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Query Topic: Nicotinamide Mononucleotide

Query Date:

ec 2 4 2 11(6)

Characterization of human nicotinate phosphoribosyltransferase: Kinetic studies, structure prediction and functional analysis by site-directed mutagenesis.
Nicotinate phosphoribosyltransferase (NaPRT, EC catalyzes the conversion of nicotinate (Na) to nicotinate mononucleotide, the first reaction of the Preiss-Handler pathway for the biosynthesis of NAD(+). Even though NaPRT activity has been described to be responsible for the ability of Na to increase NAD(+) levels in human cells more effectively than nicotinamide (Nam), so far a limited number of studies on the human NaPRT have appeared. Here, extensive characterization of a recombinant human NaPRT is reported. We determined its major kinetic parameters and assayed the influence of different compounds on its enzymatic activity. In particular, ATP showed an apparent dual stimulation/inhibition effect at low/high substrates saturation, respectively, consistent with a negative cooperativity model, whereas inorganic phosphate was found to act as an activator. Among other metabolites assayed, including nucleotides, nucleosides, and intermediates of carbohydrates metabolism, some showed inhibitory properties, i.e. CoA, several acyl-CoAs, glyceraldehyde 3-phosphate, phosphoenolpyruvate, and fructose 1,6-bisphosphate, whereas dihydroxyacetone phosphate and pyruvate exerted a stimulatory effect. Furthermore, in light of the absence of crystallographic data, we performed homology modeling to predict the protein three-dimensional structure, and molecular docking simulations to identify residues involved in the recognition and stabilization of several ligands. Most of these residues resulted universally conserved among NaPRTs, and, in this study, their importance for enzyme activity was validated through site-directed mutagenesis.
Publication Date: 2011-07-12
Journal: Biochimie

ec 2 4 2 12(3)

Weak coupling of ATP hydrolysis to the chemical equilibrium of human nicotinamide phosphoribosyltransferase.
Human nicotinamide phosphoribosyltransferase (NAMPT, EC catalyzes the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PP i) from nicotinamide (NAM) and alpha- d-5-phosphoribosyl-1-pyrophosphate (PRPP). NAMPT, by capturing the energy provided by its facultative ATPase activity, allows the production of NMN at product:substrate ratios thermodynamically forbidden in the absence of ATP. With ATP hydrolysis coupled to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold, substrate affinity dramatically increases ( K m (NAM) from 855 to 5 nM), and the K eq shifts -2.1 kcal/mol toward NMN formation. ADP-ATP isotopic exchange experiments support the formation of a high-energy phosphorylated intermediate (phospho-H247) as the mechanism for altered catalytic efficiency during ATP hydrolysis. NAMPT captures only a small portion of the energy generated by ATP hydrolysis to shift the dynamic chemical equilibrium. Although the weak energetic coupling of ATP hydrolysis appears to be a nonoptimized enzymatic function, closer analysis of this remarkable protein reveals an enzyme designed to capture NAM with high efficiency at the expense of ATP hydrolysis. NMN is a rate-limiting precursor for recycling to the essential regulatory cofactor, nicotinamide adenine dinucleotide (NAD (+)). NMN synthesis by NAMPT is powerfully inhibited by both NAD (+) ( K i = 0.14 muM) and NADH ( K i = 0.22 muM), an apparent regulatory feedback mechanism.
Publication Date: 2008-10-01
Journal: Biochemistry

ec 3 5 1 19(2)

Changes in trigonelline (N-methylnicotinic acid) content and nicotinic acid metabolism during germination of mungbean (Phaseolus aureus) seeds.
Changes in trigonelline content and in biosynthetic activity were determined in the cotyledons and embryonic axes of etiolated mungbean (Phaseolus aureus) seedlings during germination. Accumulation of trigonelline (c. 240 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds; trigonelline content decreased 2 d after imbibition. Trigonelline content in the embryonic axes increased with seedling growth and reached a peak (c. 380 nmol per embryonic axis) at day 5. Trigonelline content did not change significantly during the differentiation of hypocotyls, and the concentration was greatest in the apical 5 mm. Nicotinic acid and nicotinamide were better precursors for pyridine nucleotide synthesis than quinolinic acid, but no great differences were found in the synthesis of trigonelline from these three precursors. Trigonelline synthesis was always higher in embryonic axes than in cotyledons. Activity of quinolinate phosphoribosyltransferase (EC, nicotinate phosphoribosyltransferase (EC, and nicotinamidase (EC was found in cotyledons and embryonic axes, but no nicotinamide phosphoribosyltransferase (EC activity was detected. It follows that quinolinic acid and nicotinic acid were directly converted to nicotinic acid mononucleotide by the respective phosphoribosyltransferases, but nicotinamide appeared to be converted to nicotinic acid mononucleotide after conversion to nicotinic acid. Trigonelline synthase (nicotinate N-methyltransferase, EC activity increased in the embryonic axes, but decreased in cotyledons during germination. [14C]Nicotinic acid and trigonelline absorbed by the cotyledons were transported to the embryonic axes during germination. Trigonelline had no effect on the growth of seedlings, but nicotinic acid and nicotinamide significantly inhibited the growth of roots. Based on these findings, the role of trigonelline synthesis in mungbean seedlings is discussed.
Publication Date: 2005-04-20
Journal: Journal of experimental botany

reduced nicotinamide adenine dinucleotide(50)

Extracellular pH affects the fluorescence lifetimes of metabolic co-factors.
Autofluorescence measurements of the metabolic cofactors NADH and flavin adenine dinucleotide (FAD) provide a label-free method to quantify cellular metabolism. However, the effect of extracellular pH on flavin lifetimes is currently unknown. To quantify the relationship between extracellular pH and the fluorescence lifetimes of FAD, flavin mononucleotide (FMN), and reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]. Human breast cancer (BT474) and HeLa cells were placed in pH-adjusted media. Images of an intracellular pH indicator or endogenous fluorescence were acquired using two-photon fluorescence lifetime imaging. Fluorescence lifetimes of FAD and FMN in solutions were quantified over the same pH range. The relationship between intracellular and extracellular pH was linear in both cell lines. Between extracellular pH 4 to 9, FAD mean lifetimes increased with increasing pH. NAD(P)H mean lifetimes decreased with increasing pH between extracellular pH 5 to 9. The relationship between NAD(P)H lifetime and extracellular pH differed between the two cell lines. Fluorescence lifetimes of FAD, FAD-cholesterol oxidase, and FMN solutions decreased, showed no trend, and showed no trend, respectively, with increasing pH. Changes in endogenous fluorescence lifetimes with extracellular pH are mostly due to indirect changes within the cell rather than direct pH quenching of the endogenous molecules.
Publication Date: 2021-05-26
Journal: Journal of biomedical optics

enzyme nicotinamide mononucleotide adenylyltransferase(12)

Two for the Price of One: A Neuroprotective Chaperone Kit within NAD Synthase Protein NMNAT2.
One of the most fascinating properties of the brain is the ability to function smoothly across decades of a lifespan. Neurons are nondividing mature cells specialized in fast electrical and chemical communication at synapses. Often, neurons and synapses operate at high levels of activity through sophisticated arborizations of long axons and dendrites that nevertheless stay healthy throughout years. On the other hand, aging and activity-dependent stress strike onto the protein machineries turning proteins unfolded and prone to form pathological aggregates associated with neurodegeneration. How do neurons protect from those insults and remain healthy for their whole life? Ali and colleagues now present a molecular mechanism by which the enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) acts not only as a NAD synthase involved in axonal maintenance but as a molecular chaperone helping neurons to overcome protein unfolding and protein aggregation.
Publication Date: 2016-07-28
Journal: PLoS biology

nicotinate nicotinamide mononucleotide adenyltransferase(3)

NAD+ accumulation during pollen maturation in Arabidopsis regulating onset of germination.
Although the nicotinamide nucleotides NAD(H) and NADP(H) are essential for various metabolic reactions that play major roles in maintenance of cellular homeostasis, the significance of NAD biosynthesis is not well understood. Here, we investigated the dynamics of pollen nicotinamide nucleotides in response to imbibition, a representative germination cue. Metabolic analysis with capillary electrophoresis electrospray ionization mass spectrometry revealed that excess amount of NAD+ is accumulated in freshly harvested dry pollen, whereas it dramatically decreased immediately after contact with water. Importantly, excess of NAD+ impaired pollen tube growth. Moreover, NAD+ accumulation was retained after pollen was imbibed in the presence of NAD+-consuming reaction inhibitors and pollen germination was greatly retarded. Pollen deficient in the nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) gene, encoding a key enzyme in NAD biosynthesis, and a lack of NAD+ accumulation in the gametophyte, showed precocious pollen tube germination inside the anther locule and vigorous tube growth under high-humidity conditions. Hence, the accumulation of excess NAD+ is not essential for pollen germination, but instead participates in regulating the timing of germination onset. These results indicate that NAD+ accumulation acts to negatively regulate germination and a decrease in NAD+ plays an important role in metabolic state transition.
Publication Date: 2012-08-22
Journal: Molecular plant

adenine dinucleotide phosphate(53)

Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis.
Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP
Publication Date: 2019-11-27
Journal: Nature chemical biology

mononucleotide adenylyltransferase nmnat(50)

p53 and clock genes play an important role in memory and learning ability depression due to long-term ultraviolet A eye irradiation.
Long-term ultraviolet A (UVA) eye irradiation decreases memory and learning ability in mice. However, the underlying mechanism is still unclear. In this study, ICR mice were used to study the effects of long-term UVA eye irradiation. The eyes of mice were exposed to UVA from an FL20SBLB-A lamp three times a week for 1 year. Then, we analyzed memory and learning ability in the mice using water maze and step-through passive avoidance tests, and measured the levels of p53, Period2 (Per2), Clock, brain and muscle Arnt-like protein-1 (Bmal1), nicotinamide mononucleotide adenylyltransferase (NMNAT) activity, nicotinamide phosphoribosyltransferase (NAMPT) activity, nicotinamide adenine dinucleotide (NAD The results showed that the p53 level increased significantly following long-term UVA eye irradiation, whereas the levels of Period2, Bmal1, Clock, NMNAT and NAMPT activities, NAD These results indicate that long-term UVA eye irradiation stimulates p53, inhibits the clock gene, and reduces Sirt1 production in the NAD
Publication Date: 2021-05-20
Journal: Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology

flavin adenine dinucleotide(39)

Bioenergetic Alterations of Metabolic Redox Coenzymes as NADH, FAD and FMN by Means of Fluorescence Lifetime Imaging Techniques.
Metabolic FLIM (fluorescence lifetime imaging) is used to image bioenergetic status in cells and tissue. Whereas an attribution of the fluorescence lifetime of coenzymes as an indicator for cell metabolism is mainly accepted, it is debated whether this is valid for the redox state of cells. In this regard, an innovative algorithm using the lifetime characteristics of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) to calculate the fluorescence lifetime induced redox ratio (FLIRR) has been reported so far. We extended the FLIRR approach and present new results, which includes FLIM data of the various enzymes, such as NAD(P)H, FAD, as well as flavin mononucleotide (FMN). Our algorithm uses a two-exponential fitting procedure for the NAD(P)H autofluorescence and a three-exponential fit of the flavin signal. By extending the FLIRR approach, we introduced FLIRR1 as protein-bound NAD(P)H related to protein-bound FAD, FLIRR2 as protein-bound NAD(P)H related to free (unbound) FAD and FLIRR3 as protein-bound NAD(P)H related to protein-bound FMN. We compared the significance of extended FLIRR to the metabolic index, defined as the ratio of protein-bound NAD(P)H to free NAD(P)H. The statistically significant difference for tumor and normal cells was found to be highest for FLIRR1.
Publication Date: 2021-06-03
Journal: International journal of molecular sciences

acid mononucleotide namn(25)

Creating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide.
Nicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life. Diverse NAD mimics have been developed for applications in chemical and biological sciences. Nicotinamide cytosine dinucleotide (NCD) has emerged as a non-natural cofactor to mediate redox transformations, while cells are fed with chemically synthesized NCD. Here, we create NCD synthetase (NcdS) by reprograming the substrate binding pockets of nicotinic acid mononucleotide (NaMN) adenylyltransferase to favor cytidine triphosphate and nicotinamide mononucleotide over their regular substrates ATP and NaMN, respectively. Overexpression of NcdS alone in the model host Escherichia coli facilitated intracellular production of NCD, and higher NCD levels up to 5.0 mM were achieved upon further pathway regulation. Finally, the non-natural cofactor self-sufficiency was confirmed by mediating an NCD-linked metabolic circuit to convert L-malate into D-lactate. NcdS together with NCD-linked enzymes offer unique tools and opportunities for intriguing studies in chemical biology and synthetic biology.
Publication Date: 2021-04-11
Journal: Nature communications

mononucleotide adenylyltransferase 2(21)

Nmnat2 attenuates amyloidogenesis and up-regulates ADAM10 in AMPK activity-dependent manner.
Amyloid-β (Aβ) accumulating is considered as a causative factor for formation of senile plaque in Alzheimer's disease (AD), but its mechanism is still elusive. The Nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2), a key redox cofactor for energy metabolism, is reduced in AD. Accumulative evidence has shown that the decrease of α-secretase activity, a disintegrin and metalloprotease domain 10 (ADAM10), is responsible for the increase of Aβ productions in AD patient's brain. Here, we observe that the activity of α-secretase ADAM10 and levels of Nmnat2 are significantly decreased, meanwhile there is a simultaneous elevation of Aβ in Tg2576 mice. Over-expression of Nmnat2 increases the mRNA expression of α-secretase ADAM10 and its activity and inhibits Aβ production in N2a/APPswe cells, which can be abolished by Compound C, an AMPK antagonist, suggesting that AMPK is involved in over-expression of Nmnat2 against Aβ production. The further assays demonstrate that Nmnat2 activates AMPK by up-regulating the ratio of NAD
Publication Date: 2021-10-14
Journal: Aging

acid mononucleotide adenylyltransferase(18)

Visualizing Soluble Protein Mutants by Using Monomeric Red Fluorescent Protein as a Reporter for Directed Evolution.
Directed evolution-based protein engineering usually generates large library contained insoluble mutants because of structural disturbance by mutation. To reduce the workload and costs, it is crucial to identify and eliminate those insoluble variants prior to dedicated analysis. Here, we demonstrate a method to visualize soluble protein mutants by using monomeric red fluorescent protein (mRFP) as a fusion tag. A plasmid was devised to express nicotinic acid mononucleotide adenylyltransferase (NadD) fused with a GGGS-linked mRFP tag at the C-terminus. The plasmid was subjected to site saturation mutagenesis within the nadD gene, used to transform Escherichia coli DH10B competent cells, leading to colonies with different red intensities. It was found that the fluorescence intensity of the cell culture correlated positively with the content of NadD-mRFP mutant in the supernatant. Mutation at position 132 led to a library of which most colonies lost the red phenotype, indicating that the position had a key role for proper protein folding. Similarly, mRFP enabled identification of soluble mutants of other enzymes including 1-deoxy-D-xylulose-5-phosphate reductoisomerase and phosphite dehydrogenase. These data suggested that mRFP can serve as a fusion reporter for visualizing soluble protein mutants to facilitate more efficient library screening in directed evolution.
Publication Date: 2017-10-31
Journal: Applied biochemistry and biotechnology

pyridine nucleotide cycle(14)

Characterization and mutational analysis of a nicotinamide mononucleotide deamidase from Agrobacterium tumefaciens showing high thermal stability and catalytic efficiency.
NAD+ has emerged as a crucial element in both bioenergetic and signaling pathways since it acts as a key regulator of cellular and organismal homeostasis. Among the enzymes involved in its recycling, nicotinamide mononucleotide (NMN) deamidase is one of the key players in the bacterial pyridine nucleotide cycle, where it catalyzes the conversion of NMN into nicotinic acid mononucleotide (NaMN), which is later converted to NAD+ in the Preiss-Handler pathway. The biochemical characteristics of bacterial NMN deamidases have been poorly studied, although they have been investigated in some firmicutes, gamma-proteobacteria and actinobacteria. In this study, we present the first characterization of an NMN deamidase from an alphaproteobacterium, Agrobacterium tumefaciens (AtCinA). The enzyme was active over a broad pH range, with an optimum at pH 7.5. Moreover, the enzyme was quite stable at neutral pH, maintaining 55% of its activity after 14 days. Surprisingly, AtCinA showed the highest optimal (80°C) and melting (85°C) temperatures described for an NMN deamidase. The above described characteristics, together with its high catalytic efficiency, make AtCinA a promising biocatalyst for the production of pure NaMN. In addition, six mutants (C32A, S48A, Y58F, Y58A, T105A and R145A) were designed to study their involvement in substrate binding, and two (S31A and K63A) to determine their contribution to the catalysis. However, only four mutants (C32A, S48A Y58F and T105A) showed activity, although with reduced catalytic efficiency. These results, combined with a thermal and structural analysis, reinforce the Ser/Lys catalytic dyad mechanism as the most plausible among those proposed.
Publication Date: 2017-04-08
Journal: PloS one

reduced nicotinamide mononucleotide(12)

Reduced Nicotinamide Mononucleotide (NMNH) Potently Enhances NAD
Decreased cellular NAD
Publication Date: 2021-04-02
Journal: Journal of proteome research

human nicotinamide mononucleotide(11)

Homology modeling and deletion mutants of human nicotinamide mononucleotide adenylyltransferase isozyme 2: new insights on structure and function relationship.
Nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes the formation of NAD by means of nucleophilic attack by 5'-phosphoryl of NMN on the α-phosphoryl group of ATP. Humans possess three NMNAT isozymes (NMNAT1, NMNAT2, and NMNAT3) that differ in size and sequence, gene expression pattern, subcellular localization, oligomeric state and catalytic properties. Of these, NMNAT2, the least abundant isozyme, is the only one whose much-needed crystal structure has not been solved as yet. To fill this gap, we used the crystal structures of human NMNAT1 and NMNAT3 as templates for homology-based structural modeling of NMNAT2, and the resulting raw structure was then refined by molecular dynamics simulations in a water box to obtain a model of the final folded structure. We investigated the importance of NMNAT2's central domain, which we postulated to be dispensable for catalytic activity, instead representing an isozyme-specific control domain within the overall architecture of NMNAT2. Indeed, we experimentally confirmed that removal of different-length fragments from this central domain did not compromise the enzyme's catalytic activity or the overall tridimensional structure of the active site.
Publication Date: 2010-10-19
Journal: Protein science : a publication of the Protein Society

precursor nicotinamide mononucleotide(10)

Telomere Dysfunction Induces Sirtuin Repression that Drives Telomere-Dependent Disease.
Telomere shortening is associated with stem cell decline, fibrotic disorders, and premature aging through mechanisms that are incompletely understood. Here, we show that telomere shortening in livers of telomerase knockout mice leads to a p53-dependent repression of all seven sirtuins. P53 regulates non-mitochondrial sirtuins (Sirt1, 2, 6, and 7) post-transcriptionally through microRNAs (miR-34a, 26a, and 145), while the mitochondrial sirtuins (Sirt3, 4, and 5) are regulated in a peroxisome proliferator-activated receptor gamma co-activator 1 alpha-/beta-dependent manner at the transcriptional level. Administration of the NAD(+) precursor nicotinamide mononucleotide maintains telomere length, dampens the DNA damage response and p53, improves mitochondrial function, and, functionally, rescues liver fibrosis in a partially Sirt1-dependent manner. These studies establish sirtuins as downstream targets of dysfunctional telomeres and suggest that increasing Sirt1 activity alone or in combination with other sirtuins stabilizes telomeres and mitigates telomere-dependent disorders.
Publication Date: 2019-04-02
Journal: Cell metabolism

product nicotinamide mononucleotide(8)

Inhibition of nicotinamide phosphoribosyltransferase protects against acute pancreatitis via modulating macrophage polarization and its related metabolites.
Acute pancreatitis is a common inflammatory disorder of the exocrine pancreas with no specific therapy. Intracellular nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) salvage pathway, is involved in many inflammatory disorders. In this study, we investigated the role of NAMPT in experimental acute pancreatitis. Acute pancreatitis was induced in mice using three disparate models: (1) caerulein hyperstimulation, (2) ethanol plus palmitoleic acid, and (3) retrograde biliopancreatic ductal infusion of sodium taurocholate. The NAMPT inhibitor FK866 and NAMPT downstream product nicotinamide mononucleotide (NMN) was administered. Serum and pancreas were collected and analyzed biochemically and histologically. Bone marrow derived macrophages were isolated, cultured with cytokines or pancreatic acini, then analyzed by quantitative PCR and non-targeted metabolomics. The levels of pancreatic NAMPT and NAD were down-regulated upon acute pancreatitis. NAMPT inhibitor FK866 suppressed M1 macrophage polarization while NMN boosted it. In co-culture of macrophages with acinar cells, inhibition of NAMPT prevented M1-like macrophage differentiation induced by injured pancreatic acini. The injured pancreatic acinar milieu induced a unique metabolic signature linked to macrophage polarization, and inhibition of NAMPT reversed these metabolites changes. Furthermore, NMN supplementation aggravated caerulein hyperstimulation pancreatitis and alcoholic pancreatitis, and inhibition of NAMPT protected against caerulein hyperstimulation, alcoholic and biliary acute pancreatitis and reducing pancreatic macrophage infiltration in vivo. NAMPT inhibition protects against acute pancreatitis via preventing M1 macrophage polarization and restoring the metabolites related to macrophage polarization and that NAMPT could be a promising therapeutic target for acute pancreatitis.
Publication Date: 2021-04-04
Journal: Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.]

nicotinamide mononucleotide deamidase(7)

Structure and mechanism of the bifunctional CinA enzyme from Thermus thermophilus.
CinA is a widely distributed protein in Gram-positive and Gram-negative bacteria. It is associated with natural competence and is proposed to have a function as an enzyme participating in the pyridine nucleotide cycle, which recycles products formed by non-redox uses of NAD. Here we report the determination of the crystal structure of CinA from Thermus thermophilus, in complex with several ligands. CinA was shown to have both nicotinamide mononucleotide deamidase and ADP-ribose pyrophosphatase activities. The crystal structure shows an unusual asymmetric dimer, with three domains for each chain; the C-terminal domain harbors the nicotinamide mononucleotide deamidase activity, and the structure of a complex with the product nicotinate mononucleotide suggests a mechanism for deamidation. The N-terminal domain belongs to the COG1058 family and is associated with the ADP-ribose pyrophosphatase activity. The asymmetry in the CinA dimer arises from two alternative orientations of the COG1058 domains, only one of which forms a contact with the KH-type domain from the other chain, effectively closing the active site into, we propose, a catalytically competent state. Structures of complexes with Mg(2+)/ADP-ribose, Mg(2+)/ATP, and Mg(2+)/AMP suggest a mechanism for the ADP-ribose pyrophosphatase reaction that involves a rotation of the COG1058 domain dimer as part of the reaction cycle, so that each active site oscillates between open and closed forms, thus promoting catalysis.
Publication Date: 2014-10-15
Journal: The Journal of biological chemistry

exogenous nicotinamide mononucleotide(4)

Corneal Denervation Causes Epithelial Apoptosis Through Inhibiting NAD+ Biosynthesis.
To determine if trigeminal innervations of the corneal epithelium maintains its integrity and homeostasis through controlling the nicotinamide adenine dinucleotide (NAD) content of this tissue. Corneal denervation of C57BL/6 mice was induced by squeezing the nerve bundles that derive from the trigeminal ganglion and was confirmed by whole-mount corneal nerve staining and the sensation test. The apoptosis of the corneal epithelium was examined by TUNEL assay and annexin V/propidium iodide staining. NAD biosynthesis-related enzymes were analyzed by quantitative PCR, immunofluorescence staining, and Western blotting. FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), exogenous nicotinamide mononucleotide (NMN), and NAD+ were used to evaluate the effect of NAD+ on the apoptosis of cultured corneal epithelial cells and epithelial detachment in denervated mice. Protein expression that related to apoptosis and phosphorylation were analyzed by Western blotting. The denervated mice showed spontaneous corneal epithelial detachment and cell apoptosis accompanied with impaired epithelial NAD+ contents due to low levels of NAMPT. Similarly, inhibition of NAMPT recapitulated epithelial detachment as in denervated mice and induced apoptosis in cultured corneal epithelial cells. The replenishment of NMN or NAD+ partially slowed down corneal nerve fiber degeneration, reduced the epithelial defect in denervated mice, and improved apoptosis induction in FK866-treated cells by restoring the activation levels of SIRT1, AKT, and CREB. Corneal denervation lowered epithelial NAD+ contents through reducing the expression of NAMPT and caused cell apoptosis and epithelial defects, suggesting that corneal innervations contribute to epithelial homeostasis by regulating NAD+ biosynthesis.
Publication Date: 2019-08-16
Journal: Investigative ophthalmology & visual science

nicotinamide riboside(60)

Coronavirus 2019 (COVID-19) is caused by 'severe acute respiratory syndrome coronavirus 2' (SARS-CoV-2), first reported in Wuhan, China in December 2019, which eventually became a global disaster. Various key mediators have been reported in the pathogenesis of COVID-19. However, no effective pharmacological intervention has been available to combat COVID-19 complications. The present study screens nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) as potential inhibitors of this present generation coronavirus infection using an in-silico approach. The SARS-CoV-2 proteins (nucleocapsid, proteases, post-fusion core, phosphatase, endoriboruclease) and ACE-2 protein were selected. The 2D structure of nicotinamide ribonucleoside and nicotinamide ribonucleotide was drawn using ChemDraw 14.0 and saved in .cdx format. The results were analyzed using two parameters: full fitness energy and binding free energy (ΔG). The full fitness energy and estimated ΔG values from docking of NM, and NMN with selected SARS-CoV-2 target proteins, ADMET prediction and Target prediction indicate the interaction of NR and NMN in the treatment of COVID-19. Based on full fitness energy and estimated ΔG values from docking studies of NM and NAM with selected SARS-CoV-2 target proteins, ADME prediction, target prediction and toxicity prediction, we expect a possible therapeutic efficacy of NR in the treatment of COVID-19.
Publication Date: 2021-10-06
Journal: Saudi journal of biological sciences

mononucleotide fmn(56)

Stereospecificity of hydride transfer and molecular docking in FMN-dependent NADH-indigo reductase of Bacillus smithii.
In this study, we investigated the stereospecificity of hydride transfer from NADH to flavin mononucleotide (FMN) in reactions catalyzed by the FMN-dependent NADH-indigo reductase expressed by thermophilic Bacillus smithii. We performed
Publication Date: 2021-05-28
Journal: FEBS open bio

dinucleotide nadh(33)

Power of an Organic Electron Acceptor in Modulation of Intracellular Mitochondrial Reactive Oxygen Species: Inducing JNK- and Caspase-Dependent Apoptosis of Cancer Cells.
Here, we demonstrate an interesting strategy of modulating mitochondrial reactive oxygen species (ROS) using the organic electron acceptor molecule carbonyl-bridged bithiazole attached with bis-trifluoroacetophenone (BBT). This molecule was found to affect complex I activity. It has the propensity to bind close to the flavin mononucleotide site of complex I of mitochondria where it traps electron released from nicotinamide adenine dinucleotide (NADH) and elevates intracellular ROS, which suggests that the bridged carbonyl in BBT plays a crucial role in the acceptance of electron from NADH. We understand that the potential of the NADH/NAD+ redox couple and low-lying LUMO energy level of BBT are compatible with each other, thus favoring its entrapment of released electrons in complex I. This effect of BBT in ROS generation activates JNK and p38 stress-dependent pathways and resulted in mitochondrial-dependent apoptotic cell death with the reduction in expression of several important cyto-protecting factors (Hsp27 and NFκB), indicating its potential in inhibition of cancer cell relapse. Intriguingly, we found that BBT is not a P-glycoprotein substrate, which further reveals its excellent anticancer potential. This study enlightens us on how the power of electron acceptor ability became an emerging strategy for modulation of intracellular function.
Publication Date: 2021-03-30
Journal: ACS omega

salvage pathway(32)

Sex-specific alterations in NAD+ metabolism in 3xTg Alzheimer's disease mouse brain assessed by quantitative targeted LC-MS.
Levels of nicotinamide adenine dinucleotide (NAD+) are known to decline with age and have been associated with impaired mitochondrial function leading to neurodegeneration, a key facet of Alzheimer's disease (AD). NAD+synthesis is sustained via tryptophan-kynurenine (Trp-Kyn) pathway as de novo synthesis route, and salvage pathways dependent on the availability of nicotinic acid and nicotinamide. While being currently investigated as a multifactorial disease with a strong metabolic component, AD remains without curative treatment and important sex differences were reported in relation to disease onset and progression. The aim of this study was to reveal the potential deregulation of NAD+metabolism in AD with the direct analysis of NAD+precursors in the mouse brain tissue (wild type (WT) versus triple transgenic (3xTg) AD), using a sex-balanced design. To this end, we developed a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which allowed for the measurement of the full spectrum of NAD+precursors and intermediates in all three pathways. In brain tissue of mice with developed AD symptoms, a decrease in kynurenine (Kyn) versus increase in kynurenic acid (KA) levels were observed in both sexes with a significantly higher increment of KA in males. These alterations in Trp-Kyn pathway might be a consequence of neuroinflammation and a compensatory production of neuroprotective kynurenic acid. In the NAD+ salvage pathway, significantly lower levels of nicotinamide mononucleotide (NMN) were measured in the AD brain of males and females. Depletion of NMN implies the deregulation of salvage pathway critical for maintaining optimal NAD+ levels and mitochondrial and neuronal function.
Publication Date: 2021-04-09
Journal: Journal of neurochemistry

riboside nr(27)

Precursor comparisons for the upregulation of nicotinamide adenine dinucleotide. Novel approaches for better aging.
Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in every human cell and regulates a number of systems across multiple cellular compartments and tissue types via an endogenous and exogenous influence. NAD levels are demonstrated to decline with age and therefore measures to counteract the waning of NAD have been devised. A number of NAD precursor candidates such as nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), the reduced form of nicotinamide mononucleotide (NMNH), nicotinic acid (NA) nicotinamide (NAM), and dihydronicotinamide riboside (DNR) increase NAD levels in vitro and in vivo. This discussion will focus on the precursors NR, NMN, NMNH, and DNR in the upregulation of NAD. There are many publications on NAD precursors as it has become popular for human consumption in recent years due to its vital importance to the general consumer. However, there is no consensus between researchers and this was the aim of this review, to determine and discuss their areas of agreement versus disagreement, to highlight the gaps in research, and to give recommendations for future work. Bioavailability and potency of NR, NMNH, NMN, and DNR is also examined on the light of the most recent literature.
Publication Date: 2021-09-24
Journal: Aging medicine (Milton (N.S.W))

2 nmnat2(24)

A Novel In Vitro Assay Using Human iPSC-Derived Sensory Neurons to Evaluate the Effects of External Chemicals on Neuronal Morphology: Possible Implications in the Prediction of Abnormal Skin Sensation.
Neuronal morphological changes in the epidermis are considered to be one of causes of abnormal skin sensations in dry skin-based skin diseases. The present study aimed to develop an in vitro model optimised for human skin to test the external factors that lead to its exacerbation. Human-induced pluripotent stem cell-derived sensory neurons (hiPSC-SNs) were used as a model of human sensory neurons. The effects of chemical substances on these neurons were evaluated by observing the elongation of nerve fibers, incidence of blebs (bead-like swellings), and the expression of nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2). The nerve fiber length increased upon exposure to two common cosmetic preservatives-methylparaben and phenoxyethanol-but not to benzo[a]pyrene, an air pollutant at the estimated concentrations in the epidermis. Furthermore, the incidence of blebs increased upon exposure to benzo[a]pyrene. However, there was a decrease in the expression of NMNAT2 in nerve fibers, suggesting degenerative changes. No such degeneration was found after methylparaben or phenoxyethanol at the estimated concentrations in the epidermis. These findings suggest that methylparaben and phenoxyethanol promote nerve elongation in hiPSC-SNs, whereas benzo[a]pyrene induces nerve degeneration. Such alterations may be at least partly involved in the onset and progression of sensitive skin.
Publication Date: 2021-10-14
Journal: International journal of molecular sciences

escherichia coli(24)

Metabolic engineering of Escherichia coli for biosynthesis of β-nicotinamide mononucleotide from nicotinamide.
The β-nicotinamide mononucleotide (NMN) is a key intermediate of an essential coenzyme for cellular redox reactions, NAD. Administration of NMN is reported to improve various symptoms, such as diabetes and age-related physiological decline. Thus, NMN is attracting much attention as a promising nutraceutical. Here, we engineered an Escherichia coli strain to produce NMN from cheap substrate nicotinamide (NAM) and glucose. The supply of in vivo precursor phosphoribosyl pyrophosphate (PRPP) and ATP was enhanced by strengthening the metabolic flux from glucose. A nicotinamide phosphoribosyltransferase with high activity was newly screened, which is the key enzyme for converting NAM to NMN with PRPP as cofactor. Notably, the E. coli endogenous protein YgcS, which function is primarily in the uptake of sugars, was firstly proven to be beneficial for NMN production in this study. Fine-tuning regulation of ygcS gene expression in the engineered E. coli strain increased NMN production. Combined with process optimization of whole-cell biocatalysts reaction, a final NMN titre of 496.2 mg l
Publication Date: 2021-07-27
Journal: Microbial biotechnology

phosphate nadph(20)

Ginsenoside Rb1 Attenuates High Glucose-Induced Oxidative Injury via the NAD-PARP-SIRT Axis in Rat Retinal Capillary Endothelial Cells.
(1) Aims: The present study aimed to observe the effects of Ginsenoside Rb1 on high glucose-induced endothelial damage in rat retinal capillary endothelial cells (RCECs) and to investigate the underlying mechanism. (2) Methods: Cultured RCECs were treated with normal glucose (5.5 mM), high glucose (30 mM glucose), or high glucose plus Rb1 (20 μM). Cell viability, lactate dehydrogenase (LDH) levels, the mitochondrial DNA copy number, and the intracellular ROS content were measured to evaluate the cytotoxicity. Superoxide dismutase (SOD), catalase (CAT), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), poly(ADP-ribose) polymerase (PARP), and sirtuin (SIRT) activity was studied in cell extracts. Nicotinamide adenine dinucleotide (NAD
Publication Date: 2019-10-09
Journal: International journal of molecular sciences

wallerian degeneration(18)

Vincristine and bortezomib use distinct upstream mechanisms to activate a common SARM1-dependent axon degeneration program.
Chemotherapy-induced peripheral neuropathy is one of the most prevalent dose-limiting toxicities of anticancer therapy. Development of effective therapies to prevent chemotherapy-induced neuropathies could be enabled by a mechanistic understanding of axonal breakdown following exposure to neuropathy-causing agents. Here, we reveal the molecular mechanisms underlying axon degeneration induced by 2 widely used chemotherapeutic agents with distinct mechanisms of action: vincristine and bortezomib. We showed previously that genetic deletion of SARM1 blocks vincristine-induced neuropathy and demonstrate here that it also prevents axon destruction following administration of bortezomib in vitro and in vivo. Using cultured neurons, we found that vincristine and bortezomib converge on a core axon degeneration program consisting of nicotinamide mononucleotide NMNAT2, SARM1, and loss of NAD+ but engage different upstream mechanisms that closely resemble Wallerian degeneration after vincristine and apoptosis after bortezomib. We could inhibit the final common axon destruction pathway by preserving axonal NAD+ levels or expressing a candidate gene therapeutic that inhibits SARM1 in vitro. We suggest that these approaches may lead to therapies for vincristine- and bortezomib-induced neuropathies and possibly other forms of peripheral neuropathy.
Publication Date: 2019-09-06
Journal: JCI insight

quinolinic acid(17)

Biochemical characterization of quinolinic acid phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv and inhibition of its activity by pyrazinamide.
Quinolinic acid phosphoribosyltransferase (QAPRTase, EC is a key enzyme in the de novo pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis and a target for the development of new anti-tuberculosis drugs. QAPRTase catalyzes the synthesis of nicotinic acid mononucleotide from quinolinic acid (QA) and 5-phosphoribosyl-1-pyrophosphate (PRPP) through a phosphoribosyl transfer reaction followed by decarboxylation. The crystal structure of QAPRTase from Mycobacterium tuberculosis H37Rv (MtQAPRTase) has been determined; however, a detailed functional analysis of MtQAPRTase has not been published. Here, we analyzed the enzymatic activities of MtQAPRTase and determined the effect on catalysis of the anti-tuberculosis drug pyrazinamide (PZA). The optimum temperature and pH for MtQAPRTase activity were 60°C and pH 9.2. MtQAPRTase required bivalent metal ions and its activity was highest in the presence of Mg2+. Kinetic analyses revealed that the Km values for QA and PRPP were 0.08 and 0.39 mM, respectively, and the kcat values for QA and PRPP were 0.12 and 0.14 [s-1], respectively. When the amino acid residues of MtQAPRTase, which may interact with QA, were substituted with alanine residues, catalytic activity was undetectable. Further, PZA, which is an anti-tuberculosis drug and a structural analog of QA, markedly inhibited the catalytic activity of MtQAPRTase. The structure of PZA may provide the basis for the design of new inhibitors of MtQAPRTase. These findings provide new insights into the catalytic properties of MtQAPRTase.
Publication Date: 2014-06-21
Journal: PloS one

mitochondrial function(13)

Preconditioning of mesenchymal stem cells with ghrelin exerts superior cardioprotection in aged heart through boosting mitochondrial function and autophagy flux.
Application of mesenchymal stem cells (MSCs) is considered as a promising cell-based therapy to induce cardioprotection against ischemia-reperfusion (IR) injury. Preconditioning of MSCs is the key strategy to improve MSCs functions in vitro and their efficacy in vivo, especially in elderly subjects in whom cardioprotection is lost. This study investigated the effects of preconditioning of human umbilical cord-derived MSCs with ghrelin and their combination with nicotinamide-mononucleotide (NMN) on cardioprotection, and the role of autophagy flux and mitochondrial function in aged hearts subjected to IR injury. Aged Sprague Dawley rats (20-22 months old) were subjected to LAD occlusion-induced myocardial IR injury and treated with ghrelin-preconditioned or unconditioned-MSCs at early reperfusion. NMN (500 mg/kg, i.p) was also administered at early reperfusion and repeated 12 h later. Intra-myocardial injection of ghrelin-preconditioned MSCs reduced infarct size and cardiotroponin release of aged myocardium, and improved cardiac function following IR injury. MSCs preconditioning with ghrelin restored IR-induced mitochondrial reactive oxygen species and membrane potential depolarization and enhanced ATP production. To reveal possible mechanism, preconditioned-MSCs increased autophagy flux by downregulating the overexpression of Beclin-1 and P62 proteins and increasing the LC3-II expression and LC3-II/LC3-I ratio. Moreover, combining NMN to ghrelin-preconditioned MSCs synergistically augmented its protective effects on infarct size and mitochondrial function. All above effects were abolished by autophagy flux inhibitor, chloroquine. Thus, ghrelin may serve as a promising candidate to improve the cardioprotective efficacy of MSC-based therapy via autophagy/mitochondrial pathway and that NMN serves as a good booster in combination therapy in aged hearts.
Publication Date: 2021-05-06
Journal: European journal of pharmacology

reduced form(13)

Hyperthermophilic flavin reductase from Sulfolobus solfataricus P2: Production and biochemical characterization.
Nicotinamide adenine dinucleotide phosphate (NAD(P)H)-flavin oxidoreductases (flavin reductases) catalyze the reduction of flavin by NAD(P)H and provide the reduced form of flavin mononucleotide (FMN) to flavin-dependent monooxygenases. Based on bioinformatics analysis, we identified a putative flavin reductase gene, sso2055, in the genome of hyperthermophilic archaeon Sulfolobus solfataricus P2, and further cloned this target sequence into an expression vector. The cloned flavin reductase (EC. was purified to homogeneity and characterized further. The purified enzyme exists as a monomer of 17.8 kDa, free of chromogenic cofactors. Homology modeling revealed this enzyme as a TIM barrel, which is also supported by circular dichroism measurements revealing a beta-sheet rich content. The optimal pH for SSO2055 activity was pH 6.5 in phosphate buffer and the highest activity observed was at 120 °C within the measurable temperature. We showed that this enzyme can use FMN and flavin adenine dinucleotide (FAD) as a substrate to generate their reduced forms. The purified enzyme is predicted to be a potential flavin reductase of flavin-dependent monooxygenases that could be involved in the biodesulfurization process of S. solfataricus P2.
Publication Date: 2019-08-10
Journal: Biotechnology and applied biochemistry

phosphoribosyl transferase(12)

Adipokine Visfatin's Role in Pathogenesis of Diabesity and Related Metabolic Derangements.
Visfatin is one of the prominent adipokines secreted by adipose tissue. The level of visfatin increases significantly in persons with obesity owing to increased body mass index (BMI). During obesity, the adipocytes, which populate adipose tissue, undergo hypertrophy and hyperplasia and secrete a number of adipocytokines including visfatin. Visfatin, which also acts as an enzyme nicotinamide phosphoribosyl transferase, is one of the prominent adipokines that influence metabolic homeostasis in the body. Visfatin exists in two forms, extracellular and intracellular, and enacts a multitude of actions. The direct and indirect evidence gathered from in-vitro, in-vivo and clinical studies indicate that visfatin modulates obesity and metabolic syndrome-related pathophysiological activities including enhanced inflammation, angiogenesis, synthesis of NAD mononucleotide, and upregulation of antiapoptotic proteins in a number of cell types. It has been implicated in a number of obesity-related alterations and metabolic derangement such as diabetes, cardiovascular complications and some forms of cancers. In this review, the novel hypothesis about the role of visfatin in diabesity has been proposed which implies recent advances in studies about the pathophysiological roles of visfatin during obesity and chronic high glucose in the circulation. Visfatin at high concentration attracts immune cells and produces chronic inflammation in adipocytes. Additionally, it induces insulin resistance in many tissues and causes pancreatic beta cells dysfunction at later stages. Further, its potential as an important target to develop molecular medicine in diabesity and related metabolic syndrome has been highlighted.
Publication Date: 2018-07-06
Journal: Current molecular medicine

dinucleotide naad(8)

Structural and Functional Characterization of Plasmodium falciparum Nicotinic Acid Mononucleotide Adenylyltransferase.
Nicotinic acid mononucleotide adenylyltransferase (NaMNAT) is an indispensable enzyme for the synthesis of NAD and NAD phosphate. It catalyzes the adenylylation of nicotinic acid mononucleotide (NaMN) to yield nicotinic acid adenine dinucleotide (NaAD). Since NAD(H) and NAD phosphate(H) are essentially involved in metabolic and redox regulatory reactions, NaMNAT is an attractive drug target in the fight against bacterial and parasitic infections. Notably, NaMNAT of the malaria parasite Plasmodium falciparum possesses only 20% sequence identity with the homologous human enzyme. Here, we present for the first time the two X-ray structures of P. falciparum NaMNAT (PfNaMNAT)-in the product-bound state with NaAD and complexed with an α,β-non-hydrolizable ATP analog-the structures were determined to a resolution of 2.2Å and 2.5Å, respectively. The overall architecture of PfNaMNAT was found to be more similar to its bacterial homologs than its human counterparts although the PPHK motif conserved in bacteria is missing. Furthermore, PfNaMNAT possesses two cysteine residues within the active site that have not been described for any other NaMNATase so far and are likely to be involved in redox regulation of PfNaMNAT activity. Enzymatic studies and surface plasmon resonance data reveal that PfNaMNAT is capable of utilizing NaMN and nicotinamide mononucleotide with a slight preference for NaMN. Surprisingly, a comparison with the active site of Escherichia coli NaMNAT showed very similar architectures, despite different substrate preferences.
Publication Date: 2016-12-17
Journal: Journal of molecular biology

nmnat ec(8)

Development of a Bioluminescent High-Throughput Screening Assay for Nicotinamide Mononucleotide Adenylyltransferase (NMNAT).
Nicotinamide mononucleotide adenylyltransferase (NMNAT; EC catalyzes the reversible production of NAD
Publication Date: 2019-10-05
Journal: SLAS discovery : advancing life sciences R & D

beta-nicotinamide mononucleotide(8)

Evolutionary optimization of a modular ligase ribozyme: a small catalytic unit and a hairpin motif masking an element that could form an inactive structure.
The YFL ribozyme is an artificial ligase ribozyme isolated by a 'design and selection' strategy, in which a modular catalytic unit was generated on a rationally designed modular scaffold RNA. This ligase ribozyme has a versatile catalytic unit that accepts not only beta-nicotinamide mononucleotide (beta-NMN) but also inorganic pyrophosphate as leaving groups for template-dependent RNA ligation. Although this property is interesting from an evolutionary viewpoint regarding primitive RNA ligation/polymerization systems in the RNA world, structural analysis of the YFL ribozyme has not been continued due to apparent structural nonuniformity of its folded state. To elucidate the active structure of the YFL ribozyme, we performed in vitro evolution experiments to improve its folding ability. Biochemical and phylogenetic analyses of evolved variants indicated that the catalytic unit of the YFL ribozyme is compact and the 3' single-stranded region of the parent YFL-1 ribozyme contributes to mask an element that could form an inactive structure.
Publication Date: 2010-01-30
Journal: Nucleic acids research

acid nicotinamide(7)

The role of Phe181 in the hexamerization of Helicobacter pylori quinolinate phosphoribosyltransferase.
Quinolinic acid phosphoribosyltransferase (QAPRTase; NadC) catalyzes an indispensable step in NAD biosynthesis, one that is essential for cell survival in prokaryotes, which makes it an attractive target for antibacterial drug therapy. We recently reported the crystal structures of Helicobacter pylori QAPRTase with bound quinolinic acid, nicotinamide mononucleotide, and phthalic acid. The enzyme exists as a hexamer organized as a trimer of dimers, which is essential for full enzymatic activity. The loop between helix alpha7 and strand beta8 contributes significantly to the hydrophobic dimer-dimer interactions. Phe181Pro mutation within the alpha7-beta8 loop disrupts the hexamerization of QAPRTase, and the resultant dimer shows dramatically reduced protein stability and no activity. Our findings thus suggest that compounds able to disrupt its proper oligomerization could potentially function as selective inhibitors of Helicobacter pylori QAPRTase and represent a novel set of antibacterial agents.
Publication Date: 2007-09-04
Journal: The protein journal

mononucleotide nmnh(6)

Interactions of reduced and oxidized nicotinamide mononucleotide with wild-type and alphaD195E mutant proton-pumping nicotinamide nucleotide transhydrogenases from Escherichia coli.
The interaction of reduced nicotinamide mononucleotide (NMNH), constituting one half of NADH, with the wild-type and alphaD195E proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli was investigated. Reduction of thio-NADP+ by NMNH was catalysed at approximately 30% of the rate with NADH. Other activities including proton pumping and the cyclic reduction of 3'-acetyl-pyridine-NAD+ by NMNH in the presence of NADP+ were more strongly inhibited. The alphaD195 residue is assumed to interact with the 2'-OH moiety of the adenosine-5'-phosphate, i.e., the second nucleotide of NADH. Mutation of this residue to alphaD195E resulted in a 90% decrease in activity with NMNH as well as NADH as substrate, suggesting that it produced global structural changes of the NAD(H) binding site. The results suggest that the NMN moiety of NADH is a substrate of transhydrogenase, and that the adenine nucleotide is not required for catalysis or proton pumping.
Publication Date: 1998-10-24
Journal: Biochimica et biophysica acta

poly adp-ribose(5)

New Insights into the Roles of NAD+-Poly(ADP-ribose) Metabolism and Poly(ADP-ribose) Glycohydrolase.
Accumulating evidence has suggested the fundamental functions of NAD+-poly(ADP-ribose) metabolism in cellular and physiological processes, including energy homeostasis, signal transduction, DNA transaction, genomic stability and cell death or survival. The NAD+ biosynthesis and poly(ADP-ribose) [(ADP-R)n] turnover are tightly controlled by several key enzymes, such as nicotinamide phosphoribosyltransferase (NmPRT), nicotinamide mononucleotide adenylyltransferases (NMNATs), poly(ADP-ribose) polymerase (PARP), poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose pyrophosphorylase (ADPRPPL). Many researches investigating the roles of these enzymes in cells have revealed the physiological and pathological importance, and thereby the therapeutical values. Among these enzymes, the polymer degrading enzyme PARG has not yet been intensively studied, because of the low cellular content, lack of cell-available PARG chemical inhibitors and PARG genetic models. So, the biological roles of (ADP-R)n catabolism by PARG are still being elucidated as compared to those of synthesis by PARP. However, recent studies delineate that PARG-dependent (ADP-R)n degradation is critical for many pathological conditions, and thus PARG is an important target for chemical therapeutics for several diseases. This review will present the recent progresses about the roles of NAD+-(ADP-R)n metabolism and the structures and functions of PARG, with a focus on its role in DNA repair and cell death by apoptosis in relation to central regulatory network, and the therapeutic potentials of PARG inhibitors in cancer chemotherapy.
Publication Date: 2016-11-08
Journal: Current protein & peptide science

adenyltransferase nmnat(5)

Protective effect of nicotinamide on high glucose/palmitate-induced glucolipotoxicity to INS-1 beta cells is attributed to its inhibitory activity to sirtuins.
This study was initiated to determine whether the protective effect of nicotinamide (NAM) on high glucose/palmitate (HG/PA)-induced INS-1 beta cell death was due to its role as an anti-oxidant, nicotinamide dinucleotide (NAD+) precursor, or inhibitor of NAD+-consuming enzymes such as poly (ADP-ribose) polymerase (PARP) or sirtuins. All anti-oxidants tested were not protective against HG/PA-induced INS-1 cell death. Direct supplementation of NAD+ or indirect supplementation through NAD+ salvage or de novo pathway did not protect the death. Knockdown of the NAD+ salvage pathway enzymes such as nicotinamide phosphoribosyl transferase (NAMPT) or nicotinamide mononucleotide adenyltransferase (NMNAT) did not augment death. On the other hand, pharmacological inhibition or knockdown of PARP did not affect death. However, sirtinol as an inhibitor of NAD-dependant deacetylase or knockdown of SIRT3 or SIRT4 significantly reduced the HG/PA-induced death. These data suggest that protective effect of NAM on beta cell glucolipotoxicity is attributed to its inhibitory activity on sirtuins.
Publication Date: 2013-04-09
Journal: Archives of biochemistry and biophysics

14 c(5)

Comparison of the formation of nicotinic acid conjugates in leaves of different plant species.
There are three metabolic fates of nicotinic acid in plants: (1) nicotinic acid mononucleotide formation for NAD synthesis by the so-called salvage pathway of pyridine nucleotide biosynthesis; (2) nicotinic acid N-glucoside formation; and (3) trigonelline (N-methylnicotinic acid) formation. In the present study, the metabolism of [carbonyl-(14)C]nicotinamide was investigated in leaves of 23 wild plant species. All species readily converted nicotinamide to nicotinic acid, and only a fraction of nicotinic acid was utilised for NAD and NADP synthesis. The remaining nicotinic acid is converted to the nicotinic acid conjugates. Only one plant species, Cycas revoluta, produced both nicotinic acid N-glucoside and trigonelline; the other 22 species produced one or other of the conjugates. The nicotinic acid N-glucoside-forming plants are Cyathea lepifera, Arenga trewmula var. englri, Barringtonia racemosa, Ilex paraguariensis, Angelica japonica, Scaevola taccada and Farfugium japonicum. In contrast, trigonelline is formed in C. lepifera, Ginkgo biloba, Pinus luchuensis, Casuarina equisetifolia, Alocasia odora, Pandanus odoratissimus, Hylocereus undatus, Kalanchoe pinnata, Kalanchoe tubiflora, Populus alba, Garcinia subelliptica, Oxalis corymbosa, Leucaena leucocephala, Vigna marina, Hibiscus tiliaceus and Melicope triphylla. The diversity of nicotinic acid conjugate formation in plants is discussed using these results and our previous investigation involving a few model plants, various crops and ferns. Nicotinic acid N-glucoside formation was restricted mostly to ferns and selected orders of angiosperms, whereas other plants produce trigonelline. In most cases the formation of both nicotinic acid conjugates is incompatible, but some exceptions have been found.
Publication Date: 2012-09-18
Journal: Plant physiology and biochemistry : PPB

nmn supplementation(5)

Nicotinamide Mononucleotide Restores the Meiotic Competency of Porcine Oocytes Exposed to Ethylene Glycol Butyl Ether.
Ethylene glycol butyl ether (EGBE), a type of glycol ethers, is a common chemical used in both industrial and household products. Increasing animal studies have indicated that it produces reproductive problems, such as testicular damage, reduced female fertility, death of embryos, and birth defects. However, how it influences the female germ cells has not yet determined. Here, we found that EGBE exposure resulted in the defective porcine oocyte maturation via disruption of cytoskeleton dynamics, showing the abnormal spindle assembly, chromosome alignment, and actin organization. Meanwhile, EGBE exposure perturbed the mitochondrial distribution and function, leading to the accumulation of reactive oxygen species (ROS) and generation of DNA damage and apoptosis. Of note, nicotinamide mononucleotide (NMN) supplementation rescued the meiotic defects caused by EGBE exposure via restoring NAD
Publication Date: 2021-02-20
Journal: Frontiers in cell and developmental biology

adenylyltransferases nmnats(5)

Neuronal NMNAT2 Overexpression Does Not Achieve Significant Neuroprotection in Experimental Autoimmune Encephalomyelitis/Optic Neuritis.
Optic neuritis, inflammation, and demyelination of the optic nerve (ON), is one of the most common clinical manifestations of multiple sclerosis; affected patients suffer persistent visual symptoms due to ON degeneration and secondary retinal ganglion cell (RGC) death. The mouse experimental autoimmune encephalomyelitis (EAE) model replicates optic neuritis and significant RGC soma and axon loss. Nicotinamide mononucleotide adenylyltransferases (NMNATs) are NAD
Publication Date: 2021-10-29
Journal: Frontiers in cellular neuroscience

ca 2(4)

The mitochondrial permeability transition pore regulates endothelial bioenergetics and angiogenesis.
The mitochondrial permeability transition pore is a well-known initiator of cell death that is increasingly recognized as a physiological modulator of cellular metabolism. We sought to identify how the genetic deletion of a key regulatory subunit of the mitochondrial permeability transition pore, cyclophilin D (CypD), influenced endothelial metabolism and intracellular signaling. In cultured primary human endothelial cells, genetic targeting of CypD using siRNA or shRNA resulted in a constitutive increase in mitochondrial matrix Ca(2+) and reduced nicotinamide adenine dinucleotide (NADH). Elevated matrix NADH, in turn, diminished the cytosolic NAD(+)/NADH ratio and triggered a subsequent downregulation of the NAD(+)-dependent deacetylase sirtuin 1 (SIRT1). Downstream of SIRT1, CypD-deficient endothelial cells exhibited reduced phosphatase and tensin homolog expression and a constitutive rise in the phosphorylation of angiogenic Akt. Similar changes in SIRT1, phosphatase and tensin homolog, and Akt were also noted in the aorta and lungs of CypD knockout mice. Functionally, CypD-deficient endothelial cells and aortic tissue from CypD knockout mice exhibited a dramatic increase in angiogenesis at baseline and when exposed to vascular endothelial growth factor. The NAD(+) precursor nicotinamide mononucleotide restored the cellular NAD(+)/NADH ratio and normalized the CypD-deficient phenotype. CypD knockout mice also presented accelerated wound healing and increased neovascularization on tissue injury as monitored by optical microangiography. Our study reveals the importance of the mitochondrial permeability transition pore in the regulation of endothelial mitochondrial metabolism and vascular function. The mitochondrial regulation of SIRT1 has broad implications in the epigenetic regulation of endothelial phenotype.
Publication Date: 2015-02-28
Journal: Circulation research

nmn deamidase(4)

New insights into the phylogeny and molecular classification of nicotinamide mononucleotide deamidases.
Nicotinamide mononucleotide (NMN) deamidase is one of the key enzymes of the bacterial pyridine nucleotide cycle (PNC). It catalyzes the conversion of NMN to nicotinic acid mononucleotide, which is later converted to NAD(+) by entering the Preiss-Handler pathway. However, very few biochemical data are available regarding this enzyme. This paper represents the first complete molecular characterization of a novel NMN deamidase from the halotolerant and alkaliphilic bacterium Oceanobacillus iheyensis (OiPncC). The enzyme was active over a broad pH range, with an optimum at pH 7.4, whilst maintaining 90 % activity at pH 10.0. Surprisingly, the enzyme was quite stable at such basic pH, maintaining 61 % activity after 21 days. As regard temperature, it had an optimum at 65 °C but its stability was better below 50 °C. OiPncC was a Michaelian enzyme towards its only substrate NMN, with a K m value of 0.18 mM and a kcat/K m of 2.1 mM(-1) s(-1). To further our understanding of these enzymes, a complete phylogenetic and structural analysis was carried out taking into account the two Pfam domains usually associated with them (MocF and CinA). This analysis sheds light on the evolution of NMN deamidases, and enables the classification of NMN deamidases into 12 different subgroups, pointing to a novel domain architecture never before described. Using a Logo representation, conserved blocks were determined, providing new insights on the crucial residues involved in the binding and catalysis of both CinA and MocF domains. The analysis of these conserved blocks within new protein sequences could permit the more efficient data curation of incoming NMN deamidases.
Publication Date: 2013-12-18
Journal: PloS one

3 nmnat3(3)

[Effects of nicotinamide mononucleotide adenylyl transferase 3 on mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells via regulating nicotinamide adenine dinucleotide levels].
To investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress The bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured The rabbit BMSCs were successfully isolated and cultured NMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD 研究烟酰胺单核苷酸腺苷转移酶 3(nicotinamide mononucleotide adenylyl transferase 3,NMNAT3)调节烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD 取新西兰大白兔股骨及胫骨骨髓,采用密度梯度离心联合贴壁培养法体外分离培养 BMSCs 并传代。取第 3 代细胞经流式细胞仪、多向诱导分化鉴定后,采用增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)标记的 NMNAT3 基因过表达慢病毒(Lv-NMNAT3-EGFP)进行转染(BMSCs/Lv-NMNAT3-EGFP),采用实时荧光定量 PCR(real-time fluorescence quantitative PCR,qRT-PCR)及 Western blot 检测 BMSCs 内 NMNAT3 基因及蛋白表达情况,细胞计数试剂盒 8(cell counting kit 8,CCK-8)方法检测细胞增殖能力;以阴性对照慢病毒转染 BMSCs(BMSCs/Lv-EGFP)以及未转染 BMSCs 作为对照。另采用 H 经鉴定,体外成功分离培养兔 BMSCs;经慢病毒转染技术获得高表达 NMNAT3 基因的兔 BMSCs 稳定株,细胞内 NMNAT3 基因及蛋白明显提高( NMNAT3 通过增加线粒体内 NAD
Publication Date: 2020-05-16
Journal: Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery

x 10(3)

Synthesis of pyridine nucleotide analogs using rabbit spleen pyridine nucleotide glycohydrolase and stereospecificity of the transglycosidation reaction.
Seven pyridine nucleotide analogs were synthesized, including nicotinic acid mononucleotide, nicotinic acid-adenine dinucleotide, and nicotinic acid-adenine dinucleotidephosphate, by means of the transglycosidase activity of rabbit spleen pyridine nucleotide glycohydrolase. The velocity ratio of the transglycosidation to the hydrolysis in the presence of 16 mM nicotinamide mononucleotide and 0.3 M nicotinic acid at pH 5.2 was greater than 10 and the transglycosidation/hydrolysis partition ratio was estimated to be (12 +/- 2) x 10(3). The partition ratio values obtained with 3-acetylpyridine and nicotinylglycine were smaller than that with nicotinic acid. Configurational analysis of pyridinium C-N glycosidic linkages of the transglycosidation products by means of 1H-NMR, UV, CD, and paper chromatography indicated that the configuration was completely retained and absolutely beta. Retention of the configuration, together with the substrate specificity of the enzyme for the beta-pyridinium linkage and formation of the intermediary enzyme-phosphoribosyl (or ADP-ribosyl) complex during the enzyme catalysis, indicated that the transglycosidation reaction catalyzed by the enzyme proceeds through the double replacement mechanism.
Publication Date: 1995-07-01
Journal: Journal of biochemistry

nmn levels(2)

The female mouse is resistant to mild vitamin B
Vitamin B Female C57BL/6RccHsd mice were subjected for 18 weeks to a diet without vitamin B In the female mice, no differences between the two dietary groups were found in liver nicotinamide mononucleotide (NMN) levels, body composition, whole body energy and substrate metabolism measured by indirect calorimetry, or liver triacylglycerol metabolism. Expression of seven genes that previously were shown to respond to mild vitamin B We concluded that the female 0NR mice were not vitamin B
Publication Date: 2021-08-03
Journal: European journal of nutrition

10 -6(2)

Enzyme activities leading to NAD synthesis in human lymphocytes.
Pyridine nucleotide levels and the activities of enzymes involved in NAD synthesis (nicotinic acid phosphoribosyltransferase, nicotinic acid- and nicotinamide mononucleotide-adenylyltransferase) have been assayed in human normal lymphocytes by an HPLC method using radioactive or nonradioactive substrates. NAD concentration was 46.4 +/- 17.2 pmol 10(-6) cells, and that of NADP was 14.5 +/- 3.9 pmol 10(-6) cells (mean +/- standard deviation). The adenylyltransferase activity using nicotinic acid mononucleotide as substrate was 1.530 +/- 0.216 nmol h(-1) 10(-6) cells, using nicotinamide mononucleotide was 1.466 +/- 0.354 nmol h(-1) 10(-6) cells. The apparent K(M) values were 0.015 mM for the former substrate and 0.167 mM for the latter. The mean activity of nicotinic acid phosphoribosyltransferase was 0.038 +/- 0.014 nmol h(-1) 10(-6) cells, and the apparent K(M) for nicotinic acid was 0.165 mM. The proposed methods, easy and rapid to perform, are reliable and sensitive, avoiding the use of radiolabels except for NAPRT and displaying a very low activity. The reported findings, together with the previous ones in human erythrocytes, can provide an useful base to investigate NAD metabolism in humans through the study of blood cells.
Publication Date: 2000-07-19
Journal: Archives of biochemistry and biophysics


Nicotinamide mononucleotide augments the cytotoxic activity of natural killer cells in young and elderly mice.
Nicotinamide mononucleotide (NMN), a key nicotinamide adenine dinucleotide (NAD
Publication Date: 2021-09-22
Journal: Biomedical research (Tokyo, Japan)


Transfer hydrogenation catalysis in cells.
Hydrogenation reactions in biology are usually carried out by enzymes with nicotinamide adenine dinucleotide (NAD(P)H) or flavin mononucleotide (FAMH
Publication Date: 2021-08-31
Journal: RSC chemical biology


Modulating Sirtuin Biology and Nicotinamide Adenine Diphosphate Metabolism in Cardiovascular Disease-From Bench to Bedside.
Sirtuins (SIRT1-7) comprise a family of highly conserved deacetylases with distribution in different subcellular compartments. Sirtuins deacetylate target proteins depending on one common substrate, nicotinamide adenine diphosphate (NAD
Publication Date: 2021-10-30
Journal: Frontiers in physiology


NMN Maintains Intestinal Homeostasis by Regulating the Gut Microbiota.
The aim of this study was to determine the effects of long-term Nicotinamide mononucleotide (NMN) treatment on modulating gut microbiota diversity and composition, as well as its association with intestinal barrier function. In this study, C57BL/6J mice were fed different concentrations of NMN, and their feces were collected for detection of 16S rDNA and non-targeted metabolites to explore the effects of NMN on intestinal microbiota and metabolites. The results revealed that NMN increased the abundance of butyric acid-producing bacteria (Ruminococcae_UCG-014 and Prevotellaceae_NK3B31_group) and other probiotics (Akkermansia muciniphila), while the abundance of several harmful bacteria (Bilophila and Oscillibacter) were decreased after NMN treatment. Meanwhile, the level of bile acid-related metabolites in feces from the G1 group (0.1 mg/ml) was significantly increased compared to the control group, including cholic acid, taurodeoxycholic acid, taurocholic acid, glycocholic acid, and tauro-β-muricholic acid. In addition, long-term NMN treatment affected the permeability of the intestinal mucosa. The number of goblet cells and mucus thickness increased, as well as expression of tight junction protein. These results demonstrate that NMN reduced intestinal mucosal permeability and exerts a protective effect on the intestinal tract. This study lays the foundation for exploring NMN's utility in clinical research.
Publication Date: 2021-08-17
Journal: Frontiers in nutrition


Upregulated Nmnat2 causes neuronal death and increases seizure susceptibility in temporal lobe epilepsy.
A significant pathological feature of refractory temporal lobe epilepsy (TLE) is neuronal loss. Oxidative stress caused by repeated seizures is an important mechanism leading to neuronal loss in hippocampus. Nicotinamide-adenine dinucleotide (NAD) a coenzyme that is involved in many biochemical oxidation-reduction reactions. Nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) catalyzes an essential step in NAD (NADP) biosynthetic pathwayhas and been considered as a neuronal maintenance factor that protect neurons against insults through context-dependent mechanism. However, it is unexpected that Nmnat2 does not play a neuroprotective role in epilepsy. We found that Nmnat2 was increased in mice model of TLE. Gain-of-function approach revealed that overexpression of Nmnat2 in CA1 area enhanced seizure susceptibility and caused neuronal loss in vivo. Moreover, we found that the chaperone function was essential to increased apoptosis through the function mutation of Nmnat2. Finally, Nmnat2 overexpression in vivo reduced in expression of SOD2 and increased FoxO3a. Overall, our study discloses a new biological function of Nmnat2 in epilepsy and provides novel insights into the molecular events underlying epilepsy.
Publication Date: 2020-11-29
Journal: Brain research bulletin


β-Nicotinamide Mononucleotide (NMN) Administrated by Intraperitoneal Injection Mediates Protection Against UVB-Induced Skin Damage in Mice.
Ultraviolet light is an important environmental factor that induces skin oxidation, inflammation, and other diseases. Nicotinamide mononucleotide (NMN) has the effect of anti-oxidation and improving various physiological processes. This study explores the protective effect of NMN monomers given via intraperitoneal injection on UVB-induced photodamage. We used a murine model of UVB-induced photodamage to evaluate the effect of an NMN monomer on photoaging skin by assessing skin and liver tissue sections, serum and skin oxidative stress levels, inflammatory markers, mRNA expression, and protein expression of skin- and liver-related genes. The results showed that NMN treatment blocked UVB-induced photodamage in mice, maintaining normal structure and amount of collagen fibers, normal thickness of epidermis and dermis, reducing the production of mast cells, and maintaining complete organized skin structure. NMN intraperitoneal injection also maintained the normal morphology of the mouse liver after UVB exposure. Meanwhile, NMN intraperitoneal injection was found to increase antioxidant ability and regulate the proinflammatory response of the skin and liver to UVB irradiation by enhancing the activity of antioxidant enzymes, release of anti-inflammatory cytokines, reduction of hydrogen peroxide production (H Based on the above results, NMN monomer treatment with intraperitoneal injection also block the photodamage caused by UVB irradiation in mice by regulating the oxidative stress response and inflammatory response.
Publication Date: 2021-10-23
Journal: Journal of inflammation research


Nicotinamide mononucleotide: An emerging nutraceutical against cardiac aging?
Nicotinamide adenine dinucleotide (NAD) is essential for cellular physiological processes, directly or indirectly affecting metabolism and gene expression. The decline of NAD
Publication Date: 2021-09-11
Journal: Current opinion in pharmacology


Abnormal levels of mitochondrial proteins in plasma neuronal extracellular vesicles in major depressive disorder.
To characterize neuronal mitochondrial abnormalities in major depressive disorder (MDD), functional mitochondrial proteins (MPs) extracted from enriched plasma neuron-derived extracellular vesicles (NDEVs) of MDD participants (n = 20) were quantified before and after eight weeks of treatment with a selective serotonin reuptake inhibitor (SSRI). Pretreatment baseline NDEV levels of the transcriptional type 2 nuclear respiratory factor (NRF2) which controls mitochondrial biogenesis and many anti-oxidant gene responses, regulators of diverse neuronal mitochondrial functions cyclophilin D (CYPD) and mitofusin-2 (MFN2), leucine zipper EF-hand containing transmembrane 1 protein (LETM1) component of a calcium channel/calcium channel enhancer, mitochondrial tethering proteins syntaphilin (SNPH) and myosin VI (MY06), inner membrane electron transport complexes I (subunit 6) and III (subunit 10), the penultimate enzyme of nicotinamide adenine dinucleotide (NAD) generation nicotinamide mononucleotide adenylytransferase 2 (NMNAT2), and neuronal mitochondrial metabolic regulatory and protective factors humanin and mitochondrial open-reading frame of the 12S rRNA-c (MOTS-c) all were significantly lower than those of NDEVs from matched controls (n = 10), whereas those of pro-neurodegenerative NADase Sterile Alpha and TIR motif-containing protein 1 (SARM1) were higher. The baseline NDEV levels of transcription factor A mitochondrial (TFAM) and the transcriptional master-regulator of mitochondrial biogenesis PPAR γ coactivator-1α (PGC-1α) showed no differences between MDD participants and controls. Several of these potential biomarker proteins showed substantially different changes in untreated MDD than those we reported in untreated first-episode psychosis. NDEV levels of MPs of all functional classes, except complex I-6, NRF2 and PGC-1α were normalized in MDD participants who responded to SSRI therapy (n = 10) but not in those who failed to respond (n = 10) by psychiatric evaluation. If larger studies validate NDEV MP abnormalities, they may become useful biomarkers and identify new drug targets.
Publication Date: 2021-09-03
Journal: Molecular psychiatry


Anterograde regulation of mitochondrial genes and FGF21 signaling by hepatic LSD1.
Mitochondrial biogenesis and function are controlled by anterograde regulatory pathways involving more than 1000 nuclear-encoded proteins. Transcriptional networks controlling the nuclear-encoded mitochondrial genes remain to be fully elucidated. Here, we show that histone demethylase LSD1 KO from adult mouse liver (LSD1-LKO) reduces the expression of one-third of all nuclear-encoded mitochondrial genes and decreases mitochondrial biogenesis and function. LSD1-modulated histone methylation epigenetically regulates nuclear-encoded mitochondrial genes. Furthermore, LSD1 regulates gene expression and protein methylation of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), which controls the final step of NAD+ synthesis and limits NAD+ availability in the nucleus. Lsd1 KO reduces NAD+-dependent SIRT1 and SIRT7 deacetylase activity, leading to hyperacetylation and hypofunctioning of GABPβ and PGC-1α, the major transcriptional factor/cofactor for nuclear-encoded mitochondrial genes. Despite the reduced mitochondrial function in the liver, LSD1-LKO mice are protected from diet-induced hepatic steatosis and glucose intolerance, partially due to induction of hepatokine FGF21. Thus, LSD1 orchestrates a core regulatory network involving epigenetic modifications and NAD+ synthesis to control mitochondrial function and hepatokine production.
Publication Date: 2021-07-28
Journal: JCI insight


Nicotinic acid mononucleotide is an allosteric SARM1 inhibitor promoting axonal protection.
SARM1 is an inducible NAD
Publication Date: 2021-08-18
Journal: Experimental neurology


Signaling pathways and effectors of aging.
Aging leads to and is associated with aberrant function of multiple signaling pathways and a host of factors that maintain cellular health. Under normal conditions, the prolongevity, 5' AMP-activated protein kinase (AMPK), is dedicated to the homeostasis of metabolism and autophagy for removal of damaged cellular compartments and molecules. A host of sirtuin family of molecules, that extend life-span, regulate metabolism and repair DNA damage, and possess either mono-ADP-ribosyltransferase, or deacylase activity. Another group of pro-longevity factors, include FOX (forkhead box) proteins, a family of transcription factors that regulate the expression of genes involved in cell growth, proliferation, differentiation, and longevity. Nicotinamide phosphoribosyltransferase (NAmPRTase or Nampt) catalyzes the condensation of nicotinamide with 5-phosphoribosyl-1-pyrophosphate to yield nicotinamide mononucleotide (NMN), a requisite step for production of NAD+, which is known to increase longevity. Loss of Klotho, a transmembrane enzyme that controls the sensitivity of the organism to insulin and suppresses oxidative stress and inflammation, leads to premature aging in mice. Hydrogen sulfide and transsulfuration pathways are crucial to the long life and are required in protection of cells against damage. Aging also leads to the imbalanced activation of other pathways and factors including p53, insulin and IGF signaling, P13K/AKT, mTOR, PKA, RAS, RTK, MEK, ERK, MAPK, CRTC-1/CREB and NFkB. Such aberrant cellular functions, disturb cell metabolism, derail autophagy and other housekeeping actions, inhibit cell division, induce inflammaging and immunosenecence, cause stem cell exhaustion and induce either senescence, apoptosis or cancer.
Publication Date: 2020-10-14
Journal: Frontiers in bioscience (Landmark edition)


Exploring the binding pocket of quinone/inhibitors in mitochondrial respiratory complex I by chemical biology approaches.
NADH-quinone oxidoreductase (respiratory complex I) is a key player in mitochondrial energy metabolism. The enzyme couples electron transfer from NADH to quinone with the translocation of protons across the membrane, providing a major proton-motive force that drives ATP synthesis. Recently, X-ray crystallography and cryo-electron microscopy provided further insights into the structure and functions of the enzyme. However, little is known about the mechanism of quinone reduction, which is a crucial step in the energy coupling process. A variety of complex I inhibitors targeting the quinone-binding site have been indispensable tools for mechanistic studies on the enzyme. Using biorationally designed inhibitor probes, the author has accumulated a large amount of experimental data characterizing the actions of complex I inhibitors. On the basis of comprehensive interpretations of the data, the author reviews the structural features of the binding pocket of quinone/inhibitors in bovine mitochondrial complex I. ATP: adenosine triphosphate; BODIPY: boron dipyrromethene; complex I: proton-translocating NADH-quinone oxidoreductase; DIBO: dibenzocyclooctyne; EM: electron microscopy; FeS: iron-sulfur; FMN: flavin adenine mononucleotide; LDT: ligand-directed tosylate; NADH: nicotinamide adenine dinucleotide; ROS: reactive oxygen species; SMP: submitochondrial particle; TAMRA: 6-carboxy-
Publication Date: 2020-04-09
Journal: Bioscience, biotechnology, and biochemistry


NAMPT promotes hepatitis B virus replication and liver cancer cell proliferation through the regulation of aerobic glycolysis.
Nicotinamide phosphoribosyltransferase (NAMPT) is a critical rate-limiting enzyme involved in NAD synthesis that has been shown to contribute to the progression of liver cancer. However, the potential role and mechanism of NAMPT in hepatitis B virus (HBV)-associated liver cancer remain unclear. The present study assessed the expression of NAMPT in HBV-positive and -negative liver cancer cells, and investigated whether HBV-induced NAMPT expression is dependent on HBV X protein (HBx). In addition, the role of NAMPT in HBV replication and transcription, and in HBV-mediated liver cancer cell growth was explored. The effects of NAMPT on the glycolytic pathway were also evaluated. Reverse transcription-quantitative PCR and western blotting results revealed that NAMPT expression levels were significantly higher in HBV-positive liver cancer cells than in HBV-negative liver cancer cells, and this effect was HBx-dependent. Moreover, the activation of NAMPT was demonstrated to be required for HBV replication and transcription. The NAMPT inhibitor FK866 repressed cell survival and promoted cell death in HBV-expressing liver cancer cells, and these effects were attenuated by nicotinamide mononucleotide. Furthermore, the inhibition of NAMPT was associated with decreased glucose uptake, decreased lactate production and decreased ATP levels in HBV-expressing liver cancer cells, indicating that NAMPT may promote the aerobic glycolysis. Collectively, these findings reveal a positive feedback loop in which HBV enhances NAMPT expression and the activation of NAMPT promotes HBV replication and HBV-mediated malignant cell growth in liver cancer. The present study highlights the important role of NAMPT in the regulation of aerobic glycolysis in HBV-mediated liver cancer, and suggests that NAMPT may be a promising treatment target for patients with HBV-associated liver cancer.
Publication Date: 2021-03-30
Journal: Oncology letters


Longevity pathways in stress resistance: targeting NAD and sirtuins to treat the pathophysiology of hemorrhagic shock.
Stress resistance correlates with longevity and this pattern has been exploited to help identify genes that can influence lifespan. Reciprocally, genes and pharmacological agents that have been studied primarily in the context of longevity may be an untapped resource for treating acute stresses. Here we summarize the evidence that targeting SIRT1, studied primarily in the context of longevity, can improve outcomes in hemorrhagic shock and resuscitation. Hemorrhagic shock is a potentially fatal condition that occurs when blood loss is so severe that tissues no longer receive adequate oxygen. While stabilizing the blood pressure and reperfusing tissues are necessary, re-introducing oxygen to ischemic tissues generates a burst of reactive oxygen species that can cause secondary tissue damage. Reactive oxygen species not only exacerbate the inflammatory cascade but also can directly damage mitochondria, leading to bioenergetic failure in the affected tissues. Treatments with polyphenol resveratrol and with nicotinamide adenine dinucleotide (NAD) precursors have both shown promising results in rodent models of hemorrhagic shock and resuscitation. Although a number of different mechanisms may be at play in each case, a common theme is that resveratrol and NAD both enhance the activity of SIRT1. Moreover, many of the physiologic improvements observed with resveratrol and NAD precursors are consistent with modulation of known SIRT1 targets. Because small blood vessels and limited blood volume make mice very challenging for the development of hemorrhagic shock models, there is a paucity of direct genetic evidence testing the role of SIRT1. However, the development of more robust methods in mice as well as genetic modifications in rats should allow the study of SIRT1 transgenic and KO rodents in the near future. The potential therapeutic effect of SIRT1 in hemorrhagic shock may serve as an important example supporting the value of considering "longevity" pathways in the mitigation of acute stresses.
Publication Date: 2021-01-20
Journal: GeroScience


Correction: Pre-emptive Short-term Nicotinamide Mononucleotide Treatment in a Mouse Model of Diabetic Nephropathy.
Publication Date: 2021-10-03
Journal: Journal of the American Society of Nephrology : JASN


Lessons from Injury: How Nerve Injury Studies Reveal Basic Biological Mechanisms and Therapeutic Opportunities for Peripheral Nerve Diseases.
Since Waller and Cajal in the nineteenth and early twentieth centuries, laboratory traumatic peripheral nerve injury studies have provided great insight into cellular and molecular mechanisms governing axon degeneration and the responses of Schwann cells, the major glial cell type of peripheral nerves. It is now evident that pathways underlying injury-induced axon degeneration and the Schwann cell injury-specific state, the repair Schwann cell, are relevant to many inherited and acquired disorders of peripheral nerves. This review provides a timely update on the molecular understanding of axon degeneration and formation of the repair Schwann cell. We discuss how nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) and sterile alpha TIR motif containing protein 1 (SARM1) are required for axon survival and degeneration, respectively, how transcription factor c-JUN is essential for the Schwann cell response to nerve injury and what each tells us about disease mechanisms and potential therapies. Human genetic association with NMNAT2 and SARM1 strongly suggests aberrant activation of programmed axon death in polyneuropathies and motor neuron disorders, respectively, and animal studies suggest wider involvement including in chemotherapy-induced and diabetic neuropathies. In repair Schwann cells, cJUN is aberrantly expressed in a wide variety of human acquired and inherited neuropathies. Animal models suggest it limits axon loss in both genetic and traumatic neuropathies, whereas in contrast, Schwann cell secreted Neuregulin-1 type 1 drives onion bulb pathology in CMT1A. Finally, we discuss opportunities for drug-based and gene therapies to prevent axon loss or manipulate the repair Schwann cell state to treat acquired and inherited neuropathies and neuronopathies.
Publication Date: 2021-10-02
Journal: Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics


NMNAT Proteins that Limit Wallerian Degeneration Also Regulate Critical Period Plasticity in the Visual Cortex.
Many brain regions go through critical periods of development during which plasticity is enhanced. These critical periods are associated with extensive growth and retraction of thalamocortical and intracortical axons. Here, we investigated whether a signaling pathway that is central in Wallerian axon degeneration also regulates critical period plasticity in the primary visual cortex (V1). Wallerian degeneration is characterized by rapid disintegration of axons once they are separated from the cell body. This degenerative process is initiated by reduced presence of cytoplasmic nicotinamide mononucleotide adenylyltransferases (NMNATs) and is strongly delayed in mice overexpressing cytoplasmic NMNAT proteins, such as Wld
Publication Date: 2019-01-24
Journal: eNeuro


A Pilot Study Investigating Changes in the Human Plasma and Urine NAD+ Metabolome During a 6 Hour Intravenous Infusion of NAD.
Accumulating evidence suggests that active maintenance of optimal levels of the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD+) is beneficial in conditions of either increased NAD+ turnover or inadequate synthesis, including Alzheimer's disease and other neurodegenerative disorders and the aging process. While studies have documented the efficacy of some NAD+ precursors such as nicotinamide riboside (NR) in raising plasma NAD+, no data are currently available on the fate of directly infused NAD+ in a human cohort. This study, therefore, documented changes in plasma and urine levels of NAD+ and its metabolites during and after a 6 h 3 μmol/min NAD+ intravenous (IV) infusion. Surprisingly, no change in plasma (NAD+) or metabolites [nicotinamide, methylnicotinamide, adenosine phosphoribose ribose (ADPR) and nicotinamide mononucleotide (NMN)] were observed until after 2 h. Increased urinary excretion of methylnicotinamide and NAD+ were detected at 6 h, however, no significant rise in urinary nicotinamide was observed. This study revealed for the first time that: (i) at an infusion rate of 3 μmol/min NAD+ is rapidly and completely removed from the plasma for at least the first 2 h; (ii) the profile of metabolites is consistent with NAD+ glycohydrolase and NAD+ pyrophosphatase activity; and (iii) urinary excretion products arising from an NAD+ infusion include NAD+ itself and methyl nicotinamide (meNAM) but not NAM.
Publication Date: 2019-10-02
Journal: Frontiers in aging neuroscience


Independent AMP and NAD signaling regulates C2C12 differentiation and metabolic adaptation.
The balance of ATP production and consumption is reflected in adenosine monophosphate (AMP) and nicotinamide adenine dinucleotide (NAD) content and has been associated with phenotypic plasticity in striated muscle. Some studies have suggested that AMPK-dependent plasticity may be an indirect consequence of increased NAD synthesis and SIRT1 activity. The primary goal of this study was to assess the interaction of AMP- and NAD-dependent signaling in adaptation of C2C12 myotubes. Changes in myotube developmental and metabolic gene expression were compared following incubation with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and nicotinamide mononucleotide (NMN) to activate AMPK- and NAD-related signaling. AICAR showed no effect on NAD pool or nampt expression but significantly reduced histone H3 acetylation and GLUT1, cytochrome C oxidase subunit 2 (COX2), and MYH3 expression. In contrast, NMN supplementation for 24 h increased NAD pool by 45 % but did not reduce histone H3 acetylation nor promote mitochondrial gene expression. The combination of AMP and NAD signaling did not induce further metabolic adaptation, but NMN ameliorated AICAR-induced myotube reduction. We interpret these results as indication that AMP and NAD contribute to C2C12 differentiation and metabolic adaptation independently.
Publication Date: 2016-07-10
Journal: Journal of physiology and biochemistry


Coupled Control of Distal Axon Integrity and Somal Responses to Axonal Damage by the Palmitoyl Acyltransferase ZDHHC17.
After optic nerve crush (ONC), the cell bodies and distal axons of most retinal ganglion cells (RGCs) degenerate. RGC somal and distal axon degenerations were previously thought to be controlled by two parallel pathways, involving activation of the kinase dual leucine-zipper kinase (DLK) and loss of the axon survival factor nicotinamide mononucleotide adenylyltransferase-2 (NMNAT2), respectively. Here, we report that palmitoylation of both DLK and NMNAT2 by the palmitoyl acyltransferase ZDHHC17 couples these signals. ZDHHC17-dependent palmitoylation enables DLK-dependent somal degeneration after ONC and also ensures NMNAT-dependent distal axon integrity in healthy optic nerves. We provide evidence that ZDHHC17 also controls survival-versus-degeneration decisions in dorsal root ganglion (DRG) neurons, and we identify conserved motifs in NMNAT2 and DLK that govern their ZDHHC17-dependent regulation. These findings suggest that the control of somal and distal axon integrity should be considered as a single, holistic process, mediated by the concerted action of two palmitoylation-dependent pathways.
Publication Date: 2020-11-19
Journal: Cell reports