aeruginosa infection in vivo in a zebrafish model.

The mitochondrial NAD kinase functions as a major metabolic regulator upon increased energy demand

ObjectiveThe mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (MNADK) mediates de novo mitochondrial NADP biosynthesis by catalyzing the phosphorylation of NAD to yield NADP. In this study, we investigated the function and mechanistic basis by which MNADK regulates metabolic homeostasis. MethodsGeneralized gene set analysis by aggregating human patient genomic databases, metabolic studies with genetically engineered animal models, mitochondrial bioenergetic analysis, as well as gain- and loss- of-function studies were performed to address the functions and mechanistic basis by which MNADK regulates energy metabolism and redox state associated with metabolic disease. ResultsHuman MNADK common gene variants or decreased expression of the gene are significantly associated with the occurrence of type-2 diabetes, non-alcoholic fatty liver disease (NAFLD), or hepatocellular carcinoma (HCC). Ablation of the MNADK gene in mice led to decreased fat oxidation, coincident with increased respiratory exchange ratio (RER) and decreased energy expenditure upon energy demand triggered by endurance exercise or fasting. On an atherogenic high-fat diet (HFD), MNADK-null mice exhibited hepatic insulin resistance and glucose intolerance, indicating a type-2 diabetes-like phenotype in the absence of MNADK. MNADK deficiency led to a decrease in mitochondrial NADP(H) but an increase in cellular reactive oxygen species (ROS) in mouse livers. Consistently, protein levels of the major metabolic regulators or enzymes were decreased, while their acetylation modifications were increased in the livers of MNADK-null mice. Feeding mice with a HFD caused S-nitrosylation (SNO) modification, a posttranslational modification that represses protein activities, on MNADK protein in the liver. Reconstitution of an SNO-resistant MNADK variant, MNADK-S193, into MNADK-null mice mitigated hepatic steatosis induced by HFD. ConclusionMNADK, the only known mammalian mitochondrial NAD kinase, plays important roles in preserving energy homeostasis to mitigate the risk of metabolic disorders.

G6PD-mediated increase in de novo NADP+ biosynthesis promotes antioxidant defense and tumor metastasis.

Metastasizing cancer cells are able to withstand high levels of oxidative stress through mechanisms that are poorly understood. Here, we show that under various oxidative stress conditions, pancreatic cancer cells markedly expand NADPH and NADP+ pools. This expansion is due to up-regulation of glucose-6-phosphate dehydrogenase (G6PD), which stimulates the cytoplasmic nicotinamide adenine dinucleotide kinase (NADK1) to produce NADP+ while converting NADP+ to NADPH. G6PD is activated by the transcription factor TAp73, which is, in turn, regulated by two pathways. Nuclear factor–erythroid 2 p45-related factor-2 suppresses expression of the ubiquitin ligase PIRH2, stabilizing the TAp73 protein. Checkpoint kinases 1/2 and E2F1 induce expression of the TAp73 gene. Levels of G6PD and its upstream activators are elevated in metastatic pancreatic cancer. Knocking down G6PD impedes pancreatic cancer metastasis, whereas forced G6PD expression promotes it. These findings reveal an intracellular network that maintains redox homeostasis through G6PD-mediated increase in de novo NADP+ biosynthesis, which may be co-opted by tumor cells to enable metastasis.

Mouse models of NADK2 deficiency analyzed for metabolic and gene expression changes to elucidate pathophysiology.

NADK2 encodes the mitochondrial form of nicotinamide adenine dinucleotide (NAD) kinase, which phosphorylates NAD. Rare recessive mutations in human NADK2 are associated with a syndromic neurological mitochondrial disease that includes metabolic changes, such as hyperlysinemia and 2,4 dienoyl CoA reductase (DECR) deficiency. However, the full pathophysiology resulting from NADK2 deficiency is not known. Here, we describe two chemically induced mouse mutations in Nadk2-S326L and S330P-which cause severe neuromuscular disease and shorten lifespan. The S330P allele was characterized in detail and shown to have marked denervation of neuromuscular junctions by 5weeks of age and muscle atrophy by 11weeks of age. Cerebellar Purkinje cells also showed progressive degeneration in this model. Transcriptome profiling on brain and muscle was performed at early and late disease stages. In addition, metabolomic profiling was performed on the brain, muscle, liver and spinal cord at the same ages and on plasma at 5weeks. Combined transcriptomic and metabolomic analyses identified hyperlysinemia, DECR deficiency and generalized metabolic dysfunction in Nadk2 mutant mice, indicating relevance to the human disease. We compared findings from the Nadk model to equivalent RNA sequencing and metabolomic datasets from a mouse model of infantile neuroaxonal dystrophy, caused by recessive mutations in Pla2g6. This enabled us to identify disrupted biological processes that are common between these mouse models of neurological disease, as well as those processes that are gene-specific. These findings improve our understanding of the pathophysiology of neuromuscular diseases and describe mouse models that will be useful for future preclinical studies.

NAD kinase promotes Staphylococcus aureus pathogenesis by supporting production of virulence factors and protective enzymes.

Nicotinamide adenine dinucleotide phosphate (NADPH) is the primary electron donor for reductive reactions that are essential for the biosynthesis of major cell components in all organisms. Nicotinamide adenine dinucleotide kinase (NADK) is the only enzyme that catalyzes the synthesis of NADP(H) from NAD(H). While the enzymatic properties and physiological functions of NADK have been thoroughly studied, the role of NADK in bacterial pathogenesis remains unknown. Here, we used CRISPR interference to knock down NADK gene expression to address the role of this enzyme in Staphylococcus aureus pathogenic potential. We find that NADK inhibition drastically decreases mortality of zebrafish infected with S. aureus. Furthermore, we show that NADK promotes S. aureus survival in infected macrophages by protecting bacteria from antimicrobial defense mechanisms. Proteome-wide data analysis revealed that production of major virulence-associated factors is sustained by NADK. We demonstrate that NADK is required for expression of the quorum-sensing response regulator AgrA, which controls critical S. aureus virulence determinants. These findings support a key role for NADK in bacteria survival within innate immune cells and the host during infection.

Uncovering the Conditionally Essential Roles of NAD Kinases in Human Cells

Forward genetic screens across hundreds of human cancer cell lines have been used to identify genetic dependencies of proliferating human cells and how these vary by genotype and cell lineage. However, while it is appreciated that environmental factors influence cell physiology, there has been little investigation into how nutrient availability impacts gene essentiality. Further, most such screens have been performed in vitro using culture media that poorly reflect the metabolic composition of human blood. Previously, we developed Human Plasma‐Like Medium (HPLM), a physiologic medium designed to more closely reflect the biochemical conditions in human blood. To determine how medium composition affects gene essentiality, we recently performed CRISPR‐based screens of K562 leukemia cells in HPLM versus RPMI, the medium historically used to culture human blood cells. Among the strongest scoring HPLM‐essential genes is Nicotinamide Adenine Dinucleotide Kinase (NADK), which encodes one of two human NAD kinases (NADK, cytosolic; NADK2, mitochondrial) that can catalyze the phosphorylation of NAD(H)to NADP(H). While most human cells co‐express both isozymes, only NADK was identified as HPLM‐essential, suggesting that endogenous NADK2 expression is not sufficient to complement NADK knockout in HPLM‐cultured cells. Interestingly, NADK has been annotated as an essential gene in less than 1% of over 1000 genome‐wide CRISPR‐based screens from the Broad Institute Cancer Dependency Map project. To begin to investigate the conditional CRISPR phenotype for NADK, we first engineered NADK‐knockout cells and then found that they showed a 40% growth defect versus control cells in HPLM relative to RPMI using a short‐term growth assay. Next, to test the hypothesis that cytosolic NAD kinase activity is required for the conditionally essential role of NADK, we performed similar short‐term growth assays with NADK‐knockout cells expressing cDNAs for wild‐type and cytosol‐restricted NADK2, as well as kinase‐dead NADK. These experiments revealed that neither kinase‐dead NADK nor NADK2 overexpression could rescue the growth defect of NADK‐knockout cells, but that cytosol‐restricted NADK2 could. Collectively, these results indicate that the conditionally essential role of NADK is linked to its cytosolic NAD kinase activity. Currently, we are applying systematic approaches in media engineering and metabolomics to identify the medium component(s) that contribute to the NADK gene‐nutrient interaction, and to gain insights into the non‐redundant, context‐dependent role of NADK. Lastly, through further conditional essentiality profiling, we identified NADK2as DMEM‐essential versus HPLM. Consistent with two recently published studies, we traced this effect to the differential availability of proline between HPLM and DMEM. Collectively, our results demonstrate the human NADKs fulfill distinct cell‐essential roles and form distinct gene‐nutrient interactions that vary with their subcellular localization.

Polyphosphate-dependent nicotinamide adenine dinucleotide (NAD) kinase: A novel missing link in human mitochondria.

Polyphosphate [poly(P)] is described as a homopolymer of inorganic phosphates. Nicotinamide adenine dinucleotide kinase (NAD kinase) catalyzes the phosphorylation of NAD+ to NADP+ in the presence of ATP (ATP-NAD kinase). Novel NAD kinase that explicitly phosphorylates NAD+ to NADP+ using poly(P), besides ATP [ATP/poly(P)-NAD kinase], was found in bacteria, in particular, Gram-positive bacteria, and the gene encoding ATP/poly(P)-NAD kinase was also newly identified in Mycobacterium tuberculosis H37Rv. Both NAD kinases required multi-homopolymeric structures for activity expression. The enzymatic and genetic results, combined with their primary and tertiary structures, have led to the discovery of a long-awaited human mitochondrial NAD kinase. This discovery showed that the NAD kinase is a bacterial type of ATP/poly(P)-NAD kinase. These pioneering findings, i.e., ATP/poly(P)-NAD kinase, NAD kinase gene, and human mitochondrial NAD kinase, have significantly enhanced research on the biochemistry, molecular biology, and evolutionary biology of NAD kinase, mitochondria, and poly(P), including some biotechnological knowledge applicable to NADP+ production.

Enhancing NAD+ Metabolome in Cardiovascular Diseases: Promises and Considerations.

Nicotinamide adenine dinucleotide (NAD) is not only the master regulator of energy metabolism pathways but also an essential substrate for several key enzymes of health and disease states (1, 2). NAD+/NADH redox coupling is vital for fueling glycolysis, tricarboxylic acid (TCA) cycle and electron transport chain (ETC) for producing ATP. In addition to that, NAD+ is a substrate for NAD-dependent deacetylases called sirtuins (SIRTs), poly-ADP-ribose polymerases (PARPs), monoADP-ribosyltransferases (ARTDs), ADP ribose synthetases (CD38/CD157), and sterile alpha and toll/interleukin-1 receptor motif-containing protein 1 (SARM1), etc. (1). NAD kinase also utilizes NAD+ to generate nicotinamide adenine dinucleotide phosphate (NADP+/NADPH) which is consumed by reactive oxygen species producing enzymes, NADPH oxidases. For maintaining cellular homeostasis, NAD+ levels are tightly regulated through its salvage pathway where the by-product of enzymatic reactions, nicotinamide adenine dinucleotide (NAM), is converted back to NAD+ by the rate limiting enzyme NAM phosphoribosyl transferase (NAMPT) (1, 2). Other biosynthesis pathways include de novo synthesis from tryptophan (Trp), and from the dietary uptake of its precursors- nicotinic acid (NA) or nicotinamide riboside (NR) (1, 2).