Enhancing interacting residue prediction with integrated contact matrix prediction in protein-protein interaction.

PubMed

Du, Tianchuan; Liao, Li; Wu, Cathy H

2016-12-01

Identifying the residues in a protein that are involved in protein-protein interaction and identifying the contact matrix for a pair of interacting proteins are two computational tasks at different levels of an in-depth analysis of protein-protein interaction. Various methods for solving these two problems have been reported in the literature. However, the interacting residue prediction and contact matrix prediction were handled by and large independently in those existing methods, though intuitively good prediction of interacting residues will help with predicting the contact matrix. In this work, we developed a novel protein interacting residue prediction system, contact matrix-interaction profile hidden Markov model (CM-ipHMM), with the integration of contact matrix prediction and the ipHMM interaction residue prediction. We propose to leverage what is learned from the contact matrix prediction and utilize the predicted contact matrix as "feedback" to enhance the interaction residue prediction. The CM-ipHMM model showed significant improvement over the previous method that uses the ipHMM for predicting interaction residues only. It indicates that the downstream contact matrix prediction could help the interaction site prediction.

Plastidial NAD-Dependent Malate Dehydrogenase: A Moonlighting Protein Involved in Early Chloroplast Development Through its Interaction with an FtsH12-FtsHi Protease Complex.

PubMed

Schreier, Tina B; Antoine, Cléry; Schläfli, Michael; Galbier, Florian; Stadler, Martha; Demarsy, Emilie; Albertini, Daniele; Maier, Benjamin A; Kessler, Felix; Hörtensteiner, Stefan; Zeeman, Samuel C; Kötting, Oliver

2018-06-22

Malate dehydrogenases (MDH) convert malate to oxaloacetate using NAD(H) or NADP(H) as a cofactor. Arabidopsis thaliana mutants lacking plastidial NAD-dependent MDH (pdnad-mdh) are embryo-lethal, and constitutive silencing (miR-mdh-1) causes a pale, dwarfed phenotype. The reason for these severe phenotypes is unknown. Here, we rescued the embryo lethality of pdnad-mdh via embryo-specific expression of pdNAD-MDH. Rescued seedlings developed white leaves with aberrant chloroplasts and failed to reproduce. Inducible silencing of pdNAD-MDH at the rosette stage also resulted in white newly emerging leaves. These data suggest that pdNAD-MDH is important for early plastid development, which is consistent with the reductions in major plastidial galactolipid, carotenoid and protochlorophyllide levels in miR-mdh-1 seedlings. Surprisingly, the targeting of other NAD-dependent MDH isoforms to the plastid did not complement the embryo lethality of pdnad-mdh, while expression of enzymatically inactive pdNAD-MDH did. These complemented plants grew indistinguishably from the wild type. Both active and inactive forms of pdNAD-MDH interact with a heteromeric AAA-ATPase complex at the inner membrane of the chloroplast envelope. Silencing the expression of FtsH12, a key member of this complex, resulted in a phenotype that strongly resembles miR-mdh-1. We propose that pdNAD-MDH is essential for chloroplast development due to its moonlighting role in stabilizing FtsH12, distinct from its enzymatic function. © 2018 American Society of Plant Biologists. All rights reserved.

Nicotinamide riboside kinase structures reveal new pathways to NAD+.

PubMed

Tempel, Wolfram; Rabeh, Wael M; Bogan, Katrina L; Belenky, Peter; Wojcik, Marzena; Seidle, Heather F; Nedyalkova, Lyudmila; Yang, Tianle; Sauve, Anthony A; Park, Hee-Won; Brenner, Charles

2007-10-02

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD+) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD+ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD+.

SARM1-specific motifs in the TIR domain enable NAD+ loss and regulate injury-induced SARM1 activation.

PubMed

Summers, Daniel W; Gibson, Daniel A; DiAntonio, Aaron; Milbrandt, Jeffrey

2016-10-11

Axon injury in response to trauma or disease stimulates a self-destruction program that promotes the localized clearance of damaged axon segments. Sterile alpha and Toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) is an evolutionarily conserved executioner of this degeneration cascade, also known as Wallerian degeneration; however, the mechanism of SARM1-dependent neuronal destruction is still obscure. SARM1 possesses a TIR domain that is necessary for SARM1 activity. In other proteins, dimerized TIR domains serve as scaffolds for innate immune signaling. In contrast, dimerization of the SARM1 TIR domain promotes consumption of the essential metabolite NAD + and induces neuronal destruction. This activity is unique to the SARM1 TIR domain, yet the structural elements that enable this activity are unknown. In this study, we identify fundamental properties of the SARM1 TIR domain that promote NAD + loss and axon degeneration. Dimerization of the TIR domain from the Caenorhabditis elegans SARM1 ortholog TIR-1 leads to NAD + loss and neuronal death, indicating these activities are an evolutionarily conserved feature of SARM1 function. Detailed analysis of sequence homology identifies canonical TIR motifs as well as a SARM1-specific (SS) loop that are required for NAD + loss and axon degeneration. Furthermore, we identify a residue in the SARM1 BB loop that is dispensable for TIR activity yet required for injury-induced activation of full-length SARM1, suggesting that SARM1 function requires multidomain interactions. Indeed, we identify a physical interaction between the autoinhibitory N terminus and the TIR domain of SARM1, revealing a previously unrecognized direct connection between these domains that we propose mediates autoinhibition and activation upon injury.

Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD+-Auxotrophic Mutant ▿

PubMed Central

Zhou, Yongjin; Wang, Lei; Yang, Fan; Lin, Xinping; Zhang, Sufang; Zhao, Zongbao K.

2011-01-01

NAD (NAD+) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD+ level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD+ auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD+ de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD+. We then constructed the NAD+-auxotrophic mutant YJE003 by disrupting the essential gene nadE, which is responsible for the last step of NAD+ biosynthesis in cells harboring the ntt4 gene. The minimal NAD+ level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD+, while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD+ was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD+ concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed. PMID:21742902

Dual emission fluorescent silver nanoclusters for sensitive detection of the biological coenzyme NAD+/NADH.

PubMed

Yuan, Yufeng; Huang, Kehan; Chang, Mengfang; Qin, Cuifang; Zhang, Sanjun; Pan, Haifeng; Chen, Yan; Xu, Jianhua

2016-02-01

Fluorescent silver nanoclusters (Ag NCs) displaying dual-excitation and dual-emission properties have been developed for the specific detection of NAD(+) (nicotinamide adenine dinucleotide, oxidized form). With the increase of NAD(+) concentrations, the longer wavelength emission (with the peak at 550 nm) was gradually quenched due to the strong interactions between the NAD(+) and Ag NCs, whereas the shorter wavelength emission (peaking at 395 nm) was linearly enhanced. More important, the dual-emission intensity ratio (I395/I550), fitting by a single-exponential decay function, can efficiently detect various NAD(+) levels from 100 to 4000 μM, as well as label NAD(+)/NADH (reduced form of NAD) ratios in the range of 1-50. Copyright © 2015 Elsevier Inc. All rights reserved.

NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism.

PubMed

Xiao, Wusheng; Wang, Rui-Sheng; Handy, Diane E; Loscalzo, Joseph

2018-01-20

The nicotinamide adenine dinucleotide (NAD + )/reduced NAD + (NADH) and NADP + /reduced NADP + (NADPH) redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders. Recent Advances: Newly identified biosynthetic enzymes and newly developed genetically encoded biosensors enable us to understand better how cells maintain compartmentalized NAD(H) and NADP(H) pools. The concept of redox stress (oxidative and reductive stress) reflected by changes in NAD(H)/NADP(H) has increasingly gained attention. The emerging roles of NAD + -consuming proteins in regulating cellular redox and metabolic homeostasis are active research topics. The biosynthesis and distribution of cellular NAD(H) and NADP(H) are highly compartmentalized. It is critical to understand how cells maintain the steady levels of these redox couple pools to ensure their normal functions and simultaneously avoid inducing redox stress. In addition, it is essential to understand how NAD(H)- and NADP(H)-utilizing enzymes interact with other signaling pathways, such as those regulated by hypoxia-inducible factor, to maintain cellular redox homeostasis and energy metabolism. Additional studies are needed to investigate the inter-relationships among compartmentalized NAD(H)/NADP(H) pools and how these two dinucleotide redox couples collaboratively regulate cellular redox states and cellular metabolism under normal and pathological conditions. Furthermore, recent studies suggest the utility of using pharmacological interventions or nutrient-based bioactive NAD + precursors as therapeutic interventions for metabolic diseases. Thus, a better understanding of the cellular functions of NAD(H) and NADP(H) may facilitate efforts to address a host of pathological disorders effectively. Antioxid. Redox Signal. 28, 251-272.

Pyridine Nucleotide Complexes with Bacillus anthracis Coenzyme A-Disulfide Reductase: A Structural Analysis of Dual NAD(P)H Specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wallen,J.; Paige, C.; Mallett, T.

2008-01-01

We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis, and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 Angstroms resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides.more » The structures of the NADH and NADPH complexes at ca. 2.3 Angstroms resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367, 425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.« less

Normalization of NAD+ Redox Balance as a Therapy for Heart Failure.

PubMed

Lee, Chi Fung; Chavez, Juan D; Garcia-Menendez, Lorena; Choi, Yongseon; Roe, Nathan D; Chiao, Ying Ann; Edgar, John S; Goo, Young Ah; Goodlett, David R; Bruce, James E; Tian, Rong

2016-09-20

Impairments of mitochondrial function in the heart are linked intricately to the development of heart failure, but there is no therapy for mitochondrial dysfunction. We assessed the reduced/oxidized ratio of nicotinamide adenine dinucleotide (NADH/NAD(+) ratio) and protein acetylation in the failing heart. Proteome and acetylome analyses were followed by docking calculation, mutagenesis, and mitochondrial calcium uptake assays to determine the functional role of specific acetylation sites. The therapeutic effects of normalizing mitochondrial protein acetylation by expanding the NAD(+) pool also were tested. Increased NADH/NAD(+) and protein hyperacetylation, previously observed in genetic models of defective mitochondrial function, also are present in human failing hearts as well as in mouse hearts with pathologic hypertrophy. Elevation of NAD(+) levels by stimulating the NAD(+) salvage pathway suppressed mitochondrial protein hyperacetylation and cardiac hypertrophy, and improved cardiac function in responses to stresses. Acetylome analysis identified a subpopulation of mitochondrial proteins that was sensitive to changes in the NADH/NAD(+) ratio. Hyperacetylation of mitochondrial malate-aspartate shuttle proteins impaired the transport and oxidation of cytosolic NADH in the mitochondria, resulting in altered cytosolic redox state and energy deficiency. Furthermore, acetylation of oligomycin-sensitive conferring protein at lysine-70 in adenosine triphosphate synthase complex promoted its interaction with cyclophilin D, and sensitized the opening of mitochondrial permeability transition pore. Both could be alleviated by normalizing the NAD(+) redox balance either genetically or pharmacologically. We show that mitochondrial protein hyperacetylation due to NAD(+) redox imbalance contributes to the pathologic remodeling of the heart via 2 distinct mechanisms. Our preclinical data demonstrate a clear benefit of normalizing NADH/NAD(+) imbalance in the failing hearts

Hairpin stabilized fluorescent silver nanoclusters for quantitative detection of NAD+ and monitoring NAD+/NADH based enzymatic reactions.

PubMed

Jain, Priyamvada; Chakma, Babina; Patra, Sanjukta; Goswami, Pranab

2017-03-01

A set of 90 mer long ssDNA candidates, with different degrees of cytosine (C-levels) (% and clusters) was analyzed for their function as suitable Ag-nanocluster (AgNC) nucleation scaffolds. The sequence (P4) with highest C-level (42.2%) emerged as the only candidate supporting the nucleation process as evident from its intense fluorescence peak at λ 660 nm . Shorter DNA subsets derived from P4 with only stable hairpin structures could support the AgNC formation. The secondary hairpin structures were confirmed by PAGE, and CD studies. The number of base pairs in the stem region also contributes to the stability of the hairpins. A shorter 29 mer sequence (Sub 3) (ΔG = -1.3 kcal/mol) with 3-bp in the stem of a 7-mer loop conferred highly stable AgNC. NAD + strongly quenched the fluorescence of Sub 3-AgNC in a concentration dependent manner. Time resolved photoluminescence studies revealed the quenching involves a combined static and dynamic interaction where the binding constant and number of binding sites for NAD + were 0.201 L mol -1 and 3.6, respectively. A dynamic NAD + detection range of 50-500 μM with a limit of detection of 22.3 μM was discerned. The NAD + mediated quenching of AgNC was not interfered by NADH, NADP + , monovalent and divalent ions, or serum samples. The method was also used to follow alcohol dehydrogenase and lactate dehydrogenase catalyzed physiological reactions in a turn-on and turn-off assay, respectively. The proposed method with ssDNA-AgNC could therefore be extended to monitor other NAD + /NADH based enzyme catalyzed reactions in a turn-on/turn-off approach. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus.

PubMed

Hektor, Harm J; Kloosterman, Harm; Dijkhuizen, Lubbert

2002-12-06

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.

The Peroxisomal NAD Carrier from Arabidopsis Imports NAD in Exchange with AMP.

PubMed

van Roermund, Carlo W T; Schroers, Martin G; Wiese, Jan; Facchinelli, Fabio; Kurz, Samantha; Wilkinson, Sabrina; Charton, Lennart; Wanders, Ronald J A; Waterham, Hans R; Weber, Andreas P M; Link, Nicole

2016-07-01

Cofactors such as NAD, AMP, and Coenzyme A (CoA) are essential for a diverse set of reactions and pathways in the cell. Specific carrier proteins are required to distribute these cofactors to different cell compartments, including peroxisomes. We previously identified a peroxisomal transport protein in Arabidopsis (Arabidopsis thaliana) called the peroxisomal NAD carrier (PXN). When assayed in vitro, this carrier exhibits versatile transport functions, e.g. catalyzing the import of NAD or CoA, the exchange of NAD/NADH, and the export of CoA. These observations raise the question about the physiological function of PXN in plants. Here, we used Saccharomyces cerevisiae to address this question. First, we confirmed that PXN, when expressed in yeast, is active and targeted to yeast peroxisomes. Secondl, detailed uptake analyses revealed that the CoA transport function of PXN can be excluded under physiological conditions due to its low affinity for this substrate. Third, we expressed PXN in diverse mutant yeast strains and investigated the suppression of the mutant phenotypes. These studies provided strong evidences that PXN was not able to function as a CoA transporter or a redox shuttle by mediating a NAD/NADH exchange, but instead catalyzed the import of NAD into peroxisomes against AMP in intact yeast cells. © 2016 American Society of Plant Biologists. All Rights Reserved.

Coupling of NAD+ biosynthesis and nicotinamide ribosyl transport: characterization of NadR ribonucleotide kinase mutants of Haemophilus influenzae.

PubMed

Merdanovic, Melisa; Sauer, Elizabeta; Reidl, Joachim

2005-07-01

Previously, we characterized a pathway necessary for the processing of NAD+ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae. Here we report on the role of NadR, which is essential for NAD+ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV+ (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae. Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD+ under in vitro conditions and by NAD+ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD+ synthesis and is also involved in controlling the NAD+ biosynthesis rate.

Dynamics of NAD-metabolism: everything but constant.

PubMed

Opitz, Christiane A; Heiland, Ines

2015-12-01

NAD, as well as its phosphorylated form, NADP, are best known as electron carriers and co-substrates of various redox reactions. As such they participate in approximately one quarter of all reactions listed in the reaction database KEGG. In metabolic pathway analysis, the total amount of NAD is usually assumed to be constant. That means that changes in the redox state might be considered, but concentration changes of the NAD moiety are usually neglected. However, a growing number of NAD-consuming reactions have been identified, showing that this assumption does not hold true in general. NAD-consuming reactions are common characteristics of NAD(+)-dependent signalling pathways and include mono- and poly-ADP-ribosylation of proteins, NAD(+)-dependent deacetylation by sirtuins and the formation of messenger molecules such as cyclic ADP-ribose (cADPR) and nicotinic acid (NA)-ADP (NAADP). NAD-consuming reactions are thus involved in major signalling and gene regulation pathways such as DNA-repair or regulation of enzymes central in metabolism. All known NAD(+)-dependent signalling processes include the release of nicotinamide (Nam). Thus cellular NAD pools need to be constantly replenished, mostly by recycling Nam to NAD(+). This process is, among others, regulated by the circadian clock, causing complex dynamic changes in NAD concentration. As disturbances in NAD homoeostasis are associated with a large number of diseases ranging from cancer to diabetes, it is important to better understand the dynamics of NAD metabolism to develop efficient pharmacological invention strategies to target this pathway. © 2015 Authors; published by Portland Press Limited.

In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

PubMed Central

Zhu, Xiao-Hong; Lu, Ming; Lee, Byeong-Yeul; Ugurbil, Kamil; Chen, Wei

2015-01-01

NAD is an essential metabolite that exists in NAD+ or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD+/NADH redox state and modulating cellular signaling processes through the activity of the NAD+-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD+ and NADH contents and the NAD+/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD+, total NAD contents, and NAD+/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ. PMID:25730862

Coupling of NAD+ Biosynthesis and Nicotinamide Ribosyl Transport: Characterization of NadR Ribonucleotide Kinase Mutants of Haemophilus influenzae

PubMed Central

Merdanovic, Melisa; Sauer, Elizabeta; Reidl, Joachim

2005-01-01

Previously, we characterized a pathway necessary for the processing of NAD+ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae. Here we report on the role of NadR, which is essential for NAD+ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV+ (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae. Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD+ under in vitro conditions and by NAD+ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD+ synthesis and is also involved in controlling the NAD+ biosynthesis rate. PMID:15968050

Restoring NAD(+) Levels with NAD(+) Intermediates, the Second Law of Thermodynamics and Aging Delay.

PubMed

Poljsak, Borut; Milisav, Irina

2018-04-26

The hypothesis regarding the role of increased nicotinamide adenine dinucleotide (NAD+) levels with reference to the fundamental concepts of ageing and entropy is presented. Considering the second law of thermodynamics, NAD+ seems the appropriate candidate for reversing many aging-associated pathologies. NAD+ is presented as an essential compound that enables organisms to stay highly organized and well-maintained, with a lower entropy state.

Bioinformatic prediction and in vivo validation of residue-residue interactions in human proteins

NASA Astrophysics Data System (ADS)

Jordan, Daniel; Davis, Erica; Katsanis, Nicholas; Sunyaev, Shamil

2014-03-01

Identifying residue-residue interactions in protein molecules is important for understanding both protein structure and function in the context of evolutionary dynamics and medical genetics. Such interactions can be difficult to predict using existing empirical or physical potentials, especially when residues are far from each other in sequence space. Using a multiple sequence alignment of 46 diverse vertebrate species we explore the space of allowed sequences for orthologous protein families. Amino acid changes that are known to damage protein function allow us to identify specific changes that are likely to have interacting partners. We fit the parameters of the continuous-time Markov process used in the alignment to conclude that these interactions are primarily pairwise, rather than higher order. Candidates for sites under pairwise epistasis are predicted, which can then be tested by experiment. We report the results of an initial round of in vivo experiments in a zebrafish model that verify the presence of multiple pairwise interactions predicted by our model. These experimentally validated interactions are novel, distant in sequence, and are not readily explained by known biochemical or biophysical features.

Quantitative Analysis of NAD Synthesis-Breakdown Fluxes.

PubMed

Liu, Ling; Su, Xiaoyang; Quinn, William J; Hui, Sheng; Krukenberg, Kristin; Frederick, David W; Redpath, Philip; Zhan, Le; Chellappa, Karthikeyani; White, Eileen; Migaud, Marie; Mitchison, Timothy J; Baur, Joseph A; Rabinowitz, Joshua D

2018-05-01

The redox cofactor nicotinamide adenine dinucleotide (NAD) plays a central role in metabolism and is a substrate for signaling enzymes including poly-ADP-ribose-polymerases (PARPs) and sirtuins. NAD concentration falls during aging, which has triggered intense interest in strategies to boost NAD levels. A limitation in understanding NAD metabolism has been reliance on concentration measurements. Here, we present isotope-tracer methods for NAD flux quantitation. In cell lines, NAD was made from nicotinamide and consumed largely by PARPs and sirtuins. In vivo, NAD was made from tryptophan selectively in the liver, which then excreted nicotinamide. NAD fluxes varied widely across tissues, with high flux in the small intestine and spleen and low flux in the skeletal muscle. Intravenous administration of nicotinamide riboside or mononucleotide delivered intact molecules to multiple tissues, but the same agents given orally were metabolized to nicotinamide in the liver. Thus, flux analysis can reveal tissue-specific NAD metabolism. Copyright © 2018 Elsevier Inc. All rights reserved.

Isonicotinic Acid Hydrazide Conversion to Isonicotinyl-NAD by Catalase-peroxidases*

PubMed Central

Wiseman, Ben; Carpena, Xavi; Feliz, Miguel; Donald, Lynda J.; Pons, Miquel; Fita, Ignacio; Loewen, Peter C.

2010-01-01

Activation of the pro-drug isoniazid (INH) as an anti-tubercular drug in Mycobacterium tuberculosis involves its conversion to isonicotinyl-NAD, a reaction that requires the catalase-peroxidase KatG. This report shows that the reaction proceeds in the absence of KatG at a slow rate in a mixture of INH, NAD+, Mn2+, and O2, and that the inclusion of KatG increases the rate by >7 times. Superoxide, generated by either Mn2+- or KatG-catalyzed reduction of O2, is an essential intermediate in the reaction. Elimination of the peroxidatic process by mutation slows the rate of reaction by 60% revealing that the peroxidatic process enhances, but is not essential for isonicotinyl-NAD formation. The isonicotinyl-NAD•+ radical is identified as a reaction intermediate, and its reduction by superoxide is proposed. Binding sites for INH and its co-substrate, NAD+, are identified for the first time in crystal complexes of Burkholderia pseudomallei catalase-peroxidase with INH and NAD+ grown by co-crystallization. The best defined INH binding sites were identified, one in each subunit, on the opposite side of the protein from the entrance to the heme cavity in a funnel-shaped channel. The NAD+ binding site is ∼20 Å from the entrance to the heme cavity and involves interactions primarily with the AMP portion of the molecule in agreement with the NMR saturation transfer difference results. PMID:20554537

Prediction of interface residue based on the features of residue interaction network.

PubMed

Jiao, Xiong; Ranganathan, Shoba

2017-11-07

Protein-protein interaction plays a crucial role in the cellular biological processes. Interface prediction can improve our understanding of the molecular mechanisms of the related processes and functions. In this work, we propose a classification method to recognize the interface residue based on the features of a weighted residue interaction network. The random forest algorithm is used for the prediction and 16 network parameters and the B-factor are acting as the element of the input feature vector. Compared with other similar work, the method is feasible and effective. The relative importance of these features also be analyzed to identify the key feature for the prediction. Some biological meaning of the important feature is explained. The results of this work can be used for the related work about the structure-function relationship analysis via a residue interaction network model. Copyright © 2017 Elsevier Ltd. All rights reserved.

Permeabilization of fungal hyphae by the plant defensin NaD1 occurs through a cell wall-dependent process.

PubMed

van der Weerden, Nicole L; Hancock, Robert E W; Anderson, Marilyn A

2010-11-26

The antifungal activity of the plant defensin NaD1 involves specific interaction with the fungal cell wall, followed by permeabilization of the plasma membrane and entry of NaD1 into the cytoplasm. Prior to this study, the role of membrane permeabilization in the activity of NaD1, as well as the relevance of cell wall binding, had not been investigated. To address this, the permeabilization of Fusarium oxysporum f. sp. vasinfectum hyphae by NaD1 was investigated and compared with that by other antimicrobial peptides, including the cecropin-melittin hybrid peptide CP-29, the bovine peptide BMAP-28, and the human peptide LL-37, which are believed to act largely through membrane disruption. NaD1 appeared to permeabilize cells via a novel mechanism that required the presence of the fungal cell wall. NaD1 and Bac2A, a linear variant of the bovine peptide bactenecin, were able to enter the cytoplasm of treated hyphae, indicating that cell death is accelerated by interaction with intracellular targets.

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

PubMed

Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

2015-08-21

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

Independent AMP and NAD signaling regulates C2C12 differentiation and metabolic adaptation.

PubMed

Hsu, Chia George; Burkholder, Thomas J

2016-12-01

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.

Significance of Ser-188 in human mitochondrial NAD kinase as determined by phosphomimetic and phosphoresistant amino-acid substitutions.

PubMed

Kawabata, Yutaka; Murata, Kousaku; Kawai, Shigeyuki

2015-12-25

Human mitochondrial NAD kinase is a crucial enzyme responsible for the synthesis of mitochondrial NADP(+). Despite its significance, little is known about the regulation of this enzyme in the mitochondria. Several putative and known phosphorylation sites within the protein have been found using phosphoproteomics, and here, we examined the effect of phosphomimetic mutations at six of these sites. The enzymatic activity was downregulated by a substitution of an Asp residue at Ser-289 and Ser-376, but not a substitution of Ala, suggesting that the phosphorylation of these residues downregulates the enzyme. Moreover, the activity was completely inhibited by substituting Ser-188 with an Asp, Glu, or in particular Ala, which highlights two possibilities: first, that Ser-188 is critical for catalytic activity, and second, that phosphorylation of Ser-188 inhibits the activity. Ser-188, Ser-289, and Ser-376 were found to be highly conserved in the primary structures of mitochondrial NAD kinase homologs in higher animals. Moreover, Ser-188 has been frequently detected in human and mouse phosphorylation site studies, whereas Ser-289 and Ser-376 have not. Taken together, this indicates that Ser-188 (and perhaps the other residues) is an important phosphorylation site that can downregulate the NAD kinase activity of this critical enzyme. Copyright © 2015 Elsevier Inc. All rights reserved.

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

PubMed Central

Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

2015-01-01

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production.

PubMed

Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L; Evans, Charles R; Brenner, Charles

2011-05-11

NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.

Nrt1 and Tna1-Independent Export of NAD+ Precursor Vitamins Promotes NAD+ Homeostasis and Allows Engineering of Vitamin Production

PubMed Central

Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L.; Evans, Charles R.; Brenner, Charles

2011-01-01

NAD+ is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD+ consuming enzymes. NAD+ biosynthesis is required for two different regimens that extend lifespan in yeast. NAD+ is synthesized from tryptophan and the three vitamin precursors of NAD+: nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD+ precursors increases intracellular NAD+ levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD+ metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD+ metabolism by balancing import and export of NAD+ precursor vitamins. PMID:21589930

NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells

PubMed Central

Yamada, Shigeru; Kotake, Yaichiro; Demizu, Yosuke; Kurihara, Masaaki; Sekino, Yuko; Kanda, Yasunari

2014-01-01

Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action. PMID:25092173

NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells

NASA Astrophysics Data System (ADS)

Yamada, Shigeru; Kotake, Yaichiro; Demizu, Yosuke; Kurihara, Masaaki; Sekino, Yuko; Kanda, Yasunari

2014-08-01

Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action.

NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells.

PubMed

Yamada, Shigeru; Kotake, Yaichiro; Demizu, Yosuke; Kurihara, Masaaki; Sekino, Yuko; Kanda, Yasunari

2014-08-05

Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action.

NAD+ in Aging: Molecular Mechanisms and Translational Implications.

PubMed

Fang, Evandro F; Lautrup, Sofie; Hou, Yujun; Demarest, Tyler G; Croteau, Deborah L; Mattson, Mark P; Bohr, Vilhelm A

2017-10-01

The coenzyme NAD + is critical in cellular bioenergetics and adaptive stress responses. Its depletion has emerged as a fundamental feature of aging that may predispose to a wide range of chronic diseases. Maintenance of NAD + levels is important for cells with high energy demands and for proficient neuronal function. NAD + depletion is detected in major neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, cardiovascular disease and muscle atrophy. Emerging evidence suggests that NAD + decrements occur in various tissues during aging, and that physiological and pharmacological interventions bolstering cellular NAD + levels might retard aspects of aging and forestall some age-related diseases. Here, we discuss aspects of NAD + biosynthesis, together with putative mechanisms of NAD + action against aging, including recent preclinical and clinical trials. Published by Elsevier Ltd.

The dynamic regulation of NAD metabolism in mitochondria

PubMed Central

Stein, Liana Roberts; Imai, Shin-ichiro

2012-01-01

Mitochondria are intracellular powerhouses that produce ATP and carry out diverse functions for cellular energy metabolism. While the maintenance of an optimal NAD/NADH ratio is essential for mitochondrial function, it has recently become apparent that the maintenance of the mitochondrial NAD pool also has critical importance. The biosynthesis, transport, and catabolism of NAD and its key intermediates play an important role in the regulation of NAD-consuming mediators, such as sirtuins, poly-ADP-ribose polymerases, and CD38/157 ectoenzymes, in intra- and extracellular compartments. Mitochondrial NAD biosynthesis is also modulated in response to nutritional and environmental stimuli. In this article, we discuss this dynamic regulation of NAD metabolism in mitochondria to shed light on the intimate connection between NAD and mitochondrial function. PMID:22819213

NAD+ maintenance attenuates light induced photoreceptor degeneration Δ

PubMed Central

Bai, Shi; Sheline, Christian T.

2013-01-01

Light-induced retinal damage (LD) occurs after surgery or sun exposure. We previously showed that zinc (Zn2+) accumulated in photoreceptors and RPE cells after LD but prior to cell death, and pyruvate or nicotinamide attenuated the resultant death perhaps by restoring nicotinamide adenine dinucleotide (NAD+) levels. We first examined the levels of NAD+ and the efficacy of pyruvate or nicotinamide in oxidative toxicities using primary retinal cultures. We next manipulated NAD+ levels in vivo and tested the affect on LD to photoreceptors and RPE. NAD+ levels cycle with a 24-h rhythm in mammals, which is affected by the feeding schedule. Therefore, we tested the affect of increasing NAD+ levels on LD by giving nicotinamide, inverting the feeding schedule, or using transgenic mice which overexpress cytoplasmic nicotinamide mononucleotide adenyl-transferase-1 (cytNMNAT1), an NAD+ synthetic enzyme. Zn2+ accumulation was also assessed in culture and in retinal sections. Retinas of light damaged animals were examined by OCT and plastic sectioning, and retinal NAD levels were measured. Day fed, or nicotinamide treated rats showed less NAD+ loss, and LD compared to night fed rats or untreated rats without changing the Zn2+ staining pattern. CytNMNAT1 showed less Zn2+ staining, NAD+ loss, and cell death after LD. In conclusion, intense light, Zn2+ and oxidative toxicities caused an increase in Zn2+, NAD+ loss, and cell death which were attenuated by NAD+ restoration. Therefore, NAD+ levels play a protective role in LD-induced death of photoreceptors and RPE cells. PMID:23274583

NAD+ salvage pathway in cancer metabolism and therapy.

PubMed

Kennedy, Barry E; Sharif, Tanveer; Martell, Emma; Dai, Cathleen; Kim, Youra; Lee, Patrick W K; Gujar, Shashi A

2016-12-01

Nicotinamide adenine dinucleotide (NAD + ) is an essential coenzyme for various physiological processes including energy metabolism, DNA repair, cell growth, and cell death. Many of these pathways are typically dysregulated in cancer cells, making NAD + an intriguing target for cancer therapeutics. NAD + is mainly synthesized by the NAD + salvage pathway in cancer cells, and not surprisingly, the pharmacological targeting of the NAD + salvage pathway causes cancer cell cytotoxicity in vitro and in vivo. Several studies have described the precise consequences of NAD + depletion on cancer biology, and have demonstrated that NAD+ depletion results in depletion of energy levels through lowered rates of glycolysis, reduced citric acid cycle activity, and decreased oxidative phosphorylation. Additionally, depletion of NAD + causes sensitization of cancer cells to oxidative damage by disruption of the anti-oxidant defense system, decreased cell proliferation, and initiation of cell death through manipulation of cell signaling pathways (e.g., SIRT1 and p53). Recently, studies have explored the effect of well-known cancer therapeutics in combination with pharmacological depletion of NAD + levels, and found in many cases a synergistic effect on cancer cell cytotoxicity. In this context, we will discuss the effects of NAD + salvage pathway inhibition on cancer cell biology and provide insight on this pathway as a novel anti-cancer therapeutic target. Copyright © 2016 Elsevier Ltd. All rights reserved.

NAD+ : A key metabolic regulator with great therapeutic potential.

PubMed

Sultani, G; Samsudeen, A F; Osborne, B; Turner, N

2017-10-01

Nicotinamide adenine dinucleotide (NAD + ) is a ubiquitous metabolite that serves an essential role in the catabolism of nutrients. Recently, there has been a surge of interest in NAD + biology, with the recognition that NAD + influences many biological processes beyond metabolism, including transcription, signalling and cell survival. There are a multitude of pathways involved in the synthesis and breakdown of NAD + , and alterations in NAD + homeostasis have emerged as a common feature of a range of disease states. Here, we provide an overview of NAD + metabolism and summarise progress on the development of NAD + -related therapeutics. © 2017 British Society for Neuroendocrinology.

Agents for replacement of NAD+/NADH system in enzymatic reactions

DOEpatents

Fish, Richard H.; Kerr, John B.; Lo, Christine H.

2004-04-06

Novel agents acting as co-factors for replacement of NAD(P).sup.+ /NAD(P)H co-enzyme systems in enzymatic oxido-reductive reactions. Agents mimicking the action of NAD(P).sup.+ /NAD(P)H system in enzymatic oxidation/reduction of substrates into reduced or oxidized products. A method for selection and preparation of the mimicking agents for replacement of NAD(P).sup.+ /NAD(P)H system and a device comprising co-factors for replacement of NAD(P).sup.+ /NAD(P)H system.

Modulating NAD+ metabolism, from bench to bedside.

PubMed

Katsyuba, Elena; Auwerx, Johan

2017-09-15

Discovered in the beginning of the 20 th century, nicotinamide adenine dinucleotide (NAD + ) has evolved from a simple oxidoreductase cofactor to being an essential cosubstrate for a wide range of regulatory proteins that include the sirtuin family of NAD + -dependent protein deacylases, widely recognized regulators of metabolic function and longevity. Altered NAD + metabolism is associated with aging and many pathological conditions, such as metabolic diseases and disorders of the muscular and neuronal systems. Conversely, increased NAD + levels have shown to be beneficial in a broad spectrum of diseases. Here, we review the fundamental aspects of NAD + biochemistry and metabolism and discuss how boosting NAD + content can help ameliorate mitochondrial homeostasis and as such improve healthspan and lifespan. © 2017 The Authors.

NadN and e (P4) are essential for utilization of NAD and nicotinamide mononucleotide but not nicotinamide riboside in Haemophilus influenzae.

PubMed

Kemmer, G; Reilly, T J; Schmidt-Brauns, J; Zlotnik, G W; Green, B A; Fiske, M J; Herbert, M; Kraiss, A; Schlör, S; Smith, A; Reidl, J

2001-07-01

Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel, and a periplasmic NAD nucleotidase, encoded by nadN. It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5'-nucleotidase activity. The e (P4) protein is also shown to have NMN 5'-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed.

NadN and e (P4) Are Essential for Utilization of NAD and Nicotinamide Mononucleotide but Not Nicotinamide Riboside in Haemophilus influenzae

PubMed Central

Kemmer, Gabriele; Reilly, Thomas J.; Schmidt-Brauns, Joachim; Zlotnik, Gary W.; Green, Bruce A.; Fiske, Michael J.; Herbert, Mark; Kraiß, Anita; Schlör, Stefan; Smith, Arnold; Reidl, Joachim

2001-01-01

Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel, and a periplasmic NAD nucleotidase, encoded by nadN. It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5′-nucleotidase activity. The e (P4) protein is also shown to have NMN 5′-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed. PMID:11395461

The human NAD metabolome: Functions, metabolism and compartmentalization

PubMed Central

Nikiforov, Andrey; Kulikova, Veronika; Ziegler, Mathias

2015-01-01

Abstract The metabolism of NAD has emerged as a key regulator of cellular and organismal homeostasis. Being a major component of both bioenergetic and signaling pathways, the molecule is ideally suited to regulate metabolism and major cellular events. In humans, NAD is synthesized from vitamin B3 precursors, most prominently from nicotinamide, which is the degradation product of all NAD-dependent signaling reactions. The scope of NAD-mediated regulatory processes is wide including enzyme regulation, control of gene expression and health span, DNA repair, cell cycle regulation and calcium signaling. In these processes, nicotinamide is cleaved from NAD+ and the remaining ADP-ribosyl moiety used to modify proteins (deacetylation by sirtuins or ADP-ribosylation) or to generate calcium-mobilizing agents such as cyclic ADP-ribose. This review will also emphasize the role of the intermediates in the NAD metabolome, their intra- and extra-cellular conversions and potential contributions to subcellular compartmentalization of NAD pools. PMID:25837229

A critical analysis of computational protein design with sparse residue interaction graphs

PubMed Central

Georgiev, Ivelin S.

2017-01-01

Protein design algorithms enumerate a combinatorial number of candidate structures to compute the Global Minimum Energy Conformation (GMEC). To efficiently find the GMEC, protein design algorithms must methodically reduce the conformational search space. By applying distance and energy cutoffs, the protein system to be designed can thus be represented using a sparse residue interaction graph, where the number of interacting residue pairs is less than all pairs of mutable residues, and the corresponding GMEC is called the sparse GMEC. However, ignoring some pairwise residue interactions can lead to a change in the energy, conformation, or sequence of the sparse GMEC vs. the original or the full GMEC. Despite the widespread use of sparse residue interaction graphs in protein design, the above mentioned effects of their use have not been previously analyzed. To analyze the costs and benefits of designing with sparse residue interaction graphs, we computed the GMECs for 136 different protein design problems both with and without distance and energy cutoffs, and compared their energies, conformations, and sequences. Our analysis shows that the differences between the GMECs depend critically on whether or not the design includes core, boundary, or surface residues. Moreover, neglecting long-range interactions can alter local interactions and introduce large sequence differences, both of which can result in significant structural and functional changes. Designs on proteins with experimentally measured thermostability show it is beneficial to compute both the full and the sparse GMEC accurately and efficiently. To this end, we show that a provable, ensemble-based algorithm can efficiently compute both GMECs by enumerating a small number of conformations, usually fewer than 1000. This provides a novel way to combine sparse residue interaction graphs with provable, ensemble-based algorithms to reap the benefits of sparse residue interaction graphs while avoiding their

Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells

PubMed Central

Nikiforov, Andrey; Dölle, Christian; Niere, Marc; Ziegler, Mathias

2011-01-01

NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADP-ribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule. PMID:21504897

More to NAD+ than meets the eye: A regulator of metabolic pools and gene expression in Arabidopsis.

PubMed

Gakière, Bertrand; Fernie, Alisdair R; Pétriacq, Pierre

2018-01-05

Since its discovery more than a century ago, nicotinamide adenine dinucleotide (NAD + ) is recognised as a fascinating cornerstone of cellular metabolism. This ubiquitous energy cofactor plays vital roles in metabolic pathways and regulatory processes, a fact emphasised by the essentiality of a balanced NAD + metabolism for normal plant growth and development. Research on the role of NAD in plants has been predominantly carried out in the model plant Arabidopsis thaliana (Arabidopsis) with emphasis on the redox properties and cellular signalling functions of the metabolite. This review examines the current state of knowledge concerning how NAD can regulate both metabolic pools and gene expression in Arabidopsis. Particular focus is placed on recent studies highlighting the complexity of metabolic regulations involving NAD, more particularly in the mitochondrial compartment, and of signalling roles with respect to interactions with environmental fluctuations most specifically those involving plant immunity. Copyright © 2018 Elsevier Inc. All rights reserved.

The NAD+/PARP1/SIRT1 Axis in Aging.

PubMed

Mendelsohn, Andrew R; Larrick, James W

2017-06-01

NAD+ levels decline with age in diverse animals from Caenorhabditis elegans to mice. Raising NAD+ levels by dietary supplementation with NAD+ precursors, nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN), improves mitochondrial function and muscle and neural and melanocyte stem cell function in mice, as well as increases murine life span. Decreased NAD+ levels with age reduce SIRT1 function and reduce the mitochondrial unfolded protein response, which can be overcome by NR supplementation. Decreased NAD+ levels cause NAD+-binding protein DBC1 to form a complex with PARP1, inhibiting poly(adenosine diphosphate-ribose) polymerase (PARP) catalytic activity. Old mice have increased amounts of DBC1-PARP1 complexes, lower PARP activity, increased DNA damage, and reduced nonhomologous end joining and homologous recombination repair. DBC1-PARP1 complexes in old mice can be broken by increasing NAD+ levels through treatment with NMN, reducing DNA damage and restoring PARP activity to youthful levels. The mechanism of declining NAD+ levels and its fundamental importance to aging are yet to be elucidated. There is a correlation of PARP activity with mammalian life span that suggests that NAD+/SIRT1/PARP1 may be more significant than the modest effects on life span observed for NR supplementation in old mice. The NAD+/PARP1/SIRT1 axis may link NAD+ levels and DNA damage with the apparent epigenomic DNA methylation clocks that have been described.

Prediction of residue-residue contact matrix for protein-protein interaction with Fisher score features and deep learning.

PubMed

Du, Tianchuan; Liao, Li; Wu, Cathy H; Sun, Bilin

2016-11-01

Protein-protein interactions play essential roles in many biological processes. Acquiring knowledge of the residue-residue contact information of two interacting proteins is not only helpful in annotating functions for proteins, but also critical for structure-based drug design. The prediction of the protein residue-residue contact matrix of the interfacial regions is challenging. In this work, we introduced deep learning techniques (specifically, stacked autoencoders) to build deep neural network models to tackled the residue-residue contact prediction problem. In tandem with interaction profile Hidden Markov Models, which was used first to extract Fisher score features from protein sequences, stacked autoencoders were deployed to extract and learn hidden abstract features. The deep learning model showed significant improvement over the traditional machine learning model, Support Vector Machines (SVM), with the overall accuracy increased by 15% from 65.40% to 80.82%. We showed that the stacked autoencoders could extract novel features, which can be utilized by deep neural networks and other classifiers to enhance learning, out of the Fisher score features. It is further shown that deep neural networks have significant advantages over SVM in making use of the newly extracted features. Copyright © 2016. Published by Elsevier Inc.

Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios.

PubMed Central

Rutter, G A; Denton, R M

1988-01-01

1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system. PMID:3421900

NAD Acts as an Integral Regulator of Multiple Defense Layers.

PubMed

Pétriacq, Pierre; Ton, Jurriaan; Patrit, Oriane; Tcherkez, Guillaume; Gakière, Bertrand

2016-11-01

Pyridine nucleotides, such as NAD, are crucial redox carriers and have emerged as important signaling molecules in stress responses. Previously, we have demonstrated in Arabidopsis (Arabidopsis thaliana) that the inducible NAD-overproducing nadC lines are more resistant to an avirulent strain of Pseudomonas syringae pv tomato (Pst-AvrRpm1), which was associated with salicylic acid-dependent defense. Here, we have further characterized the NAD-dependent immune response in Arabidopsis. Quinolinate-induced stimulation of intracellular NAD in transgenic nadC plants enhanced resistance against a diverse range of (a)virulent pathogens, including Pst-AvrRpt2, Dickeya dadantii, and Botrytis cinerea Characterization of the redox status demonstrated that elevated NAD levels induce reactive oxygen species (ROS) production and the expression of redox marker genes of the cytosol and mitochondrion. Using pharmacological and reverse genetics approaches, we show that NAD-induced ROS production functions independently of NADPH oxidase activity and light metabolism but depends on mitochondrial respiration, which was increased at higher NAD. We further demonstrate that NAD primes pathogen-induced callose deposition and cell death. Mass spectrometry analysis reveals that NAD simultaneously induces different defense hormones and that the NAD-induced metabolic profiles are similar to those of defense-expressing plants after treatment with pathogen-associated molecular patterns. We thus conclude that NAD triggers metabolic profiles rather similar to that of pathogen-associated molecular patterns and discuss how signaling cross talk between defense hormones, ROS, and NAD explains the observed resistance to pathogens. © 2016 American Society of Plant Biologists. All Rights Reserved.

In vivo (31) P MRS assessment of intracellular NAD metabolites and NAD(+) /NADH redox state in human brain at 4 T.

PubMed

Lu, Ming; Zhu, Xiao-Hong; Chen, Wei

2016-07-01

NAD(+) and NADH play key roles in cellular respiration. Intracellular redox state defined by the NAD(+) /NADH ratio (RX) reflects the cellular metabolic and physiopathological status. By taking advantage of high/ultrahigh magnetic field strengths, we have recently established a novel in vivo (31) P MRS-based NAD assay for noninvasive and quantitative measurements of intracellular NAD concentrations and redox state in animal and human brains at 16.4 T, 9.4 T and 7 T. To explore its potential for clinical application, in this study we investigated the feasibility of assessing the NAD metabolism and redox state in human brain at a lower field of 4 T by incorporating the (1) H-decoupling technique with the in vivo (31) P NAD assay. The use of (1) H decoupling significantly narrowed the linewidths of NAD and α-ATP resonances, resulting in higher sensitivity and better spectral resolution as compared with the (1) H-coupled (31) P spectrum. These improvements made it possible to reliably quantify cerebral NAD concentrations and RX, consistent with previously reported results obtained from similar age human subjects at 7 T. In summary, this work demonstrates the capability and utility of the (1) H-decoupled (31) P MRS-based NAD assay at lower field strength; thus, it opens new opportunities for studying intracellular NAD metabolism and redox state in human brain at clinical settings. This conclusion is supported by the simulation results, indicating that similar performance and reliability as observed at 4T can be achieved at 3 T with the same signal-to-noise ratio. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

NAD+ glycohydrolase, an ecto-enzyme of calf spleen cells.

PubMed Central

Muller, H M; Muller, C D; Schuber, F

1983-01-01

By using a sensitive fluorimetric assay of NAD+ glycohydrolase (EC 3.2.2.6), we showed that calf spleen cells are able to hydrolyse 1,N6-etheno-NAD+ given in the medium. The observed rates of substrate hydrolysis and product accumulation in the medium are equivalent. Moreover, the splenocytes are able to cleave the nicotinamide-ribose bond of a water-soluble polymer of NAD+, and their NAD+ glycohydrolase activity is fully inhibited by a high-molecular-weight Blue Dextran. Selective permeation of the cellular membrane digitonin revealed an intracellular pool of NAD+ glycohydrolase, which accounts for 25% of the total activity. We conclude that NAD+ glycohydrolase associated with the splenocytes has the characteristics of an ecto-enzyme. PMID:6192807

Boosting NAD to spare hearing.

PubMed

Brenner, Charles

2014-12-02

Ex vivo experiments have strangely shown that inhibition or stimulation of NAD metabolism can be neuroprotective. In this issue of Cell Metabolism, Brown et al. (2014) demonstrate that cochlear NAD is diminished by deafening noise but protected by nicotinamide riboside or WldS mutation. Hearing protection by nicotinamide riboside depends on Sirt3. Copyright © 2014 Elsevier Inc. All rights reserved.

NAD+ and vitamin B3: from metabolism to therapies.

PubMed

Sauve, Anthony A

2008-03-01

The role of NAD(+) metabolism in health and disease is of increased interest as the use of niacin (nicotinic acid) has emerged as a major therapy for treatment of hyperlipidemias and with the recognition that nicotinamide can protect tissues and NAD(+) metabolism in a variety of disease states, including ischemia/reperfusion. In addition, a growing body of evidence supports the view that NAD(+) metabolism regulates important biological effects, including lifespan. NAD(+) exerts potent effects through the poly(ADP-ribose) polymerases, mono-ADP-ribosyltransferases, and the recently characterized sirtuin enzymes. These enzymes catalyze protein modifications, such as ADP-ribosylation and deacetylation, leading to changes in protein function. These enzymes regulate apoptosis, DNA repair, stress resistance, metabolism, and endocrine signaling, suggesting that these enzymes and/or NAD(+) metabolism could be targeted for therapeutic benefit. This review considers current knowledge of NAD(+) metabolism in humans and microbes, including new insights into mechanisms that regulate NAD(+) biosynthetic pathways, current use of nicotinamide and nicotinic acid as pharmacological agents, and opportunities for drug design that are directed at modulation of NAD(+) biosynthesis for treatment of human disorders and infections.

Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment

PubMed Central

Zhou, Minghai; Ottenberg, Gregory; Sferrazza, Gian Franco; Hubbs, Christopher; Fallahi, Mohammad; Rumbaugh, Gavin; Brantley, Alicia F.

2015-01-01

The mechanisms of neuronal death in protein misfolding neurodegenerative diseases such as Alzheimer’s, Parkinson’s and prion diseases are poorly understood. We used a highly toxic misfolded prion protein (TPrP) model to understand neurotoxicity induced by prion protein misfolding. We show that abnormal autophagy activation and neuronal demise is due to severe, neuron-specific, nicotinamide adenine dinucleotide (NAD+) depletion. Toxic prion protein-exposed neuronal cells exhibit dramatic reductions of intracellular NAD+ followed by decreased ATP production, and are completely rescued by treatment with NAD+ or its precursor nicotinamide because of restoration of physiological NAD+ levels. Toxic prion protein-induced NAD+ depletion results from PARP1-independent excessive protein ADP-ribosylations. In vivo, toxic prion protein-induced degeneration of hippocampal neurons is prevented dose-dependently by intracerebral injection of NAD+. Intranasal NAD+ treatment of prion-infected sick mice significantly improves activity and delays motor impairment. Our study reveals NAD+ starvation as a novel mechanism of autophagy activation and neurodegeneration induced by a misfolded amyloidogenic protein. We propose the development of NAD+ replenishment strategies for neuroprotection in prion diseases and possibly other protein misfolding neurodegenerative diseases. PMID:25678560

Flavonoid apigenin is an inhibitor of the NAD+ ase CD38: implications for cellular NAD+ metabolism, protein acetylation, and treatment of metabolic syndrome.

PubMed

Escande, Carlos; Nin, Veronica; Price, Nathan L; Capellini, Verena; Gomes, Ana P; Barbosa, Maria Thereza; O'Neil, Luke; White, Thomas A; Sinclair, David A; Chini, Eduardo N

2013-04-01

Metabolic syndrome is a growing health problem worldwide. It is therefore imperative to develop new strategies to treat this pathology. In the past years, the manipulation of NAD(+) metabolism has emerged as a plausible strategy to ameliorate metabolic syndrome. In particular, an increase in cellular NAD(+) levels has beneficial effects, likely because of the activation of sirtuins. Previously, we reported that CD38 is the primary NAD(+)ase in mammals. Moreover, CD38 knockout mice have higher NAD(+) levels and are protected against obesity and metabolic syndrome. Here, we show that CD38 regulates global protein acetylation through changes in NAD(+) levels and sirtuin activity. In addition, we characterize two CD38 inhibitors: quercetin and apigenin. We show that pharmacological inhibition of CD38 results in higher intracellular NAD(+) levels and that treatment of cell cultures with apigenin decreases global acetylation as well as the acetylation of p53 and RelA-p65. Finally, apigenin administration to obese mice increases NAD(+) levels, decreases global protein acetylation, and improves several aspects of glucose and lipid homeostasis. Our results show that CD38 is a novel pharmacological target to treat metabolic diseases via NAD(+)-dependent pathways.

Assimilation of NAD(+) precursors in Candida glabrata.

PubMed

Ma, Biao; Pan, Shih-Jung; Zupancic, Margaret L; Cormack, Brendan P

2007-10-01

The yeast pathogen Candida glabrata is a nicotinamide adenine dinucleotide (NAD(+)) auxotroph and its growth depends on the environmental supply of vitamin precursors of NAD(+). C. glabrata salvage pathways defined in this article allow NAD(+) to be synthesized from three compounds - nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NA is salvaged through a functional Preiss-Handler pathway. NAM is first converted to NA by nicotinamidase and then salvaged by the Preiss-Handler pathway. Salvage of NR in C. glabrata occurs via two routes. The first, in which NR is phosphorylated by the NR kinase Nrk1, is independent of the Preiss-Handler pathway. The second is a novel pathway in which NR is degraded by the nucleosidases Pnp1 and Urh1, with a minor role for Meu1, and ultimately converted to NAD(+) via the nicotinamidase Pnc1 and the Preiss-Handler pathway. Using C. glabrata mutants whose growth depends exclusively on the external NA or NR supply, we also show that C. glabrata utilizes NR and to a lesser extent NA as NAD(+) sources during disseminated infection.

Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

PubMed

Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

2014-11-01

A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases. Copyright © 2014 Elsevier B.V. All rights reserved.

Primary structure of inorganic polyphosphate/ATP-NAD kinase from Micrococcus flavus, and occurrence of substrate inorganic polyphosphate for the enzyme.

PubMed

Kawai, Shigeyuki; Mori, Shigetarou; Murata, Kousaku

2003-08-01

The gene encoding an inorganic polyphosphate/ATP-NAD kinase was cloned from Micrococcus flavus, and its primary structure was analyzed. Alignment of the primary structure with those of other characterized NAD kinases revealed candidate amino acid residues, mainly charged ones, that would be related to inorganic polyphosphate use. The alignment also showed that the primary structure found carried a protruding C-terminal polypeptide. Although the C-terminal polypeptide was demonstrated to be dispensable for the kinase activities, and was proposed to be removed in M. flavus, the entire primary structure including the C-terminal polypeptide was homologous with that of the ATP synthase beta chain. The inorganic polyphosphate used by the inorganic polyphosphate/ATP-NAD kinase as a phosphoryl donor was isolated from cells of M. flavus, suggesting that the ability of the enzyme to use inorganic polyphosphate is of physiological significance and is not an evolutionary trait alone.

Role of NAD+ and mitochondrial sirtuins in cardiac and renal diseases

PubMed Central

Hershberger, Kathleen A.; Martin, Angelical S.; Hirschey, Matthew D.

2017-01-01

The coenzyme nicotinamide adenine dinucleotide (NAD+) has key roles in the regulation of redox status and energy metabolism. NAD+ depletion is emerging as a major contributor to the pathogenesis of cardiac and renal diseases and NAD+ repletion strategies have shown therapeutic potential as a means to restore healthy metabolism and physiological function. The pleotropic roles of NAD+ enable several possible avenues by which repletion of this coenzyme could have therapeutic efficacy. In particular, NAD+ functions as a co-substrate in deacylation reactions carried out by the sirtuin family of enzymes. These NAD+-dependent deacylases control several aspects of metabolism and a wealth of data suggests that boosting sirtuin activity via NAD+ supplementation might be a promising therapy for cardiac and renal pathologies. This Review summarizes the role of NAD+ metabolism in the heart and kidney, and highlights the mitochondrial sirtuins as mediators of some of the beneficial effects of NAD+-boosting therapies in preclinical animal models. We surmise that modulating the NAD+–sirtuin axis is a clinically relevant approach to develop new therapies for cardiac and renal diseases. PMID:28163307

Role of NAD+ and mitochondrial sirtuins in cardiac and renal diseases.

PubMed

Hershberger, Kathleen A; Martin, Angelical S; Hirschey, Matthew D

2017-04-01

The coenzyme nicotinamide adenine dinucleotide (NAD + ) has key roles in the regulation of redox status and energy metabolism. NAD + depletion is emerging as a major contributor to the pathogenesis of cardiac and renal diseases and NAD + repletion strategies have shown therapeutic potential as a means to restore healthy metabolism and physiological function. The pleotropic roles of NAD + enable several possible avenues by which repletion of this coenzyme could have therapeutic efficacy. In particular, NAD + functions as a co-substrate in deacylation reactions carried out by the sirtuin family of enzymes. These NAD + -dependent deacylases control several aspects of metabolism and a wealth of data suggests that boosting sirtuin activity via NAD + supplementation might be a promising therapy for cardiac and renal pathologies. This Review summarizes the role of NAD + metabolism in the heart and kidney, and highlights the mitochondrial sirtuins as mediators of some of the beneficial effects of NAD + -boosting therapies in preclinical animal models. We surmise that modulating the NAD + -sirtuin axis is a clinically relevant approach to develop new therapies for cardiac and renal diseases.

A bionanohybrid ZnAl-NADS ecological pesticide as a treatment for soft rot disease in potato (Solanum tuberosum L.).

PubMed

Morales-Irigoyen, Erika Elizabeth; de Las Mercedes Gómez-Y-Gómez, Yolanda; Flores-Moreno, Jorge Luis; Franco-Hernández, Marina Olivia

2017-09-18

Pectobacterium carotovorum (Pc) is a phytopathogenic strain that causes soft rot disease in potato (Solanum tuberosum L.), resulting in postharvest losses. Chemical control is effective for managing this disease, but overdoses cause adverse effects. Because farmers insist on using chemical agents for crop protection, it is necessary to develop more effective pesticides in which the active compound released can be regulated. In this context, we proposed the synthesis of ZnAl-NADS, in which nalidixic acid sodium salt (NADS) is linked to a ZnAl-NO 3 layered double hydroxide (LDH) host as a nanocarrier. XRD, FT-IR, and SEM analyses confirmed the successful intercalation of NADS into the interplanar LDH space. The drug release profile indicated that the maximum release was completed in 70 or 170 min for free NADS (alone) or for NADS released from ZnAl-NADS, respectively. This slow release was attributed to strong electrostatic interactions between the drug and the anion exchanger. A modulated release is preferable to the action of the bulk NADS, showing increased effectiveness and minimizing the amount of the chemical available to pollute the soil and the water. The fitting data from modified Freundlich and parabolic diffusion models explain the release behavior of the NADS, suggesting that the drug released from ZnAl-NADS bionanohybrid was carried out from the interlamellar sites, according to the ion exchange diffusion process also involving intraparticle diffusion (coeffect). ZnAl-NADS was tested in vitro against Escherichia coli (Ec) and Pc and exhibited bacteriostatic and biocidal effects at 0.025 and 0.075 mg mL -1 , respectively. ZnAl-NADS was also tested in vivo as an ecological pesticide for combating potato soft rot and was found to delay typical disease symptoms. In conclusion, ZnAl-NADS can potentially be used to control pests, infestation, and plant disease.

Structure of the adenylation domain of NAD[superscript +]-dependent DNA ligase from Staphylococcus aureus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Seungil; Chang, Jeanne S.; Griffor, Matt

DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3''-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD{sup +}-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD{sup +}-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD{sup +}-binding pocket and the 'C2more » tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.« less

Detection of cerebral NAD+ in humans at 7T.

PubMed

de Graaf, Robin A; De Feyter, Henk M; Brown, Peter B; Nixon, Terence W; Rothman, Douglas L; Behar, Kevin L

2017-09-01

To develop 1 H-based MR detection of nicotinamide adenine dinucleotide (NAD + ) in the human brain at 7T and validate the 1 H results with NAD + detection based on 31 P-MRS. 1 H-MR detection of NAD + was achieved with a one-dimensional double-spin-echo method on a slice parallel to the surface coil transceiver. Perturbation of the water resonance was avoided through the use of frequency-selective excitation. 31 P-MR detection of NAD + was performed with an unlocalized pulse-acquire sequence. Both 1 H- and 31 P-MRS allowed the detection of NAD + signals on every subject in 16 min. Spectral fitting provided an NAD + concentration of 107 ± 28 μM for 1 H-MRS and 367 ± 78 μM and 312 ± 65 μM for 31 P-MRS when uridine diphosphate glucose (UDPG) was excluded and included, respectively, as an overlapping signal. NAD + detection by 1 H-MRS is a simple method that comes at the price of reduced NMR visibility. NAD + detection by 31 P-MRS has near-complete NMR visibility, but it is complicated by spectral overlap with NADH and UDPG. Overall, the 1 H- and 31 P-MR methods both provide exciting opportunities to study NAD + metabolism on human brain in vivo. © 2016 International Society for Magnetic Resonance in Medicine. Magn Reson Med 78:828-835, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

NAD1 Controls Defense-Like Responses in Medicago truncatula Symbiotic Nitrogen Fixing Nodules Following Rhizobial Colonization in a BacA-Independent Manner

PubMed Central

Domonkos, Ágota; Kovács, Szilárd; Gombár, Anikó; Kiss, Ernő; Horváth, Beatrix; Kováts, Gyöngyi Z.; Farkas, Attila; Tóth, Mónika T.; Ayaydin, Ferhan; Bóka, Károly; Fodor, Lili; Endre, Gabriella; Kaló, Péter

2017-01-01

Legumes form endosymbiotic interaction with host compatible rhizobia, resulting in the development of nitrogen-fixing root nodules. Within symbiotic nodules, rhizobia are intracellularly accommodated in plant-derived membrane compartments, termed symbiosomes. In mature nodule, the massively colonized cells tolerate the existence of rhizobia without manifestation of visible defense responses, indicating the suppression of plant immunity in the nodule in the favur of the symbiotic partner. Medicago truncatula DNF2 (defective in nitrogen fixation 2) and NAD1 (nodules with activated defense 1) genes are essential for the control of plant defense during the colonization of the nitrogen-fixing nodule and are required for bacteroid persistence. The previously identified nodule-specific NAD1 gene encodes a protein of unknown function. Herein, we present the analysis of novel NAD1 mutant alleles to better understand the function of NAD1 in the repression of immune responses in symbiotic nodules. By exploiting the advantage of plant double and rhizobial mutants defective in establishing nitrogen-fixing symbiotic interaction, we show that NAD1 functions following the release of rhizobia from the infection threads and colonization of nodule cells. The suppression of plant defense is self-dependent of the differentiation status of the rhizobia. The corresponding phenotype of nad1 and dnf2 mutants and the similarity in the induction of defense-associated genes in both mutants suggest that NAD1 and DNF2 operate close together in the same pathway controlling defense responses in symbiotic nodules. PMID:29240711

NAD1 Controls Defense-Like Responses in Medicago truncatula Symbiotic Nitrogen Fixing Nodules Following Rhizobial Colonization in a BacA-Independent Manner.

PubMed

Domonkos, Ágota; Kovács, Szilárd; Gombár, Anikó; Kiss, Ernő; Horváth, Beatrix; Kováts, Gyöngyi Z; Farkas, Attila; Tóth, Mónika T; Ayaydin, Ferhan; Bóka, Károly; Fodor, Lili; Ratet, Pascal; Kereszt, Attila; Endre, Gabriella; Kaló, Péter

2017-12-14

Legumes form endosymbiotic interaction with host compatible rhizobia, resulting in the development of nitrogen-fixing root nodules. Within symbiotic nodules, rhizobia are intracellularly accommodated in plant-derived membrane compartments, termed symbiosomes. In mature nodule, the massively colonized cells tolerate the existence of rhizobia without manifestation of visible defense responses, indicating the suppression of plant immunity in the nodule in the favur of the symbiotic partner. Medicago truncatula DNF2 (defective in nitrogen fixation 2) and NAD1 (nodules with activated defense 1) genes are essential for the control of plant defense during the colonization of the nitrogen-fixing nodule and are required for bacteroid persistence. The previously identified nodule-specific NAD1 gene encodes a protein of unknown function. Herein, we present the analysis of novel NAD1 mutant alleles to better understand the function of NAD1 in the repression of immune responses in symbiotic nodules. By exploiting the advantage of plant double and rhizobial mutants defective in establishing nitrogen-fixing symbiotic interaction, we show that NAD1 functions following the release of rhizobia from the infection threads and colonization of nodule cells. The suppression of plant defense is self-dependent of the differentiation status of the rhizobia. The corresponding phenotype of nad1 and dnf2 mutants and the similarity in the induction of defense-associated genes in both mutants suggest that NAD1 and DNF2 operate close together in the same pathway controlling defense responses in symbiotic nodules.

The crystal structure of NAD(P)H oxidase from Lactobacillus sanfranciscensis: insights into the conversion of O2 into two water molecules by the flavoenzyme.

PubMed

Lountos, George T; Jiang, Rongrong; Wellborn, William B; Thaler, Tracey L; Bommarius, Andreas S; Orville, Allen M

2006-08-15

The FAD-dependent NAD(P)H oxidase from Lactobacillus sanfrancisensis (L.san-Nox2) catalyzes the oxidation of 2 equivalents of either NADH or NADPH and reduces 1 equivalent of O(2) to yield 2 equivalents of water. During steady-state turnover only 0.5% of the reducing equivalents are detected in solution as hydrogen peroxide, suggesting that it is not released from the enzyme after the oxidation of the first equivalent of NAD(P)H and reaction with O(2). Here we report the crystal structure of L.san-Nox2 to 1.8 A resolution. The enzyme crystallizes as a dimer with each monomer consisting of a FAD binding domain (residues 1-120), a NAD(P)H binding domain (residues 150-250), and a dimerization domain (residues 325-451). The electron density for the redox-active Cys42 residue located adjacent to the si-face FAD is consistent with oxidation to the sulfenic acid (Cys-SOH) state. The side chain of Cys42 is also observed in two conformations; in one the sulfenic acid is hydrogen bonded to His10 and in the other it hydrogen bonds with the FAD O2' atom. Surprisingly, the NAD(P)H binding domains each contain an ADP ligand as established by electron density maps and MALDI-TOF analysis of the ligands released from heat-denatured enzyme. The ADP ligand copurifies with the enzyme, and its presence does not inhibit enzyme activity. Consequently, we hypothesize that either NADPH or NADH substrates bind via a long channel that extends from the enzyme exterior and terminates at the FAD re-face. A homology model of the NADH oxidase from Lactococcus lactis (L.lac-Nox2) was also generated using the crystal structure of L.san-Nox2, which reveals several important similarities and differences between the two enzymes. HPLC analysis of ligands released from denatured L.lac-Nox2 indicates that it does not bind ADP, which correlates with the specificity of the enzyme for oxidation of NADH.

NAD Acts as an Integral Regulator of Multiple Defense Layers1[OPEN

PubMed Central

Patrit, Oriane; Tcherkez, Guillaume; Gakière, Bertrand

2016-01-01

Pyridine nucleotides, such as NAD, are crucial redox carriers and have emerged as important signaling molecules in stress responses. Previously, we have demonstrated in Arabidopsis (Arabidopsis thaliana) that the inducible NAD-overproducing nadC lines are more resistant to an avirulent strain of Pseudomonas syringae pv tomato (Pst-AvrRpm1), which was associated with salicylic acid-dependent defense. Here, we have further characterized the NAD-dependent immune response in Arabidopsis. Quinolinate-induced stimulation of intracellular NAD in transgenic nadC plants enhanced resistance against a diverse range of (a)virulent pathogens, including Pst-AvrRpt2, Dickeya dadantii, and Botrytis cinerea. Characterization of the redox status demonstrated that elevated NAD levels induce reactive oxygen species (ROS) production and the expression of redox marker genes of the cytosol and mitochondrion. Using pharmacological and reverse genetics approaches, we show that NAD-induced ROS production functions independently of NADPH oxidase activity and light metabolism but depends on mitochondrial respiration, which was increased at higher NAD. We further demonstrate that NAD primes pathogen-induced callose deposition and cell death. Mass spectrometry analysis reveals that NAD simultaneously induces different defense hormones and that the NAD-induced metabolic profiles are similar to those of defense-expressing plants after treatment with pathogen-associated molecular patterns. We thus conclude that NAD triggers metabolic profiles rather similar to that of pathogen-associated molecular patterns and discuss how signaling cross talk between defense hormones, ROS, and NAD explains the observed resistance to pathogens. PMID:27621425

The potential regulatory roles of NAD(+) and its metabolism in autophagy.

PubMed

Zhang, Dong-Xia; Zhang, Jia-Ping; Hu, Jiong-Yu; Huang, Yue-Sheng

2016-04-01

(Macro)autophagy mediates the bulk degradation of defective organelles, long-lived proteins and protein aggregates in lysosomes and plays a critical role in cellular and tissue homeostasis. Defective autophagy processes have been found to contribute to a variety of metabolic diseases. However, the regulatory mechanisms of autophagy are not fully understood. Increasing data indicate that nicotinamide adenine nucleotide (NAD(+)) homeostasis correlates intimately with autophagy. NAD(+) is a ubiquitous coenzyme that functions primarily as an electron carrier of oxidoreductase in multiple redox reactions. Both NAD(+) homeostasis and its metabolism are thought to play critical roles in regulating autophagy. In this review, we discuss how the regulation of NAD(+) and its metabolism can influence autophagy. We focus on the regulation of NAD(+)/NADH homeostasis and the effects of NAD(+) consumption by poly(ADP-ribose) (PAR) polymerase-1 (PARP-1), NAD(+)-dependent deacetylation by sirtuins and NAD(+) metabolites on autophagy processes and the underlying mechanisms. Future studies should provide more direct evidence for the regulation of autophagy processes by NAD(+). A better understanding of the critical roles of NAD(+) and its metabolites on autophagy will shed light on the complexity of autophagy regulation, which is essential for the discovery of new therapeutic tools for autophagy-related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Redesign of Schistosoma mansoni NAD+ catabolizing enzyme : the active site H103W mutation restores ADP-ribosyl cyclase activity†

PubMed Central

Kuhn, Isabelle; Kellenberger, Esther; Rognan, Didier; Lund, Frances E.; Muller-Steffner, Hélène; Schuber, Francis

2008-01-01

Schistosoma mansoni NAD(P)+ catabolizing enzyme (SmNACE) is a new member of the ADP-ribosyl cyclase family. In contrast to all the other enzymes which are involved in the production of metabolites that elicit Ca2+ mobilization, SmNACE is virtually unable to transform NAD+ into the second messenger cyclic ADP-ribose (cADPR). Sequence alignments revealed that one of four conserved residues within the active site of these enzymes was replaced in SmNACE by a histidine (His103) instead of the highly conserved tryptophan. To find out whether the inability of SmNACE to catalyze the canonical ADP-ribosyl cyclase reaction is linked to this change we have replaced His103 with a tryptophan. The H103W mutation in SmNACE was indeed found to restore ADP-ribosyl cyclase activity as cADPR amounts for 7% of the reaction products, i.e., a value larger than observed for other members of this family such as CD38. Introduction of a Trp103 residue provides some of the binding characteristics of mammalian ADP-ribosyl cyclases such as increased affinity for Cibacron blue and slow-binding inhibition by araF-NAD+. Homology modeling of wild-type and H103W mutant three-dimensional structures, and docking of substrates within the active sites, provide new insight into the catalytic mechanism of SmNACE. Both residue side chains share similar roles in the nicotinamide-ribose bond cleavage step leading to an E.ADP-ribosyl reaction intermediate. They diverge however in the evolution of this intermediate; His103 provides a more polar environment favoring the accessibility to water and hydrolysis leading to ADP-ribose at the expense of the intramolecular cyclization pathway resulting in cADPR. PMID:17002287

The NAD/NARB System: Advertising Self-Regulation at Work.

ERIC Educational Resources Information Center

Hays, Robert

Self-regulation, as defined by the National Advertising Division/National Advertising Review Board (NAD/NARB), is a process whereby the advertising industry regulates itself and turns to the federal government only if the system fails. The NAD/NARB system involves a two-step process: complaints are initially handled by the NAD and then are either…

NAD+/NADH and skeletal muscle mitochondrial adaptations to exercise

PubMed Central

White, Amanda T.

2012-01-01

The pyridine nucleotides, NAD+ and NADH, are coenzymes that provide oxidoreductive power for the generation of ATP by mitochondria. In skeletal muscle, exercise perturbs the levels of NAD+, NADH, and consequently, the NAD+/NADH ratio, and initial research in this area focused on the contribution of redox control to ATP production. More recently, numerous signaling pathways that are sensitive to perturbations in NAD+(H) have come to the fore, as has an appreciation for the potential importance of compartmentation of NAD+(H) metabolism and its subsequent effects on various signaling pathways. These pathways, which include the sirtuin (SIRT) proteins SIRT1 and SIRT3, the poly(ADP-ribose) polymerase (PARP) proteins PARP1 and PARP2, and COOH-terminal binding protein (CtBP), are of particular interest because they potentially link changes in cellular redox state to both immediate, metabolic-related changes and transcriptional adaptations to exercise. In this review, we discuss what is known, and not known, about the contribution of NAD+(H) metabolism and these aforementioned proteins to mitochondrial adaptations to acute and chronic endurance exercise. PMID:22436696

NAMPT and NAMPT-controlled NAD Metabolism in Vascular Repair.

PubMed

Wang, Pei; Li, Wen-Lin; Liu, Jian-Min; Miao, Chao-Yu

2016-06-01

Vascular repair plays important roles in postischemic remodeling and rehabilitation in cardiovascular and cerebrovascular disease, such as stroke and myocardial infarction. Nicotinamide adenine dinucleotide (NAD), a well-known coenzyme involved in electron transport chain for generation of adenosine triphosphate, has emerged as an important controller regulating various biological signaling pathways. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme for NAD biosynthesis in mammals. NAMPT may also act in a nonenzymatic manner, presumably mediated by unknown receptor(s). Rapidly accumulating data in the past decade show that NAMPT and NAMPT-controlled NAD metabolism regulate fundamental biological functions in endothelial cells, vascular smooth muscle cells, and endothelial progenitor cells. The NAD-consuming proteins, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38, may contribute to the regulatory effects of NAMPT-NAD axis in these cells and vascular repair. This review discusses the current data regarding NAMPT and NAMPT-controlled NAD metabolism in vascular repair and the clinical potential translational application of NAMPT-related products in treatment of cardiovascular and cerebrovascular disease.

Effect of the R119G mutation on human P5CR structure and its interactions with NAD: Insights derived from molecular dynamics simulation and free energy analysis.

PubMed

Sang, Peng; Xie, Yue-Hui; Li, Lin-Hua; Ye, Yu-Jia; Hu, Wei; Wang, Jing; Wan, Wen; Li, Rui; Li, Long-Jun; Ma, Lin-Ling; Li, Zhi; Liu, Shu-Qun; Meng, Zhao-Hui

2017-04-01

Pyrroline-5-carboxylate reductase (P5CR), an enzyme with conserved housekeeping roles, is involved in the etiology of cutis laxa. While previous work has shown that the R119G point mutation in the P5CR protein is involved, the structural mechanism behind the pathology remains to be elucidated. In order to probe the role of the R119G mutation in cutis laxa, we performed molecular dynamics (MD) simulations, essential dynamics (ED) analysis, and Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations on wild type (WT) and mutant P5CR-NAD complex. These MD simulations and ED analyses suggest that the R119G mutation decreases the flexibility of P5CR, specifically in the substrate binding pocket, which could decrease the kinetics of the cofactor entrance and egress. Furthermore, the MM-PBSA calculations suggest the R119G mutant has a lower cofactor binding affinity for NAD than WT. Our study provides insight into the possible role of the R119G mutation during interactions between P5CR and NAD, thus bettering our understanding of how the mutation promotes cutis laxa. Copyright © 2017 Elsevier Ltd. All rights reserved.

Investigation of the NADH/NAD+ ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox.

PubMed

Tejwani, Vijay; Schmitt, Franz-Josef; Wilkening, Svea; Zebger, Ingo; Horch, Marius; Lenz, Oliver; Friedrich, Thomas

2017-01-01

Ralstonia eutropha is a hydrogen-oxidizing ("Knallgas") bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H 2 -driven production of biodegradable polymers and hydrocarbons. H 2 oxidation by R. eutropha takes place in the presence of O 2 and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H 2 utilization. The so-called soluble hydrogenase (SH) couples reversibly H 2 oxidation with the reduction of NAD + to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD + pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD + ] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD + ] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD + ] ratios represents a novel and sensitive tool to determine the redox state of cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Uridine monophosphate synthetase enables eukaryotic de novo NAD+ biosynthesis from quinolinic acid.

PubMed

McReynolds, Melanie R; Wang, Wenqing; Holleran, Lauren M; Hanna-Rose, Wendy

2017-07-07

NAD + biosynthesis is an attractive and promising therapeutic target for influencing health span and obesity-related phenotypes as well as tumor growth. Full and effective use of this target for therapeutic benefit requires a complete understanding of NAD + biosynthetic pathways. Here, we report a previously unrecognized role for a conserved phosphoribosyltransferase in NAD + biosynthesis. Because a required quinolinic acid phosphoribosyltransferase (QPRTase) is not encoded in its genome, Caenorhabditis elegans are reported to lack a de novo NAD + biosynthetic pathway. However, all the genes of the kynurenine pathway required for quinolinic acid (QA) production from tryptophan are present. Thus, we investigated the presence of de novo NAD + biosynthesis in this organism. By combining isotope-tracing and genetic experiments, we have demonstrated the presence of an intact de novo biosynthesis pathway for NAD + from tryptophan via QA, highlighting the functional conservation of this important biosynthetic activity. Supplementation with kynurenine pathway intermediates also boosted NAD + levels and partially reversed NAD + -dependent phenotypes caused by mutation of pnc-1 , which encodes a nicotinamidase required for NAD + salvage biosynthesis, demonstrating contribution of de novo synthesis to NAD + homeostasis. By investigating candidate phosphoribosyltransferase genes in the genome, we determined that the conserved uridine monophosphate phosphoribosyltransferase (UMPS), which acts in pyrimidine biosynthesis, is required for NAD + biosynthesis in place of the missing QPRTase. We suggest that similar underground metabolic activity of UMPS may function in other organisms. This mechanism for NAD + biosynthesis creates novel possibilities for manipulating NAD + biosynthetic pathways, which is key for the future of therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sporty, J; Lin, S; Kato, M

2009-02-18

Nicotinamide adenine dinucleotide (NAD{sup +}) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or by the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD{sup +} decomposition products. NAD{sup +} biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD{sup +} biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD{sup +} and NADH (the reduced form of NAD{sup +}) analyses onmore » BY4742 wild type, NAD+ salvage pathway knockout (npt1{Delta}), and NAD+ de novo pathway knockout (qpt1{Delta}) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized {sup 14}C labeled nicotinic acid in the culture media combined with HPLC speciation and both UV and {sup 14}C detection to quantitate the total amounts of NAD{sup +} and NADH and the amounts derived from the salvage pathway. We observe that wild type and qpt1{Delta} yeast exclusively utilize extracellular nicotinic acid for NAD{sup +} and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observe that NAD{sup +} concentrations decrease in all three strains under CR. However, unlike the wild type strain, NADH concentrations do not decrease and NAD{sup +}:NADH ratios do not increase under CR for either knockout strain. Lifespan analyses reveal that CR results in a lifespan increase of approximately 25% for the wild type and qpt1{Delta} strains, while no increase in lifespan is observed for the npt1{Delta} strain. In combination these data suggest that having a functional salvage pathway is more important than the absolute levels of

Sequence divergence and diversity suggests ongoing functional diversification of vertebrate NAD metabolism

PubMed Central

Gossmann, Toni I.; Ziegler, Mathias

2014-01-01

NAD is not only an important cofactor in redox reactions but has also received attention in recent years because of its physiological importance in metabolic regulation, DNA repair and signaling. In contrast to the redox reactions, these regulatory processes involve degradation of NAD and therefore necessitate a constant replenishment of its cellular pool. NAD biosynthetic enzymes are common to almost all species in all clades, but the number of NAD degrading enzymes varies substantially across taxa. In particular, vertebrates, including humans, have a manifold of NAD degrading enzymes which require a high turnover of NAD. As there is currently a lack of a systematic study of how natural selection has shaped enzymes involved in NAD metabolism we conducted a comprehensive evolutionary analysis based on intraspecific variation and interspecific divergence. We compare NAD biosynthetic and degrading enzymes in four eukaryotic model species and subsequently focus on human NAD metabolic enzymes and their orthologs in other vertebrates. We find that the majority of enzymes involved in NAD metabolism are subject to varying levels of purifying selection. While NAD biosynthetic enzymes appear to experience a rather high level of evolutionary constraint, there is evidence for positive selection among enzymes mediating NAD-dependent signaling. This is particularly evident for members of the PARP family, a diverse protein family involved in DNA damage repair and programmed cell death. Based on haplotype information and substitution rate analysis we pinpoint sites that are potential targets of positive selection. We also link our findings to a three dimensional structure, which suggests that positive selection occurs in domains responsible for DNA binding and polymerization rather than the NAD catalytic domain. Taken together, our results indicate that vertebrate NAD metabolism is still undergoing functional diversification. PMID:25084685

Sequence divergence and diversity suggests ongoing functional diversification of vertebrate NAD metabolism.

PubMed

Gossmann, Toni I; Ziegler, Mathias

2014-11-01

NAD is not only an important cofactor in redox reactions but has also received attention in recent years because of its physiological importance in metabolic regulation, DNA repair and signaling. In contrast to the redox reactions, these regulatory processes involve degradation of NAD and therefore necessitate a constant replenishment of its cellular pool. NAD biosynthetic enzymes are common to almost all species in all clades, but the number of NAD degrading enzymes varies substantially across taxa. In particular, vertebrates, including humans, have a manifold of NAD degrading enzymes which require a high turnover of NAD. As there is currently a lack of a systematic study of how natural selection has shaped enzymes involved in NAD metabolism we conducted a comprehensive evolutionary analysis based on intraspecific variation and interspecific divergence. We compare NAD biosynthetic and degrading enzymes in four eukaryotic model species and subsequently focus on human NAD metabolic enzymes and their orthologs in other vertebrates. We find that the majority of enzymes involved in NAD metabolism are subject to varying levels of purifying selection. While NAD biosynthetic enzymes appear to experience a rather high level of evolutionary constraint, there is evidence for positive selection among enzymes mediating NAD-dependent signaling. This is particularly evident for members of the PARP family, a diverse protein family involved in DNA damage repair and programmed cell death. Based on haplotype information and substitution rate analysis we pinpoint sites that are potential targets of positive selection. We also link our findings to a three dimensional structure, which suggests that positive selection occurs in domains responsible for DNA binding and polymerization rather than the NAD catalytic domain. Taken together, our results indicate that vertebrate NAD metabolism is still undergoing functional diversification. Crown Copyright © 2014. Published by Elsevier B

Anatomy of an engineered NAD-binding site.

PubMed Central

Mittl, P. R.; Berry, A.; Scrutton, N. S.; Perham, R. N.; Schulz, G. E.

1994-01-01

The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2'-phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38-43). In order to analyze the structural changes involved, we have determined 4 high-resolution crystal structures, i.e., the structures of the wild-type enzyme (1.86 A resolution, R-factor of 16.8%), of the wild-type enzyme ligated with NADP (2.0 A, 20.8%), of the NAD-dependent mutant (1.74 A, 16.8%), and of the NAD-dependent mutant ligated with NAD (2.2 A, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NAD-binding site assumes a less rigid structure than the NADP site of the wild-type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 A from the sites of the mutations, this observation reminds us that mutations may have important long-range consequences that are difficult to anticipate. PMID:7833810

NAMPT-Mediated Salvage Synthesis of NAD+ Controls Morphofunctional Changes of Macrophages

PubMed Central

Venter, Gerda; Oerlemans, Frank T. J. J.; Willemse, Marieke; Wijers, Mietske; Fransen, Jack A. M.; Wieringa, Bé

2014-01-01

Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H) (i.e. NAD+ and NADH) and NADP(H) (i.e. NADP+ and NADPH) play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD+-supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT), found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD+ in macrophages. Inhibition of NAD+ salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD+ levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD+ induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD+ levels by NAD+, NMN, or NADP+ supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD+’s cytosolic role in the regulation of morphofunctional characteristics of macrophages. PMID:24824795

Porcine CD38 exhibits prominent secondary NAD(+) cyclase activity.

PubMed

Ting, Kai Yiu; Leung, Christina F P; Graeff, Richard M; Lee, Hon Cheung; Hao, Quan; Kotaka, Masayo

2016-03-01

Cyclic ADP-ribose (cADPR) mobilizes intracellular Ca(2+) stores and activates Ca(2+) influx to regulate a wide range of physiological processes. It is one of the products produced from the catalysis of NAD(+) by the multifunctional CD38/ADP-ribosyl cyclase superfamily. After elimination of the nicotinamide ring by the enzyme, the reaction intermediate of NAD(+) can either be hydrolyzed to form linear ADPR or cyclized to form cADPR. We have previously shown that human CD38 exhibits a higher preference towards the hydrolysis of NAD(+) to form linear ADPR while Aplysia ADP-ribosyl cyclase prefers cyclizing NAD(+) to form cADPR. In this study, we characterized the enzymatic properties of porcine CD38 and revealed that it has a prominent secondary NAD(+) cyclase activity producing cADPR. We also determined the X-ray crystallographic structures of porcine CD38 and were able to observe conformational flexibility at the base of the active site of the enzyme which allow the NAD(+) reaction intermediate to adopt conformations resulting in both hydrolysis and cyclization forming linear ADPR and cADPR respectively. © 2016 The Protein Society.

Vitamins and aging: pathways to NAD+ synthesis.

PubMed

Denu, John M

2007-05-04

Recent genetic evidence reveals additional salvage pathways for NAD(+) synthesis. In this issue, Belenky et al. (2007) report that nicotinamide riboside, a new NAD(+) precursor, regulates Sir2 deacetylase activity and life span in yeast. The ability of nicotinamide riboside to enhance life span does not depend on calorie restriction.

Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation.

PubMed

Kalous, Kelsey S; Wynia-Smith, Sarah L; Olp, Michael D; Smith, Brian C

2016-12-02

The sirtuin family of proteins catalyze the NAD + -dependent deacylation of acyl-lysine residues. Humans encode seven sirtuins (Sirt1-7), and recent studies have suggested that post-translational modification of Sirt1 by cysteine S-nitrosation correlates with increased acetylation of Sirt1 deacetylase substrates. However, the mechanism of Sirt1 inhibition by S-nitrosation was unknown. Here, we show that Sirt1 is transnitrosated and inhibited by the physiologically relevant nitrosothiol S-nitrosoglutathione. Steady-state kinetic analyses and binding assays were consistent with Sirt1 S-nitrosation inhibiting binding of both the NAD + and acetyl-lysine substrates. Sirt1 S-nitrosation correlated with Zn 2+ release from the conserved sirtuin Zn 2+ -tetrathiolate and a loss of α-helical structure without overall thermal destabilization of the enzyme. Molecular dynamics simulations suggested that Zn 2+ loss due to Sirt1 S-nitrosation results in repositioning of the tetrathiolate subdomain away from the rest of the catalytic domain, thereby disrupting the NAD + and acetyl-lysine-binding sites. Sirt1 S-nitrosation was reversed upon exposure to the thiol-based reducing agents, including physiologically relevant concentrations of the cellular reducing agent glutathione. Reversal of S-nitrosation resulted in full restoration of Sirt1 activity only in the presence of Zn 2+ , consistent with S-nitrosation of the Zn 2+ -tetrathiolate as the primary source of Sirt1 inhibition upon S-nitrosoglutathione treatment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation*

PubMed Central

Kalous, Kelsey S.; Wynia-Smith, Sarah L.; Olp, Michael D.

2016-01-01

The sirtuin family of proteins catalyze the NAD+-dependent deacylation of acyl-lysine residues. Humans encode seven sirtuins (Sirt1–7), and recent studies have suggested that post-translational modification of Sirt1 by cysteine S-nitrosation correlates with increased acetylation of Sirt1 deacetylase substrates. However, the mechanism of Sirt1 inhibition by S-nitrosation was unknown. Here, we show that Sirt1 is transnitrosated and inhibited by the physiologically relevant nitrosothiol S-nitrosoglutathione. Steady-state kinetic analyses and binding assays were consistent with Sirt1 S-nitrosation inhibiting binding of both the NAD+ and acetyl-lysine substrates. Sirt1 S-nitrosation correlated with Zn2+ release from the conserved sirtuin Zn2+-tetrathiolate and a loss of α-helical structure without overall thermal destabilization of the enzyme. Molecular dynamics simulations suggested that Zn2+ loss due to Sirt1 S-nitrosation results in repositioning of the tetrathiolate subdomain away from the rest of the catalytic domain, thereby disrupting the NAD+ and acetyl-lysine-binding sites. Sirt1 S-nitrosation was reversed upon exposure to the thiol-based reducing agents, including physiologically relevant concentrations of the cellular reducing agent glutathione. Reversal of S-nitrosation resulted in full restoration of Sirt1 activity only in the presence of Zn2+, consistent with S-nitrosation of the Zn2+-tetrathiolate as the primary source of Sirt1 inhibition upon S-nitrosoglutathione treatment. PMID:27756843

Flavonoid Apigenin Is an Inhibitor of the NAD+ase CD38

PubMed Central

Escande, Carlos; Nin, Veronica; Price, Nathan L.; Capellini, Verena; Gomes, Ana P.; Barbosa, Maria Thereza; O’Neil, Luke; White, Thomas A.; Sinclair, David A.; Chini, Eduardo N.

2013-01-01

Metabolic syndrome is a growing health problem worldwide. It is therefore imperative to develop new strategies to treat this pathology. In the past years, the manipulation of NAD+ metabolism has emerged as a plausible strategy to ameliorate metabolic syndrome. In particular, an increase in cellular NAD+ levels has beneficial effects, likely because of the activation of sirtuins. Previously, we reported that CD38 is the primary NAD+ase in mammals. Moreover, CD38 knockout mice have higher NAD+ levels and are protected against obesity and metabolic syndrome. Here, we show that CD38 regulates global protein acetylation through changes in NAD+ levels and sirtuin activity. In addition, we characterize two CD38 inhibitors: quercetin and apigenin. We show that pharmacological inhibition of CD38 results in higher intracellular NAD+ levels and that treatment of cell cultures with apigenin decreases global acetylation as well as the acetylation of p53 and RelA-p65. Finally, apigenin administration to obese mice increases NAD+ levels, decreases global protein acetylation, and improves several aspects of glucose and lipid homeostasis. Our results show that CD38 is a novel pharmacological target to treat metabolic diseases via NAD+-dependent pathways. PMID:23172919

Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis.

PubMed

Wang, Xueying; Zhou, Yongjin J; Wang, Lei; Liu, Wujun; Liu, Yuxue; Peng, Chang; Zhao, Zongbao K

2017-07-01

NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli , NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering. IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

PubMed

Shi, Hongjun; Enriquez, Annabelle; Rapadas, Melissa; Martin, Ella M M A; Wang, Roni; Moreau, Julie; Lim, Chai K; Szot, Justin O; Ip, Eddie; Hughes, James N; Sugimoto, Kotaro; Humphreys, David T; McInerney-Leo, Aideen M; Leo, Paul J; Maghzal, Ghassan J; Halliday, Jake; Smith, Janine; Colley, Alison; Mark, Paul R; Collins, Felicity; Sillence, David O; Winlaw, David S; Ho, Joshua W K; Guillemin, Gilles J; Brown, Matthew A; Kikuchi, Kazu; Thomas, Paul Q; Stocker, Roland; Giannoulatou, Eleni; Chapman, Gavin; Duncan, Emma L; Sparrow, Duncan B; Dunwoodie, Sally L

2017-08-10

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

Vibrio Phage KVP40 Encodes a Functional NAD+ Salvage Pathway.

PubMed

Lee, Jae Yun; Li, Zhiqun; Miller, Eric S

2017-05-01

The genome of T4-type Vibrio bacteriophage KVP40 has five genes predicted to encode proteins of pyridine nucleotide metabolism, of which two, nadV and natV , would suffice for an NAD + salvage pathway. NadV is an apparent nicotinamide phosphoribosyltransferase (NAmPRTase), and NatV is an apparent bifunctional nicotinamide mononucleotide adenylyltransferase (NMNATase) and nicotinamide-adenine dinucleotide pyrophosphatase (Nudix hydrolase). Genes encoding the predicted salvage pathway were cloned and expressed in Escherichia coli , the proteins were purified, and their enzymatic properties were examined. KVP40 NadV NAmPRTase is active in vitro , and a clone complements a Salmonella mutant defective in both the bacterial de novo and salvage pathways. Similar to other NAmPRTases, the KVP40 enzyme displayed ATPase activity indicative of energy coupling in the reaction mechanism. The NatV NMNATase activity was measured in a coupled reaction system demonstrating NAD + biosynthesis from nicotinamide, phosphoribosyl pyrophosphate, and ATP. The NatV Nudix hydrolase domain was also shown to be active, with preferred substrates of ADP-ribose, NAD + , and NADH. Expression analysis using reverse transcription-quantitative PCR (qRT-PCR) and enzyme assays of infected Vibrio parahaemolyticus cells demonstrated nadV and natV transcription during the early and delayed-early periods of infection when other KVP40 genes of nucleotide precursor metabolism are expressed. The distribution and phylogeny of NadV and NatV proteins among several large double-stranded DNA (dsDNA) myophages, and also those from some very large siphophages, suggest broad relevance of pyridine nucleotide scavenging in virus-infected cells. NAD + biosynthesis presents another important metabolic resource control point by large, rapidly replicating dsDNA bacteriophages. IMPORTANCE T4-type bacteriophages enhance DNA precursor synthesis through reductive reactions that use NADH/NADPH as the electron donor and NAD

NAMPT-Mediated NAD(+) Biosynthesis Is Essential for Vision In Mice.

PubMed

Lin, Jonathan B; Kubota, Shunsuke; Ban, Norimitsu; Yoshida, Mitsukuni; Santeford, Andrea; Sene, Abdoulaye; Nakamura, Rei; Zapata, Nicole; Kubota, Miyuki; Tsubota, Kazuo; Yoshino, Jun; Imai, Shin-Ichiro; Apte, Rajendra S

2016-09-27

Photoreceptor death is the endpoint of many blinding diseases. Identifying unifying pathogenic mechanisms in these diseases may offer global approaches for facilitating photoreceptor survival. We found that rod or cone photoreceptor-specific deletion of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the major NAD(+) biosynthetic pathway beginning with nicotinamide, caused retinal degeneration. In both cases, we could rescue vision with nicotinamide mononucleotide (NMN). Significantly, retinal NAD(+) deficiency was an early feature of multiple mouse models of retinal dysfunction, including light-induced degeneration, streptozotocin-induced diabetic retinopathy, and age-associated dysfunction. Mechanistically, NAD(+) deficiency caused metabolic dysfunction and consequent photoreceptor death. We further demonstrate that the NAD(+)-dependent mitochondrial deacylases SIRT3 and SIRT5 play important roles in retinal homeostasis and that NAD(+) deficiency causes SIRT3 dysfunction. These findings demonstrate that NAD(+) biosynthesis is essential for vision, provide a foundation for future work to further clarify the mechanisms involved, and identify a unifying therapeutic target for diverse blinding diseases. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Loss of NAD Homeostasis Leads to Progressive and Reversible Degeneration of Skeletal Muscle.

PubMed

Frederick, David W; Loro, Emanuele; Liu, Ling; Davila, Antonio; Chellappa, Karthikeyani; Silverman, Ian M; Quinn, William J; Gosai, Sager J; Tichy, Elisia D; Davis, James G; Mourkioti, Foteini; Gregory, Brian D; Dellinger, Ryan W; Redpath, Philip; Migaud, Marie E; Nakamaru-Ogiso, Eiko; Rabinowitz, Joshua D; Khurana, Tejvir S; Baur, Joseph A

2016-08-09

NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function. Copyright © 2016 Elsevier Inc. All rights reserved.

Crystal structure of human aldehyde dehydrogenase 1A3 complexed with NAD+ and retinoic acid

PubMed Central

Moretti, Andrea; Li, Jianfeng; Donini, Stefano; Sobol, Robert W.; Rizzi, Menico; Garavaglia, Silvia

2016-01-01

The aldehyde dehydrogenase family 1 member A3 (ALDH1A3) catalyzes the oxidation of retinal to the pleiotropic factor retinoic acid using NAD+. The level of ALDHs enzymatic activity has been used as a cancer stem cell marker and seems to correlate with tumour aggressiveness. Elevated ALDH1A3 expression in mesenchymal glioma stem cells highlights the potential of this isozyme as a prognosis marker and drug target. Here we report the first crystal structure of human ALDH1A3 complexed with NAD+ and the product all-trans retinoic acid (REA). The tetrameric ALDH1A3 folds into a three domain-based architecture highly conserved along the ALDHs family. The structural analysis revealed two different and coupled conformations for NAD+ and REA that we propose to represent two snapshots along the catalytic cycle. Indeed, the isoprenic moiety of REA points either toward the active site cysteine, or moves away adopting the product release conformation. Although ALDH1A3 shares high sequence identity with other members of the ALDH1A family, our structural analysis revealed few peculiar residues in the 1A3 isozyme active site. Our data provide information into the ALDH1As catalytic process and can be used for the structure-based design of selective inhibitors of potential medical interest. PMID:27759097

Increased Rate of NAD Metabolism Shortens Plant Longevity by Accelerating Developmental Senescence in Arabidopsis.

PubMed

Hashida, Shin-Nosuke; Itami, Taketo; Takahara, Kentaro; Hirabayashi, Takayuki; Uchimiya, Hirofumi; Kawai-Yamada, Maki

2016-11-01

NAD is a well-known co-enzyme that mediates hundreds of redox reactions and is the basis of various processes regulating cell responses to different environmental and developmental cues. The regulatory mechanism that determines the amount of cellular NAD and the rate of NAD metabolism remains unclear. We created Arabidopsis thaliana plants overexpressing the NAD synthase (NADS) gene that participates in the final step of NAD biosynthesis. NADS overexpression enhanced the activity of NAD biosynthesis but not the amounts of NAD + , NADH, NADP + or NADPH. However, the amounts of some intermediates were elevated, suggesting that NAD metabolism increased. The NAD redox state was greatly facilitated by an imbalance between NAD generation and degradation in response to bolting. Metabolite profiling and transcriptional analysis revealed that the drastic modulation of NAD redox homeostasis increased tricarboxylic acid flux, causing the ectopic generation of reactive oxygen species. Vascular bundles suffered from oxidative stress, leading to a malfunction in amino acid and organic acid transportation that caused early wilting of the flower stalk and shortened plant longevity, probably due to malnutrition. We concluded that the mechanism regulating the balance between NAD synthesis and degradation is important in the systemic plant response to developmental cues during the growth-phase transition. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Gene Cloning and Characterization of the Very Large NAD-Dependent l-Glutamate Dehydrogenase from the Psychrophile Janthinobacterium lividum, Isolated from Cold Soil▿

PubMed Central

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2007-01-01

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids. PMID:17526698

Gene cloning and characterization of the very large NAD-dependent l-glutamate dehydrogenase from the psychrophile Janthinobacterium lividum, isolated from cold soil.

PubMed

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2007-08-01

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids.

NAD+ : A big player in cardiac and skeletal muscle remodeling and aging.

PubMed

Chaturvedi, Pankaj; Tyagi, Suresh C

2018-03-01

In the past decade, NAD+ has gained importance for its beneficial effects as antioxidant and anti-aging molecule. A paper in science by Zhang et al. () has described that NAD+ when replenished, ameliorates muscle dystrophy in mice by improving mitochondrial function. NAD+ was also demonstrated by the authors to improve the life span of mice. Cox et al. () demonstrated the cardiac effects of NAD+ which mitigated chronic heart failure via mitochondrial redox state mechanism. Cox et al. () also demonstrated that NAD+ is provided in the drinking water, it improves cardiac relaxation in volume overload model of heart failure. Although NAD+ has a profound anti-aging and anti-oxidant effects, its effect on humans and use as a dietary supplement needs more exploration. © 2017 Wiley Periodicals, Inc.

Nads FSK Modem, LEA 74-2248

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, K.R.

1976-01-12

The Nads FSK Modem is a compact unit designed to operate in conjunction with EIA standard interfacing and the data terminal equipment of the 1200 Baud digital communications network of the Nevada Automated Diagnostics System (NADS). The modem is constructed in a Nuclear Instrumentation Module System (NIMS) module for compatability with the NADS system. The modulator section of the modem accepts serial, digital signals at 1200 Baud which may be either standard TTL levels or bipolar signals meeting either the EIA RS-232C or RS-232B standards. The output of the modulator is a Frequency-Shift Keyed (FSK) signal having frequencies of 2.2more » kHz for Mark and 1.2 kHz for Space. The demodulator section accepts the above FSK signal as input, and outputs serial, digital signals at 1200 Baud at either TTL or EIA RS-232C levels. Specifications and operation and calibration instructions are given. (WHK)« less

New Therapeutic Concept of NAD Redox Balance for Cisplatin Nephrotoxicity

PubMed Central

Oh, Gi-Su; Kim, Hyung-Jin; Shen, AiHua; Lee, Su-Bin; Yang, Sei-Hoon; Shim, Hyeok; Cho, Eun-Young; Kwon, Kang-Beom; Kwak, Tae Hwan; So, Hong-Seob

2016-01-01

Cisplatin is a widely used chemotherapeutic agent for the treatment of various tumors. In addition to its antitumor activity, cisplatin affects normal cells and may induce adverse effects such as ototoxicity, nephrotoxicity, and peripheral neuropathy. Various mechanisms such as DNA adduct formation, mitochondrial dysfunction, oxidative stress, and inflammatory responses are closely associated with cisplatin-induced nephrotoxicity; however, the precise mechanism remains unclear. The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of cellular energy metabolism and homeostasis. Recent studies have demonstrated associations between disturbance in intracellular NAD+ levels and clinical progression of various diseases through the production of reactive oxygen species and inflammation. Furthermore, we demonstrated that reduction of the intracellular NAD+/NADH ratio is critically involved in cisplatin-induced kidney damage through inflammation and oxidative stress and that increase of the cellular NAD+/NADH ratio suppresses cisplatin-induced kidney damage by modulation of potential damage mediators such as oxidative stress and inflammatory responses. In this review, we describe the role of NAD+ metabolism in cisplatin-induced nephrotoxicity and discuss a potential strategy for the prevention or treatment of cisplatin-induced adverse effects with a particular focus on NAD+-dependent cellular pathways. PMID:26881219

Involvement of cytosolic NAD+ glycohydrolase in cyclic ADP-ribose metabolism.

PubMed

Matsumura, N; Tanuma, S

1998-12-18

The NAD+ glycohydrolase homogeneously purified from bovine brain cytosol was found to catalyze the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). Although the formation of cADPR from NAD+ does not exceed about 2% of the reaction products, the cyclase activity is clearly evidenced by its conversion of NGD+ to cyclic GDP-ribose (cGDPR), which cannot be hydrolyzed to GDPR. Importantly, a steep increase in cADPR hydrolytic activity was observed at cADPR concentrations above 60 microM, which could be reproduced on a Hill curve with a Hill coefficient of 2. Thus, the allosteric binding of cADPR to the NAD+ glycohydrolase (E) molecule promotes the hydrolysis of cADPR. These results suggest that NAD+ hydrolysis to ADPR and nicotinamide catalyzed by the NAD+ glycohydrolase occurs through the formation of a cADPR. E. cADP-ribosyl complex. The low production of cADPR by NAD+ glycohydrolase compared with invertebrate ADP-ribosyl cyclase is believed to be attributable to the fast hydrolysis of cADPR by the allosteric effect of cADPR bound to the same enzyme that produces it. Copyright 1998 Academic Press.

Genetically encoded probes for NAD+/NADH monitoring.

PubMed

Bilan, Dmitry S; Belousov, Vsevolod V

2016-11-01

NAD + and NADH participate in many metabolic reactions. The NAD + /NADH ratio is an important parameter reflecting the general metabolic and redox state of different types of cells. For a long time, in situ and in vivo NAD + /NADH monitoring has been hampered by the lack of suitable tools. The recent development of genetically encoded indicators based on fluorescent proteins linked to specific nucleotide-binding domains has already helped to address this monitoring problem. In this review, we will focus on four available indicators: Peredox, Frex family probes, RexYFP and SoNar. Each indicator has advantages and limitations. We will also discuss the most important points that should be considered when selecting a suitable indicator for certain experimental conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

Nicotinamide Adenine Dinucleotide (NAD+) and Nicotinamide: Sex Differences in Cerebral Ischemia

PubMed Central

Siegel, Chad S.; McCullough, Louise D.

2013-01-01

Background Previous literature suggests that cell death pathways activated after cerebral ischemia differ between the sexes. While caspase-dependent mechanisms predominate in the female brain, caspase-independent cell death induced by activation of Poly (ADP-ribose) polymerase (PARP) predominates in the male brain. PARP-1 gene deletion decreases infarction volume in the male brain, but paradoxically increases damage in PARP-1 knockout females. Purpose This study examined stroke induced changes in NAD+, a key energy molecule involved in PARP-1 activation in both sexes. Methods Mice were subjected to Middle Cerebral Artery Occlusion and NAD+ levels were assessed. Caspase-3 activity and nuclear translocation was assessed 6 hours after ischemia. In additional cohorts, Nicotinamide (500mg/kg i.p.) a precursor of NAD+ or vehicle was administered and infarction volume was measured 24 hours after ischemia. Results Males have higher baseline NAD+ levels than females. Significant stroke-induced NAD+ depletion occurred in males and ovariectomized females but not in intact females. PARP-1 deletion prevented the stroke induced loss in NAD+ in males, but worsened NAD+ loss in PARP-1 deficient females. Preventing NAD+ loss with nicotinamide reduced infarct in wild-type males and PARP-1 knockout mice of both sexes, with no effect in WT females. Caspase-3 activity was significantly increased in PARP-1 knockout females compared to males and wild-type females, this was reversed with nicotinamide. Conclusions Sex differences exist in baseline and stroke-induced NAD+ levels. Nicotinamide protected males and PARP knockout mice, but had minimal effects in the wild-type female brain. This may be secondary to differences in energy metabolism between the sexes. PMID:23403179

Nicotinamide Riboside Is a Major NAD+ Precursor Vitamin in Cow Milk.

PubMed

Trammell, Samuel Aj; Yu, Liping; Redpath, Philip; Migaud, Marie E; Brenner, Charles

2016-05-01

Nicotinamide riboside (NR) is a recently discovered NAD(+) precursor vitamin with a unique biosynthetic pathway. Although the presence of NR in cow milk has been known for more than a decade, the concentration of NR with respect to the other NAD(+) precursors was unknown. We aimed to determine NAD(+) precursor vitamin concentration in raw samples of milk from individual cows and from commercially available cow milk. LC tandem mass spectrometry and isotope dilution technologies were used to quantify NAD(+) precursor vitamin concentration and to measure NR stability in raw and commercial milk. Nuclear magnetic resonance (NMR) spectroscopy was used to test for NR binding to substances in milk. Cow milk typically contained ∼12 μmol NAD(+) precursor vitamins/L, of which 60% was present as nicotinamide and 40% was present as NR. Nicotinic acid and other NAD(+) metabolites were below the limits of detection. Milk from samples testing positive for Staphylococcus aureus contained lower concentrations of NR (Spearman ρ = -0.58, P = 0.014), and NR was degraded by S. aureus Conventional milk contained more NR than milk sold as organic. Nonetheless, NR was stable in organic milk and exhibited an NMR spectrum consistent with association with a protein fraction in skim milk. NR is a major NAD(+) precursor vitamin in cow milk. Control of S. aureus may be important to preserve the NAD(+) precursor vitamin concentration of milk. © 2016 American Society for Nutrition.

The 2.5 Å Crystal Structure of the SIRT1 Catalytic Domain Bound to Nicotinamide Adenine Dinucleotide (NAD + ) and an Indole (EX527 Analogue) Reveals a Novel Mechanism of Histone Deacetylase Inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Xun; Allison, Dagart; Condon, Bradley

2013-02-14

The sirtuin SIRT1 is a NAD+-dependent histone deacetylase, a Sir2 family member, and one of seven human sirtuins. Sirtuins are conserved from archaea to mammals and regulate transcription, genome stability, longevity, and metabolism. SIRT1 regulates transcription via deacetylation of transcription factors such as PPARγ, NFκB, and the tumor suppressor protein p53. EX527 (27) is a nanomolar SIRT1 inhibitor and a micromolar SIRT2 inhibitor. To elucidate the mechanism of SIRT inhibition by 27, we determined the 2.5 Å crystal structure of the SIRT1 catalytic domain (residues 241–516) bound to NAD+ and the 27 analogue compound 35. 35 binds deep in themore » catalytic cleft, displacing the NAD+ nicotinamide and forcing the cofactor into an extended conformation. The extended NAD+ conformation sterically prevents substrate binding. The SIRT1/NAD+/35 crystal structure defines a novel mechanism of histone deacetylase inhibition and provides a basis for understanding, and rationally improving, inhibition of this therapeutically important target by drug-like molecules.« less

Mechanistic Links Between PARP, NAD, and Brain Inflammation After TBI

DTIC Science & Technology

2015-10-01

1 AWARD NUMBER: W81XWH-13-2-0091 TITLE: Mechanistic Links Between PARP, NAD , and Brain Inflammation After TBI PRINCIPAL INVESTIGATOR...COVERED 25 Sep 2014 - 24 Sep 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Mechanistic Links Between PARP, NAD , and Brain Inflammation After TBI 5b. GRANT...efficacy of veliparib and NAD as agents for suppressing inflammation and improving outcomes after traumatic brain injury. The animal models include

Redox Specificity of 2-Hydroxyacid-Coupled NAD+/NADH Dehydrogenases: A Study Exploiting “Reactive” Arginine as a Reporter of Protein Electrostatics

PubMed Central

Durani, Susheel

2013-01-01

With “reactive” arginine as a kinetic reporter, 2-hydroxyacid dehydrogenases are assessed in basis of their specialization as NAD+-reducing or NADH-oxidizing enzymes. Specifically, M4 and H4 lactate dehydrogenases (LDHs) and cytoplasmic and mitochondrial malate dehydrogenases (MDHs) are compared to assess if their coenzyme specificity may involve electrostatics of cationic or neutral nicotinamide structure as the basis. The enzymes from diverse eukaryote and prokaryote sources thus are assessed in “reactivity” of functionally-critical arginine as a function of salt concentration and pH. Electrostatic calculations were performed on “reactive” arginines and found good correspondence with experiment. The reductive and oxidative LDHs and MDHs are assessed in their count over ionizable residues and in placement details of the residues in their structures as proteins. The variants found to be high or low in ΔpKa of “reactive” arginine are found to be also strong or weak cations that preferentially oxidize NADH (neutral nicotinamide structure) or reduce NAD+ (cationic nicotinamide structure). The ionized groups of protein structure may thus be important to redox specificity of the enzyme on basis of electrostatic preference for the oxidized (cationic nicotinamide) or reduced (neutral nicotinamide) coenzyme. Detailed comparisons of isozymes establish that the residues contributing in their redox specificity are scrambled in structure of the reductive enzyme. PMID:24391777

Inhibition of NAD glycohydrolase and ADP-ribosyl transferases by carbocyclic analogues of oxidized nicotinamide adenine dinucleotide.

PubMed

Slama, J T; Simmons, A M

1989-09-19

Analogues of oxidized nicotinamide adenine dinucleotide (NAD+) in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ have recently been synthesized [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183]. Carbocyclic NAD+ analogues have been shown to inhibit NAD glycohydrolases and ADP-ribosyl transferases such as cholera toxin A subunit. In this study, the diastereomeric mixture of dinucleotides was separated, and the inhibitory capacity of each of the purified diastereomers was defined. The NAD+ analogue in which the D-dihydroxycyclopentane is substituted for the D-ribose is designated carba-NAD and was demonstrated to be a poor inhibitor of the Bungarus fasciatus venom NAD glycohydrolase. The diastereomeric dinucleotide pseudo-carbocyclic-NAD (psi-carba-NAD), containing L-dihydroxycyclopentane in place of the D-ribose of NAD+, was shown, however, to be a potent competitive inhibitor of the venom NAD glycohydrolase with an inhibitor dissociation constant (Ki) of 35 microM. This was surprising since psi-carba-NAD contains the carbocyclic analogue of the unnatural L-ribotide and was therefore expected to be a biologically inactive diastereomer. psi-Carba-NAD also competitively inhibited the insoluble brain NAD glycohydrolase from cow (Ki = 6.7 microM) and sheep (Ki = 31 microM) enzyme against which carba-NAD is ineffective. Sensitivity to psi-carba-NAD was found to parallel sensitivity to inhibition by isonicotinic acid hydrazide, another NADase inhibitor. psi-Carba-NAD is neither a substrate for nor an inhibitor of alcohol dehydrogenase, whereas carba-NAD is an efficient dehydrogenase substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells*

PubMed Central

VanLinden, Magali R.; Dölle, Christian; Pettersen, Ina K. N.; Kulikova, Veronika A.; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E.; Palmieri, Ferdinando; Nikiforov, Andrey A.; Tronstad, Karl Johan; Ziegler, Mathias

2015-01-01

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD+ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD+ in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD+ content, we have expressed plant and yeast mitochondrial NAD+ carriers in human cells and observed a profound increase in mitochondrial NAD+. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD+ content. Surprisingly, constitutive redistribution of NAD+ from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD+ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD+ levels. These results suggest that a mitochondrial NAD+ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD+ synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. PMID:26432643

Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius.

PubMed

Pennacchio, Angela; Giordano, Assunta; Pucci, Biagio; Rossi, Mosè; Raia, Carlo A

2010-03-01

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

NMNAT1 inhibits axon degeneration via blockade of SARM1-mediated NAD+ depletion

PubMed Central

Sasaki, Yo; Nakagawa, Takashi; Mao, Xianrong; DiAntonio, Aaron; Milbrandt, Jeffrey

2016-01-01

Overexpression of the NAD+ biosynthetic enzyme NMNAT1 leads to preservation of injured axons. While increased NAD+ or decreased NMN levels are thought to be critical to this process, the mechanism(s) of this axon protection remain obscure. Using steady-state and flux analysis of NAD+ metabolites in healthy and injured mouse dorsal root ganglion axons, we find that rather than altering NAD+ synthesis, NMNAT1 instead blocks the injury-induced, SARM1-dependent NAD+ consumption that is central to axon degeneration. DOI: http://dx.doi.org/10.7554/eLife.19749.001 PMID:27735788

Position-dependent interactions between cysteine residues and the helix dipole

PubMed Central

Miranda, JJ L.

2003-01-01

A protein model was developed for studying the interaction between cysteine residues and the helix dipole. Site-directed mutagenesis was used to introduce cysteine residues at the N-terminus of helix H in recombinant sperm whale myoglobin. Based on the difference in thiol pKa between folded proteins and an unfolded peptide, the energy of interaction between the thiolate and the helix dipole was determined. Thiolates at the N1 and N2 positions of the helix were stabilized by 0.3 kcal/mole and 0.7 kcal/mole, respectively. A thiolate at the Ncap position was stabilized by 2.8 kcal/mole, and may involve a hydrogen bond. In context with other studies, an experimentally observed helix dipole effect may be defined in terms of two distinct components. A charge-dipole component involves electrostatic interactions with peptide bond dipoles in the first two turns of the helix and affects residues at all positions of the terminus; a hydrogen bond component involves one or more backbone amide groups and is only possible at the capping position due to conformational restraints elsewhere. The nature and magnitude of the helix dipole effect is, therefore, position-dependent. Results from this model system were used to interpret cysteine reactivity in rodent hemoglobins and the thioredoxin family. PMID:12493830

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

PubMed

VanLinden, Magali R; Dölle, Christian; Pettersen, Ina K N; Kulikova, Veronika A; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E; Palmieri, Ferdinando; Nikiforov, Andrey A; Tronstad, Karl Johan; Ziegler, Mathias

2015-11-13

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of residue pairing in interacting β-strands from a predicted residue contact map.

PubMed

Mao, Wenzhi; Wang, Tong; Zhang, Wenxuan; Gong, Haipeng

2018-04-19

Despite the rapid progress of protein residue contact prediction, predicted residue contact maps frequently contain many errors. However, information of residue pairing in β strands could be extracted from a noisy contact map, due to the presence of characteristic contact patterns in β-β interactions. This information may benefit the tertiary structure prediction of mainly β proteins. In this work, we propose a novel ridge-detection-based β-β contact predictor to identify residue pairing in β strands from any predicted residue contact map. Our algorithm RDb 2 C adopts ridge detection, a well-developed technique in computer image processing, to capture consecutive residue contacts, and then utilizes a novel multi-stage random forest framework to integrate the ridge information and additional features for prediction. Starting from the predicted contact map of CCMpred, RDb 2 C remarkably outperforms all state-of-the-art methods on two conventional test sets of β proteins (BetaSheet916 and BetaSheet1452), and achieves F1-scores of ~ 62% and ~ 76% at the residue level and strand level, respectively. Taking the prediction of the more advanced RaptorX-Contact as input, RDb 2 C achieves impressively higher performance, with F1-scores reaching ~ 76% and ~ 86% at the residue level and strand level, respectively. In a test of structural modeling using the top 1 L predicted contacts as constraints, for 61 mainly β proteins, the average TM-score achieves 0.442 when using the raw RaptorX-Contact prediction, but increases to 0.506 when using the improved prediction by RDb 2 C. Our method can significantly improve the prediction of β-β contacts from any predicted residue contact maps. Prediction results of our algorithm could be directly applied to effectively facilitate the practical structure prediction of mainly β proteins. All source data and codes are available at http://166.111.152.91/Downloads.html or the GitHub address of https://github.com/wzmao/RDb2C .

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maeda, Tomoji, E-mail: t-maeda@nichiyaku.ac.jp; Tanabe-Fujimura, Chiaki; Fujita, Yu

2016-05-13

Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targetingmore » of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin. -- Highlights: •Herp interacts with NQO1. •NQO1 regulates Herp degradation.« less

NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

PubMed Central

Harkcom, William T.; Ghosh, Ananda K.; Sung, Matthew S.; Matov, Alexandre; Brown, Kevin D.; Giannakakou, Paraskevi; Jaffrey, Samie R.

2014-01-01

Nicotinamide adenine dinucleotide (NAD+) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD+-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD+. Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD+ levels. We find that these effects of NAD+ are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD+ on microtubule polymers. Taken together, these data demonstrate that NAD+ and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents. PMID:24889606

Kinetics and structural features of dimeric glutamine-dependent bacterial NAD+ synthetases suggest evolutionary adaptation to available metabolites.

PubMed

Santos, Adrian Richard Schenberger; Gerhardt, Edileusa Cristina Marques; Moure, Vivian Rotuno; Pedrosa, Fábio Oliveira; Souza, Emanuel Maltempi; Diamanti, Riccardo; Högbom, Martin; Huergo, Luciano Fernandes

2018-05-11

NADH (NAD + ) and its reduced form NADH serve as cofactors for a variety of oxidoreductases that participate in many metabolic pathways. NAD + also is used as substrate by ADP-ribosyl transferases and by sirtuins. NAD + biosynthesis is one of the most fundamental biochemical pathways in nature, and the ubiquitous NAD + synthetase (NadE) catalyzes the final step in this biosynthetic route. Two different classes of NadE have been described to date: dimeric single-domain ammonium-dependent NadE NH3 and octameric glutamine-dependent NadE Gln , and the presence of multiple NadE isoforms is relatively common in prokaryotes. Here, we identified a novel dimeric group of NadE Gln in bacteria. Substrate preferences and structural analyses suggested that dimeric NadE Gln enzymes may constitute evolutionary intermediates between dimeric NadE NH3 and octameric NadE Gln The characterization of additional NadE isoforms in the diazotrophic bacterium Azospirillum brasilense along with the determination of intracellular glutamine levels in response to an ammonium shock led us to propose a model in which these different NadE isoforms became active accordingly to the availability of nitrogen. These data may explain the selective pressures that support the coexistence of multiple isoforms of NadE in some prokaryotes. © 2018 Santos et al.

Coenzyme engineering of a hyperthermophilic 6-phosphogluconate dehydrogenase from NADP + to NAD + with its application to biobatteries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Hui; Zhu, Zhiguang; Huang, Rui

Engineering the coenzyme specificity of redox enzymes plays an important role in metabolic engineering, synthetic biology, and biocatalysis, but it has rarely been applied to bioelectrochemistry. Here we develop a rational design strategy to change the coenzyme specificity of 6-phosphogluconate dehydrogenase (6PGDH) from a hyperthermophilic bacterium Thermotoga maritima from its natural coenzyme NADP + to NAD +. Through amino acid-sequence alignment of NADP +- and NAD +-preferred 6PGDH enzymes and computer-aided substrate-coenzyme docking, the key amino acid residues responsible for binding the phosphate group of NADP + were identified. Four mutants were obtained via site-directed mutagenesis. The best mutant N32E/R33I/T34Imore » exhibited a ~6.4 × 10 4-fold reversal of the coenzyme selectivity from NADP + to NAD +. The maximum power density and current density of the biobattery catalyzed by the mutant were 0.135 mW cm -2 and 0.255 mA cm -2, ~25% higher than those obtained from the wide-type 6PGDH-based biobattery at the room temperature. By using this 6PGDH mutant, the optimal temperature of running the biobattery was as high as 65 °C, leading to a high power density of 1.75 mW cm -2. As a result, this study demonstrates coenzyme engineering of a hyperthermophilic 6PGDH and its application to high-temperature biobatteries.« less

Coenzyme engineering of a hyperthermophilic 6-phosphogluconate dehydrogenase from NADP + to NAD + with its application to biobatteries

DOE PAGES

Chen, Hui; Zhu, Zhiguang; Huang, Rui; ...

2016-11-02

Engineering the coenzyme specificity of redox enzymes plays an important role in metabolic engineering, synthetic biology, and biocatalysis, but it has rarely been applied to bioelectrochemistry. Here we develop a rational design strategy to change the coenzyme specificity of 6-phosphogluconate dehydrogenase (6PGDH) from a hyperthermophilic bacterium Thermotoga maritima from its natural coenzyme NADP + to NAD +. Through amino acid-sequence alignment of NADP +- and NAD +-preferred 6PGDH enzymes and computer-aided substrate-coenzyme docking, the key amino acid residues responsible for binding the phosphate group of NADP + were identified. Four mutants were obtained via site-directed mutagenesis. The best mutant N32E/R33I/T34Imore » exhibited a ~6.4 × 10 4-fold reversal of the coenzyme selectivity from NADP + to NAD +. The maximum power density and current density of the biobattery catalyzed by the mutant were 0.135 mW cm -2 and 0.255 mA cm -2, ~25% higher than those obtained from the wide-type 6PGDH-based biobattery at the room temperature. By using this 6PGDH mutant, the optimal temperature of running the biobattery was as high as 65 °C, leading to a high power density of 1.75 mW cm -2. As a result, this study demonstrates coenzyme engineering of a hyperthermophilic 6PGDH and its application to high-temperature biobatteries.« less

Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries

NASA Astrophysics Data System (ADS)

Chen, Hui; Zhu, Zhiguang; Huang, Rui; Zhang, Yi-Heng Percival

2016-11-01

Engineering the coenzyme specificity of redox enzymes plays an important role in metabolic engineering, synthetic biology, and biocatalysis, but it has rarely been applied to bioelectrochemistry. Here we develop a rational design strategy to change the coenzyme specificity of 6-phosphogluconate dehydrogenase (6PGDH) from a hyperthermophilic bacterium Thermotoga maritima from its natural coenzyme NADP+ to NAD+. Through amino acid-sequence alignment of NADP+- and NAD+-preferred 6PGDH enzymes and computer-aided substrate-coenzyme docking, the key amino acid residues responsible for binding the phosphate group of NADP+ were identified. Four mutants were obtained via site-directed mutagenesis. The best mutant N32E/R33I/T34I exhibited a ~6.4 × 104-fold reversal of the coenzyme selectivity from NADP+ to NAD+. The maximum power density and current density of the biobattery catalyzed by the mutant were 0.135 mW cm-2 and 0.255 mA cm-2, ~25% higher than those obtained from the wide-type 6PGDH-based biobattery at the room temperature. By using this 6PGDH mutant, the optimal temperature of running the biobattery was as high as 65 °C, leading to a high power density of 1.75 mW cm-2. This study demonstrates coenzyme engineering of a hyperthermophilic 6PGDH and its application to high-temperature biobatteries.

Simultaneous measurement of NAD metabolome in aged mice tissue using liquid chromatography tandem-mass spectrometry.

PubMed

Yaku, Keisuke; Okabe, Keisuke; Nakagawa, Takashi

2018-06-01

Nicotinamide adenine dinucleotide (NAD) is a major co-factor that mediates multiple biological processes including redox reaction and gene expression. Recently, NAD metabolism has received considerable attention because administration of NAD precursors exhibited beneficial effects against aging-related metabolic disorders in animals. Although numerous studies have reported that NAD levels decline with aging in multiple animal tissues, the pathway and kinetics of NAD metabolism in aged organs are not completely understood. To determine the NAD metabolism upon aging, we developed targeted metabolomics based on an LC/MS/MS system. Our method is simple and applicable to crude biological samples, including culture cells and animal tissues. Unlike a conventional enzymatic cycling assay, our approach can determine NAD and NADH (reduced form of NAD) by performing a single sample preparation. Further, we validated our method using biological samples and investigated the alteration of the NAD metabolome during aging. Consistent with previous reports, the NAD levels in the liver and skeletal muscle decreased with aging. Further, we detected a significant increase in nicotinamide mononucleotide and nicotinamide riboside in the kidney upon aging. The LC/MS/MS-based NAD metabolomics that we have developed is extensively applicable to biomedical studies, and the results will present innovative ideas for the aging studies, especially for that of NAD metabolism. Copyright © 2018 John Wiley & Sons, Ltd.

Biofuel cell anode: NAD +/glucose dehydrogenase-coimmobilized ketjenblack electrode

NASA Astrophysics Data System (ADS)

Miyake, T.; Oike, M.; Yoshino, S.; Yatagawa, Y.; Haneda, K.; Kaji, H.; Nishizawa, M.

2009-09-01

We have studied the coimmobilization of glucose dehydrogenase (GDH) and its cofactor, oxidized nicotinamide adenine dinucleotide (NAD +), on a ketjenblack (KB) electrode as a step toward a biofuel cell anode that works without mediators. A KB electrode was first treated with a sulfuric acid/nitric acid/water mixture to lower the overvoltage for NADH oxidation, and was next chemically modified with NAD + and GDH. The improved GDH/NAD +/KB electrode is found to oxidize glucose around 0 V vs. Ag/AgCl. A biofuel cell constructed with a bilirubin oxidase-immobilized KB cathode showed a maximum power density of 52 μW/cm 2 at 0.3 V.

Elongator Plays a Positive Role in Exogenous NAD-Induced Defense Responses in Arabidopsis.

PubMed

An, Chuanfu; Ding, Yezhang; Zhang, Xudong; Wang, Chenggang; Mou, Zhonglin

2016-05-01

Extracellular NAD is emerging as an important signal molecule in animal cells, but its role in plants has not been well-established. Although it has been shown that exogenous NAD(+) activates defense responses in Arabidopsis, components in the exogenous NAD(+)-activated defense pathway remain to be fully discovered. In a genetic screen for mutants insensitive to exogenous NAD(+) (ien), we isolated a mutant named ien2. Map-based cloning revealed that IEN2 encodes ELONGATA3 (ELO3)/AtELP3, a subunit of the Arabidopsis Elongator complex, which functions in multiple biological processes, including histone modification, DNA (de)methylation, and transfer RNA modification. Mutations in the ELO3/AtELP3 gene compromise exogenous NAD(+)-induced expression of pathogenesis-related (PR) genes and resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326, and transgenic expression of the coding region of ELO3/AtELP3 in elo3/Atelp3 restores NAD(+) responsiveness to the mutant plants, demonstrating that ELO3/AtELP3 is required for exogenous NAD(+)-induced defense responses. Furthermore, mutations in genes encoding the other five Arabidopsis Elongator subunits (ELO2/AtELP1, AtELP2, ELO1/AtELP4, AtELP5, and AtELP6) also compromise exogenous NAD(+)-induced PR gene expression and resistance to P. syringae pv. maculicola ES4326. These results indicate that the Elongator complex functions as a whole in exogenous NAD(+)-activated defense signaling in Arabidopsis.

Metabolic control by sirtuins and other enzymes that sense NAD+, NADH, or their ratio.

PubMed

Anderson, Kristin A; Madsen, Andreas S; Olsen, Christian A; Hirschey, Matthew D

2017-12-01

NAD + is a dinucleotide cofactor with the potential to accept electrons in a variety of cellular reduction-oxidation (redox) reactions. In its reduced form, NADH is a ubiquitous cellular electron donor. NAD + , NADH, and the NAD + /NADH ratio have long been known to control the activity of several oxidoreductase enzymes. More recently, enzymes outside those participating directly in redox control have been identified that sense these dinucleotides, including the sirtuin family of NAD + -dependent protein deacylases. In this review, we highlight examples of non-redox enzymes that are controlled by NAD + , NADH, or NAD + /NADH. In particular, we focus on the sirtuin family and assess the current evidence that the sirtuin enzymes sense these dinucleotides and discuss the biological conditions under which this might occur; we conclude that sirtuins sense NAD + , but neither NADH nor the ratio. Finally, we identify future studies that might be informative to further interrogate physiological and pathophysiological changes in NAD + and NADH, as well as enzymes like sirtuins that sense and respond to redox changes in the cell. Copyright © 2017 Elsevier B.V. All rights reserved.

The structural requirements of organophosphorus insecticides (OPI) for reducing chicken embryo NAD(+) content in OPI-induced teratogenesis in chickens.

PubMed

Seifert, Josef

2016-05-01

The objective of this study was to determine the structural requirements of organophosphorus insecticides (OPI) for reducing chicken embryo nicotinamide adenine dinucleotide (NAD(+)) content in OPI-induced teratogenesis and compare them with those needed for OPI inhibition of yolk sac membrane kynurenine formamidase (KFase), the proposed primary target for OPI teratogens in chicken embryos. The comparative molecular field analysis (COMFA) of three-dimensional quantitative structure-activity relationship (3D QSAR) revealed the electrostatic and steric fields as good predictors of OPI structural requirements to reduce NAD(+) content in chicken embryos. The dominant electrostatic interactions were localized at nitrogen-1, nitrogen-3, nitrogen of 2-amino substituent of the pyrimidinyl of pyrimidinyl phosphorothioates, and at the oxygen of crotonamide carbonyl in crotonamide phosphates. Bulkiness of the substituents at carbon-6 of the pyrimidinyls and/or N-substituents of crotonamides was the steric structural component that contributed to superiority of those OPI for reducing embryonic NAD(+) levels. Both electrostatic and steric requirements are similar to those defined in our previous study for OPI inhibition of chicken embryo yolk sac membrane KFase. The findings of this study provide another piece of evidence for the cause-and-effect relationship between yolk sac membrane KFase inhibition and reduced embryo NAD(+) content in NAD-associated OPI-induced teratogenesis in chickens. Copyright © 2015 Elsevier Inc. All rights reserved.

Increasing NAD Synthesis in Muscle via Nicotinamide Phosphoribosyltransferase Is Not Sufficient to Promote Oxidative Metabolism*

PubMed Central

Frederick, David W.; Davis, James G.; Dávila, Antonio; Agarwal, Beamon; Michan, Shaday; Puchowicz, Michelle A.; Nakamaru-Ogiso, Eiko; Baur, Joseph A.

2015-01-01

The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle. PMID:25411251

Increasing NAD synthesis in muscle via nicotinamide phosphoribosyltransferase is not sufficient to promote oxidative metabolism.

PubMed

Frederick, David W; Davis, James G; Dávila, Antonio; Agarwal, Beamon; Michan, Shaday; Puchowicz, Michelle A; Nakamaru-Ogiso, Eiko; Baur, Joseph A

2015-01-16

The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Depletion of NAD pool contributes to impairment of endothelial progenitor cell mobilization in diabetes.

PubMed

Wang, Pei; Yang, Xi; Zhang, Zheng; Song, Jie; Guan, Yun-Feng; Zou, Da-Jin; Miao, Chao-Yu

2016-06-01

The impaired mobilization of endothelial progenitor cells (EPCs) from bone marrow (BM) critically contributes to the diabetes-associated vascular complications. Here, we investigated the relationship between the nicotinamide phosphoribosyltransferase (NAMPT)-controlled nicotinamide adenine dinucleotide (NAD) metabolism and the impaired mobilization of BM-derived EPCs in diabetic condition. The NAMPT-NAD pool in BM and BM-derived EPCs in wild-type (WT) and diabetic db/db mice was determined. Nicotinamide, a natural substrate for NAD biosynthesis, was administrated for 2weeks in db/db mice to examine the influence of enhancing NAD pool on BM and blood EPCs number. The modulations of stromal cell-derived factor-1α (SDF-1α) and endothelial nitric oxide synthase (eNOS) protein in BM were measured using immunoblotting. The EPCs intracellular NAMPT level and NAD concentration, as well as the blood EPCs number, were compared between 9 healthy people and 16 patients with type 2 diabetes mellitus (T2DM). The T2DM patients were treated with nicotinamide for two weeks and then the blood EPCs number was determined. Moreover, the association between blood EPCs numbers and EPCs intracellular NAD(+)/NAMPT protein levels in 21 healthy individuals was determined. We found that NAD concentration and NAMPT expression in BM and BM-derived EPCs of db/db mice were significantly lower than those in WT mice BM. Enhancing NAD pool not only increased the EPCs intracellular NAD concentration and blood EPCs number, but also improved post-ischemic wound healing and blood reperfusion in db/db mice with hind-limb ischemia model. Enhancing NAD pool rescued the impaired modulations of stromal cell-derived factor-1α (SDF-1α) and endothelial nitric oxide synthase (eNOS) protein levels in db/db mice BM upon hind-limb ischemia. In addition, enhancing NAD pool significantly inhibited PARP and caspase-3 activates in db/db mice BM. The intracellular NAMPT-NAD pool was positively associated with blood

Expression of the human NAD(P)-metabolizing ectoenzyme CD38 compromises systemic acquired resistance in Arabidopsis.

PubMed

Zhang, Xudong; Mou, Zhonglin

2012-09-01

Plant systemic acquired resistance (SAR) is a long-lasting, broad-spectrum immune response that is mounted after primary pathogen infection. Although SAR has been extensively researched, the molecular mechanisms underlying its activation have not been completely understood. We have previously shown that the electron carrier NAD(P) leaks into the plant extracellular compartment upon pathogen attack and that exogenous NAD(P) activates defense gene expression and disease resistance in local treated leaves, suggesting that extracellular NAD(P) [eNAD(P)] might function as a signal molecule activating plant immune responses. To further establish the function of eNAD(P) in plant immunity, we tested the effect of exogenous NAD(P) on resistance gene-mediated hypersensitive response (HR) and SAR. We found that exogenous NAD(P) completely suppresses HR-mediated cell death but does not affect HR-mediated disease resistance. Local application of exogenous NAD(P) is unable to induce SAR in distal tissues, indicating that eNAD(P) is not a sufficient signal for SAR activation. Using transgenic Arabidopsis plants expressing the human NAD(P)-metabolizing ectoenzyme CD38, we demonstrated that altering eNAD(P) concentration or signaling compromises biological induction of SAR. This result suggests that eNAD(P) may play a critical signaling role in activation of SAR.

NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation

PubMed Central

Ryu, Dongryeol; Zhang, Hongbo; Ropelle, Eduardo R.; Sorrentino, Vincenzo; Mázala, Davi A. G.; Mouchiroud, Laurent; Marshall, Philip L.; Campbell, Matthew D.; Ali, Amir Safi; Knowels, Gary M.; Bellemin, Stéphanie; Iyer, Shama R.; Wang, Xu; Gariani, Karim; Sauve, Anthony A.; Cantó, Carles; Conley, Kevin E.; Walter, Ludivine; Lovering, Richard M.; Chin, Eva R.; Jasmin, Bernard J.; Marcinek, David J.; Menzies, Keir J.; Auwerx, Johan

2017-01-01

Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD+) synthesis, consistent with a potential role for the essential cofactor NAD+ in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene’s muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5’-diphosphate (ADP)–ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD+ and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD+ levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ biosynthesis. Replenishing NAD+ stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr−/− mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD+ repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD+ may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures. PMID:27798264

NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation.

PubMed

Ryu, Dongryeol; Zhang, Hongbo; Ropelle, Eduardo R; Sorrentino, Vincenzo; Mázala, Davi A G; Mouchiroud, Laurent; Marshall, Philip L; Campbell, Matthew D; Ali, Amir Safi; Knowels, Gary M; Bellemin, Stéphanie; Iyer, Shama R; Wang, Xu; Gariani, Karim; Sauve, Anthony A; Cantó, Carles; Conley, Kevin E; Walter, Ludivine; Lovering, Richard M; Chin, Eva R; Jasmin, Bernard J; Marcinek, David J; Menzies, Keir J; Auwerx, Johan

2016-10-19

Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD + ) synthesis, consistent with a potential role for the essential cofactor NAD + in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene's muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5'-diphosphate (ADP)-ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD + and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD + levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD + biosynthesis. Replenishing NAD + stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr -/- mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD + repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD + may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures. Copyright © 2016, American Association for the Advancement of Science.

Adipose tissue NAD+ biology in obesity and insulin resistance: From mechanism to therapy.

PubMed

Yamaguchi, Shintaro; Yoshino, Jun

2017-05-01

Nicotinamide adenine dinucleotide (NAD + ) biosynthetic pathway, mediated by nicotinamide phosphoribosyltransferase (NAMPT), a key NAD + biosynthetic enzyme, plays a pivotal role in controlling many biological processes, such as metabolism, circadian rhythm, inflammation, and aging. Over the past decade, NAMPT-mediated NAD + biosynthesis, together with its key downstream mediator, namely the NAD + -dependent protein deacetylase SIRT1, has been demonstrated to regulate glucose and lipid metabolism in a tissue-dependent manner. These discoveries have provided novel mechanistic and therapeutic insights into obesity and its metabolic complications, such as insulin resistance, an important risk factor for developing type 2 diabetes and cardiovascular disease. This review will focus on the importance of adipose tissue NAMPT-mediated NAD + biosynthesis and SIRT1 in the pathophysiology of obesity and insulin resistance. We will also critically explore translational and clinical aspects of adipose tissue NAD + biology. © 2017 WILEY Periodicals, Inc.

NAD+ Biosynthesis Ameliorates a Zebrafish Model of Muscular Dystrophy

PubMed Central

Goody, Michelle F.; Kelly, Meghan W.; Reynolds, Christine J.; Khalil, Andre; Crawford, Bryan D.; Henry, Clarissa A.

2012-01-01

Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result from genetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrix microenvironment. Cell-matrix adhesions are exquisite sensors of physiological conditions and mediate responses that allow cells to adapt to changing conditions. Thus, one approach towards finding targets for future therapeutic applications is to identify cell adhesion pathways that mediate these dynamic, adaptive responses in vivo. We find that nicotinamide riboside kinase 2b-mediated NAD+ biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrix adhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of the dystrophin-glycoprotein complex or integrin alpha7. Exogenous NAD+ or a vitamin precursor to NAD+ reduces muscle fiber degeneration and results in significantly faster escape responses in dystrophic embryos. Overexpression of paxillin, a cell adhesion protein downstream of NAD+ in this novel cell adhesion pathway, reduces muscle degeneration in zebrafish with intact integrin receptors but does not improve motility. Activation of this pathway significantly increases organization of laminin, a major component of the extracellular matrix basement membrane. Our results indicate that the primary protective effects of NAD+ result from changes to the basement membrane, as a wild-type basement membrane is sufficient to increase resilience of dystrophic muscle fibers to damage. The surprising result that NAD+ supplementation ameliorates dystrophy in dystrophin-glycoprotein complex– or integrin alpha7–deficient zebrafish suggests the existence of an additional laminin receptor complex that anchors muscle fibers to the basement membrane. We find that integrin alpha6 participates in this pathway, but either integrin alpha7 or the dystrophin-glycoprotein complex is required in conjunction with integrin

NAD and the aging process: Role in life, death and everything in between.

PubMed

Chini, Claudia C S; Tarragó, Mariana G; Chini, Eduardo N

2017-11-05

Life as we know it cannot exist without the nucleotide nicotinamide adenine dinucleotide (NAD). From the simplest organism, such as bacteria, to the most complex multicellular organisms, NAD is a key cellular component. NAD is extremely abundant in most living cells and has traditionally been described to be a cofactor in electron transfer during oxidation-reduction reactions. In addition to participating in these reactions, NAD has also been shown to play a key role in cell signaling, regulating several pathways from intracellular calcium transients to the epigenetic status of chromatin. Thus, NAD is a molecule that provides an important link between signaling and metabolism, and serves as a key molecule in cellular metabolic sensoring pathways. Importantly, it has now been clearly demonstrated that cellular NAD levels decline during chronological aging. This decline appears to play a crucial role in the development of metabolic dysfunction and age-related diseases. In this review we will discuss the molecular mechanisms responsible for the decrease in NAD levels during aging. Since other reviews on this subject have been recently published, we will concentrate on presenting a critical appraisal of the current status of the literature and will highlight some controversial topics in the field. In particular, we will discuss the potential role of the NADase CD38 as a driver of age-related NAD decline. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Intracellular NAD+ levels are associated with LPS-induced TNF-α release in pro-inflammatory macrophages

PubMed Central

Al-Shabany, Abbas Jawad; Moody, Alan John; Foey, Andrew David; Billington, Richard Andrew

2016-01-01

Metabolism and immune responses have been shown to be closely linked and as our understanding increases, so do the intricacies of the level of linkage. NAD+ has previously been shown to regulate tumour necrosis factor-α (TNF-α) synthesis and TNF-α has been shown to regulate NAD+ homoeostasis providing a link between a pro-inflammatory response and redox status. In the present study, we have used THP-1 differentiation into pro- (M1-like) and anti- (M2-like) inflammatory macrophage subset models to investigate this link further. Pro- and anti-inflammatory macrophages showed different resting NAD+ levels and expression levels of NAD+ homoeostasis enzymes. Challenge with bacterial lipopolysaccharide, a pro-inflammatory stimulus for macrophages, caused a large, biphasic and transient increase in NAD+ levels in pro- but not anti-inflammatory macrophages that were correlated with TNF-α release and inhibition of certain NAD+ synthesis pathways blocked TNF-α release. Lipopolysaccharide stimulation also caused changes in mRNA levels of some NAD+ homoeostasis enzymes in M1-like cells. Surprisingly, despite M2-like cells not releasing TNF-α or changing NAD+ levels in response to lipopolysaccharide, they showed similar mRNA changes compared with M1-like cells. These data further strengthen the link between pro-inflammatory responses in macrophages and NAD+. The agonist-induced rise in NAD+ shows striking parallels to well-known second messengers and raises the possibility that NAD+ is acting in a similar manner in this model. PMID:26764408

Protein-bound NAD(P)H Lifetime is Sensitive to Multiple Fates of Glucose Carbon.

PubMed

Sharick, Joe T; Favreau, Peter F; Gillette, Amani A; Sdao, Sophia M; Merrins, Matthew J; Skala, Melissa C

2018-04-03

While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ 2 ) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ 2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.

The NAD+ metabolism of Leishmania, notably the enzyme nicotinamidase involved in NAD+ salvage, offers prospects for development of anti-parasite chemotherapy.

PubMed

Michels, Paul A M; Avilán, Luisana

2011-10-01

NAD+ plays multiple, essential roles in the cell. As a cofactor in many redox reactions it is key in the cellular energy metabolism and as a substrate it participates in many reactions leading to a variety of covalent modifications of enzymes with major roles in regulation of expression and metabolism. Cells may have the ability to produce this metabolite either via alternative de novo synthesis pathways and/or by different salvage pathways. In this issue of Molecular Microbiology, Gazanion et al. (2011) demonstrate that Leishmania species can only rely on the salvage of NAD+ building blocks. One of the enzymes involved, nicotinamidase, is absent from human cells. The enzyme is important for growth of Leishmania infantum and essential for establishing an infection. The crystal structure of the parasite protein has been solved and shows prospects for design of inhibitors to be used as leads for development of new drugs. Indeed, NAD+ metabolism is currently being considered as a promising drug target in various diseases and the vulnerability of Leishmania for interference of this metabolism has been proved in previous work by the same group, by showing that administration of NAD+ precursors has detrimental effect on the pathogenic, amastigote stage of this parasite. © 2011 Blackwell Publishing Ltd.

Empirical solvent-mediated potentials hold for both intra-molecular and inter-molecular inter-residue interactions.

PubMed Central

Keskin, O.; Bahar, I.; Badretdinov, A. Y.; Ptitsyn, O. B.; Jernigan, R. L.

1998-01-01

Whether knowledge-based intra-molecular inter-residue potentials are valid to represent inter-molecular interactions taking place at protein-protein interfaces has been questioned in several studies. Differences in the chain connectivity effect and in residue packing geometry between interfaces and single chain monomers have been pointed out as possible sources of distinct energetics for the two cases. In the present study, the interfacial regions of protein-protein complexes are examined to extract inter-molecular inter-residue potentials, using the same statistical methods as those previously adopted for intra-molecular residue pairs. Two sets of energy parameters are derived, corresponding to solvent-mediation and "average residue" mediation. The former set is shown to be highly correlated (correlation coefficient 0.89) with that previously obtained for inter-residue interactions within single chain monomers, while the latter exhibits a weaker correlation (0.69) with its intra-molecular counterpart. In addition to the close similarity of intra- and inter-molecular solvent-mediated potentials, they are shown to be significantly more residue-specific and thereby discriminative compared to the residue-mediated ones, indicating that solvent-mediation plays a major role in controlling the effective inter-residue interactions, either at interfaces, or within single monomers. Based on this observation, a reduced set of energy parameters comprising 20 one-body and 3 two-body terms is proposed (as opposed to the 20 x 20 tables of inter-residue potentials), which reproduces the conventional 20 x 20 tables with a correlation coefficient of 0.99. PMID:9865952

Molecular interactions and residues involved in force generation in the T4 viral DNA packaging motor.

PubMed

Migliori, Amy D; Smith, Douglas E; Arya, Gaurav

2014-12-12

Many viruses utilize molecular motors to package their genomes into preformed capsids. A striking feature of these motors is their ability to generate large forces to drive DNA translocation against entropic, electrostatic, and bending forces resisting DNA confinement. A model based on recently resolved structures of the bacteriophage T4 motor protein gp17 suggests that this motor generates large forces by undergoing a conformational change from an extended to a compact state. This transition is proposed to be driven by electrostatic interactions between complementarily charged residues across the interface between the N- and C-terminal domains of gp17. Here we use atomistic molecular dynamics simulations to investigate in detail the molecular interactions and residues involved in such a compaction transition of gp17. We find that although electrostatic interactions between charged residues contribute significantly to the overall free energy change of compaction, interactions mediated by the uncharged residues are equally if not more important. We identify five charged residues and six uncharged residues at the interface that play a dominant role in the compaction transition and also reveal salt bridging, van der Waals, and solvent hydrogen-bonding interactions mediated by these residues in stabilizing the compact form of gp17. The formation of a salt bridge between Glu309 and Arg494 is found to be particularly crucial, consistent with experiments showing complete abrogation in packaging upon Glu309Lys mutation. The computed contributions of several other residues are also found to correlate well with single-molecule measurements of impairments in DNA translocation activity caused by site-directed mutations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hepatic NAD(+) deficiency as a therapeutic target for non-alcoholic fatty liver disease in ageing.

PubMed

Zhou, Can-Can; Yang, Xi; Hua, Xia; Liu, Jian; Fan, Mao-Bing; Li, Guo-Qiang; Song, Jie; Xu, Tian-Ying; Li, Zhi-Yong; Guan, Yun-Feng; Wang, Pei; Miao, Chao-Yu

2016-08-01

Ageing is an important risk factor of non-alcoholic fatty liver disease (NAFLD). Here, we investigated whether the deficiency of nicotinamide adenine dinucleotide (NAD(+) ), a ubiquitous coenzyme, links ageing with NAFLD. Hepatic concentrations of NAD(+) , protein levels of nicotinamide phosphoribosyltransferase (NAMPT) and several other critical enzymes regulating NAD(+) biosynthesis, were compared in middle-aged and aged mice or patients. The influences of NAD(+) decline on the steatosis and steatohepatitis were evaluated in wild-type and H247A dominant-negative, enzymically-inactive NAMPT transgenic mice (DN-NAMPT) given normal or high-fat diet (HFD). Hepatic NAD(+) level decreased in aged mice and humans. NAMPT-controlled NAD(+) salvage, but not de novo biosynthesis pathway, was compromised in liver of elderly mice and humans. Given normal chow, middle-age DN-NAMPT mice displayed systemic NAD(+) reduction and had moderate NAFLD phenotypes, including lipid accumulation, enhanced oxidative stress, triggered inflammation and impaired insulin sensitivity in liver. All these NAFLD phenotypes, especially release of pro-inflammatory factors, Kupffer cell accumulation, monocytes infiltration, NLRP3 inflammasome pathway and hepatic fibrosis (Masson's staining and α-SMA staining), deteriorated further under HFD challenge. Oral administration of nicotinamide riboside, a natural NAD(+) precursor, completely corrected these NAFLD phenotypes induced by NAD(+) deficiency alone or HFD, whereas adenovirus-mediated SIRT1 overexpression only partially rescued these phenotypes. These results provide the first evidence that ageing-associated NAD(+) deficiency is a critical risk factor for NAFLD, and suggest that supplementation with NAD(+) substrates may be a promising therapeutic strategy to prevent and treat NAFLD. © 2016 The British Pharmacological Society.

TEMPOL increases NAD(+) and improves redox imbalance in obese mice.

PubMed

Yamato, Mayumi; Kawano, Kimika; Yamanaka, Yuki; Saiga, Misako; Yamada, Ken-Ichi

2016-08-01

Continuous energy conversion is controlled by reduction-oxidation (redox) processes. NAD(+) and NADH represent an important redox couple in energy metabolism. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) is a redox-cycling nitroxide that promotes the scavenging of several reactive oxygen species (ROS) and is reduced to hydroxylamine by NADH. TEMPOL is also involved in NAD(+) production in the ascorbic acid-glutathione redox cycle. We utilized the chemical properties of TEMPOL to investigate the effects of antioxidants and NAD(+)/NADH modulators on the metabolic imbalance in obese mice. Increases in the NAD(+)/NADH ratio by TEMPOL ameliorated the metabolic imbalance when combined with a dietary intervention, changing from a high-fat diet to a normal diet. Plasma levels of the superoxide marker dihydroethidium were higher in mice receiving the dietary intervention compared with a control diet, but were normalized with TEMPOL consumption. These findings provide novel insights into redox regulation in obesity. Copyright © 2016. Published by Elsevier B.V.

Studies of lysine cyclodeaminase from Streptomyces pristinaespiralis: Insights into the complex transition NAD+ state.

PubMed

Ying, Hanxiao; Wang, Jing; Shi, Ting; Zhao, Yilei; Wang, Xin; Ouyang, Pingkai; Chen, Kequan

2018-01-01

Lysine cyclodeaminase (LCD) catalyzes the piperidine ring formation in macrolide-pipecolate natural products metabolic pathways from a lysine substrate through a combination of cyclization and deamination. This enzyme belongs to a unique enzyme class, which uses NAD + as the catalytic prosthetic group instead of as the co-substrate. To understand the molecular details of NAD + functions in lysine cyclodeaminase, we have determined four ternary crystal structure complexes of LCD-NAD + with pipecolic acid (LCD-PA), lysine (LCD-LYS), and an intermediate (LCD-INT) as ligands at 2.26-, 2.00-, 2.17- and 1.80 Å resolutions, respectively. By combining computational studies, a NAD + -mediated "gate keeper" function involving NAD + /NADH and Arg49 that control the binding and entry of the ligand lysine was revealed, confirming the critical roles of NAD + in the substrate access process. Further, in the gate opening form, a substrate delivery tunnel between ε-carboxyl moiety of Glu264 and the α-carboxyl moiety of Asp236 was observed through a comparison of four structure complexes. The LCD structure details including NAD + -mediated "gate keeper" and substrate tunnel may assist in the exploration the NAD + function in this unique enzyme class, and in regulation of macrolide-pipecolate natural product synthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Monocyte-derived extracellular Nampt-dependent biosynthesis of NAD+ protects the heart against pressure overload

PubMed Central

Yano, Masamichi; Akazawa, Hiroshi; Oka, Toru; Yabumoto, Chizuru; Kudo-Sakamoto, Yoko; Kamo, Takehiro; Shimizu, Yu; Yagi, Hiroki; Naito, Atsuhiko T.; Lee, Jong-Kook; Suzuki, Jun-ichi; Sakata, Yasushi; Komuro, Issei

2015-01-01

Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the rate-limiting step in the salvage pathway for nicotinamide adenine dinucleotide (NAD+) biosynthesis, and thereby regulates the deacetylase activity of sirtuins. Here we show accommodative regulation of myocardial NAD+ by monocyte-derived extracellular Nampt (eNampt), which is essential for hemodynamic compensation to pressure overload. Although intracellular Nampt (iNampt) expression was decreased in pressure-overloaded hearts, myocardial NAD+ concentration and Sirt1 activity were preserved. In contrast, iNampt was up-regulated in spleen and monocytes, and circulating eNampt protein and nicotinamide mononucleotide (NMN), a key precursor of NAD+, were significantly increased. Pharmacological inhibition of Nampt by FK866 or depletion of monocytes/macrophages by clodronate liposomes disrupted the homeostatic mechanism of myocardial NAD+ levels and NAD+-dependent Sirt1 activity, leading to susceptibility to cardiomyocyte apoptosis and cardiac decompensation in pressure-overloaded mice. These biochemical and hemodynamic defects were prevented by systemic administration of NMN. Our studies uncover a crucial role of monocyte-derived eNampt in myocardial adaptation to pressure overload, and highlight a potential intervention controlling myocardial NAD+ against heart failure. PMID:26522369

Dunnione ameliorates cisplatin ototoxicity through modulation of NAD(+) metabolism.

PubMed

Kim, Hyung-Jin; Pandit, Arpana; Oh, Gi-Su; Shen, AiHua; Lee, Su-Bin; Khadka, Dipendra; Lee, SeungHoon; Shim, Hyeok; Yang, Sei-Hoon; Cho, Eun-Young; Kwak, Tae Hwan; Choe, Seong-Kyu; Park, Raekil; So, Hong-Seob

2016-03-01

Ototoxicity is an important issue in patients receiving cisplatin chemotherapy. Numerous studies have demonstrated that cisplatin-induced ototoxicity is related to oxidative stress and DNA damage. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. The cofactor nicotinamide adenine dinucleotide (NAD(+)) has emerged as an important regulator of energy metabolism and cellular homeostasis. Here, we demonstrate that the levels and activities of sirtuin-1 (SIRT1) are suppressed by the reduction of intracellular NAD(+) levels in cisplatin-mediated ototoxicity. We provide evidence that the decreases in SIRT1 activity and expression facilitated by increasing poly(ADP-ribose) polymerase-1 (PARP-1) activation and microRNA-34a levels through cisplatin-mediated p53 activation aggravate the associated ototoxicity. Furthermore, we show that the induction of cellular NAD(+) levels using dunnione, which targets intracellular NQO1, prevents the toxic effects of cisplatin through the regulation of PARP-1 and SIRT1 activity. These results suggest that direct modulation of cellular NAD(+) levels by pharmacological agents could be a promising therapeutic approach for protection from cisplatin-induced ototoxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

ARTD1 (PARP1) activation and NAD+ in DNA repair and cell death

PubMed Central

Fouquerel, Elise; Sobol, Robert W.

2014-01-01

Nicotinamide adenine dinucleotide, NAD+, is a small metabolite coenzyme that is essential for the progress of crucial cellular pathways including glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial respiration. These processes consume and produce both oxidative and reduced forms of NAD (NAD+ and NADH). NAD+ is also important for ADP(ribosyl)ation reactions mediated by the ADP-ribosyltransferase enzymes (ARTDs) or deacetylation reactions catalysed by the sirtuins (SIRTs) which use NAD+ as a substrate. In this review, we highlight the significance of NAD+ catabolism in DNA repair and cell death through its utilization by ARTDs and SIRTs. We summarize the current findings on the involvement of ARTD1 activity in DNA repair and most specifically its involvement in the trigger of cell death mediated by energy depletion. By sharing the same substrate, the activities of ARTDs and SIRTs are tightly linked and dependent on each other and are thereby involved in the same cellular processes that play an important role in cancer biology, inflammatory diseases and ischemia/reperfusion. PMID:25283336

Hepatic NAD+ deficiency as a therapeutic target for non‐alcoholic fatty liver disease in ageing

PubMed Central

Zhou, Can‐Can; Yang, Xi; Hua, Xia; Liu, Jian; Fan, Mao‐Bing; Li, Guo‐Qiang; Song, Jie; Xu, Tian‐Ying; Li, Zhi‐Yong; Guan, Yun‐Feng

2016-01-01

Abstract Background and Purpose Ageing is an important risk factor of non‐alcoholic fatty liver disease (NAFLD). Here, we investigated whether the deficiency of nicotinamide adenine dinucleotide (NAD+), a ubiquitous coenzyme, links ageing with NAFLD. Experimental Approach Hepatic concentrations of NAD+, protein levels of nicotinamide phosphoribosyltransferase (NAMPT) and several other critical enzymes regulating NAD+ biosynthesis, were compared in middle‐aged and aged mice or patients. The influences of NAD+ decline on the steatosis and steatohepatitis were evaluated in wild‐type and H247A dominant‐negative, enzymically‐inactive NAMPT transgenic mice (DN‐NAMPT) given normal or high‐fat diet (HFD). Key Results Hepatic NAD+ level decreased in aged mice and humans. NAMPT‐controlled NAD+ salvage, but not de novo biosynthesis pathway, was compromised in liver of elderly mice and humans. Given normal chow, middle‐age DN‐NAMPT mice displayed systemic NAD+ reduction and had moderate NAFLD phenotypes, including lipid accumulation, enhanced oxidative stress, triggered inflammation and impaired insulin sensitivity in liver. All these NAFLD phenotypes, especially release of pro‐inflammatory factors, Kupffer cell accumulation, monocytes infiltration, NLRP3 inflammasome pathway and hepatic fibrosis (Masson's staining and α‐SMA staining), deteriorated further under HFD challenge. Oral administration of nicotinamide riboside, a natural NAD+ precursor, completely corrected these NAFLD phenotypes induced by NAD+ deficiency alone or HFD, whereas adenovirus‐mediated SIRT1 overexpression only partially rescued these phenotypes. Conclusions and Implications These results provide the first evidence that ageing‐associated NAD+ deficiency is a critical risk factor for NAFLD, and suggest that supplementation with NAD+ substrates may be a promising therapeutic strategy to prevent and treat NAFLD. PMID:27174364

Overcoming temozolomide resistance in glioblastoma via dual inhibition of NAD+ biosynthesis and base excision repair

PubMed Central

Goellner, Eva M.; Grimme, Bradford; Brown, Ashley R.; Lin, Ying-Chih; Wang, Xiao-Hong; Sugrue, Kelsey F.; Mitchell, Leah; Trivedi, Ram N.; Tang, Jiang-bo; Sobol, Robert W.

2011-01-01

Glioblastoma multiforme (GBM) is a devastating brain tumor with poor prognosis and low median survival time. Standard treatment includes radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). However, a large percentage of tumors are resistant to the cytotoxic effects of the TMZ-induced DNA lesion O6-methylguanine (O6-MeG) due to elevated expression of the repair protein O6-methylguanine-DNA methyltransferase (MGMT) or a defect in the mismatch repair (MMR) pathway. Although a majority of the TMZ induced lesions (N7-methylguanine and N3-methyladenine) are base excision repair (BER) substrates, these DNA lesions are also readily repaired. However, blocking BER can enhance response to TMZ and therefore the BER pathway has emerged as an attractive target for reversing TMZ resistance. Our lab has recently reported that inhibition of BER leads to the accumulation of repair intermediates that induce energy depletion-mediated cell death via hyperactivation of poly(ADP-ribose) polymerase. Based on our observation that TMZ-induced cell death via BER inhibition is dependent on the availability of NAD+, we have hypothesized that combined BER and NAD+ biosynthesis inhibition will increase TMZ efficacy in glioblastoma cell lines greater than BER inhibition alone. Importantly, we find that the combination of BER and NAD+ biosynthesis inhibition significantly sensitizes glioma cells with elevated expression of MGMT and those deficient in MMR, two genotypes normally associated with TMZ resistance. Dual targeting of these two interacting pathways (DNA repair and NAD+ biosynthesis) may prove to be an effective treatment combination for patients with resistant and recurrent GBM. PMID:21406402

Metabolic Response to NAD Depletion across Cell Lines Is Highly Variable.

PubMed

Xiao, Yang; Kwong, Mandy; Daemen, Anneleen; Belvin, Marcia; Liang, Xiaorong; Hatzivassiliou, Georgia; O'Brien, Thomas

2016-01-01

Nicotinamide adenine dinucleotide (NAD) is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide phosphoribosyltransferase, which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth in vivo. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334), one that shows intermediate sensitivity (NCI-H441), and one that is insensitive (LC-KJ). Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP) and had lower reactive oxygen species (ROS) levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition.

gRINN: a tool for calculation of residue interaction energies and protein energy network analysis of molecular dynamics simulations.

PubMed

Serçinoglu, Onur; Ozbek, Pemra

2018-05-25

Atomistic molecular dynamics (MD) simulations generate a wealth of information related to the dynamics of proteins. If properly analyzed, this information can lead to new insights regarding protein function and assist wet-lab experiments. Aiming to identify interactions between individual amino acid residues and the role played by each in the context of MD simulations, we present a stand-alone software called gRINN (get Residue Interaction eNergies and Networks). gRINN features graphical user interfaces (GUIs) and a command-line interface for generating and analyzing pairwise residue interaction energies and energy correlations from protein MD simulation trajectories. gRINN utilizes the features of NAMD or GROMACS MD simulation packages and automatizes the steps necessary to extract residue-residue interaction energies from user-supplied simulation trajectories, greatly simplifying the analysis for the end-user. A GUI, including an embedded molecular viewer, is provided for visualization of interaction energy time-series, distributions, an interaction energy matrix, interaction energy correlations and a residue correlation matrix. gRINN additionally offers construction and analysis of Protein Energy Networks, providing residue-based metrics such as degrees, betweenness-centralities, closeness centralities as well as shortest path analysis. gRINN is free and open to all users without login requirement at http://grinn.readthedocs.io.

Ultra-fast HPM detectors improve NAD(P)H FLIM

NASA Astrophysics Data System (ADS)

Becker, Wolfgang; Wetzker, Cornelia; Benda, Aleš

2018-02-01

Metabolic imaging by NAD(P)H FLIM requires the decay functions in the individual pixels to be resolved into the decay components of bound and unbound NAD(P)H. Metabolic information is contained in the lifetime and relative amplitudes of the components. The separation of the decay components and the accuracy of the amplitudes and lifetimes improves substantially by using ultra-fast HPM-100-06 and HPM-100-07 hybrid detectors. The IRF width in combination with the Becker & Hickl SPC-150N and SPC-150NX TCSPC modules is less than 20 ps. An IRF this fast does not interfere with the fluorescence decay. The usual deconvolution process in the data analysis then virtually becomes a simple curve fitting, and the parameters of the NAD(P)H decay components are obtained at unprecedented accuracy.

[Influence exogenous nicotinamide adenine dinucleotide (NAD+) on contractile and bioelectric activity of the rat heart].

PubMed

Pustovit, K B; Kuz'min, V S; Sukhova, G S

2014-04-01

This study is aimed to the investigation of the nicotinamide adenine dinucleotide (NAD+) effects and mechanisms of action in a heart. NAD+ (mcM) induces multiphase alternation of contractile activity of isolated rat heart: short positive inotropic action is followed by a negative inotropic phase. NAD+ (1-100 mcM) induces decreasing of action potential duration (APD) in rat atrial myocardium (from 45 +/- 0.82 ms in control experiments to 39 +/- 1.05 (n = 8) and 32 +/- 2 (n = 8) during application of 10 and 100 mcM of NAD+, respectively). Significant APD increase (from 45 +/- 0.82 ms to 74 +/- 1.89 (n = 8) ms) was observed during washing out of NAD+ (100 mcM). ATP or adenosine was unable to increase APD both during application or washing out. NAD+ induced APD decrease was not suppressed by P1-antagonist theophylline. P1-purinoreceptor and metabolite independent direct action of NAD+ in rat heart is suggested. Activation of P2X or P2Y receptors, cyclic ADP-ribose accumulation in cardiomyocytes is proposed as a main mechanism of NAD(+)-induced effects in the heart.

Novel concept of enzyme selective nicotinamide adenine dinucleotide (NAD)-modified inhibitors based on enzyme taxonomy from the diphosphate conformation of NAD.

PubMed

Fujii, Mikio; Kitagawa, Yasuyuki; Iida, Shui; Kato, Keisuke; Ono, Machiko

2015-11-15

The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enzymatic assay for calmodulins based on plant NAD kinase activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required formore » 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.« less

In vitro metabolic engineering for the salvage synthesis of NAD(.).

PubMed

Honda, Kohsuke; Hara, Naoya; Cheng, Maria; Nakamura, Anna; Mandai, Komako; Okano, Kenji; Ohtake, Hisao

2016-05-01

Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)(+) and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD(+) from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat-treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60°C, the NAD(+) concentration could be kept almost constant for 15h. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

NAD+ protects against EAE by regulating CD4+ T-cell differentiation

PubMed Central

Tullius, Stefan G.; Biefer, Hector Rodriguez Cetina; Li, Suyan; Trachtenberg, Alexander J.; Edtinger, Karoline; Quante, Markus; Krenzien, Felix; Uehara, Hirofumi; Yang, Xiaoyong; Kissick, Haydn T.; Kuo, Winston P.; Ghiran, Ionita; de la Fuente, Miguel A.; Arredouani, Mohamed S.; Camacho, Virginia; Tigges, John C.; Toxavidis, Vasilis; El Fatimy, Rachid; Smith, Brian D.; Vasudevan, Anju; ElKhal, Abdallah

2014-01-01

CD4+ T cells are involved in the development of autoimmunity, including multiple sclerosis (MS). Here we show that nicotinamide adenine dinucleotide (NAD+) blocks experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by inducing immune homeostasis through CD4+IFNγ+IL-10+ T cells and reverses disease progression by restoring tissue integrity via remyelination and neuroregeneration. We show that NAD+ regulates CD4+ T-cell differentiation through tryptophan hydroxylase-1 (Tph1), independently of well-established transcription factors. In the presence of NAD+, the frequency of T-bet−/− CD4+IFNγ+ T cells was twofold higher than wild-type CD4+ T cells cultured in conventional T helper 1 polarizing conditions. Our findings unravel a new pathway orchestrating CD4+ T-cell differentiation and demonstrate that NAD+ may serve as a powerful therapeutic agent for the treatment of autoimmune and other diseases. PMID:25290058

Isonicotinamide Enhances Sir2 Protein-mediated Silencing and Longevity in Yeast by Raising Intracellular NAD+ Concentration*

PubMed Central

McClure, Julie M.; Wierman, Margaret B.; Maqani, Nazif; Smith, Jeffrey S.

2012-01-01

Sirtuins are an evolutionarily conserved family of NAD+-dependent protein deacetylases that function in the regulation of gene transcription, cellular metabolism, and aging. Their activity requires the maintenance of an adequate intracellular NAD+ concentration through the combined action of NAD+ biosynthesis and salvage pathways. Nicotinamide (NAM) is a key NAD+ precursor that is also a byproduct and feedback inhibitor of the deacetylation reaction. In Saccharomyces cerevisiae, the nicotinamidase Pnc1 converts NAM to nicotinic acid (NA), which is then used as a substrate by the NAD+ salvage pathway enzyme NA phosphoribosyltransferase (Npt1). Isonicotinamide (INAM) is an isostere of NAM that stimulates yeast Sir2 deacetylase activity in vitro by alleviating the NAM inhibition. In this study, we determined that INAM stimulates Sir2 through an additional mechanism in vivo, which involves elevation of the intracellular NAD+ concentration. INAM enhanced normal silencing at the rDNA locus but only partially suppressed the silencing defects of an npt1Δ mutant. Yeast cells grown in media lacking NA had a short replicative life span, which was extended by INAM in a SIR2-dependent manner and correlated with increased NAD+. The INAM-induced increase in NAD+ was strongly dependent on Pnc1 and Npt1, suggesting that INAM increases flux through the NAD+ salvage pathway. Part of this effect was mediated by the NR salvage pathways, which generate NAM as a product and require Pnc1 to produce NAD+. We also provide evidence suggesting that INAM influences the expression of multiple NAD+ biosynthesis and salvage pathways to promote homeostasis during stationary phase. PMID:22539348

Site and significance of chemically modifiable cysteine residues in glutamate dehydrogenase of Clostridium symbiosum and the use of protection studies to measure coenzyme binding.

PubMed Central

Syed, S E; Hornby, D P; Brown, P E; Fitton, J E; Engel, P C

1994-01-01

Protein chemical studies of NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.2) from Clostridium symbiosum indicate only two cysteine residues/subunit, in good agreement with the gene sequence. Experiments with various thiol-modifying reagents reveal that in native clostridial GDH only one of these two cysteines is accessible for reaction. This residue does not react with iodoacetate, iodoacetamide, N-ethylmaleimide or N-phenylmaleimide, but reaction with either p-chloromercuribenzene sulphonate or 5,5'-dithiobis(2-nitrobenzoic acid) causes complete inactivation, preventable by NAD+ or NADH but not by glutamate or 2-oxoglutarate. Protection studies with combinations of substrates show that glutamate enhances protection by NADH, whereas 2-oxoglutarate diminishes it. These studies were also used to determine a dissociation constant (0.69 mM) for the enzyme-NAD+ complex. Similar data for NADH indicated mildly cooperative binding with a Hill coefficient of 1.32. The significance of these results is discussed in the light of the high-resolution crystallographic structure for clostridial GDH and in relation to information for GDH from other sources. PMID:8129708

Nicotinamide dependence of uropathogenic Escherichia coli UTI89 and application of nadB as a neutral insertion site.

PubMed

Li, Zhaoli; Bouckaert, Julie; Deboeck, Francine; De Greve, Henri; Hernalsteens, Jean-Pierre

2012-03-01

NAD and NADP are ubiquitous in the metabolism of Escherichia coli K-12. NAD auxotrophy can be rendered by mutation in any of the three genes nadB, nadA and nadC. The nadB and nadA genes were defined as antivirulence loci in Shigella spp., as a mutation (mainly in nadB) disrupting the synthesis of quinolinate is required for virulence. Uropathogenic E. coli (UPEC) isolates from acute cystitis patients, exhibiting nicotinamide auxotrophy, were of serotype O18 : K1 : H7. E. coli UTI89, the model uropathogenic and O18 : K1 : H7 strain, requires nicotinamide or quinolinate for growth. A mutation in the nadB gene, encoding L-aspartate oxidase, was shown to be responsible for the nicotinamide requirement of UTI89. This was further confirmed by complementation of UTI89 with a recombinant plasmid harbouring the nadB gene of E. coli K-12. An Ala28Val point mutant of the recombinant plasmid failed to support the growth of UTI89 in minimal medium. This proves that the Ala28Val mutation in the NadB gene of UTI89 completely impedes de novo synthesis of nicotinamide. In spontaneous prototrophic revertants of UTI89, the nadB gene has a Val28Ala mutation. Both analyses implicate that the nicotinamide auxotrophy of UTI89 is caused by a single Ala28Val mutation in NadB. We showed that the same mutation is also present in other NAD auxotrophic E. coli O18 strains. No significant differences were observed between the virulence of isogenic NAD auxotrophic and prototrophic strains in the murine ascending urinary tract infection model. Considering these data, we applied the nadB locus as a neutral site for DNA insertions in the bacterial chromosome. We successfully restored the parental phenotype of a fimH mutant by inserting fimH, with a synthetic em7 promoter, into the nadB gene. This neutral insertion site is of significance for further research on the pathogenicity of UPEC.

Computational Analysis of the Interaction Energies between Amino Acid Residues of the Measles Virus Hemagglutinin and Its Receptors.

PubMed

Xu, Fengqi; Tanaka, Shigenori; Watanabe, Hirofumi; Shimane, Yasuhiro; Iwasawa, Misako; Ohishi, Kazue; Maruyama, Tadashi

2018-05-03

Measles virus (MV) causes an acute and highly devastating contagious disease in humans. Employing the crystal structures of three human receptors, signaling lymphocyte-activation molecule (SLAM), CD46, and Nectin-4, in complex with the measles virus hemagglutinin (MVH), we elucidated computationally the details of binding energies between the amino acid residues of MVH and those of the receptors with an ab initio fragment molecular orbital (FMO) method. The calculated inter-fragment interaction energies (IFIEs) revealed a number of significantly interacting amino acid residues of MVH that played essential roles in binding to the receptors. As predicted from previously reported experiments, some important amino-acid residues of MVH were shown to be common but others were specific to interactions with the three receptors. Particularly, some of the (non-polar) hydrophobic residues of MVH were found to be attractively interacting with multiple receptors, thus indicating the importance of the hydrophobic pocket for intermolecular interactions (especially in the case of Nectin-4). In contrast, the electrostatic interactions tended to be used for specific molecular recognition. Furthermore, we carried out FMO calculations for in silico experiments of amino acid mutations, finding reasonable agreements with virological experiments concerning the substitution effect of residues. Thus, the present study demonstrates that the electron-correlated FMO method is a powerful tool to search exhaustively for amino acid residues that contribute to interactions with receptor molecules. It is also applicable for designing inhibitors of MVH and engineered MVs for cancer therapy.

Intracellular Redox State Revealed by In Vivo 31P MRS Measurement of NAD+ and NADH Contents in Brains

PubMed Central

Lu, Ming; Zhu, Xiao-Hong; Zhang, Yi; Chen, Wei

2015-01-01

Purpose Nicotinamide adenine dinucleotide (NAD), in oxidized (NAD+) or reduced (NADH) form, plays key roles in cellular metabolism. Intracellular NAD+/NADH ratio represents the cellular redox state; however, it is difficult to measure in vivo. We report here a novel in vivo 31P MRS method for noninvasive measurement of intracellular NAD concentrations and NAD+/NADH ratio in the brain. Methods It uses a theoretical model to describe the NAD spectral patterns at a given field for quantification. Standard NAD solutions and independent cat brain measurements at 9.4 T and 16.4 T were used to evaluate this method. We also measured T1 values of brain NAD. Results Model simulation and studies of solutions and brains indicate that the proposed method can quantify submillimolar NAD concentrations with reasonable accuracy if adequate 31P MRS signal-to-noise ratio and linewidth were obtained. The NAD concentrations and NAD+/NADH ratio of cat brains measured at 16.4 T and 9.4 T were consistent despite the significantly different T1 values and NAD spectra patterns at two fields. Conclusion This newly established 31P MRS method makes it possible for the first time to noninvasively study the intracellular redox state and its roles in brain functions and diseases, and it can potentially be applied to other organs. PMID:23843330

NAD+-Carrying Mesoporous Silica Nanoparticles Can Prevent Oxidative Stress-Induced Energy Failures of Both Rodent Astrocytes and PC12 Cells

PubMed Central

Chen, Heyu; Wang, Yao; Zhang, Jixi; Ma, Yingxin; Wang, Caixia; Zhou, Ying; Gu, Hongchen; Ying, Weihai

2013-01-01

Aim To test the hypothesis that NAD+-carrying mesoporous silica nanoparticles (M-MSNs@NAD+) can effectively deliver NAD+ into cells to produce cytoprotective effects. Methods & Materials NAD+ was incorporated into M-MSNs. Primary rat astrocyte cultures and PC12 cells were treated with H2O2, followed by post-treatment with M-MSNs@NAD+. After various durations of the post-treatment, intracellular NAD+ levels, intracellular ATP levels and lactate dehydrogenase (LDH) release were determined. Results & Discussion M-MSNs can be effectively loaded with NAD+. The M-MSNs@NAD+ can significantly attenuate H2O2-induced NAD+ and ATP decreases in both astrocyte cultures and PC12 cells. M-MSNs@NAD+ can also partially prevent the H2O2-induced LDH release from both astrocyte cultures and PC12 cells. In contrast, the NAD+ that is spontaneously released from the M-MSNs@NAD+ is insufficient to prevent the H2O2-induced damage. Conclusions Our study has suggested the first approach that can effectively deliver NAD+ into cells, which provides an important basis both for elucidating the roles of intracellular NAD+ in biological functions and for therapeutic applications of NAD+. Our study has also provided the first direct evidence demonstrating a key role of NAD+ depletion in oxidative stress-induced ATP decreases. PMID:24040179

MacroH2A1.1 regulates mitochondrial respiration by limiting nuclear NAD+ consumption.

PubMed

Posavec Marjanović, Melanija; Hurtado-Bagès, Sarah; Lassi, Maximilian; Valero, Vanesa; Malinverni, Roberto; Delage, Hélène; Navarro, Miriam; Corujo, David; Guberovic, Iva; Douet, Julien; Gama-Perez, Pau; Garcia-Roves, Pablo M; Ahel, Ivan; Ladurner, Andreas G; Yanes, Oscar; Bouvet, Philippe; Suelves, Mònica; Teperino, Raffaele; Pospisilik, J Andrew; Buschbeck, Marcus

2017-11-01

Histone variants are structural components of eukaryotic chromatin that can replace replication-coupled histones in the nucleosome. The histone variant macroH2A1.1 contains a macrodomain capable of binding NAD + -derived metabolites. Here we report that macroH2A1.1 is rapidly induced during myogenic differentiation through a switch in alternative splicing, and that myotubes that lack macroH2A1.1 have a defect in mitochondrial respiratory capacity. We found that the metabolite-binding macrodomain was essential for sustained optimal mitochondrial function but dispensable for gene regulation. Through direct binding, macroH2A1.1 inhibits basal poly-ADP ribose polymerase 1 (PARP-1) activity and thus reduces nuclear NAD + consumption. The resultant accumulation of the NAD + precursor NMN allows for maintenance of mitochondrial NAD + pools that are critical for respiration. Our data indicate that macroH2A1.1-containing chromatin regulates mitochondrial respiration by limiting nuclear NAD + consumption and establishing a buffer of NAD + precursors in differentiated cells.

Tributyltin induces mitochondrial fission through NAD-IDH dependent mitofusin degradation in human embryonic carcinoma cells.

PubMed

Yamada, Shigeru; Kotake, Yaichiro; Nakano, Mizuho; Sekino, Yuko; Kanda, Yasunari

2015-08-01

Organotin compounds, such as tributyltin (TBT), are well-known endocrine disruptors. TBT acts at the nanomolar level through genomic pathways via the peroxisome proliferator activated receptor (PPAR)/retinoid X receptor (RXR). We recently reported that TBT inhibits cell growth and the ATP content in the human embryonic carcinoma cell line NT2/D1 via a non-genomic pathway involving NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which metabolizes isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we evaluated the effects of TBT on mitochondrial NAD-IDH and energy production. Staining with MitoTracker revealed that nanomolar TBT levels induced mitochondrial fragmentation. TBT also degraded the mitochondrial fusion proteins, mitofusins 1 and 2. Interestingly, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. Incubation with an α-ketoglutarate analogue partially recovered TBT-induced mitochondrial dysfunction, supporting the involvement of NAD-IDH. Our data suggest that nanomolar TBT levels impair mitochondrial quality control via NAD-IDH in NT2/D1 cells. Thus, mitochondrial function in embryonic cells could be used to assess cytotoxicity associated with metal exposure.

NAMPT-Mediated NAD Biosynthesis as the Internal Timing Mechanism: In NAD+ World, Time Is Running in Its Own Way.

PubMed

Poljsak, Borut

2017-09-08

The biological age of organisms differs from the chronological age and is determined by internal aging clock(s). How cells estimate time on a scale of 24 hours is relatively well studied; however, how biological time is measured by cells, tissues, organs, or organisms in longer time periods (years and decades) is largely unknown. What is clear and widely agreed upon is that the link to age and age-related diseases is not chronological, as it does not depend on a fixed passage of time. Rather, this link depends on the biological age of an individual cell, tissue, organ, or organism and not on time in a strictly chronological sense. Biological evolution does not invent new methods as often as improving upon already existing ones. It should be easier to evolve and remodel the existing (circadian) time clock mechanism to use it for measurement or regulation of longer time periods than to invent a new time mechanism/clock. Specifically, it will be demonstrated that the circadian clock can also be used to regulate circannual or even longer time periods. Nicotinamide phosphoribosyltransferase (NAMPT)-mediated nicotinamide adenine dinucleotide (NAD+) levels, being regulated by the circadian clock, might be the missing link between aging, cell cycle control, DNA damage repair, cellular metabolism and the aging clock, which is responsible for the biological age of an organism. The hypothesis that NAMPT/NAD+/SIRT1 might represent the time regulator that determines the organismal biological age will be presented. The biological age of tissues and organs might be regulated and synchronized through eNAMPT blood secretion. The "NAD World 2.0" concept will be upgraded with detailed insights into mechanisms that regulate NAD + -mediated aging clock ticking, the duration and amplitude of which are responsible for the aging rate of humans.

CapZyme-Seq Comprehensively Defines Promoter-Sequence Determinants for RNA 5' Capping with NAD.

PubMed

Vvedenskaya, Irina O; Bird, Jeremy G; Zhang, Yuanchao; Zhang, Yu; Jiao, Xinfu; Barvík, Ivan; Krásný, Libor; Kiledjian, Megerditch; Taylor, Deanne M; Ebright, Richard H; Nickels, Bryce E

2018-05-03

Nucleoside-containing metabolites such as NAD + can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD + capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD + capping and define a consensus promoter sequence for NAD + capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD + -capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD + and provide a general method for analysis of NCIN capping in vitro and in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.

The Effects of NAD+ on Apoptotic Neuronal Death and Mitochondrial Biogenesis and Function after Glutamate Excitotoxicity

PubMed Central

Wang, Xiaowan; Li, Hailong; Ding, Shinghua

2014-01-01

NAD+ is an essential co-enzyme for cellular energy metabolism and is also involved as a substrate for many cellular enzymatic reactions. It has been shown that NAD+ has a beneficial effect on neuronal survival and brain injury in in vitro and in vivo ischemic models. However, the effect of NAD+ on mitochondrial biogenesis and function in ischemia has not been well investigated. In the present study, we used an in vitro glutamate excitotoxicity model of primary cultured cortical neurons to study the effect of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function. Our results show that supplementation of NAD+ could effectively reduce apoptotic neuronal death, and apoptotic inducing factor translocation after neurons were challenged with excitotoxic glutamate stimulation. Using different approaches including confocal imaging, mitochondrial DNA measurement and Western blot analysis of PGC-1 and NRF-1, we also found that NAD+ could significantly attenuate glutamate-induced mitochondrial fragmentation and the impairment of mitochondrial biogenesis. Furthermore, NAD+ treatment effectively inhibited mitochondrial membrane potential depolarization and NADH redistribution after excitotoxic glutamate stimulation. Taken together, our results demonstrated that NAD+ is capable of inhibiting apoptotic neuronal death after glutamate excitotoxicity via preserving mitochondrial biogenesis and integrity. Our findings provide insights into potential neuroprotective strategies in ischemic stroke. PMID:25387075

Unifying mechanism for Aplysia ADP-ribosyl cyclase and CD38/NAD(+) glycohydrolases.

PubMed Central

Cakir-Kiefer, C; Muller-Steffner, H; Schuber, F

2000-01-01

Highly purified Aplysia californica ADP-ribosyl cyclase was found to be a multifunctional enzyme. In addition to the known transformation of NAD(+) into cADP-ribose this enzyme is able to catalyse the solvolysis (hydrolysis and methanolysis) of cADP-ribose. This cADP-ribose hydrolase activity, which becomes detectable only at high concentrations of the enzyme, is amplified with analogues such as pyridine adenine dinucleotide, in which the cleavage rate of the pyridinium-ribose bond is much reduced compared with NAD(+). Although the specificity ratio V(max)/K(m) is in favour of NAD(+) by 4 orders of magnitude, this multifunctionality allowed us to propose a 'partitioning' reaction scheme for the Aplysia enzyme, similar to that established previously for mammalian CD38/NAD(+) glycohydrolases. This mechanism involves the formation of a single oxocarbenium-type intermediate that partitions to cADP-ribose and solvolytic products via competing pathways. In favour of this mechanism was the finding that the enzyme also catalysed the hydrolysis of NMN(+), a substrate that cannot undergo cyclization. The major difference between the mammalian and the invertebrate enzymes resides in their relative cyclization/hydrolysis rate-constant ratios, which dictate their respective yields of cADP-ribose (ADP-ribosyl cyclase activity) and ADP-ribose (NAD(+) glycohydrolase activity). For the Aplysia enzyme's catalysed transformation of NAD(+) we favour a mechanism where the formation of cADP-ribose precedes that of ADP-ribose; i.e. macroscopically the invertebrate ADP-ribosyl cyclase conforms to a sequential reaction pathway as a limiting form of the partitioning mechanism. PMID:10861229

Structure of Escherichia coli AdhP (ethanol-inducible dehydrogenase) with bound NAD.

PubMed

Thomas, Leonard M; Harper, Angelica R; Miner, Whitney A; Ajufo, Helen O; Branscum, Katie M; Kao, Lydia; Sims, Paul A

2013-07-01

The crystal structure of AdhP, a recombinantly expressed alcohol dehydrogenase from Escherichia coli K-12 (substrain MG1655), was determined to 2.01 Å resolution. The structure, which was solved using molecular replacement, also included the structural and catalytic zinc ions and the cofactor nicotinamide adenine dinucleotide (NAD). The crystals belonged to space group P21, with unit-cell parameters a = 68.18, b = 118.92, c = 97.87 Å, β = 106.41°. The final R factor and Rfree were 0.138 and 0.184, respectively. The structure of the active site of AdhP suggested a number of residues that may participate in a proton relay, and the overall structure of AdhP, including the coordination to structural and active-site zinc ions, is similar to those of other tetrameric alcohol dehydrogenase enzymes.

Effects of exogenous nicotinamide adenine dinucleotide (NAD+) in the rat heart are mediated by P2 purine receptors.

PubMed

Kuzmin, Vladislav S; Pustovit, Ksenia B; Abramochkin, Denis V

2016-06-27

Recently, NAD+ has been considered as an essential factor, participating in nerve control of physiological functions and intercellular communication. NAD+ also has been supposed as endogenous activator of P1 and P2 purinoreceptors. Effects of extracellular NAD+ remain poorly investigated in cardiac tissue. This study aims to investigate the effects of extracellular NAD+ in different types of supraventricular and ventricular working myocardium from rat and their potential mechanisms. The standard technique of sharp microelectrode action potential recording in cardiac multicellular preparations was used to study the effects of NAD+. Extracellular NAD+ induced significant changes in bioelectrical activity of left auricle (LA), right auricle (RA), pulmonary veins (PV) and right ventricular wall (RV) myocardial preparations. 10-100 μM NAD+ produced two opposite effects in LA and RA - quickly developing and transient prolongation of action potentials (AP) and delayed sustained AP shortening, which follows the initial positive effect. In PV and RV only AP shortening was observed in response to NAD+ application. In PV preparations AP shortening induced by NAD+ may be considered as a potential proarrhythmic effect. Revealed cardiotropic effects of NAD+ are likely to be mediated by P2 purine receptors, since P1 blocker DPCPX failed to affect them and P2 antagonist suramin abolished NAD + -induced alterations of electrical activity. P2X receptors may be responsible for NAD + -induced short-lasting AP prolongation, while P2Y receptors mediate persistent AP shortening. The latter effect is partially removed by PLC inhibitor U73122 showing the potential involvement of phosphoinositide signaling pathway in mediation of NAD+ cardiotropic effects. Extracellular NAD+ is supposed to be a novel regulator of cardiac electrical activity. P2 receptors represent the main target of NAD+ at least in the rat heart.

ERp57 interacts with conserved cysteine residues in the MHC class I peptide-binding groove.

PubMed

Antoniou, Antony N; Santos, Susana G; Campbell, Elaine C; Lynch, Sarah; Arosa, Fernando A; Powis, Simon J

2007-05-15

The oxidoreductase ERp57 is a component of the major histocompatibility complex (MHC) class I peptide-loading complex. ERp57 can interact directly with MHC class I molecules, however, little is known about which of the cysteine residues within the MHC class I molecule are relevant to this interaction. MHC class I molecules possess conserved disulfide bonds between cysteines 101-164, and 203-259 in the peptide-binding and alpha3 domain, respectively. By studying a series of mutants of these conserved residues, we demonstrate that ERp57 predominantly associates with cysteine residues in the peptide-binding domain, thus indicating ERp57 has direct access to the peptide-binding groove of MHC class I molecules during assembly.

Changes in Oxidative Damage, Inflammation and [NAD(H)] with Age in Cerebrospinal Fluid

PubMed Central

Guest, Jade; Grant, Ross; Mori, Trevor A.; Croft, Kevin D.

2014-01-01

An extensive body of evidence indicates that oxidative stress and inflammation play a central role in the degenerative changes of systemic tissues in aging. However a comparatively limited amount of data is available to verify whether these processes also contribute to normal aging within the brain. High levels of oxidative damage results in key cellular changes including a reduction in available nicotinamide adenine dinucleotide (NAD+), an essential molecule required for a number of vital cellular processes including DNA repair, immune signaling and epigenetic processing. In this study we quantified changes in [NAD(H)] and markers of inflammation and oxidative damage (F2-isoprostanes, 8-OHdG, total antioxidant capacity) in the cerebrospinal fluid (CSF) of healthy humans across a wide age range (24–91 years). CSF was collected from consenting patients who required a spinal tap for the administration of anesthetic. CSF of participants aged >45 years was found to contain increased levels of lipid peroxidation (F2-isoprostanes) (p = 0.04) and inflammation (IL-6) (p = 0.00) and decreased levels of both total antioxidant capacity (p = 0.00) and NAD(H) (p = 0.05), compared to their younger counterparts. A positive association was also observed between plasma [NAD(H)] and CSF NAD(H) levels (p = 0.03). Further analysis of the data identified a relationship between alcohol intake and CSF [NAD(H)] and markers of inflammation. The CSF of participants who consumed >1 standard drink of alcohol per day contained lower levels of NAD(H) compared to those who consumed no alcohol (p<0.05). An increase in CSF IL-6 was observed in participants who reported drinking >0–1 (p<0.05) and >1 (p<0.05) standard alcoholic drinks per day compared to those who did not drink alcohol. Taken together these data suggest a progressive age associated increase in oxidative damage, inflammation and reduced [NAD(H)] in the brain which may be exacerbated by alcohol intake. PMID

Pharmacological Inhibitors of NAD Biosynthesis as Potential An ticancer Agents.

PubMed

Lucas, Stephanie; Soave, Claire; Nabil, Ghazal; Ahmed, Zainab Sabry Othman; Chen, Guohua; El-Banna, Hossny Awad; Dou, Q Ping; Wang, Jian

2017-01-01

Alteration of cellular metabolism is a hallmark of cancer, which underlies exciting opportunities to develop effective, anti-cancer therapeutics through inhibition of cancer metabolism. Nicotinamide Adenine Dinucleotide (NAD+), an essential coenzyme of energy metabolism and a signaling molecule linking cellular energy status to a spectrum of molecular regulation, has been shown to be in high demand in a variety of cancer cells. Depletion of NAD+ by inhibition of its key biosynthetic enzymes has become an attractive strategy to target cancer. The main objective of this article is to review the recent patents which develop and implicate the chemical inhibitors of the key NAD+ biosynthetic enzymes for cancer treatment. We first discuss the biological principles of NAD+ metabolism in normal and malignant cells, with a focus on the feasibility of selectively targeting cancer cells by pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT) and indoleamine/tryptophan 2,3-dioxygenases (IDO/TDO), the rate-limiting salvage and de novo NAD+ biosynthetic enzymes, respectively. We then analyze a series of recent patents on development and optimization of chemical scaffolds for inhibiting NAMPT or IDO/TDO enzymes as potential anticancer drugs. Conclusion and Results: We have reviewed 16 relevant patents published since 2015, and summarized the chemical properties, mechanisms of action and proposed applications of the patented compounds. Without a better understanding of the properties of these compounds, their utility for further optimization and clinical use is unknown. For the compounds that have been tested using cell and mouse models of cancer, results look promising and clinical trials are currently ongoing to see if these results translate to improved cancer treatments. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The NAD(+) precursor nicotinamide riboside decreases exercise performance in rats.

PubMed

Kourtzidis, Ioannis A; Stoupas, Andreas T; Gioris, Ioannis S; Veskoukis, Aristidis S; Margaritelis, Nikos V; Tsantarliotou, Maria; Taitzoglou, Ioannis; Vrabas, Ioannis S; Paschalis, Vassilis; Kyparos, Antonios; Nikolaidis, Michalis G

2016-01-01

Nicotinamide adenine dinucleotide (NAD(+)) and its phosphorylated form (NADP(+)) are key molecules in ubiquitous bioenergetic and cellular signaling pathways, regulating cellular metabolism and homeostasis. Thus, supplementation with NAD(+) and NADP(+) precursors emerged as a promising strategy to gain many and multifaceted health benefits. In this proof-of-concept study, we sought to investigate whether chronic nicotinamide riboside administration (an NAD(+) precursor) affects exercise performance. Eighteen Wistar rats were equally divided in two groups that received either saline vehicle or nicotinamide riboside at a dose of 300 mg/kg body weight/day for 21 days via gavage. At the end of the 21-day administration protocol, both groups performed an incremental swimming performance test. The nicotinamide riboside group showed a tendency towards worse physical performance by 35 % compared to the control group at the final 10 % load (94 ± 53 s for the nicotinamide riboside group and 145 ± 59 s for the control group; P = 0.071). Our results do not confirm the previously reported ergogenic effect of nicotinamide riboside. The potentially negative effect of nicotinamide riboside administration on physical performance may be attributed to the pleiotropic metabolic and redox properties of NAD(+) and NADP(+).

NAD+ metabolism and the control of energy homeostasis - a balancing act between mitochondria and the nucleus

PubMed Central

Cantó, Carles; Menzies, Keir; Auwerx, Johan

2015-01-01

NAD+ has emerged as a vital cofactor that can rewire metabolism, activate sirtuins and maintain mitochondrial fitness through mechanisms such as the mitochondrial unfolded protein response. This improved understanding of NAD+ metabolism revived interest in NAD+ boosting strategies to manage a wide spectrum of diseases, ranging from diabetes to cancer. In this review, we summarize how NAD+ metabolism links energy status with adaptive cellular and organismal responses and how this knowledge can be therapeutically exploited. PMID:26118927

The RING 2.0 web server for high quality residue interaction networks.

PubMed

Piovesan, Damiano; Minervini, Giovanni; Tosatto, Silvio C E

2016-07-08

Residue interaction networks (RINs) are an alternative way of representing protein structures where nodes are residues and arcs physico-chemical interactions. RINs have been extensively and successfully used for analysing mutation effects, protein folding, domain-domain communication and catalytic activity. Here we present RING 2.0, a new version of the RING software for the identification of covalent and non-covalent bonds in protein structures, including π-π stacking and π-cation interactions. RING 2.0 is extremely fast and generates both intra and inter-chain interactions including solvent and ligand atoms. The generated networks are very accurate and reliable thanks to a complex empirical re-parameterization of distance thresholds performed on the entire Protein Data Bank. By default, RING output is generated with optimal parameters but the web server provides an exhaustive interface to customize the calculation. The network can be visualized directly in the browser or in Cytoscape. Alternatively, the RING-Viz script for Pymol allows visualizing the interactions at atomic level in the structure. The web server and RING-Viz, together with an extensive help and tutorial, are available from URL: http://protein.bio.unipd.it/ring. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Enhancement of succinate yield by manipulating NADH/NAD+ ratio and ATP generation.

PubMed

Li, Jiaojiao; Li, Yikui; Cui, Zhiyong; Liang, Quanfeng; Qi, Qingsheng

2017-04-01

We previously engineered Escherichia coli YL104 to efficiently produce succinate from glucose. In this study, we investigated the relationships between the NADH/NAD + ratio, ATP level, and overall yield of succinate production by using glucose as the carbon source in YL104. First, the use of sole NADH dehydrogenases increased the overall yield of succinate by 7% and substantially decreased the NADH/NAD + ratio. Second, the soluble fumarate reductase from Saccharomyces cerevisiae was overexpressed to manipulate the anaerobic NADH/NAD + ratio and ATP level. Third, another strategy for reducing the ATP level was applied by introducing ATP futile cycling for improving succinate production. Finally, a combination of these methods exerted a synergistic effect on improving the overall yield of succinate, which was 39% higher than that of the previously engineered strain YL104. The study results indicated that regulation of the NADH/NAD + ratio and ATP level is an efficient strategy for succinate production.

Study the effect of Vitamin K on intracellular NAD level in yeast by fluorescence spectrum

NASA Astrophysics Data System (ADS)

Yahong, Chen; Ruxiu, Cai; Ke, Zhang

2007-05-01

The intracellular NAD level plays a pivotal role in numerous biological processes such as rhythm, senescence, cancer and death. The study of the intracellular NAD level has been one of the "hotspots" in biomedical research. We investigated the effect of Vitamin K on intracellular NAD level in yeast by fluorescence spectrum in this paper. Plasma membrane redox system of yeast was found to be greatly promoted by the addition of Vitamin K 3 or Vitamin K 1. Ferricyanide reduction catalyzed by Vitamin K was accompanied by the decrease in intracellular NADH concentration and the increase in intracellular NAD level of yeast cells.

Mitochondrial NAD(P)H In vivo: Identifying Natural Indicators of Oxidative Phosphorylation in the (31)P Magnetic Resonance Spectrum.

PubMed

Conley, Kevin E; Ali, Amir S; Flores, Brandon; Jubrias, Sharon A; Shankland, Eric G

2016-01-01

Natural indicators provide intrinsic probes of metabolism, biogenesis and oxidative protection. Nicotinamide adenine dinucleotide metabolites (NAD(P)) are one class of indicators that have roles as co-factors in oxidative phosphorylation, glycolysis, and anti-oxidant protection, as well as signaling in the mitochondrial biogenesis pathway. These many roles are made possible by the distinct redox states (NAD(P)(+) and NAD(P)H), which are compartmentalized between cytosol and mitochondria. Here we provide evidence for detection of NAD(P)(+) and NAD(P)H in separate mitochondrial and cytosol pools in vivo in human tissue by phosphorus magnetic resonance spectroscopy ((31)P MRS). These NAD(P) pools are identified by chemical standards (NAD(+), NADP(+), and NADH) and by physiological tests. A unique resonance reflecting mitochondrial NAD(P)H is revealed by the changes elicited by elevation of mitochondrial oxidation. The decline of NAD(P)H with oxidation is matched by a stoichiometric rise in the NAD(P)(+) peak. This unique resonance also provides a measure of the improvement in mitochondrial oxidation that parallels the greater phosphorylation found after exercise training in these elderly subjects. The implication is that the dynamics of the mitochondrial NAD(P)H peak provides an intrinsic probe of the reversal of mitochondrial dysfunction in elderly muscle. Thus, non-invasive detection of NAD(P)(+) and NAD(P)H in cytosol vs. mitochondria yields natural indicators of redox compartmentalization and sensitive intrinsic probes of the improvement of mitochondrial function with an intervention in human tissues in vivo. These natural indicators hold the promise of providing mechanistic insight into metabolism and mitochondrial function in vivo in a range of tissues in health, disease and with treatment.

Dunnione ameliorates cisplatin-induced small intestinal damage by modulating NAD{sup +} metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandit, Arpana; Kim, Hyung-Jin; Oh, Gi-Su

2015-11-27

Although cisplatin is a widely used anticancer drug for the treatment of a variety of tumors, its use is critically limited because of adverse effects such as ototoxicity, nephrotoxicity, neuropathy, and gastrointestinal damage. Cisplatin treatment increases oxidative stress biomarkers in the small intestine, which may induce apoptosis of epithelial cells and thereby elicit damage to the small intestine. Nicotinamide adenine dinucleotide (NAD{sup +}) is a cofactor for various enzymes associated with cellular homeostasis. In the present study, we demonstrated that the hyper-activation of poly(ADP-ribose) polymerase-1 (PARP-1) is closely associated with the depletion of NAD{sup +} in the small intestine aftermore » cisplatin treatment, which results in downregulation of sirtuin1 (SIRT1) activity. Furthermore, a decrease in SIRT1 activity was found to play an important role in cisplatin-mediated small intestinal damage through nuclear factor (NF)-κB p65 activation, facilitated by its acetylation increase. However, use of dunnione as a strong substrate for the NADH:quinone oxidoreductase 1 (NQO1) enzyme led to an increase in intracellular NAD{sup +} levels and prevented the cisplatin-induced small intestinal damage correlating with the modulation of PARP-1, SIRT1, and NF-κB. These results suggest that direct modulation of cellular NAD{sup +} levels by pharmacological NQO1 substrates could be a promising therapeutic approach for protecting against cisplatin-induced small intestinal damage. - Highlights: • NAD{sup +} acts as a cofactor for numerous enzymes including Sirtuins and PARP. • Up-regulation of SIRT1 could attenuate the cisplatin-induced intestinal damage. • Modulation of the cellular NAD{sup +} could be a promising therapeutic approach.« less

The NAD+ precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet induced obesity

PubMed Central

Cantó, Carles; Houtkooper, Riekelt H.; Pirinen, Eija; Youn, Dou Y.; Oosterveer, Maaike H.; Cen, Yana; Fernandez-Marcos, Pablo J.; Yamamoto, Hiroyasu; Andreux, Pénélope A.; Cettour-Rose, Philippe; Gademann, Karl; Rinsch, Chris; Schoonjans, Kristina; Sauve, Anthony A.; Auwerx, Johan

2013-01-01

SUMMARY As NAD+ is a rate-limiting co-substrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38 —both NAD+ consumers— increases NAD+ bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD+ precursor with the ability to increase NAD+ levels, Sir2-dependent gene silencing and replicative lifespan in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD+ levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin, NR, could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function. PMID:22682224

Predicting helix–helix interactions from residue contacts in membrane proteins

PubMed Central

Lo, Allan; Chiu, Yi-Yuan; Rødland, Einar Andreas; Lyu, Ping-Chiang; Sung, Ting-Yi; Hsu, Wen-Lian

2009-01-01

Motivation: Helix–helix interactions play a critical role in the structure assembly, stability and function of membrane proteins. On the molecular level, the interactions are mediated by one or more residue contacts. Although previous studies focused on helix-packing patterns and sequence motifs, few of them developed methods specifically for contact prediction. Results: We present a new hierarchical framework for contact prediction, with an application in membrane proteins. The hierarchical scheme consists of two levels: in the first level, contact residues are predicted from the sequence and their pairing relationships are further predicted in the second level. Statistical analyses on contact propensities are combined with other sequence and structural information for training the support vector machine classifiers. Evaluated on 52 protein chains using leave-one-out cross validation (LOOCV) and an independent test set of 14 protein chains, the two-level approach consistently improves the conventional direct approach in prediction accuracy, with 80% reduction of input for prediction. Furthermore, the predicted contacts are then used to infer interactions between pairs of helices. When at least three predicted contacts are required for an inferred interaction, the accuracy, sensitivity and specificity are 56%, 40% and 89%, respectively. Our results demonstrate that a hierarchical framework can be applied to eliminate false positives (FP) while reducing computational complexity in predicting contacts. Together with the estimated contact propensities, this method can be used to gain insights into helix-packing in membrane proteins. Availability: http://bio-cluster.iis.sinica.edu.tw/TMhit/ Contact: tsung@iis.sinica.edu.tw Supplementary information:Supplementary data are available at Bioinformatics online. PMID:19244388

Regulation of the Nampt-mediated NAD salvage pathway and its therapeutic implications in pancreatic cancer.

PubMed

Ju, Huai-Qiang; Zhuang, Zhuo-Nan; Li, Hao; Tian, Tian; Lu, Yun-Xin; Fan, Xiao-Qiang; Zhou, Hai-Jun; Mo, Hai-Yu; Sheng, Hui; Chiao, Paul J; Xu, Rui-Hua

2016-08-28

Nicotinamide adenine dinucleotide (NAD) is a crucial cofactor for the redox reactions in the metabolic pathways of cancer cells that have elevated aerobic glycolysis (Warburg effect). Cancer cells are reported to rely on NAD recycling and inhibition of the NAD salvage pathway causes metabolic collapse and cell death. However, the underlying regulatory mechanisms and clinical implications for the NAD salvage pathway in pancreatic ductal adenocarcinoma (PDAC) remain unclear. This study showed that the expression of Nampt, the rate-limiting enzyme of the NAD salvage pathway, was significantly increased in PDAC cells and PDAC tissues. Additionally, inhibition of Nampt impaired tumor growth in vitro and tumorigenesis in vivo, which was accompanied by a decreased cellular NAD level and glycolytic activity. Mechanistically, the Nampt expression was independent of Kras and p16 status, but it was directly regulated by miR-206, which was inversely correlated with the expression of Nampt in PDAC tissues. Importantly, pharmacological inhibition of Nampt by its inhibitor, FK866, significantly enhanced the antitumor activity of gemcitabine in PDAC cells and in orthotopic xenograft mouse models. In conclusion, the present study revealed a novel regulatory mechanism for Nampt in PDAC and suggested that Nampt inhibition may override gemcitabine resistance by decreasing the NAD level and suppressing glycolytic activity, warranting further clinical investigation for pancreatic cancer treatment. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

CD38 Dictates Age-Related NAD Decline and Mitochondrial Dysfunction through an SIRT3-Dependent Mechanism.

PubMed

Camacho-Pereira, Juliana; Tarragó, Mariana G; Chini, Claudia C S; Nin, Veronica; Escande, Carlos; Warner, Gina M; Puranik, Amrutesh S; Schoon, Renee A; Reid, Joel M; Galina, Antonio; Chini, Eduardo N

2016-06-14

Nicotinamide adenine dinucleotide (NAD) levels decrease during aging and are involved in age-related metabolic decline. To date, the mechanism responsible for the age-related reduction in NAD has not been elucidated. Here we demonstrate that expression and activity of the NADase CD38 increase with aging and that CD38 is required for the age-related NAD decline and mitochondrial dysfunction via a pathway mediated at least in part by regulation of SIRT3 activity. We also identified CD38 as the main enzyme involved in the degradation of the NAD precursor nicotinamide mononucleotide (NMN) in vivo, indicating that CD38 has a key role in the modulation of NAD-replacement therapy for aging and metabolic diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Unconstrained Structural Equation Models of Latent Interactions: Contrasting Residual- and Mean-Centered Approaches

ERIC Educational Resources Information Center

Marsh, Herbert W.; Wen, Zhonglin; Hau, Kit-Tai; Little, Todd D.; Bovaird, James A.; Widaman, Keith F.

2007-01-01

Little, Bovaird and Widaman (2006) proposed an unconstrained approach with residual centering for estimating latent interaction effects as an alternative to the mean-centered approach proposed by Marsh, Wen, and Hau (2004, 2006). Little et al. also differed from Marsh et al. in the number of indicators used to infer the latent interaction factor…

Regulation of NAD+- and NADP+-linked isocitrate dehydrogenase in the obligate methylotrophic bacterium Pseudomonas W6.

PubMed

Hofmann, K H; Babel, W

1980-01-01

Cell-free extracts of the obligate methanol-utilizing bacterium Pseudomonas W6 catalyze the oxydation of isocitrate to alpha-ketoglutarate in the presence of NAD+ and NADP+. After electro-focusing of the crude extract of Pseudomonas W6 actually two distinct bands each of NAD+-linked isocitrate dehydrogenase (NAD+-IDH) and of NADP+-linked isocitrate dehydrogenase (NADP+-IDH) could be observed. The NAD+-IDH was completely separated from the NADP+-IDH by employing DEAE ion exchange chromatography and further purified by affinity chromatography using Cibacron blue F 3G-A. The NAD+-IDH was inhibited by a high energy charge, whereas the NADP+-IDH was found to be independent of energy charge. Consequently the NAD+-IDH showed the control behaviour of an enzyme of an energy-generating sequence which, however, equally fulfils a catabolic and an anabolic function. With respect to the inhibition by reduced pyridine nucleotides and alpha-ketoglutarate differences between NAD+-IDH and NADP+-IDH were also found. Only the NADP+-linked enzyme exhibited a feedback inhibition by its reaction products alpha-ketoglutarate and NADPH. This control behaviour gives evidence for the biosynthetic function of the NADP+-IDH. These results confer an amphibolic character to the sequence from citrate to alpha-ketoglutarate in the incomplete citric-acid cycle of Pseudomonas W6.

MNADK, a novel liver-enriched mitochondrion-localized NAD kinase

PubMed Central

Zhang, Ren

2013-01-01

Summary NADP+ and its reducing equivalent NADPH are essential for counteracting oxidative damage. Mitochondria are the major source of oxidative stress, since the majority of superoxide is generated from the mitochondrial respiratory chain. Because NADP+ cannot pass through the mitochondrial membrane, NADP+ generation within mitochondria is critical. However, only a single human NAD kinase (NADK) has been identified, and it is localized to the cytosol. Therefore, sources of mitochondrial NADP+ and mechanisms for maintaining its redox balance remain largely unknown. Here, we show that the uncharacterized human gene C5ORF33, named MNADK (mouse homologue 1110020G09Rik), encodes a novel mitochondrion-localized NAD kinase. In mice MNADK is mostly expressed in the liver, and also abundant in brown fat, heart, muscle and kidney, all being mitochondrion-rich. Indeed, MNADK is localized to mitochondria in Hep G2 cells, a human liver cell line, as demonstrated by fluorescence imaging. Having a conserved NAD kinase domain, a recombinant MNADK showed NAD kinase activity, confirmed by mass spectrometry analysis. Consistent with a role of NADP+ as a coenzyme in anabolic reactions, such as lipid synthesis, MNADK is nutritionally regulated in mice. Fasting increased MNADK levels in liver and fat, and obesity dramatically reduced its level in fat. MNADK expression was suppressed in human liver tumors. Identification of MNADK immediately suggests a model in which NADK and MNADK are responsible for de novo synthesis of NADP+ in cytosol and mitochondria, respectively, and therefore provides novel insights into understanding the sources and mechanisms of mitochondrial NADP+ and NADH production in human cells. PMID:23616928

NAD+ Deficits in Age-Related Diseases and Cancer.

PubMed

Garrido, Amanda; Djouder, Nabil

2017-08-01

The phenomenon of aging has gained widespread attention in recent times. Although significant advances have been made to better understand aging and its related pathologies including cancer, there is not yet a clear mechanism explaining why diseases and cancer are inherent parts of the aging process. Finding a unifying equation that could bridge aging and its related diseases would allow therapeutic development and solve an immense human health problem to live longer and better. In this review, we discuss NAD + reduction as the central mechanism that may connect aging to its related pathologies and cancer. NAD + boosters would ensure and ameliorate health quality during aging. Copyright © 2017 Elsevier Inc. All rights reserved.

Nicotinamide pre-treatment ameliorates NAD(H) hyperoxidation and improves neuronal function after severe hypoxia

PubMed Central

Shetty, Pavan K; Galeffi, Francesca; Turner, Dennis A.

2014-01-01

Prolonged hypoxia leads to irreversible loss of neuronal function and metabolic impairment of nicotinamide adenine dinucleotide recycling (between NAD+ and NADH) immediately after reoxygenation, resulting in NADH hyperoxidation. We test whether addition of nicotinamide (to enhance NAD+ levels) or PARP-1 inhibition (to prevent consumption of NAD+) can be effective in improving either loss of neuronal function or hyperoxidation following severe hypoxic injury in hippocampal slices. After severe, prolonged hypoxia (maintained for 3 min after spreading depression) there was hyperoxidation of NADH following reoxygenation, an increased soluble NAD+/NADH ratio, loss of neuronal field excitatory post-synaptic potential (fEPSP) and decreased ATP content. Nicotinamide incubation (5 mM) 2 hr prior to hypoxia significantly increased total NAD(H) content, improved neuronal recovery, enhanced ATP content, and prevented NADH hyperoxidation. The nicotinamide-induced increase in total soluble NAD(H) was more significant in the cytosolic compartment than within mitochondria. Prolonged incubation with PJ-34 (>1hr) led to enhanced baseline NADH fluorescence prior to hypoxia, as well as improved neuronal recovery, NADH hyperoxidation and ATP content on recovery from severe hypoxia and reoxygenation. In this acute model of severe neuronal dysfunction prolonged incubation with either nicotinamide or PJ-34 prior to hypoxia improved recovery of neuronal function, enhanced NADH reduction and ATP content, but neither treatment restored function when administered during or after prolonged hypoxia and reoxygenation. PMID:24184921

The role of charged surface residues in the binding ability of small hubs in protein-protein interaction networks

PubMed Central

Patil, Ashwini; Nakamura, Haruki

2007-01-01

Hubs are highly connected proteins in a protein-protein interaction network. Previous work has implicated disordered domains and high surface charge as the properties significant in the ability of hubs to bind multiple proteins. While conformational flexibility of disordered domains plays an important role in the binding ability of large hubs, high surface charge is the dominant property in small hubs. In this study, we further investigate the role of the high surface charge in the binding ability of small hubs in the absence of disordered domains. Using multipole expansion, we find that the charges are highly distributed over the hub surfaces. Residue enrichment studies show that the charged residues in hubs are more prevalent on the exposed surface, with the exception of Arg, which is predominantly found at the interface, as compared to non-hubs. This suggests that the charged residues act primarily from the exposed surface rather than the interface to affect the binding ability of small hubs. They do this through (i) enhanced intra-molecular electrostatic interactions to lower the desolvation penalty, (ii) indirect long – range intermolecular interactions with charged residues on the partner proteins for better complementarity and electrostatic steering, and (iii) increased solubility for enhanced diffusion-controlled rate of binding. Along with Arg, we also find a high prevalence of polar residues Tyr, Gln and His and the hydrophobic residue Met at the interfaces of hubs, all of which have the ability to form multiple types of interactions, indicating that the interfaces of hubs are optimized to participate in multiple interactions. PMID:27857564

The role of charged surface residues in the binding ability of small hubs in protein-protein interaction networks.

PubMed

Patil, Ashwini; Nakamura, Haruki

2007-01-01

Hubs are highly connected proteins in a protein-protein interaction network. Previous work has implicated disordered domains and high surface charge as the properties significant in the ability of hubs to bind multiple proteins. While conformational flexibility of disordered domains plays an important role in the binding ability of large hubs, high surface charge is the dominant property in small hubs. In this study, we further investigate the role of the high surface charge in the binding ability of small hubs in the absence of disordered domains. Using multipole expansion, we find that the charges are highly distributed over the hub surfaces. Residue enrichment studies show that the charged residues in hubs are more prevalent on the exposed surface, with the exception of Arg, which is predominantly found at the interface, as compared to non-hubs. This suggests that the charged residues act primarily from the exposed surface rather than the interface to affect the binding ability of small hubs. They do this through (i) enhanced intra-molecular electrostatic interactions to lower the desolvation penalty, (ii) indirect long - range intermolecular interactions with charged residues on the partner proteins for better complementarity and electrostatic steering, and (iii) increased solubility for enhanced diffusion-controlled rate of binding. Along with Arg, we also find a high prevalence of polar residues Tyr, Gln and His and the hydrophobic residue Met at the interfaces of hubs, all of which have the ability to form multiple types of interactions, indicating that the interfaces of hubs are optimized to participate in multiple interactions.

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD(+) regeneration.

PubMed

Wang, Jizhong; Yang, Chengli; Chen, Xing; Bao, Bingxin; Zhang, Xuan; Li, Dali; Du, Xingfan; Shi, Ruofu; Yang, Junfang; Zhu, Ronghui

2016-08-01

To find an efficient and cheap system for NAD(+) regeneration A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD(+) regeneration was established. The system is attractive in that the O2 consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD(+) was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C CONCLUSION: The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD(+) regeneration.

Interaction between dimer interface residues of native and mutated SOD1 protein: a theoretical study.

PubMed

Keerthana, S P; Kolandaivel, P

2015-04-01

Cu-Zn superoxide dismutase 1 (SOD1) is a highly conserved bimetallic protein enzyme, used for the scavenging the superoxide radicals (O2 (-)) produced due to aerobic metabolism in the mitochondrial respiratory chain. Over 100 mutations have been identified and found to be in the homodimeric structure of SOD1. The enzyme has to be maintained in its dimeric state for the structural stability and enzymatic activity. From our investigation, we found that the mutations apart from the dimer interface residues are found to affect the dimer stability of protein and hence enhancing the aggregation and misfolding tendency of mutated protein. The homodimeric state of SOD1 is found to be held together by the non-covalent interactions. The molecular dynamics simulation has been used to study the hydrogen bond interactions between the dimer interface residues of the monomers in native and mutated forms of SOD1 in apo- and holo-states. The results obtained by this analysis reveal the fact that the loss of hydrogen bond interactions between the monomers of the dimer is responsible for the reduced stability of the apo- and holo-mutant forms of SOD1. The conformers with dimer interface residues in native and mutated protein obtained by the molecular dynamics simulation is subjected to quantum mechanical study using M052X/6-31G(d) level of theory. The charge transfer between N-H···O interactions in the dimer interface residues were studied. The weak interaction between the monomers of the dimer accounts for the reduced dimerization and enhanced deformation energy in the mutated SOD1 protein.

The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity.

PubMed

Cantó, Carles; Houtkooper, Riekelt H; Pirinen, Eija; Youn, Dou Y; Oosterveer, Maaike H; Cen, Yana; Fernandez-Marcos, Pablo J; Yamamoto, Hiroyasu; Andreux, Pénélope A; Cettour-Rose, Philippe; Gademann, Karl; Rinsch, Chris; Schoonjans, Kristina; Sauve, Anthony A; Auwerx, Johan

2012-06-06

As NAD(+) is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38-both NAD(+) consumers-increases NAD(+) bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD(+) precursor with the ability to increase NAD(+) levels, Sir2-dependent gene silencing, and replicative life span in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD(+) levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high-fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin NR could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function. Copyright © 2012 Elsevier Inc. All rights reserved.

An NAD+-dependent transcriptional program governs self-renewal and radiation resistance in glioblastoma.

PubMed

Gujar, Amit D; Le, Son; Mao, Diane D; Dadey, David Y A; Turski, Alice; Sasaki, Yo; Aum, Diane; Luo, Jingqin; Dahiya, Sonika; Yuan, Liya; Rich, Keith M; Milbrandt, Jeffrey; Hallahan, Dennis E; Yano, Hiroko; Tran, David D; Kim, Albert H

2016-12-20

Accumulating evidence suggests cancer cells exhibit a dependency on metabolic pathways regulated by nicotinamide adenine dinucleotide (NAD + ). Nevertheless, how the regulation of this metabolic cofactor interfaces with signal transduction networks remains poorly understood in glioblastoma. Here, we report nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD + synthesis, is highly expressed in glioblastoma tumors and patient-derived glioblastoma stem-like cells (GSCs). High NAMPT expression in tumors correlates with decreased patient survival. Pharmacological and genetic inhibition of NAMPT decreased NAD + levels and GSC self-renewal capacity, and NAMPT knockdown inhibited the in vivo tumorigenicity of GSCs. Regulatory network analysis of RNA sequencing data using GSCs treated with NAMPT inhibitor identified transcription factor E2F2 as the center of a transcriptional hub in the NAD + -dependent network. Accordingly, we demonstrate E2F2 is required for GSC self-renewal. Downstream, E2F2 drives the transcription of members of the inhibitor of differentiation (ID) helix-loop-helix gene family. Finally, we find NAMPT mediates GSC radiation resistance. The identification of a NAMPT-E2F2-ID axis establishes a link between NAD + metabolism and a self-renewal transcriptional program in glioblastoma, with therapeutic implications for this formidable cancer.

Dynamic single-cell NAD(P)H measurement reveals oscillatory metabolism throughout the E. coli cell division cycle.

PubMed

Zhang, Zheng; Milias-Argeitis, Andreas; Heinemann, Matthias

2018-02-01

Recent work has shown that metabolism between individual bacterial cells in an otherwise isogenetic population can be different. To investigate such heterogeneity, experimental methods to zoom into the metabolism of individual cells are required. To this end, the autofluoresence of the redox cofactors NADH and NADPH offers great potential for single-cell dynamic NAD(P)H measurements. However, NAD(P)H excitation requires UV light, which can cause cell damage. In this work, we developed a method for time-lapse NAD(P)H imaging in single E. coli cells. Our method combines a setup with reduced background emission, UV-enhanced microscopy equipment and optimized exposure settings, overall generating acceptable NAD(P)H signals from single cells, with minimal negative effect on cell growth. Through different experiments, in which we perturb E. coli's redox metabolism, we demonstrated that the acquired fluorescence signal indeed corresponds to NAD(P)H. Using this new method, for the first time, we report that intracellular NAD(P)H levels oscillate along the bacterial cell division cycle. The developed method for dynamic measurement of NAD(P)H in single bacterial cells will be an important tool to zoom into metabolism of individual cells.

Mutations that Allow SIR2 Orthologs to Function in a NAD+-Depleted Environment.

PubMed

Ondracek, Caitlin R; Frappier, Vincent; Ringel, Alison E; Wolberger, Cynthia; Guarente, Leonard

2017-03-07

Sirtuin enzymes depend on NAD + to catalyze protein deacetylation. Therefore, the lowering of NAD + during aging leads to decreased sirtuin activity and may speed up aging processes in laboratory animals and humans. In this study, we used a genetic screen to identify two mutations in the catalytic domain of yeast Sir2 that allow the enzyme to function in an NAD + -depleted environment. These mutant enzymes give rise to a significant increase of yeast replicative lifespan and increase deacetylation by the Sir2 ortholog, SIRT1, in mammalian cells. Our data suggest that these mutations increase the stability of the conserved catalytic sirtuin domain, thereby increasing the catalytic efficiency of the mutant enzymes. Our approach to identifying sirtuin mutants that permit function in NAD + -limited environments may inform the design of small molecules that can maintain sirtuin activity in aging organisms. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Kynurenine Pathway Metabolism is Involved in the Maintenance of the Intracellular NAD+ Concentration in Human Primary Astrocytes

PubMed Central

Grant, Ross; Nguyen, Susan; Guillemin, Gilles

2010-01-01

Efficient synthesis of NAD+ is critical to maintaining cell viability in all organs of the body. However, little is known of the pathway(s) by which cells of the central nervous system produce NAD+. The aim of this study was to investigate the relationship, between tryptophan degradation via the kynurenine pathway (KP) and de novo NAD+ synthesis in human astrocytes, a major cell type within the brain. In this study we observed that inhibition of single enzymes of the KP resulted in significant decreases in NAD+ levels in astroglial cells after a 24 hr period. We also observed that astrocytes cultured in media deficient in tryptophan, nicotinic acid and nicotinamide resulted in a 50% decrease in NAD+ levels after 24 hrs. This decrease in NAD+ was partially restored by supplementation of the culture media with either tryptophan or kynurenine, or nicotinic acid or with supply of the salvage pathway precursor nicotinamide. PMID:22084595

RIBEYE(B)-domain binds to lipid components of synaptic vesicles in an NAD(H)-dependent, redox-sensitive manner.

PubMed

Schwarz, Karin; Schmitz, Frank

2017-03-20

Synaptic ribbons are needed for fast and continuous exocytosis in ribbon synapses. RIBEYE is a main protein component of synaptic ribbons and is necessary to build the synaptic ribbon. RIBEYE consists of a unique A-domain and a carboxyterminal B-domain, which binds NAD(H). Within the presynaptic terminal, the synaptic ribbons are in physical contact with large numbers of synaptic vesicle (SV)s. How this physical contact between ribbons and synaptic vesicles is established at a molecular level is not well understood. In the present study, we demonstrate that the RIBEYE(B)-domain can directly interact with lipid components of SVs using two different sedimentation assays with liposomes of defined chemical composition. Similar binding results were obtained with a SV-containing membrane fraction. The binding of liposomes to RIBEYE(B) depends upon the presence of a small amount of lysophospholipids present in the liposomes. Interestingly, binding of liposomes to RIBEYE(B) depends on NAD(H) in a redox-sensitive manner. The binding is enhanced by NADH, the reduced form, and is inhibited by NAD + , the oxidized form. Lipid-mediated attachment of vesicles is probably part of a multi-step process that also involves additional, protein-dependent processes. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Purification of a NAD(P) reductase-like protein from the thermogenic appendix of the Sauromatum guttatum inflorescence.

PubMed

Skubatz, Hanna; Howald, William N

2013-03-01

A NAD(P) reductase-like protein with a molecular mass of 34.146 ± 34 Da was purified to homogeneity from the appendix of the inflorescence of the Sauromatum guttatum. On-line liquid chromatography/electrospray ionization-mass spectrometry was used to isolate and quantify the protein. For the identification of the protein, liquid chromatography/electrospray ionization-tandem mass spectrometry analysis of tryptic digests of the protein was carried out. The acquired mass spectra were used for database searching, which led to the identification of a single tryptic peptide. The 12 amino acid tryptic peptide (FLPSEFGNDVDR) was found to be identical to amino acid residues at the positions 108-120 of isoflavone reductase in the Arabidopsis genome. A BLAST search identified this sequence region as unique and specific to a class of NAD(P)-dependent reductases involved in phenylpropanoid biosynthesis. Edman degradation revealed that the protein was N-terminally blocked. The amount of the protein (termed RL, NAD(P) reductase-like protein) increased 60-fold from D-4 (4 days before inflorescence-opening, designated as D-day) to D-Day, and declined the following day, when heat-production ceased. When salicylic acid, the endogenous trigger of heat-production in the Sauromatum appendix, was applied to premature appendices, a fivefold decrease in the amount of RL was detected in the treated section relative to the non-treated section. About 40 % of RL was found in the cytoplasm. Another 30 % was detected in Percoll-purified mitochondria and the rest, about 30 % was associated with a low speed centrifugation pellet due to nuclei and amyloplast localization. RL was also found in other thermogenic plants and detected in Arabidopsis leaves. The function of RL in thermogenic and non-thermogenic plants requires further investigation.

Nicotinamide mononucleotide inhibits post-ischemic NAD(+) degradation and dramatically ameliorates brain damage following global cerebral ischemia.

PubMed

Park, Ji H; Long, Aaron; Owens, Katrina; Kristian, Tibor

2016-11-01

Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor for multiple cellular metabolic reactions and has a central role in energy production. Brain ischemia depletes NAD(+) pools leading to bioenergetics failure and cell death. Nicotinamide mononucleotide (NMN) is utilized by the NAD(+) salvage pathway enzyme, nicotinamide adenylyltransferase (Nmnat) to generate NAD(+). Therefore, we examined whether NMN could protect against ischemic brain damage. Mice were subjected to transient forebrain ischemia and treated with NMN or vehicle at the start of reperfusion or 30min after the ischemic insult. At 2, 4, and 24h of recovery, the proteins poly-ADP-ribosylation (PAR), hippocampal NAD(+) levels, and expression levels of NAD(+) salvage pathway enzymes were determined. Furthermore, animal's neurologic outcome and hippocampal CA1 neuronal death was assessed after six days of reperfusion. NMN (62.5mg/kg) dramatically ameliorated the hippocampal CA1 injury and significantly improved the neurological outcome. Additionally, the post-ischemic NMN treatment prevented the increase in PAR formation and NAD(+) catabolism. Since the NMN administration did not affect animal's temperature, blood gases or regional cerebral blood flow during recovery, the protective effect was not a result of altered reperfusion conditions. These data suggest that administration of NMN at a proper dosage has a strong protective effect against ischemic brain injury. Published by Elsevier Inc.

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.

PubMed

Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E; Ziegler, Mathias; Nikiforov, Andrey

2015-11-06

NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells*

PubMed Central

Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E.; Ziegler, Mathias; Nikiforov, Andrey

2015-01-01

NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5′-nucleotidases (5′-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5′-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5′-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors. PMID:26385918

33 CFR 110.168 - Hampton Roads, Virginia and adjacent waters (Datum: NAD 83).

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Hampton Roads, Virginia and adjacent waters (Datum: NAD 83). 110.168 Section 110.168 Navigation and Navigable Waters COAST GUARD..., Virginia and adjacent waters (Datum: NAD 83). (a) Anchorage Grounds—(1) Anchorage A [Naval Anchorage]. The...

Phosphoribosyl diphosphate synthetase-independent NAD de novo synthesis in Escherichia coli: a new phenotype of phosphate regulon mutants.

PubMed Central

Hove-Jensen, B

1996-01-01

Phosphoribosyl diphosphate-lacking (delta prs) mutant strains of Escherichia coli require NAD, guanosine, uridine, histidine, and tryptophan for growth. NAD is required by phosphoribosyl diphosphate-lacking mutants because of lack of one of the substrates for the quinolinate phosphoribosyltransferase reaction, an enzyme of the NAD de novo pathway. Several NAD-independent mutants of a host from which prs had been deleted were isolated; all of them were shown to have lesions in the pstSCAB-phoU operon, in which mutations lead to derepression of the Pho regulon. In addition NAD-independent growth was dependent on a functional quinolinate phosphoribosyltransferase. The prs suppressor mutations led to the synthesis of a new phosphoryl compound that may act as a precursor for a new NAD biosynthetic pathway. This compound may be synthesized by the product of an unknown phosphate starvation-inducible gene of the Pho regulon because the ability of pst or phoU mutations to suppress the NAD requirement requires PhoB, the transcriptional activator of the Pho regulon. PMID:8550505

Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

PubMed

Hu, Liyan; Ibrahim, Khalid; Stucki, Martin; Frapolli, Michele; Shahbeck, Noora; Chaudhry, Farrukh A; Görg, Boris; Häussinger, Dieter; Penberthy, W Todd; Ben-Omran, Tawfeg; Häberle, Johannes

2015-11-01

Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

The Emergence of the Nicotinamide Riboside Kinases in the regulation of NAD+ Metabolism.

PubMed

Fletcher, Rachel S; Lavery, Gareth

2018-05-30

The concept of replenishing or elevating nicotinamide adenine dinucleotide (NAD+) availability to combat metabolic disease and ageing (described extensively in recent reviews [1, 2]) is an area of intense research. This has led to a need to define the endogenous regulatory pathways and mechanisms cell and tissues utilise to maximise NAD+ availability such that strategies to intervene in the clinical setting are able to be fully realised. This review discusses the importance of different salvage pathways involved in metabolising the vitamin B3 class of NAD+ precursor molecules, with a particular focus on the recently identified nicotinamide riboside kinase (NRK) pathway at both a tissue-specific and systemic level.

Metabolism Dealing with Thermal Degradation of NAD+ in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

PubMed

Hachisuka, Shin-Ichi; Sato, Takaaki; Atomi, Haruyuki

2017-10-01

NAD + is an important cofactor for enzymatic oxidation reactions in all living organisms, including (hyper)thermophiles. However, NAD + is susceptible to thermal degradation at high temperatures. It can thus be expected that (hyper)thermophiles harbor mechanisms that maintain in vivo NAD + concentrations and possibly remove and/or reuse undesirable degradation products of NAD + Here we confirmed that at 85°C, thermal degradation of NAD + results mostly in the generation of nicotinamide and ADP-ribose, the latter known to display toxicity by spontaneously linking to proteins. The hyperthermophilic archaeon Thermococcus kodakarensis possesses a putative ADP-ribose pyrophosphatase (ADPR-PPase) encoded by the TK2284 gene. ADPR-PPase hydrolyzes ADP-ribose to ribose 5-phosphate (R5P) and AMP. The purified recombinant TK2284 protein exhibited activity toward ADP-ribose as well as ADP-glucose. Kinetic analyses revealed a much higher catalytic efficiency toward ADP-ribose, suggesting that ADP-ribose was the physiological substrate. To gain insight into the physiological function of TK2284, a TK2284 gene disruption strain was constructed and examined. Incubation of NAD + in the cell extract of the mutant strain at 85°C resulted in higher ADP-ribose accumulation and lower AMP production compared with those in experiments with the host strain cell extract. The mutant strain also exhibited lower cell yield and specific growth rates in a synthetic amino acid medium compared with those of the host strain. The results obtained here suggest that the ADPR-PPase in T. kodakarensis is responsible for the cleavage of ADP-ribose to R5P and AMP, providing a means to utilize the otherwise dead-end product of NAD + breakdown. IMPORTANCE Hyperthermophilic microorganisms living under high temperature conditions should have mechanisms that deal with the degradation of thermolabile molecules. NAD + is an important cofactor for enzymatic oxidation reactions and is susceptible to thermal

A Method to Identify Nucleolus-Associated Chromatin Domains (NADs).

PubMed

Carpentier, Marie-Christine; Picart-Picolo, Ariadna; Pontvianne, Frédéric

2018-01-01

The nuclear context needs to be taken into consideration to better understand the mechanisms shaping the epigenome and its organization, and therefore its impact on gene expression. For example, in Arabidopsis, heterochromatin is preferentially localized at the nuclear and the nucleolar periphery. Although chromatin domains associating with the nuclear periphery remain to be identified in plant cells, Nucleolus Associated chromatin Domains (NADs) can be identified thanks to a protocol allowing the isolation of pure nucleoli. We describe here the protocol enabling the identification of NADs in Arabidopsis. Providing the transfer of a nucleolus marker as described here in other crop species, this protocol is broadly applicable.

Determination of NAD + and NADH level in a Single Cell Under H 2O 2 Stress by Capillary Electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xi, Wenjun

2008-01-01

A capillary electrophoresis (CE) method is developed to determine both NAD + and NADH levels in a single cell, based on an enzymatic cycling reaction. The detection limit can reach down to 0.2 amol NAD + and 1 amol NADH on a home-made CE-LIF setup. The method showed good reproducibility and specificity. After an intact cell was injected into the inlet of a capillary and lysed using a Tesla coil, intracellular NAD + and NADH were separated, incubated with the cycling buffer, and quantified by the amount of fluorescent product generated. NADH and NAD + levels of single cells ofmore » three cell lines and primary astrocyte culture were determined using this method. Comparing cellular NAD + and NADH levels with and without exposure to oxidative stress induced by H 2O 2, it was found that H9c2 cells respond to the stress by reducing both cellular NAD + and NADH levels, while astrocytes respond by increasing cellular NADH/NAD + ratio.« less

Gene Transfers Shaped the Evolution of De Novo NAD+ Biosynthesis in Eukaryotes

PubMed Central

Ternes, Chad M.; Schönknecht, Gerald

2014-01-01

NAD+ is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD+ biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD+ biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD+ biosynthesis in eukaryotes was shaped by numerous gene transfers. PMID:25169983

Sulphur Atoms from Methionines Interacting with Aromatic Residues Are Less Prone to Oxidation

PubMed Central

Aledo, Juan C.; Cantón, Francisco R.; Veredas, Francisco J.

2015-01-01

Methionine residues exhibit different degrees of susceptibility to oxidation. Although solvent accessibility is a relevant factor, oxidation at particular sites cannot be unequivocally explained by accessibility alone. To explore other possible structural determinants, we assembled different sets of oxidation-sensitive and oxidation-resistant methionines contained in human proteins. Comparisons of the proteins containing oxidized methionines with all proteins in the human proteome led to the conclusion that the former exhibit a significantly higher mean value of methionine content than the latter. Within a given protein, an examination of the sequence surrounding the non-oxidized methionine revealed a preference for neighbouring tyrosine and tryptophan residues, but not for phenylalanine residues. However, because the interaction between sulphur atoms and aromatic residues has been reported to be important for the stabilization of protein structure, we carried out an analysis of the spatial interatomic distances between methionines and aromatic residues, including phenylalanine. The results of these analyses uncovered a new determinant for methionine oxidation: the S-aromatic motif, which decreases the reactivity of the involved sulphur towards oxidants. PMID:26597773

Elevated microRNA-34a in obesity reduces NAD+ levels and SIRT1 activity by directly targeting NAMPT.

PubMed

Choi, Sung-E; Fu, Ting; Seok, Sunmi; Kim, Dong-Hyun; Yu, Eunkyung; Lee, Kwan-Woo; Kang, Yup; Li, Xiaoling; Kemper, Byron; Kemper, Jongsook Kim

2013-12-01

SIRT1 is an NAD(+)-dependent deacetylase that is implicated in prevention of many age-related diseases including metabolic disorders. As SIRT1 deacetylase activity is dependent on NAD(+) levels and the development of compounds that directly activate SIRT1 has been controversial, indirectly activating SIRT1 through enhancing NAD(+) bioavailability has received increasing attention. NAD(+) levels are reduced in obesity and the aged, but the underlying mechanisms remain unclear. We recently showed that hepatic microRNA-34a (miR-34a), which is elevated in obesity, directly targets and decreases SIRT1 expression. Here, we further show that miR-34a reduces NAD(+) levels and SIRT1 activity by targeting NAMPT, the rate-limiting enzyme for NAD(+) biosynthesis. A functional binding site for miR-34a is present in the 3' UTR of NAMPT mRNA. Hepatic overexpression of miR-34a reduced NAMPT/NAD(+) levels, increased acetylation of the SIRT1 target transcriptional regulators, PGC-1α, SREBP-1c, FXR, and NF-κB, and resulted in obesity-mimetic outcomes. The decreased NAMPT/NAD(+) levels were independent of miR-34a effects on SIRT1 levels as they were also observed in SIRT1 liver-specific knockout mice. Further, the miR-34a-mediated decreases were reversed by treatment with the NAD(+) intermediate, nicotinamide mononucleotide. Conversely, antagonism of miR-34a in diet-induced obese mice restored NAMPT/NAD(+) levels and alleviated steatosis, inflammation, and glucose intolerance. Anti-miR-34a-mediated increases in NAD(+) levels were attenuated when NAMPT was downregulated. Our findings reveal a novel function of miR-34a in reducing both SIRT1 expression and activity in obesity. The miR-34a/NAMPT axis presents a potential target for treating obesity- and aging-related diseases involving SIRT1 dysfunction like steatosis and type 2 diabetes. © 2013 the Anatomical Society and John Wiley & Sons Ltd.

Identification of a Novel Pathway of Transforming Growth Factor-β1 Regulation by Extracellular NAD+ in Mouse Macrophages

PubMed Central

Zamora, Ruben; Azhar, Nabil; Namas, Rajaie; Metukuri, Mallikarjuna R.; Clermont, Thierry; Gladstone, Chase; Namas, Rami A.; Hermus, Linda; Megas, Cristina; Constantine, Gregory; Billiar, Timothy R.; Fink, Mitchell P.; Vodovotz, Yoram

2012-01-01

Extracellular β-nicotinamide adenine dinucleotide (NAD+) is anti-inflammatory. We hypothesized that NAD+ would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-β1. Indeed, NAD+ led to increases in both active and latent cell-associated TGF-β1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD+ acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca2+. Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca2+ ionophores recapitulated the effects of NAD+ on TGF-β1, whereas the cADPR antagonist 8-Br-cADPR, Ca2+ chelation, and antagonism of L-type Ca2+ channels suppressed these effects. The time and dose effects of NAD+ on TGF-β1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-β1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-β1 by NAD+. Thus, in vitro and in silico evidence points to NAD+ as a novel modulator of TGF-β1. PMID:22829588

Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD+ Analogue.

PubMed

Rovira, Alexander R; Fin, Andrea; Tor, Yitzhak

2017-11-08

The synthesis, photophysics, and biochemical utility of a fluorescent NAD + analogue based on an isothiazolo[4,3-d]pyrimidine core (N tz AD + ) are described. Enzymatic reactions, photophysically monitored in real time, show N tz AD + and N tz ADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as N tz AD + is converted to N tz ADH, reflecting a complementary photophysical behavior to that of the native NAD + /NADH. N tz AD + and N tz ADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding tz ADP-ribose. N tz AD + also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer tz ADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD + .

NAD+-Dependent Activation of Sirt1 Corrects the Phenotype in a Mouse Model of Mitochondrial Disease

PubMed Central

Cerutti, Raffaele; Pirinen, Eija; Lamperti, Costanza; Marchet, Silvia; Sauve, Anthony A.; Li, Wei; Leoni, Valerio; Schon, Eric A.; Dantzer, Françoise; Auwerx, Johan; Viscomi, Carlo; Zeviani, Massimo

2014-01-01

Summary Mitochondrial disorders are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain. Pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling, often fatal, disorders. Pathways related to mitochondrial biogenesis are targets of Sirtuin1, a NAD+-dependent protein deacetylase. As NAD+ boosts the activity of Sirtuin1 and other sirtuins, intracellular levels of NAD+ play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell. We show here that supplementation with nicotinamide riboside, a natural NAD+ precursor, or reduction of NAD+ consumption by inhibiting the poly(ADP-ribose) polymerases, leads to marked improvement of the respiratory chain defect and exercise intolerance of the Sco2 knockout/knockin mouse, a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis. This strategy is potentially translatable into therapy of mitochondrial disorders in humans. PMID:24814483

Extracellular NAD+ Suppresses Adrenergic Effects in the Atrial Myocardium of Rats during the Early Postnatal Ontogeny.

PubMed

Pustovit, K B; Ivanova, A D; Kuz'min, V S

2018-05-01

The effects of sympathetic cotransmitter NAD+ (10 μM) on bioelectric activity of the heart under conditions of adrenergic stimulation were studied on isolated spontaneously contracting preparations (without stimulation) of the right atrium from 2-7-day-old rats. Action potentials were recorded in the working myocardium using standard microelectrode technique. Perfusion of the right atrium with norepinephrine solution (1 μM) altered the configuration and significantly lengthened the action potentials. NAD + against the background of norepinephrine stimulation significantly decreased the duration of action potentials, in particular, at 25% repolarization. The effect of purine compounds NAD + , ATP, and adenosine on bioelectrical activity of the heart of newborn rats was studied under basal conditions (without norepinephrine stimulation). The effect of NAD + against the background of adrenergic stimulation was more pronounced than under basal conditions and was probably determined by suppression of I CaL , which can be the main mechanism of NAD + action on rat heart.

Pharmacological NAD-Boosting Strategies Improve Mitochondrial Homeostasis in Human Complex I-Mutant Fibroblasts.

PubMed

Felici, Roberta; Lapucci, Andrea; Cavone, Leonardo; Pratesi, Sara; Berlinguer-Palmini, Rolando; Chiarugi, Alberto

2015-06-01

Mitochondrial disorders are devastating genetic diseases for which efficacious therapies are still an unmet need. Recent studies report that increased availability of intracellular NAD obtained by inhibition of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 or supplementation with the NAD-precursor nicotinamide riboside (NR) ameliorates energetic derangement and symptoms in mouse models of mitochondrial disorders. Whether these pharmacological approaches also improve bioenergetics of human cells harboring mitochondrial defects is unknown. It is also unclear whether the same signaling cascade is prompted by PARP-1 inhibitors and NR supplementation to improve mitochondrial homeostasis. Here, we show that human fibroblasts mutant for the NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) subunit of respiratory complex I have similar ATP, NAD, and mitochondrial content compared with control cells, but show reduced mitochondrial membrane potential. Interestingly, mutant cells also show increased transcript levels of mitochondrial DNA but not nuclear DNA respiratory complex subunits, suggesting activation of a compensatory response. At variance with prior work in mice, however, NR supplementation, but not PARP-1 inhibition, increased intracellular NAD content in NDUFS1 mutant human fibroblasts. Conversely, PARP-1 inhibitors, but not NR supplementation, increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes constitutively induced in mutant cells. Still, both NR and PARP-1 inhibitors restored mitochondrial membrane potential and increased organelle content as well as oxidative activity of NDUFS1-deficient fibroblasts. Overall, data provide the first evidence that in human cells harboring a mitochondrial respiratory defect exposure to NR or PARP-1, inhibitors activate different signaling pathways that are not invariantly prompted by NAD increases, but equally able to improve energetic

Imaging the NADH:NAD+ Homeostasis for Understanding the Metabolic Response of Mycobacterium to Physiologically Relevant Stresses.

PubMed

Bhat, Shabir A; Iqbal, Iram K; Kumar, Ashwani

2016-01-01

The NADH:NAD + ratio is the primary indicator of the metabolic state of bacteria. NAD(H) homeostasis is critical for Mycobacterium tuberculosis (Mtb) survival and is thus considered an important drug target, but the spatio-temporal measurements of NAD(H) remain a challenge. Genetically encoded fluorescent biosensors of the NADH:NAD + ratios were recently described, paving the way for investigations of the metabolic state of pathogens during infection. Here we have adapted the genetically encoded biosensor Peredox for measurement of the metabolic state of Mtb in vitro and during infection of macrophage cells. Using Peredox, here we show that inhibition of the electron transport chain, disruption of the membrane potential and proton gradient, exposure to reactive oxygen species and treatment with antimycobacterial drugs led to the accumulation of NADH in mycobacterial cells. We have further demonstrated that Mtb residing in macrophages displays higher NADH:NAD + ratios, that may indicate a metabolic stress faced by the intracellular Mtb. We also demonstrate that the Mtb residing in macrophages display a metabolic heterogeneity, which may perhaps explain the tolerance displayed by intracellular Mtb. Next we studied the effect of immunological modulation by interferon gamma on metabolism of intracellular Mtb, since macrophage activation is known to restrict mycobacterial growth. We observed that activation of resting macrophages with interferon-gamma results in higher NADH:NAD + levels in resident Mtb cells. We have further demonstrated that exposure of Isoniazid, Bedaquiline, Rifampicin, and O-floxacin results in higher NADH:NAD + ratios in the Mtb residing in macrophages. However, intracellular Mtb displays lower NADH:NAD + ratio upon exposure to clofazimine. In summary, we have generated reporter strains capable of measuring the metabolic state of Mtb cells in vitro and in vivo with spatio-temporal resolution. We believe that this tool will facilitate further

Embryo yolk sac membrane kynurenine formamidase of l-tryptophan to NAD+ pathway as a primary target for organophosphorus insecticides (OPI) in OPI-induced NAD-associated avian teratogenesis.

PubMed

Seifert, Josef

2017-10-01

The objective of this study was to provide in ovo evidence for the proposed role of kynurenine formamidase of l-tryptophan to NAD + pathway in embryo yolk sac membranes as a primary target for organophosphorus insecticide (OPI) teratogens in OPI-induced NAD-associated avian teratogenesis. Slices prepared from yolk sac membranes or embryo livers of chicken eggs treated with the OPI dicrotophos and/or methyl parathion were incubated with l-tryptophan. Yolk sac membrane slices metabolized l-tryptophan in the pathway to NAD + before that function was established in livers. OPI interfered in ovo with the second step of l-tryptophan to NAD + biosynthesis by inhibiting kynurenine formamidase. Its inhibition due to the teratogen dicrotophos occurred in yolk sac membranes during the period of embryo highest susceptibility to OPI teratogens in contrast to delayed and lower inhibition caused by the nonteratogen methyl parathion. Both OPI affected liver kynurenine formamidase in a similar manner. The onsets of liver enzyme inhibition, however, were delayed by about two days and occurred at the time of the reduced embryo susceptibility to teratogens. The early disruption of l-tryptophan metabolism and higher inhibition of kynurenine formamidase in yolk sac membranes may be the factors that determine action of OPI as teratogens in chicken embryos. Copyright © 2017 Elsevier Ltd. All rights reserved.

Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

PubMed

Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

2018-01-01

Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

The NAD+ precursor nicotinic acid improves genomic integrity in human peripheral blood mononuclear cells after X-irradiation.

PubMed

Weidele, Kathrin; Beneke, Sascha; Bürkle, Alexander

2017-04-01

NAD + is an essential cofactor for enzymes catalyzing redox-reactions as well as an electron carrier in energy metabolism. Aside from this, NAD + consuming enzymes like poly(ADP-ribose) polymerases and sirtuins are important regulators involved in chromatin-restructuring processes during repair and epigenetics/transcriptional adaption. In order to replenish cellular NAD + levels after cleavage, synthesis starts from precursors such as nicotinamide, nicotinamide riboside or nicotinic acid to match the need for this essential molecule. In the present study, we investigated the impact of supplementation with nicotinic acid on resting and proliferating human mononuclear blood cells with a focus on DNA damage and repair processes. We observed that nicotinic acid supplementation increased NAD + levels as well as DNA repair efficiency and enhanced genomic stability evaluated by micronucleus test after x-ray treatment. Interestingly, resting cells displayed lower basal levels of DNA breaks compared to proliferating cells, but break-induction rates were identical. Despite similar levels of p53 protein upregulation after irradiation, higher NAD + concentrations led to reduced acetylation of this protein, suggesting enhanced SIRT1 activity. Our data reveal that even in normal primary human cells cellular NAD + levels may be limiting under conditions of genotoxic stress and that boosting the NAD + system with nicotinic acid can improve genomic stability. Copyright © 2017 Elsevier B.V. All rights reserved.

Theophylline prevents NAD{sup +} depletion via PARP-1 inhibition in human pulmonary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moonen, Harald J.J.; Geraets, Liesbeth; Vaarhorst, Anika

2005-12-30

Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD{sup +}, resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD{sup +} pool, and of NAD{sup +}-dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD{sup +} levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzymemore » inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies.« less

NAD deamidation "a new reaction" by an enzyme from Aspergillus terreus DSM 826.

PubMed

Elzainy, Tahany A; Ali, Thanaa H

2005-02-01

NAD deamidation is a non-previously recognized reaction. This reaction has been found to be catalyzed by extracts of Aspergillus terreus DSM 826. Conversion of NAD to the biosynthetic intermediate, deamido NAD, by these extracts, at the optimum pH and temperature did not exceed about 55 of the amount of the substrate added. Completion of the reaction was achieved when the extracts were pre-heated at 50 degrees C for 15 min in absence of the substrate. In a very similar manner, the extracts catalyzed hydrolytic cleavage of the amide linkages of different biomolecules such as nicotinamide, nicotinamide riboside, nicotinamide mononucleotide, L-glutamine, L-asparagine and acetamide. Polyacrylamide was also deamidated under the same conditions. In addition, complete dephosphorylation of the dinucleotide molecule was also effected by the same extracts. Separation of the NAD deamidating enzyme from the NAD dephosphorylating enzyme was achieved on using either DEAE - Sephadex A-25 or Sephadex G-200 column chromatography. The obtained phosphohydrolase-free-deamidase showed optimum activity at pH 8 of 0.1 M phosphate buffer and 50 degrees C. It exhibited broad substrate specificity and hyperbolic substrate saturation kinetics. It was isosterically inhibited by the product of its activity and this inhibition was prevented by heating the extracts at 50 degrees C for 15 min. Its activity was not affected in presence of sodium fluoride, partially inhibited in presence of magnesium chloride and was retained in the freezer for some months.

Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1)*

PubMed Central

Harlan, Benjamin A.; Pehar, Mariana; Sharma, Deep R.; Beeson, Gyda; Beeson, Craig C.; Vargas, Marcelo R.

2016-01-01

Nicotinamide adenine dinucleotide (NAD+) participates in redox reactions and NAD+-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD+-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD+ as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1–7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD+ salvage pathway capable of resynthesizing NAD+ from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD+ levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD+ salvage pathway in astrocytes. Supplementation with the NAD+ precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD+ levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1–2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD+ content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD+ salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS. PMID:27002158

Organomineral interactions as an important mechanism for stabilisation of bacterial residues in soil

NASA Astrophysics Data System (ADS)

Miltner, Anja; Achtenhagen, Jan; Kästner, Matthias

2017-04-01

Although plant material is the original input of organic matter to soils, microbial residues have been identified to contribute to a large extent to soil organic matter. However, until now it is unclear how microbial residues are stabilised in soil and protected from degradation. We hypothesised that organomineral interactions, in particular encrustation by oxides, may play an important role, which might vary depending on environmental conditions, e.g. redox potential. Therefore we produced 14C-labelled Escherichia coli cells and cell envelope fragments and coprecipitated these materials with Fe oxide or Al oxide. Mineral-free (control) and mineral-encrusted bacterial residues were incubated for 345 days at 20˚ C under either oxic or oxygen-limited conditions, and mineralisation was quantified by scintillation counting of the CO2 produced during incubation. Oxygen limitation was achieved by first exchanging the atmosphere in the incubation vessels with dinitrogen gas. After 100 days of incubation, the anoxic treatments were waterlogged to further decrease the redox potential, and after 290 days, glucose and nutrients were supplied to all treatments in order to foster microbial activity and consumption of electron acceptors. The mineralisation curves were fitted by double-exponential (0-100 days), first-order kinetic (100-290 days) and linear (290-345 days) models. The model parameters were tested for significant differences between the treatments by three-way ANOVA with post-hoc Bonferroni t-test. We found that encrustation by the oxides significantly reduced mineralisation of the bacterial residues. This effect was inversed by reductive dissolution of Fe oxides after substrate and nutrient addition to the oxygen-limited treatments, suggesting a significant role of the encrustation in stabilisation of the bacterial residues. We also observed that bacterial cell envelope fragments were generally slightly more resistant to mineralisation than whole cells. The

Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody.

PubMed

Malito, Enrico; Biancucci, Marco; Faleri, Agnese; Ferlenghi, Ilaria; Scarselli, Maria; Maruggi, Giulietta; Lo Surdo, Paola; Veggi, Daniele; Liguori, Alessia; Santini, Laura; Bertoldi, Isabella; Petracca, Roberto; Marchi, Sara; Romagnoli, Giacomo; Cartocci, Elena; Vercellino, Irene; Savino, Silvana; Spraggon, Glen; Norais, Nathalie; Pizza, Mariagrazia; Rappuoli, Rino; Masignani, Vega; Bottomley, Matthew James

2014-12-02

Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5-15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.

33 CFR 110.235 - Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83).

Code of Federal Regulations, 2011 CFR

2011-07-01

... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). 110.235 Section 110.235 Navigation and Navigable Waters COAST... Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). (a) The anchorage grounds—(1...

33 CFR 110.235 - Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83).

Code of Federal Regulations, 2014 CFR

2014-07-01

... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). 110.235 Section 110.235 Navigation and Navigable Waters COAST... Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). (a) The anchorage grounds—(1...

33 CFR 110.235 - Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83).

Code of Federal Regulations, 2012 CFR

2012-07-01

... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). 110.235 Section 110.235 Navigation and Navigable Waters COAST... Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). (a) The anchorage grounds—(1...

33 CFR 110.235 - Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83).

Code of Federal Regulations, 2013 CFR

2013-07-01

... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). 110.235 Section 110.235 Navigation and Navigable Waters COAST... Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). (a) The anchorage grounds—(1...

33 CFR 110.235 - Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83).

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). 110.235 Section 110.235 Navigation and Navigable Waters COAST... Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). (a) The anchorage grounds—(1...

Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD[superscript +] synthetase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuenchor, Watchalee; Doukov, Tzanko I.; Resto, Melissa

Glutamine-dependent NAD{sup +} synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD{sup +} from NaAD{sup +} (nicotinic acid-adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 {angstrom} (1 {angstrom} = 0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase-ligandmore » complexes with substrates (NaAD{sup +}/ATP), substrate analogue {l_brace}NaAD{sup +}/AMP-CPP (adenosine 5'-[{alpha},{beta}-methylene]triphosphate){r_brace} and intermediate analogues (NaAD{sup +}/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD{sup +}/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.« less

Protein kinase C epsilon regulates mitochondrial pools of Nampt and NAD following resveratrol and ischemic preconditioning in the rat cortex

PubMed Central

Morris-Blanco, Kahlilia C; Cohan, Charles H; Neumann, Jake T; Sick, Thomas J; Perez-Pinzon, Miguel A

2014-01-01

Preserving mitochondrial pools of nicotinamide adenine dinucleotide (NAD) or nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in NAD production, maintains mitochondrial function and confers neuroprotection after ischemic stress. However, the mechanisms involved in regulating mitochondrial-localized Nampt or NAD have not been defined. In this study, we investigated the roles of protein kinase C epsilon (PKCɛ) and AMP-activated protein kinase (AMPK) in regulating mitochondrial pools of Nampt and NAD after resveratrol or ischemic preconditioning (IPC) in the cortex and in primary neuronal-glial cortical cultures. Using the specific PKCɛ agonist ψɛRACK, we found that PKCɛ induced robust activation of AMPK in vitro and in vivo and that AMPK was required for PKCɛ-mediated ischemic neuroprotection. In purified mitochondrial fractions, PKCɛ enhanced Nampt levels in an AMPK-dependent manner and was required for increased mitochondrial Nampt after IPC or resveratrol treatment. Analysis of intrinsic NAD autofluorescence using two-photon microscopy revealed that PKCɛ modulated NAD in the mitochondrial fraction. Further assessments of mitochondrial NAD concentrations showed that PKCɛ has a key role in regulating the mitochondrial NAD+/nicotinamide adenine dinucleotide reduced (NADH) ratio after IPC and resveratrol treatment in an AMPK- and Nampt-dependent manner. These findings indicate that PKCɛ is critical to increase or maintain mitochondrial Nampt and NAD after pathways of ischemic neuroprotection in the brain. PMID:24667915

Comparative Metabolomic Profiling Reveals That Dysregulated Glycolysis Stemming from Lack of Salvage NAD+ Biosynthesis Impairs Reproductive Development in Caenorhabditis elegans*

PubMed Central

Wang, Wenqing; McReynolds, Melanie R.; Goncalves, Jimmy F.; Shu, Muya; Dhondt, Ineke; Braeckman, Bart P.; Lange, Stephanie E.; Kho, Kelvin; Detwiler, Ariana C.; Pacella, Marisa J.; Hanna-Rose, Wendy

2015-01-01

Temporal developmental progression is highly coordinated in Caenorhabditis elegans. However, loss of nicotinamidase PNC-1 activity slows reproductive development, uncoupling it from its typical progression relative to the soma. Using LC/MS we demonstrate that pnc-1 mutants do not salvage the nicotinamide released by NAD+ consumers to resynthesize NAD+, resulting in a reduction in global NAD+ bioavailability. We manipulate NAD+ levels to demonstrate that a minor deficit in NAD+ availability is incompatible with a normal pace of gonad development. The NAD+ deficit compromises NAD+ consumer activity, but we surprisingly found no functional link between consumer activity and reproductive development. As a result we turned to a comparative metabolomics approach to identify the cause of the developmental phenotype. We reveal widespread metabolic perturbations, and using complementary pharmacological and genetic approaches, we demonstrate that a glycolytic block accounts for the slow pace of reproductive development. Interestingly, mitochondria are protected from both the deficiency in NAD+ biosynthesis and the effects of reduced glycolytic output. We suggest that compensatory metabolic processes that maintain mitochondrial activity in the absence of efficient glycolysis are incompatible with the requirements for reproductive development, which requires high levels of cell division. In addition to demonstrating metabolic requirements for reproductive development, this work also has implications for understanding the mechanisms behind therapeutic interventions that target NAD+ salvage biosynthesis for the purposes of inhibiting tumor growth. PMID:26350462

Oleate ameliorates palmitate-induced reduction of NAMPT activity and NAD levels in primary human hepatocytes and hepatocarcinoma cells.

PubMed

Penke, Melanie; Schuster, Susanne; Gorski, Theresa; Gebhardt, Rolf; Kiess, Wieland; Garten, Antje

2017-10-03

Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide adenine dinucleotide (NAD) levels are crucial for liver function. The saturated fatty acid palmitate and the unsaturated fatty acid oleate are the main free fatty acids in adipose tissue and human diet. We asked how these fatty acids affect cell survival, NAMPT and NAD levels in HepG2 cells and primary human hepatocytes. HepG2 cells were stimulated with palmitate (0.5mM), oleate (1mM) or a combination of both (0.5mM/1mM) as well as nicotinamide mononucleotide (NMN) (0.5 mM) or the specific NAMPT inhibitor FK866 (10nM). Cell survival was measured by WST-1 assay and Annexin V/propidium iodide staining. NAD levels were determined by NAD/NADH Assay or HPLC. Protein and mRNA levels were analysed by Western blot analyses and qPCR, respectively. NAMPT enzyme activity was measured using radiolabelled 14 C-nicotinamide. Lipids were stained by Oil red O staining. Palmitate significantly reduced cell survival and induced apoptosis at physiological doses. NAMPT activity and NAD levels significantly declined after 48h of palmitate. In addition, NAMPT mRNA expression was enhanced which was associated with increased NAMPT release into the supernatant, while intracellular NAMPT protein levels remained stable. Oleate alone did not influence cell viability and NAMPT activity but ameliorated the negative impact of palmitate on cell survival, NAMPT activity and NAD levels, as well as the increased NAMPT mRNA expression and secretion. NMN was able to normalize intracellular NAD levels but did not ameliorate cell viability after co-stimulation with palmitate. FK866, a specific NAMPT inhibitor did not influence lipid accumulation after oleate-treatment. Palmitate targets NAMPT activity with a consequent cellular depletion of NAD. Oleate protects from palmitate-induced apoptosis and variation of NAMPT and NAD levels. Palmitate-induced cell stress leads to an increase of NAMPT mRNA and accumulation in the supernatant. However

New function for Escherichia coli xanthosine phophorylase (xapA): genetic and biochemical evidences on its participation in NAD+ salvage from nicotinamide

PubMed Central

2014-01-01

Background In an effort to reconstitute the NAD+ synthetic pathway in Escherichia coli (E. coli), we produced a set of gene knockout mutants with deficiencies in previously well-defined NAD+de novo and salvage pathways. Unexpectedly, the mutant deficient in NAD+de novo and salvage pathway I could grow in M9/nicotinamide medium, which was contradictory to the proposed classic NAD+ metabolism of E. coli. Such E. coli mutagenesis assay suggested the presence of an undefined machinery to feed nicotinamide into the NAD+ biosynthesis. We wanted to verify whether xanthosine phophorylase (xapA) contributed to a new NAD+ salvage pathway from nicotinamide. Results Additional knockout of xapA further slowed down the bacterial growth in M9/nicotinamide medium, whereas the complementation of xapA restored the growth phenotype. To further validate the new function of xapA, we cloned and expressed E. coli xapA as a recombinant soluble protein. Biochemical assay confirmed that xapA was capable of using nicotinamide as a substrate for nicotinamide riboside formation. Conclusions Both the genetic and biochemical evidences indicated that xapA could convert nicotinamide to nicotinamide riboside in E. coli, albeit with relatively weak activity, indicating that xapA may contribute to a second NAD+ salvage pathway from nicotinamide. We speculate that this xapA-mediated NAD+ salvage pathway might be significant in some bacteria lacking NAD+de novo and NAD+ salvage pathway I or II, to not only use nicotinamide riboside, but also nicotinamide as precursors to synthesize NAD+. However, this speculation needs to be experimentally tested. PMID:24506841

New function for Escherichia coli xanthosine phophorylase (xapA): genetic and biochemical evidences on its participation in NAD(+) salvage from nicotinamide.

PubMed

Dong, Wei-Ren; Sun, Cen-Cen; Zhu, Guan; Hu, Shi-Hua; Xiang, Li-Xin; Shao, Jian-Zhong

2014-02-08

In an effort to reconstitute the NAD(+) synthetic pathway in Escherichia coli (E. coli), we produced a set of gene knockout mutants with deficiencies in previously well-defined NAD(+)de novo and salvage pathways. Unexpectedly, the mutant deficient in NAD(+) de novo and salvage pathway I could grow in M9/nicotinamide medium, which was contradictory to the proposed classic NAD(+) metabolism of E. coli. Such E. coli mutagenesis assay suggested the presence of an undefined machinery to feed nicotinamide into the NAD(+) biosynthesis. We wanted to verify whether xanthosine phophorylase (xapA) contributed to a new NAD(+) salvage pathway from nicotinamide. Additional knockout of xapA further slowed down the bacterial growth in M9/nicotinamide medium, whereas the complementation of xapA restored the growth phenotype. To further validate the new function of xapA, we cloned and expressed E. coli xapA as a recombinant soluble protein. Biochemical assay confirmed that xapA was capable of using nicotinamide as a substrate for nicotinamide riboside formation. Both the genetic and biochemical evidences indicated that xapA could convert nicotinamide to nicotinamide riboside in E. coli, albeit with relatively weak activity, indicating that xapA may contribute to a second NAD(+) salvage pathway from nicotinamide. We speculate that this xapA-mediated NAD(+) salvage pathway might be significant in some bacteria lacking NAD(+) de novo and NAD(+) salvage pathway I or II, to not only use nicotinamide riboside, but also nicotinamide as precursors to synthesize NAD(+). However, this speculation needs to be experimentally tested.

NAD(+)-dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease.

PubMed

Cerutti, Raffaele; Pirinen, Eija; Lamperti, Costanza; Marchet, Silvia; Sauve, Anthony A; Li, Wei; Leoni, Valerio; Schon, Eric A; Dantzer, Françoise; Auwerx, Johan; Viscomi, Carlo; Zeviani, Massimo

2014-06-03

Mitochondrial disorders are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain. Pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling, often fatal, disorders. Pathways related to mitochondrial biogenesis are targets of Sirtuin1, a NAD(+)-dependent protein deacetylase. As NAD(+) boosts the activity of Sirtuin1 and other sirtuins, intracellular levels of NAD(+) play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell. We show here that supplementation with nicotinamide riboside, a natural NAD(+) precursor, or reduction of NAD(+) consumption by inhibiting the poly(ADP-ribose) polymerases, leads to marked improvement of the respiratory chain defect and exercise intolerance of the Sco2 knockout/knockin mouse, a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis. This strategy is potentially translatable into therapy of mitochondrial disorders in humans. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1).

PubMed

Harlan, Benjamin A; Pehar, Mariana; Sharma, Deep R; Beeson, Gyda; Beeson, Craig C; Vargas, Marcelo R

2016-05-13

Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS. © 2016 by The American Society for Biochemistry and Molecular

Mitochondrial nad2 gene is co-transcripted with CMS-associated orfB gene in cytoplasmic male-sterile stem mustard (Brassica juncea).

PubMed

Yang, Jing-Hua; Zhang, Ming-Fang; Yu, Jing-Quan

2009-02-01

The transcriptional patterns of mitochondrial respiratory related genes were investigated in cytoplasmic male-sterile and fertile maintainer lines of stem mustard, Brassica juncea. There were numerous differences in nad2 (subunit 2 of NADH dehydrogenase) between stem mustard CMS and its maintainer line. One novel open reading frame, hereafter named orfB gene, was located at the downstream of mitochondrial nad2 gene in the CMS. The novel orfB gene had high similarity with YMF19 family protein, orfB in Raphanus sativus, Helianthus annuus, Nicotiana tabacum and Beta vulgaris, orfB-CMS in Daucus carota, atp8 gene in Arabidopsis thaliana, 5' flanking of orf224 in B. napus (nap CMS) and 5' flanking of orf220 gene in CMS Brassica juncea. Three copies probed by specific fragment (amplified by primers of nad2F and nad2R from CMS) were found in the CMS line following Southern blotting digested with HindIII, but only a single copy in its maintainer line. Meanwhile, two transcripts were shown in the CMS line following Northern blotting while only one transcript was detected in the maintainer line, which were probed by specific fragment (amplified by primers of nad2F and nad2R from CMS). Meanwhile, the expression of nad2 gene was reduced in CMS bud compared to that in its maintainer line. We thus suggested that nad2 gene may be co-transcripted with CMS-associated orfB gene in the CMS. In addition, the specific fragment that was amplified by primers of nad2F and nad2R just spanned partial sequences of nad2 gene and orfB gene. Such alterations in the nad2 gene would impact the activity of NADH dehydrogenase, and subsequently signaling, inducing the expression of nuclear genes involved in male sterility in this type of cytoplasmic male sterility.

High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(.).

PubMed

Huang, Rui; Chen, Hui; Zhong, Chao; Kim, Jae Eung; Zhang, Yi-Heng Percival

2016-09-02

Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP(+) to NAD(+). Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD(+), and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP(+) to NAD(+). This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.

Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sporty, J; Kabir, M M; Turteltaub, K

A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, approximately 10{sup 8} yeast cells were harvested by centrifugation and then lysed under non-oxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50-mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH{sub 3}CN + 50-mM ammonium acetate (3:1; v:v) was added to the cell lysates. After sample centrifugation to pellet precipitated proteins, organic solvent removal was performed on supernatants by chloroform extraction. Themore » remaining aqueous phase was dried and resuspended in 50-mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-VIS absorbance detection. Applicability of this procedure for quantifying NAD and NADH levels was evaluated by culturing yeast under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. NAD and NADH contents are similar to previously reported levels in yeast obtained using enzymatic assays performed separately on acid (for NAD) and alkali (for NADH) extracts. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (1) applicable to quantification of these metabolites in mammalian and bacterial cell cultures; and (2) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.« less

Imaging the NADH:NAD+ Homeostasis for Understanding the Metabolic Response of Mycobacterium to Physiologically Relevant Stresses

PubMed Central

Bhat, Shabir A.; Iqbal, Iram K.; Kumar, Ashwani

2016-01-01

The NADH:NAD+ ratio is the primary indicator of the metabolic state of bacteria. NAD(H) homeostasis is critical for Mycobacterium tuberculosis (Mtb) survival and is thus considered an important drug target, but the spatio-temporal measurements of NAD(H) remain a challenge. Genetically encoded fluorescent biosensors of the NADH:NAD+ ratios were recently described, paving the way for investigations of the metabolic state of pathogens during infection. Here we have adapted the genetically encoded biosensor Peredox for measurement of the metabolic state of Mtb in vitro and during infection of macrophage cells. Using Peredox, here we show that inhibition of the electron transport chain, disruption of the membrane potential and proton gradient, exposure to reactive oxygen species and treatment with antimycobacterial drugs led to the accumulation of NADH in mycobacterial cells. We have further demonstrated that Mtb residing in macrophages displays higher NADH:NAD+ ratios, that may indicate a metabolic stress faced by the intracellular Mtb. We also demonstrate that the Mtb residing in macrophages display a metabolic heterogeneity, which may perhaps explain the tolerance displayed by intracellular Mtb. Next we studied the effect of immunological modulation by interferon gamma on metabolism of intracellular Mtb, since macrophage activation is known to restrict mycobacterial growth. We observed that activation of resting macrophages with interferon-gamma results in higher NADH:NAD+ levels in resident Mtb cells. We have further demonstrated that exposure of Isoniazid, Bedaquiline, Rifampicin, and O-floxacin results in higher NADH:NAD+ ratios in the Mtb residing in macrophages. However, intracellular Mtb displays lower NADH:NAD+ ratio upon exposure to clofazimine. In summary, we have generated reporter strains capable of measuring the metabolic state of Mtb cells in vitro and in vivo with spatio-temporal resolution. We believe that this tool will facilitate further studies on

The Mitochondrion-Targeted PENTATRICOPEPTIDE REPEAT78 Protein Is Required for nad5 Mature mRNA Stability and Seed Development in Maize.

PubMed

Zhang, Ya-Feng; Suzuki, Masaharu; Sun, Feng; Tan, Bao-Cai

2017-10-09

Pentatricopepetide repeat (PPR) proteins are a large family of RNA-binding proteins involved in RNA metabolism in plant organelles. Although many PPR proteins have been functionally studied, few of them are identified with a function in mitochondrial RNA stability. By using a reverse genetic approach, we characterized the role of the mitochondrion-targeted PPR78 protein in nad5 mature mRNA stability and maize (Zea mays) seed development. Loss of PPR78 function leads to a dramatic reduction in the steady-state level of mitochondrial nad5 mature mRNA, blocks the assembly of complex I in the electron transport chain, and causes an arrest in embryogenesis and endosperm development. Characterization of a second strong allele confirms the function of PPR78 in nad5 mRNA accumulation and maize seed development. The generation of mature nad5 requires the assembly of three distinct precursor RNAs via trans-splicing reactions, and the accumulation of nad5T1 precursor is reduced in the ppr78 mutants. However, it is the instability of mature nad5 rather than nad5T1 causing loss of the full-length nad5 transcript, and degradation of nad5 losing both translation start and stop codons is enriched in the mutant. Our data imply the assembly of mature nad5 mRNA precedes the protection of PPR78. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition.

PubMed

Bogan, Katrina L; Brenner, Charles

2008-01-01

Although baseline requirements for nicotinamide adenine dinucleotide (NAD+) synthesis can be met either with dietary tryptophan or with less than 20 mg of daily niacin, which consists of nicotinic acid and/or nicotinamide, there is growing evidence that substantially greater rates of NAD+ synthesis may be beneficial to protect against neurological degeneration, Candida glabrata infection, and possibly to enhance reverse cholesterol transport. The distinct and tissue-specific biosynthetic and/or ligand activities of tryptophan, nicotinic acid, nicotinamide, and the newly identified NAD+ precursor, nicotinamide riboside, reviewed herein, are responsible for vitamin-specific effects and side effects. Because current data suggest that nicotinamide riboside may be the only vitamin precursor that supports neuronal NAD+ synthesis, we present prospects for human nicotinamide riboside supplementation and propose areas for future research.

Comparative Metabolomic Profiling Reveals That Dysregulated Glycolysis Stemming from Lack of Salvage NAD+ Biosynthesis Impairs Reproductive Development in Caenorhabditis elegans.

PubMed

Wang, Wenqing; McReynolds, Melanie R; Goncalves, Jimmy F; Shu, Muya; Dhondt, Ineke; Braeckman, Bart P; Lange, Stephanie E; Kho, Kelvin; Detwiler, Ariana C; Pacella, Marisa J; Hanna-Rose, Wendy

2015-10-23

Temporal developmental progression is highly coordinated in Caenorhabditis elegans. However, loss of nicotinamidase PNC-1 activity slows reproductive development, uncoupling it from its typical progression relative to the soma. Using LC/MS we demonstrate that pnc-1 mutants do not salvage the nicotinamide released by NAD(+) consumers to resynthesize NAD(+), resulting in a reduction in global NAD(+) bioavailability. We manipulate NAD(+) levels to demonstrate that a minor deficit in NAD(+) availability is incompatible with a normal pace of gonad development. The NAD(+) deficit compromises NAD(+) consumer activity, but we surprisingly found no functional link between consumer activity and reproductive development. As a result we turned to a comparative metabolomics approach to identify the cause of the developmental phenotype. We reveal widespread metabolic perturbations, and using complementary pharmacological and genetic approaches, we demonstrate that a glycolytic block accounts for the slow pace of reproductive development. Interestingly, mitochondria are protected from both the deficiency in NAD(+) biosynthesis and the effects of reduced glycolytic output. We suggest that compensatory metabolic processes that maintain mitochondrial activity in the absence of efficient glycolysis are incompatible with the requirements for reproductive development, which requires high levels of cell division. In addition to demonstrating metabolic requirements for reproductive development, this work also has implications for understanding the mechanisms behind therapeutic interventions that target NAD(+) salvage biosynthesis for the purposes of inhibiting tumor growth. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD+ in healthy middle-aged and older adults.

PubMed

Martens, Christopher R; Denman, Blair A; Mazzo, Melissa R; Armstrong, Michael L; Reisdorph, Nichole; McQueen, Matthew B; Chonchol, Michel; Seals, Douglas R

2018-03-29

Nicotinamide adenine dinucleotide (NAD + ) has emerged as a critical co-substrate for enzymes involved in the beneficial effects of regular calorie restriction on healthspan. As such, the use of NAD + precursors to augment NAD + bioavailability has been proposed as a strategy for improving cardiovascular and other physiological functions with aging in humans. Here we provide the evidence in a 2 × 6-week randomized, double-blind, placebo-controlled, crossover clinical trial that chronic supplementation with the NAD + precursor vitamin, nicotinamide riboside (NR), is well tolerated and effectively stimulates NAD + metabolism in healthy middle-aged and older adults. Our results also provide initial insight into the effects of chronic NR supplementation on physiological function in humans, and suggest that, in particular, future clinical trials should further assess the potential benefits of NR for reducing blood pressure and arterial stiffness in this group.

The NAD+ Precursor Nicotinamide Riboside Rescues Mitochondrial Defects and Neuronal Loss in iPSC and Fly Models of Parkinson's Disease.

PubMed

Schöndorf, David C; Ivanyuk, Dina; Baden, Pascale; Sanchez-Martinez, Alvaro; De Cicco, Silvia; Yu, Cong; Giunta, Ivana; Schwarz, Lukas K; Di Napoli, Gabriele; Panagiotakopoulou, Vasiliki; Nestel, Sigrun; Keatinge, Marcus; Pruszak, Jan; Bandmann, Oliver; Heimrich, Bernd; Gasser, Thomas; Whitworth, Alexander J; Deleidi, Michela

2018-06-05

While mitochondrial dysfunction is emerging as key in Parkinson's disease (PD), a central question remains whether mitochondria are actual disease drivers and whether boosting mitochondrial biogenesis and function ameliorates pathology. We address these questions using patient-derived induced pluripotent stem cells and Drosophila models of GBA-related PD (GBA-PD), the most common PD genetic risk. Patient neurons display stress responses, mitochondrial demise, and changes in NAD+ metabolism. NAD+ precursors have been proposed to ameliorate age-related metabolic decline and disease. We report that increasing NAD+ via the NAD+ precursor nicotinamide riboside (NR) significantly ameliorates mitochondrial function in patient neurons. Human neurons require nicotinamide phosphoribosyltransferase (NAMPT) to maintain the NAD+ pool and utilize NRK1 to synthesize NAD+ from NAD+ precursors. Remarkably, NR prevents the age-related dopaminergic neuronal loss and motor decline in fly models of GBA-PD. Our findings suggest NR as a viable clinical avenue for neuroprotection in PD and other neurodegenerative diseases. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Identification of novel resistance mechanisms to NAMPT inhibition via the de novo NAD+ biosynthesis pathway and NAMPT mutation.

PubMed

Guo, Jun; Lam, Lloyd T; Longenecker, Kenton L; Bui, Mai H; Idler, Kenneth B; Glaser, Keith B; Wilsbacher, Julie L; Tse, Chris; Pappano, William N; Huang, Tzu-Hsuan

2017-09-23

Cancer cells have an unusually high requirement for the central and intermediary metabolite nicotinamide adenine dinucleotide (NAD + ), and NAD + depletion ultimately results in cell death. The rate limiting step within the NAD + salvage pathway required for converting nicotinamide to NAD + is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). Targeting NAMPT has been investigated as an anti-cancer strategy, and several highly selective small molecule inhibitors have been found to potently inhibit NAMPT in cancer cells, resulting in NAD + depletion and cytotoxicity. To identify mechanisms that could cause resistance to NAMPT inhibitor treatment, we generated a human fibrosarcoma cell line refractory to the highly potent and selective NAMPT small molecule inhibitor, GMX1778. We uncovered novel and unexpected mechanisms of resistance including significantly increased expression of quinolinate phosphoribosyl transferase (QPRT), a key enzyme in the de novo NAD + synthesis pathway. Additionally, exome sequencing of the NAMPT gene in the resistant cells identified a single heterozygous point mutation that was not present in the parental cell line. The combination of upregulation of the NAD + de novo synthesis pathway through QPRT over-expression and NAMPT mutation confers resistance to GMX1778, but the cells are only partially resistant to next-generation NAMPT inhibitors. The resistance mechanisms uncovered herein provide a potential avenue to continue exploration of next generation NAMPT inhibitors to treat neoplasms in the clinic. Copyright © 2017 Elsevier Inc. All rights reserved.

OXPHOS-Mediated Induction of NAD+ Promotes Complete Oxidation of Fatty Acids and Interdicts Non-Alcoholic Fatty Liver Disease.

PubMed

Akie, Thomas E; Liu, Lijun; Nam, Minwoo; Lei, Shi; Cooper, Marcus P

2015-01-01

OXPHOS is believed to play an important role in non-alcoholic fatty liver disease (NAFLD), however, precise mechanisms whereby OXPHOS influences lipid homeostasis are incompletely understood. We previously reported that ectopic expression of LRPPRC, a protein that increases cristae density and OXPHOS, promoted fatty acid oxidation in cultured primary hepatocytes. To determine the biological significance of that observation and define underlying mechanisms, we have ectopically expressed LRPPRC in mouse liver in the setting of NAFLD. Interestingly, ectopic expression of LRPPRC in mouse liver completely interdicted NAFLD, including inflammation. Consistent with mitigation of NAFLD, two markers of hepatic insulin resistance--ROS and PKCε activity--were both modestly reduced. As reported by others, improvement of NAFLD was associated with improved whole-body insulin sensitivity. Regarding hepatic lipid homeostasis, the ratio of NAD+ to NADH was dramatically increased in mouse liver replete with LRPPRC. Pharmacological activators and inhibitors of the cellular respiration respectively increased and decreased the [NAD+]/[NADH] ratio, indicating respiration-mediated control of the [NAD+]/[NADH] ratio. Supporting a prominent role for NAD+, increasing the concentration of NAD+ stimulated complete oxidation of fatty acids. Importantly, NAD+ rescued impaired fatty acid oxidation in hepatocytes deficient for either OXPHOS or SIRT3. These data are consistent with a model whereby augmented hepatic OXPHOS increases NAD+, which in turn promotes complete oxidation of fatty acids and protects against NAFLD.

Tributyltin induces G2/M cell cycle arrest via NAD(+)-dependent isocitrate dehydrogenase in human embryonic carcinoma cells.

PubMed

Asanagi, Miki; Yamada, Shigeru; Hirata, Naoya; Itagaki, Hiroshi; Kotake, Yaichiro; Sekino, Yuko; Kanda, Yasunari

2016-04-01

Organotin compounds, such as tributyltin (TBT), are well-known endocrine-disrupting chemicals (EDCs). We have recently reported that TBT induces growth arrest in the human embryonic carcinoma cell line NT2/D1 at nanomolar levels by inhibiting NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the irreversible conversion of isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we examined whether TBT at nanomolar levels affects cell cycle progression in NT2/D1 cells. Propidium iodide staining revealed that TBT reduced the ratio of cells in the G1 phase and increased the ratio of cells in the G2/M phase. TBT also reduced cell division cycle 25C (cdc25C) and cyclin B1, which are key regulators of G2/M progression. Furthermore, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. The G2/M arrest induced by TBT was abolished by NAD-IDHα knockdown. Treatment with a cell-permeable α-ketoglutarate analogue recovered the effect of TBT, suggesting the involvement of NAD-IDH. Taken together, our data suggest that TBT at nanomolar levels induced G2/M cell cycle arrest via NAD-IDH in NT2/D1 cells. Thus, cell cycle analysis in embryonic cells could be used to assess cytotoxicity associated with nanomolar level exposure of EDCs.

Mobile phones electromagnetic radiation and NAD+-dependent isocitrate dehydrogenase as a mitochondrial marker in asthenozoospermia.

PubMed

Hagras, Abeer M; Toraih, Eman A; Fawzy, Manal S

2016-12-01

NAD + -dependent Isocitrate Dehydrogenase (NAD + -IDH) could be one of the cell phone radiation targets. Enzyme activity alteration may lead to decline in sperm motility during radio-frequency electromagnetic waves (RF-EMW) exposure. The current case control study aimed to investigate the possible relationship between mitochondrial NAD + -IDH activity in human seminal plasma and sperm motility among asthenozoospermic cellular phone users. A total number of ninety idiopathic infertile males referred from the Department of Dermatology and Andrology, were enrolled in this study. NAD + -IDH activity was measured in human seminal plasma by spectrophotometer. Computer-aided sperm analysis (CASA) following WHO criteria has been used for semen analyses. The results showed that IDH activity was increased in patients with prolonged cell phone daily use ≥4 h/day. Its level, correlated negatively with either the motility ratio percentages (r = -0.46, p  < 0.001) or the progressive motility percentages (r = -0.50, p  < 0.001) in the study groups. The current study suggests that NAD + -IDH in human seminal plasma could be one of seminal plasma biomarkers reflecting the mitochondrial function of spermatozoa. Alteration of its level could reflect the defective motility of sperms among some cases of cellular phone users.

Upregulation of mitochondrial NAD+ levels impairs the clonogenicity of SSEA1+ glioblastoma tumor-initiating cells.

PubMed

Son, Myung Jin; Ryu, Jae-Sung; Kim, Jae Yun; Kwon, Youjeong; Chung, Kyung-Sook; Mun, Seon Ju; Cho, Yee Sook

2017-06-09

Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD + ) metabolism. However, the functional role of NAD + metabolism in regulating the characteristics of TICs is not known. In this study, we provide evidence that the mitochondrial NAD + levels affect the characteristics of glioma-driven SSEA1 + TICs, including clonogenic growth potential. An increase in the mitochondrial NAD + levels by the overexpression of the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) significantly suppressed the sphere-forming ability and induced differentiation of TICs, suggesting a loss of the characteristics of TICs. In addition, increased SIRT3 activity and reduced lactate production, which are mainly observed in healthy and young cells, appeared following NNT-overexpressed TICs. Moreover, in vivo tumorigenic potential was substantially abolished by NNT overexpression. Conversely, the short interfering RNA-mediated knockdown of NNT facilitated the maintenance of TIC characteristics, as evidenced by the increased numbers of large tumor spheres and in vivo tumorigenic potential. Our results demonstrated that targeting the maintenance of healthy mitochondria with increased mitochondrial NAD + levels and SIRT3 activity could be a promising strategy for abolishing the development of TICs as a new therapeutic approach to treating aging-associated tumors.

Extremely high intracellular concentration of glucose-6-phosphate and NAD(H) in Deinococcus radiodurans.

PubMed

Yamashiro, Takumi; Murata, Kousaku; Kawai, Shigeyuki

2017-03-01

Deinococcus radiodurans is highly resistant to ionizing radiation and UV radiation, and oxidative stress caused by such radiations. NADP(H) seems to be important for this resistance (Slade and Radman, Microbiol Mol Biol Rev 75:133-191; Slade, Radman, Microbiol Mol Biol Rev 75:133-191, 2011), but the mechanism underlying the generation of NADP(H) or NAD(H) in D. radiodurans has not fully been addressed. Intracellular concentrations of NAD + , NADH, NADP + , and NADPH in D. radiodurans are also not determined yet. We found that cell extracts of D. radiodurans catalyzed reduction of NAD(P) + in vitro, indicating that D. radiodurans cells contain both enzymes and a high concentration of substrates for this activity. The enzyme and the substrate were attributed to glucose-6-phosphate dehydrogenase and glucose-6-phosphate of which intracellular concentration was extremely high. Unexpectedly, the intracellular concentration of NAD(H) was also much greater than that of NADP(H), suggesting some significant roles of NADH. These unusual features of this bacterium would shed light on a new aspect of physiology of this bacterium.

Molecular mechanism of the allosteric regulation of the αγ heterodimer of human NAD-dependent isocitrate dehydrogenase

PubMed Central

Ma, Tengfei; Peng, Yingjie; Huang, Wei; Ding, Jianping

2017-01-01

Human NAD-dependent isocitrate dehydrogenase catalyzes the decarboxylation of isocitrate (ICT) into α-ketoglutarate in the Krebs cycle. It exists as the α2βγ heterotetramer composed of the αβ and αγ heterodimers. Previously, we have demonstrated biochemically that the α2βγ heterotetramer and αγ heterodimer can be allosterically activated by citrate (CIT) and ADP. In this work, we report the crystal structures of the αγ heterodimer with the γ subunit bound without or with different activators. Structural analyses show that CIT, ADP and Mg2+ bind adjacent to each other at the allosteric site. The CIT binding induces conformational changes at the allosteric site, which are transmitted to the active site through the heterodimer interface, leading to stabilization of the ICT binding at the active site and thus activation of the enzyme. The ADP binding induces no further conformational changes but enhances the CIT binding through Mg2+-mediated interactions, yielding a synergistic activation effect. ICT can also bind to the CIT-binding subsite, which induces similar conformational changes but exhibits a weaker activation effect. The functional roles of the key residues are verified by mutagenesis, kinetic and structural studies. Our structural and functional data together reveal the molecular mechanism of the allosteric regulation of the αγ heterodimer. PMID:28098230

CD38-NAD+Axis Regulates Immunotherapeutic Anti-Tumor T Cell Response.

PubMed

Chatterjee, Shilpak; Daenthanasanmak, Anusara; Chakraborty, Paramita; Wyatt, Megan W; Dhar, Payal; Selvam, Shanmugam Panneer; Fu, Jianing; Zhang, Jinyu; Nguyen, Hung; Kang, Inhong; Toth, Kyle; Al-Homrani, Mazen; Husain, Mahvash; Beeson, Gyda; Ball, Lauren; Helke, Kristi; Husain, Shahid; Garrett-Mayer, Elizabeth; Hardiman, Gary; Mehrotra, Meenal; Nishimura, Michael I; Beeson, Craig C; Bupp, Melanie Gubbels; Wu, Jennifer; Ogretmen, Besim; Paulos, Chrystal M; Rathmell, Jeffery; Yu, Xue-Zhong; Mehrotra, Shikhar

2018-01-09

Heightened effector function and prolonged persistence, the key attributes of Th1 and Th17 cells, respectively, are key features of potent anti-tumor T cells. Here, we established ex vivo culture conditions to generate hybrid Th1/17 cells, which persisted long-term in vivo while maintaining their effector function. Using transcriptomics and metabolic profiling approaches, we showed that the enhanced anti-tumor property of Th1/17 cells was dependent on the increased NAD + -dependent activity of the histone deacetylase Sirt1. Pharmacological or genetic inhibition of Sirt1 activity impaired the anti-tumor potential of Th1/17 cells. Importantly, T cells with reduced surface expression of the NADase CD38 exhibited intrinsically higher NAD + , enhanced oxidative phosphorylation, higher glutaminolysis, and altered mitochondrial dynamics that vastly improved tumor control. Lastly, blocking CD38 expression improved tumor control even when using Th0 anti-tumor T cells. Thus, strategies targeting the CD38-NAD + axis could increase the efficacy of anti-tumor adoptive T cell therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

Targeting SIRT1 to improve metabolism: all you need is NAD+?

PubMed Central

Cantó, Carles; Auwerx, Johan

2013-01-01

SIRT1 is an evolutionary conserved NAD+-dependent deacetylase that is at the pinnacle of metabolic control, all the way from yeast to humans. SIRT1 senses changes in intracellular NAD+ levels, which reflect energy level, and uses this information to adapt the cellular energy output, such that the it matches cellular energy requirements. Generally, but not exclusively, the changes induced by SIRT1 activation are transcriptional in nature and are related to an increase in mitochondrial metabolism and antioxidant protection. These attractive features have validated SIRT1 as a therapeutic target in the management of metabolic disease and prompted an intensive search to identify pharmacological SIRT1 activators. In this review we will first give an overview of the SIRT1 biology with a particular focus on its role in metabolic control. We will then analyze the pros and cons of the current strategies used to activate SIRT1 and explore the emerging evidence indicating that modulation of NAD+ levels could provide an effective way to achieve such goals. PMID:22106091

PARP inhibitor rucaparib induces changes in NAD levels in cells and liver tissues as assessed by MRS.

PubMed

Almeida, Gilberto S; Bawn, Carlo M; Galler, Martin; Wilson, Ian; Thomas, Huw D; Kyle, Suzanne; Curtin, Nicola J; Newell, David R; Maxwell, Ross J

2017-09-01

Poly(adenosine diphosphate ribose) polymerases (PARPs) are multifunctional proteins which play a role in many cellular processes. Namely, PARP1 and PARP2 have been shown to be involved in DNA repair, and therefore are valid targets in cancer treatment with PARP inhibitors, such as rucaparib, currently in clinical trials. Proton magnetic resonance spectroscopy ( 1 H-MRS) was used to study the impact of rucaparib in vitro and ex vivo in liver tissue from mice, via quantitative analysis of nicotinamide adenosine diphosphate (NAD + ) spectra, to assess the potential of MRS as a biomarker of the PARP inhibitor response. SW620 (colorectal) and A2780 (ovarian) cancer cell lines, and PARP1 wild-type (WT) and PARP1 knock-out (KO) mice, were treated with rucaparib, temozolomide (methylating agent) or a combination of both drugs. 1 H-MRS spectra were obtained from perchloric acid extracts of tumour cells and mouse liver. Both cell lines showed an increase in NAD + levels following PARP inhibitor treatment in comparison with temozolomide treatment. Liver extracts from PARP1 WT mice showed a significant increase in NAD + levels after rucaparib treatment compared with untreated mouse liver, and a significant decrease in NAD + levels in the temozolomide-treated group. The combination of rucaparib and temozolomide did not prevent the NAD + depletion caused by temozolomide treatment. The 1 H-MRS results show that NAD + levels can be used as a biomarker of PARP inhibitor and methylating agent treatments, and suggest that in vivo measurement of NAD + would be valuable. Copyright © 2017 John Wiley & Sons, Ltd.

High-throughput screening of coenzyme preference change of thermophilic 6-phosphogluconate dehydrogenase from NADP + to NAD +

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Rui; Chen, Hui; Zhong, Chao

Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP + to NAD +. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfatemore » (PMS), NAD +, and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP + to NAD +. Furthermore, this screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.« less

High-throughput screening of coenzyme preference change of thermophilic 6-phosphogluconate dehydrogenase from NADP + to NAD +

DOE PAGES

Huang, Rui; Chen, Hui; Zhong, Chao; ...

2016-09-02

Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP + to NAD +. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfatemore » (PMS), NAD +, and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP + to NAD +. Furthermore, this screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.« less

Atomic-Resolution Structures of Horse Liver Alcohol Dehydrogenase with NAD[superscript +] and Fluoroalcohols Define Strained Michaelis Complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Plapp, Bryce V.; Ramaswamy, S.; Iowa)

2013-01-16

Structures of horse liver alcohol dehydrogenase complexed with NAD{sup +} and unreactive substrate analogues, 2,2,2-trifluoroethanol or 2,3,4,5,6-pentafluorobenzyl alcohol, were determined at 100 K at 1.12 or 1.14 {angstrom} resolution, providing estimates of atomic positions with overall errors of 0.02 {angstrom}, the geometry of ligand binding, descriptions of alternative conformations of amino acid residues and waters, and evidence of a strained nicotinamide ring. The four independent subunits from the two homodimeric structures differ only slightly in the peptide backbone conformation. Alternative conformations for amino acid side chains were identified for 50 of the 748 residues in each complex, and Leu-57 andmore » Leu-116 adopt different conformations to accommodate the different alcohols at the active site. Each fluoroalcohol occupies one position, and the fluorines of the alcohols are well-resolved. These structures closely resemble the expected Michaelis complexes with the pro-R hydrogens of the methylene carbons of the alcohols directed toward the re face of C4N of the nicotinamide rings with a C-C distance of 3.40 {angstrom}. The oxygens of the alcohols are ligated to the catalytic zinc at a distance expected for a zinc alkoxide (1.96 {angstrom}) and participate in a low-barrier hydrogen bond (2.52 {angstrom}) with the hydroxyl group of Ser-48 in a proton relay system. As determined by X-ray refinement with no restraints on bond distances and planarity, the nicotinamide rings in the two complexes are slightly puckered (quasi-boat conformation, with torsion angles of 5.9{sup o} for C4N and 4.8{sup o} for N1N relative to the plane of the other atoms) and have bond distances that are somewhat different compared to those found for NAD(P){sup +}. It appears that the nicotinamide ring is strained toward the transition state on the path to alcohol oxidation.« less

A computational tool to predict the evolutionarily conserved protein-protein interaction hot-spot residues from the structure of the unbound protein.

PubMed

Agrawal, Neeraj J; Helk, Bernhard; Trout, Bernhardt L

2014-01-21

Identifying hot-spot residues - residues that are critical to protein-protein binding - can help to elucidate a protein's function and assist in designing therapeutic molecules to target those residues. We present a novel computational tool, termed spatial-interaction-map (SIM), to predict the hot-spot residues of an evolutionarily conserved protein-protein interaction from the structure of an unbound protein alone. SIM can predict the protein hot-spot residues with an accuracy of 36-57%. Thus, the SIM tool can be used to predict the yet unknown hot-spot residues for many proteins for which the structure of the protein-protein complexes are not available, thereby providing a clue to their functions and an opportunity to design therapeutic molecules to target these proteins. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray analysis of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1

PubMed Central

Taketa, Midori; Nakagawa, Hanae; Habukawa, Mao; Osuka, Hisao; Kihira, Kiyohito; Komori, Hirofumi; Shibata, Naoki; Ishii, Masaharu; Igarashi, Yasuo; Nishihara, Hirofumi; Yoon, Ki-Seok; Ogo, Seiji; Shomura, Yasuhito; Higuchi, Yoshiki

2015-01-01

NAD+-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD+ and NADH. Here, the isolation, purification and crystallization of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1 are reported. Crystals of the NAD+-reducing [NiFe] hydrogenase were obtained within one week from a solution containing polyethylene glycol using the sitting-drop vapour-diffusion method and micro-seeding. The crystal diffracted to 2.58 Å resolution and belonged to space group C2, with unit-cell parameters a = 131.43, b = 189.71, c = 124.59 Å, β = 109.42°. Assuming the presence of two NAD+-reducing [NiFe] hydrogenase molecules in the asymmetric unit, V M was calculated to be 2.2 Å3  Da−1, which corresponds to a solvent content of 43%. Initial phases were determined by the single-wavelength anomalous dispersion method using the anomalous signal from the Fe atoms. PMID:25615977

Modulating Transmembrane α-Helix Interactions through pH-Sensitive Boundary Residues.

PubMed

Ng, Derek P; Deber, Charles M

2016-08-09

Changes in pH can alter the structure and activity of proteins and may be used by the cell to control molecular function. This coupling can also be used in non-native applications through the design of pH-sensitive biomolecules. For example, the pH (low) insertion peptide (pHLIP) can spontaneously insert into a lipid bilayer when the pH decreases. We have previously shown that the α-helicity and helix-helix interactions of the TM2 α-helix of the proteolipid protein (PLP) are sensitive to the local hydrophobicity at its C-terminus. Given that there is an ionizable residue (Glu-88) at the C-terminus of this transmembrane (TM) segment, we hypothesized that changing the ionization state of this residue through pH may alter the local hydrophobicity of the peptide enough to affect both its secondary structure and helix-helix interactions. To examine this phenomenon, we synthesized peptide analogues of the PLP TM2 α-helix (wild-type sequence (66)AFQYVIYGTASFFFLYGALLLAEGF(90)). Using circular dichroism and Förster resonance energy transfer in the membrane-mimetic detergent sodium dodecyl sulfate, we found that a decrease in pH increases both peptide α-helicity and the extent of self-association. This pH-dependent effect is due specifically to the presence of Glu-88 at the C-terminus. Additional experiments in which Phe-90 was mutated to residues of varying hydrophobicities indicated that the strength of this effect is dependent on the local hydrophobicity near Glu-88. Our results have implications for the design of TM peptide switches and improve our understanding of how membrane protein structure and activity can be regulated through local molecular environmental changes.

Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication

PubMed Central

Stetz, Gabrielle; Verkhivker, Gennady M.

2017-01-01

Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions. Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood. In this work, we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions. A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways. We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability, proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues. These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues. The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites. Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone. The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling, suggesting that these sites could be integral to the network organization and coordination of structural changes. Using a network-based formalism of allostery, we

Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication.

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2017-01-01

Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions. Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood. In this work, we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions. A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways. We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability, proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues. These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues. The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites. Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone. The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling, suggesting that these sites could be integral to the network organization and coordination of structural changes. Using a network-based formalism of allostery, we

Upregulation of mitochondrial NAD+ levels impairs the clonogenicity of SSEA1+ glioblastoma tumor-initiating cells

PubMed Central

Son, Myung Jin; Ryu, Jae-Sung; Kim, Jae Yun; Kwon, Youjeong; Chung, Kyung-Sook; Mun, Seon Ju; Cho, Yee Sook

2017-01-01

Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD+) metabolism. However, the functional role of NAD+ metabolism in regulating the characteristics of TICs is not known. In this study, we provide evidence that the mitochondrial NAD+ levels affect the characteristics of glioma-driven SSEA1+ TICs, including clonogenic growth potential. An increase in the mitochondrial NAD+ levels by the overexpression of the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) significantly suppressed the sphere-forming ability and induced differentiation of TICs, suggesting a loss of the characteristics of TICs. In addition, increased SIRT3 activity and reduced lactate production, which are mainly observed in healthy and young cells, appeared following NNT-overexpressed TICs. Moreover, in vivo tumorigenic potential was substantially abolished by NNT overexpression. Conversely, the short interfering RNA-mediated knockdown of NNT facilitated the maintenance of TIC characteristics, as evidenced by the increased numbers of large tumor spheres and in vivo tumorigenic potential. Our results demonstrated that targeting the maintenance of healthy mitochondria with increased mitochondrial NAD+ levels and SIRT3 activity could be a promising strategy for abolishing the development of TICs as a new therapeutic approach to treating aging-associated tumors. PMID:28604662

P7C3 neuroprotective chemicals function by activating the rate-limiting enzyme in NAD salvage.

PubMed

Wang, Gelin; Han, Ting; Nijhawan, Deepak; Theodoropoulos, Pano; Naidoo, Jacinth; Yadavalli, Sivaramakrishnan; Mirzaei, Hamid; Pieper, Andrew A; Ready, Joseph M; McKnight, Steven L

2014-09-11

The P7C3 class of aminopropyl carbazole chemicals fosters the survival of neurons in a variety of rodent models of neurodegeneration or nerve cell injury. To uncover its mechanism of action, an active derivative of P7C3 was modified to contain both a benzophenone for photocrosslinking and an alkyne for CLICK chemistry. This derivative was found to bind nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme involved in the conversion of nicotinamide into nicotinamide adenine dinucleotide (NAD). Administration of active P7C3 chemicals to cells treated with doxorubicin, which induces NAD depletion, led to a rebound in intracellular levels of NAD and concomitant protection from doxorubicin-mediated toxicity. Active P7C3 variants likewise enhanced the activity of the purified NAMPT enzyme, providing further evidence that they act by increasing NAD levels through its NAMPT-mediated salvage. Copyright © 2014 Elsevier Inc. All rights reserved.

Investigation of the Ionization Mechanism of NAD+/NADH-Modified Gold Electrodes in ToF-SIMS Analysis.

PubMed

Hua, Xin; Zhao, Li-Jun; Long, Yi-Tao

2018-06-04

Analysis of nicotinamide adenine dinucleotide (NAD + /NADH)-modified electrodes is important for in vitro monitoring of key biological processes. In this work, time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to analyze NAD + /NADH-modified gold electrodes. Interestingly, no obvious characteristic peaks of nicotinamide fragment could be observed in the mass spectra of NAD + /NADH in their neutral sodium pyrophosphate form. However, after acidification, the characteristic peaks for both NAD + and NADH were detected. This was due to the suppression effect of inner pyrophosphoric salts in both neutral molecules. Besides, it was proved that the suppression by inner salt was intramolecular. No obvious suppression was found between neighboring molecules. These results demonstrated the suppression effect of inner salts in ToF-SIMS analysis, providing useful evidence for the study of ToF-SIMS ionization mechanism of organic molecule-modified electrodes. Graphical Abstract ᅟ.

Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA).

PubMed

Zhu, Hui; Shuman, Stewart

2005-04-01

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.

Key role of an ADP - ribose - dependent transcriptional regulator of NAD metabolism for fitness and virulence of Pseudomonas aeruginosa.

PubMed

Okon, Elza; Dethlefsen, Sarah; Pelnikevich, Anna; Barneveld, Andrea van; Munder, Antje; Tümmler, Burkhard

2017-01-01

NAD is an essential co-factor of redox reactions and metabolic conversions of NAD-dependent enzymes. NAD biosynthesis in the opportunistic pathogen Pseudomonas aeruginosa has yet not been experimentally explored. The in silico search for orthologs in the P. aeruginosa PAO1 genome identified the operon pncA - pncB1-nadE (PA4918-PA4920) to encode the nicotinamidase, nicotinate phosporibosyltransferase and Nad synthase of salvage pathway I. The functional role of the preceding genes PA4917 and PA4916 was resolved by the characterization of recombinant protein. PA4917 turned out to encode the nicotinate mononucleotide adenylyltransferase NadD2 and PA4916 was determined to encode the transcriptional repressor NrtR that binds to an intergenic sequence between nadD2 and pncA. Complex formation between the catalytically inactive Nudix protein NrtR and its DNA binding site was suppressed by the antirepressor ADP-ribose. NrtR plasposon mutagenesis abrogated virulence of P. aeruginosa TBCF10839 in a murine acute airway infection model and constrained its metabolite profile. When grown together with other isogenic plasposon mutants, the nrtR knock-out was most compromised in competitive fitness to persist in nutrient-rich medium in vitro or murine airways in vivo. This example demonstrates how tightly metabolism and virulence can be intertwined by key elements of metabolic control. Copyright © 2016 Elsevier GmbH. All rights reserved.

Functions of key residues in the ligand-binding pocket of vitamin D receptor: Fragment molecular orbital interfragment interaction energy analysis

NASA Astrophysics Data System (ADS)

Yamagishi, Kenji; Yamamoto, Keiko; Yamada, Sachiko; Tokiwa, Hiroaki

2006-03-01

Fragment molecular orbital-interfragment interaction energy calculations of the vitamin D receptor (VDR)/1α,25-dihydroxyvitamin D 3 complex were utilized to assign functions of key residues of the VDR. Only one residue forms a significant interaction with the corresponding hydroxy group of the ligand, although two residues are located around each hydroxy group. The degradation of binding affinity for derivatives upon removal of a hydroxy group is closely related to the trend in the strength of the hydrogen bonds. Type II hereditary rickets due to an Arg274 point mutation is caused by the lack of the strongest hydrogen bond.

78 FR 36571 - North American Datum of 1983 (NAD 83) Outer Continental Shelf (OCS) Provisional Official...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-18

... DEPARTMENT OF THE INTERIOR Bureau of Ocean Energy Management [MMAA104000] North American Datum of 1983 (NAD 83) Outer Continental Shelf (OCS) Provisional Official Protraction Diagram (OPDs) AGENCY... OPDs. SUMMARY: Notice is hereby given that effective with this publication two NAD 83-based OCS...

Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+.

PubMed

Belenky, Peter; Racette, Frances G; Bogan, Katrina L; McClure, Julie M; Smith, Jeffrey S; Brenner, Charles

2007-05-04

Although NAD(+) biosynthesis is required for Sir2 functions and replicative lifespan in yeast, alterations in NAD(+) precursors have been reported to accelerate aging but not to extend lifespan. In eukaryotes, nicotinamide riboside is a newly discovered NAD(+) precursor that is converted to nicotinamide mononucleotide by specific nicotinamide riboside kinases, Nrk1 and Nrk2. In this study, we discovered that exogenous nicotinamide riboside promotes Sir2-dependent repression of recombination, improves gene silencing, and extends lifespan without calorie restriction. The mechanism of action of nicotinamide riboside is totally dependent on increased net NAD(+) synthesis through two pathways, the Nrk1 pathway and the Urh1/Pnp1/Meu1 pathway, which is Nrk1 independent. Additionally, the two nicotinamide riboside salvage pathways contribute to NAD(+) metabolism in the absence of nicotinamide-riboside supplementation. Thus, like calorie restriction in the mouse, nicotinamide riboside elevates NAD(+) and increases Sir2 function.

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes*

PubMed Central

Christensen, Caspar Elo; Karlsson, Magnus; Winther, Jakob R.; Jensen, Pernille Rose; Lerche, Mathilde H.

2014-01-01

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD+]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD+]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD+]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD+]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD+]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments. PMID:24302737

Crosstalk of Signaling and Metabolism Mediated by the NAD(+)/NADH Redox State in Brain Cells.

PubMed

Winkler, Ulrike; Hirrlinger, Johannes

2015-12-01

The energy metabolism of the brain has to be precisely adjusted to activity to cope with the organ's energy demand, implying that signaling regulates metabolism and metabolic states feedback to signaling. The NAD(+)/NADH redox state constitutes a metabolic node well suited for integration of metabolic and signaling events. It is affected by flux through metabolic pathways within a cell, but also by the metabolic state of neighboring cells, for example by lactate transferred between cells. Furthermore, signaling events both in neurons and astrocytes have been reported to change the NAD(+)/NADH redox state. Vice versa, a number of signaling events like astroglial Ca(2+) signals, neuronal NMDA-receptors as well as the activity of transcription factors are modulated by the NAD(+)/NADH redox state. In this short review, this bidirectional interdependence of signaling and metabolism involving the NAD(+)/NADH redox state as well as its potential relevance for the physiology of the brain and the whole organism in respect to blood glucose regulation and body weight control are discussed.

Origin of a sensitive dependence of calculated {beta}{beta}-decay amplitudes on the particle-particle residual interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodin, Vadim; Faessler, Amand

2011-07-15

In the present work the sensitivity of calculated {beta}{beta}-decay amplitudes to a realistic residual interaction is analyzed in the framework of the approach of O. A. Rumyantsev and M. H. Urin, Phys. Lett. B 443, 51 (1998). and V. A. Rodin, M. H. Urin, and A. Faessler, Nucl. Phys. A 747, 297 (2005). Both the Gamow-Teller (GT) and Fermi (F) matrix elements M{sup 2}{nu} for two-neutrino {beta}{beta} decay (2{nu}{beta}{beta} decay), along with the monopole transition contributions to the total matrix elements M{sup 0{nu}} of neutrinoless {beta}{beta} decay (0{nu}{beta}{beta} decay), are calculated within the quasiparticle random-phase approximation (QRPA). In the aforementionedmore » approach decompositions of M{sup 2{nu}} and M{sup 0{nu}} can be obtained in terms of the corresponding energy-weighted sum rules S. It is shown that in most of the cases almost the whole dependence of M{sup 2{nu}} and M{sup 0{nu}} on the particle-particle (p-p) renormalization parameter g{sub pp} is accounted for by the g{sub pp} dependence of the corresponding sum rules S. General expressions relating S to a realistic residual particle-particle interaction are derived, which show a pronounced sensitivity of S to the singlet-channel interaction in the case of F transitions and to the triplet-channel interaction in the case of GT transitions. Thus, the sensitivity of M{sup 2{nu}} and M{sup 0{nu}} to the SU(4)-symmetry-breaking part of the p-p residual interaction is dictated by the generic structure of the {beta}{beta}-decay amplitudes. Therefore, a choice of this part in a particular calculation needs a special caution. Finally, a better isospin-consistent way of renormalization of a realistic residual p-p interaction to use in QRPA calculations is suggested.« less

Improved strategies for electrochemical 1,4-NAD(P)H2 regeneration: A new era of bioreactors for industrial biocatalysis.

PubMed

Morrison, Clifford S; Armiger, William B; Dodds, David R; Dordick, Jonathan S; Koffas, Mattheos A G

Industrial enzymatic reactions requiring 1,4-NAD(P)H 2 to perform redox transformations often require convoluted coupled enzyme regeneration systems to regenerate 1,4-NAD(P)H 2 from NAD(P) and recycle the cofactor for as many turnovers as possible. Renewed interest in recycling the cofactor via electrochemical means is motivated by the low cost of performing electrochemical reactions, easy monitoring of the reaction progress, and straightforward product recovery. However, electrochemical cofactor regeneration methods invariably produce adventitious reduced cofactor side products which result in unproductive loss of input NAD(P). We review various literature strategies for mitigating adventitious product formation by electrochemical cofactor regeneration systems, and offer insight as to how a successful electrochemical bioreactor system could be constructed to engineer efficient 1,4-NAD(P)H 2 -dependent enzyme reactions of interest to the industrial biocatalysis community. Copyright © 2017 Elsevier Inc. All rights reserved.

Adenosine mimetics as inhibitors of NAD+-dependent histone deacetylases, from kinase to sirtuin inhibition.

PubMed

Trapp, Johannes; Jochum, Anne; Meier, Rene; Saunders, Laura; Marshall, Brett; Kunick, Conrad; Verdin, Eric; Goekjian, Peter; Sippl, Wolfgang; Jung, Manfred

2006-12-14

NAD+-dependent histone deacetylases, sirtuins, cleave acetyl groups from lysines of histones and other proteins to regulate their activity. Identification of potent selective inhibitors would help to elucidate sirtuin biology and could lead to useful therapeutic agents. NAD+ has an adenosine moiety that is also present in the kinase cofactor ATP. Kinase inhibitors based upon adenosine mimesis may thus also target NAD+-dependent enzymes. We present a systematic approach using adenosine mimics from one cofactor class (kinase inhibitors) as a viable method to generate new lead structures in another cofactor class (sirtuin inhibitors). Our findings have broad implications for medicinal chemistry and specifically for sirtuin inhibitor design. Our results also raise a question as to whether selectivity profiling for kinase inhibitors should be limited to ATP-dependent targets.

Treatment with NAD(+) inhibited experimental autoimmune encephalomyelitis by activating AMPK/SIRT1 signaling pathway and modulating Th1/Th17 immune responses in mice.

PubMed

Wang, Jueqiong; Zhao, Congying; Kong, Peng; Sun, Huanhuan; Sun, Zhe; Bian, Guanyun; Sun, Yafei; Guo, Li

2016-10-01

Nicotinamide adenine dinucleotide (NAD(+)) plays vital roles in mitochondrial functions, cellular energy metabolism and calcium homeostasis. In this study, we investigated the effect of NAD(+) administration for the treatment of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. EAE, a classical animal model of multiple sclerosis (MS), was induced by subcutaneous injection of myelin oligodendrocyteglycoprotein (MOG). The mice were treated with 250mg/kg (body weight) NAD(+) in PBS administered intraperitoneally once daily. We observed that NAD(+) treatment could lessen the severity of EAE. Additionally, NAD(+) treatment attenuated pathological injuries of EAE mice. We also found that the AMP-activated protein kinase (AMPK)/silent mating-type information regulation 2 homolog 1(SIRT1) pathway was activated in the NAD(+)-treated mice and NAD(+) treatment suppressed pro-inflammatory T cell responses. Our findings demonstrated that NAD(+) could be an effective and promising agent to treat multiple sclerosis and its effects on other autoimmune diseases should be explored. Copyright © 2016 Elsevier B.V. All rights reserved.

Converting NADH to NAD+ by nicotinamide nucleotide transhydrogenase as a novel strategy against mitochondrial pathologies during aging.

PubMed

Olgun, Abdullah

2009-08-01

Mitochondrial DNA defects are involved supposedly via free radicals in many pathologies including aging and cancer. But, interestingly, free radical production was not found increased in prematurely aging mice having higher mutation rate in mtDNA. Therefore, some other mechanisms like the increase of mitochondrial NADH/NAD(+) and ubiquinol/ubiquinone ratios, can be in action in respiratory chain defects. NADH/NAD(+) ratio can be normalized by the activation or overexpression of nicotinamide nucleotide transhydrogenase (NNT), a mitochondrial enzyme catalyzing the following very important reaction: NADH + NADP(+ )<--> NADPH + NAD(+). The products NAD(+) and NADPH are required in many critical biological processes, e.g., NAD(+) is used by histone deacetylase Sir2 which regulates longevity in different species. NADPH is used in a number of biosynthesis reactions (e.g., reduced glutathione synthesis), and processes like apoptosis. Increased ubiquinol/ubiquinone ratio interferes the function of dihydroorotate dehydrogenase, the only mitochondrial enzyme involved in ubiquinone mediated de novo pyrimidine synthesis. Uridine and its prodrug triacetyluridine are used to compensate pyrimidine deficiency but their bioavailability is limited. Therefore, the normalization of the ubiquinol/ubiquinone ratio can be accomplished by allotopic expression of alternative oxidase, a mitochondrial ubiquinol oxidase which converts ubiquinol to ubiquinone.

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising "Hot-Spot" Lysine Residues.

PubMed

van den Biggelaar, Maartje; Madsen, Jesper J; Faber, Johan H; Zuurveld, Marleen G; van der Zwaan, Carmen; Olsen, Ole H; Stennicke, Henning R; Mertens, Koen; Meijer, Alexander B

2015-07-03

Lysine residues are implicated in driving the ligand binding to the LDL receptor family. However, it has remained unclear how specificity is regulated. Using coagulation factor VIII as a model ligand, we now study the contribution of individual lysine residues in the interaction with the largest member of the LDL receptor family, low-density lipoprotein receptor-related protein (LRP1). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and SPR interaction analysis on a library of lysine replacement variants as two independent approaches, we demonstrate that the interaction between factor VIII (FVIII) and LRP1 occurs over an extended surface containing multiple lysine residues. None of the individual lysine residues account completely for LRP1 binding, suggesting an additive binding model. Together with structural docking studies, our data suggest that FVIII interacts with LRP1 via an extended surface of multiple lysine residues that starts at the bottom of the C1 domain and winds around the FVIII molecule. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Transcriptional regulation of NAD metabolism in bacteria: genomic reconstruction of NiaR (YrxA) regulon

PubMed Central

Rodionov, Dmitry A.; Li, Xiaoqing; Rodionova, Irina A.; Yang, Chen; Sorci, Leonardo; Dervyn, Etienne; Martynowski, Dariusz; Zhang, Hong; Gelfand, Mikhail S.; Osterman, Andrei L.

2008-01-01

A comparative genomic approach was used to reconstruct transcriptional regulation of NAD biosynthesis in bacteria containing orthologs of Bacillus subtilis gene yrxA, a previously identified niacin-responsive repressor of NAD de novo synthesis. Members of YrxA family (re-named here NiaR) are broadly conserved in the Bacillus/Clostridium group and in the deeply branching Fusobacteria and Thermotogales lineages. We analyzed upstream regions of genes associated with NAD biosynthesis to identify candidate NiaR-binding DNA motifs and assess the NiaR regulon content in these species. Representatives of the two distinct types of candidate NiaR-binding sites, characteristic of the Firmicutes and Thermotogales, were verified by an electrophoretic mobility shift assay. In addition to transcriptional control of the nadABC genes, the NiaR regulon in some species extends to niacin salvage (the pncAB genes) and includes uncharacterized membrane proteins possibly involved in niacin transport. The involvement in niacin uptake proposed for one of these proteins (re-named NiaP), encoded by the B. subtilis gene yceI, was experimentally verified. In addition to bacteria, members of the NiaP family are conserved in multicellular eukaryotes, including human, pointing to possible NaiP involvement in niacin utilization in these organisms. Overall, the analysis of the NiaR and NrtR regulons (described in the accompanying paper) revealed mechanisms of transcriptional regulation of NAD metabolism in nearly a hundred diverse bacteria. PMID:18276644

Computational Prediction of Hot Spot Residues

PubMed Central

Morrow, John Kenneth; Zhang, Shuxing

2013-01-01

Most biological processes involve multiple proteins interacting with each other. It has been recently discovered that certain residues in these protein-protein interactions, which are called hot spots, contribute more significantly to binding affinity than others. Hot spot residues have unique and diverse energetic properties that make them challenging yet important targets in the modulation of protein-protein complexes. Design of therapeutic agents that interact with hot spot residues has proven to be a valid methodology in disrupting unwanted protein-protein interactions. Using biological methods to determine which residues are hot spots can be costly and time consuming. Recent advances in computational approaches to predict hot spots have incorporated a myriad of features, and have shown increasing predictive successes. Here we review the state of knowledge around protein-protein interactions, hot spots, and give an overview of multiple in silico prediction techniques of hot spot residues. PMID:22316154

Coulometric determination of NAD+ and NADH in normal and cancer cells using LDH, RVC and a polymer mediator.

PubMed

Torabi, F; Ramanathan, K; Larsson, P O; Gorton, L; Svanberg, K; Okamoto, Y; Danielsson, B; Khayyami, M

1999-11-15

An electrochemical method for the measurement of NAD(+) and NADH in normal and cancer tissues using flow injection analysis (FIA) is reported. Reticulated vitreous carbon (RVC) electrodes with entrapped l-lactate dehydrogenase (LDH) and a new redox polymer containing covalently bound toluidine blue O (TBO) were employed for this purpose. Both NAD(+) and NADH were estimated coulometrically based on their reaction with LDH. The latter was immobilized on controlled pore glass (CPG) by cross-linking with glutaraldehyde and packed within the RVC. The concentrations of NAD(+) and NADH in the tissues, estimated using different electron mediators such as ferricyanide (FCN), meldola blue (MB) and TBO have also been compared. The effects of flow rate, pH, applied potential (versus Ag/AgCl reference) and adsorption of the mediators have also been investigated. Based on the measurements of NAD(+) and NADH in normal and cancer tissues it has been concluded that the NADH concentration is lower, while the NAD(+) concentration is higher in cancer tissues. Amongst the electron mediators TBO was found to be a more stable mediator for such measurements.

Chemically engineered papain as artificial formate dehydrogenase for NAD(P)H regeneration.

PubMed

Haquette, Pierre; Talbi, Barisa; Barilleau, Laure; Madern, Nathalie; Fosse, Céline; Salmain, Michèle

2011-08-21

Organometallic complexes of the general formula [(η(6)-arene)Ru(N⁁N)Cl](+) and [(η(5)-Cp*)Rh(N⁁N)Cl](+) where N⁁N is a 2,2'-dipyridylamine (DPA) derivative carrying a thiol-targeted maleimide group, 2,2'-bispyridyl (bpy), 1,10-phenanthroline (phen) or ethylenediamine (en) and arene is benzene, 2-chloro-N-[2-(phenyl)ethyl]acetamide or p-cymene were identified as catalysts for the stereoselective reduction of the enzyme cofactors NAD(P)(+) into NAD(P)H with formate as a hydride donor. A thorough comparison of their effectiveness towards NAD(+) (expressed as TOF) revealed that the Rh(III) complexes were much more potent catalysts than the Ru(II) complexes. Within the Ru(II) complex series, both the N⁁N and arene ligands forming the coordination sphere had a noticeable influence on the activity of the complexes. Covalent anchoring of the maleimide-functionalized Ru(II) and Rh(III) complexes to the cysteine endoproteinase papain yielded hybrid metalloproteins, some of them displaying formate dehydrogenase activity with potentially interesting kinetic parameters.

Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

NASA Astrophysics Data System (ADS)

Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

2015-05-01

Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs.

Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

PubMed Central

Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

2015-01-01

Abstract. Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs. PMID:25688541

A LED-based method for monitoring NAD(P)H and FAD fluorescence in cell cultures and brain slices.

PubMed

Rösner, Jörg; Liotta, Agustin; Schmitz, Dietmar; Heinemann, Uwe; Kovács, Richard

2013-01-30

Nicotinamide- and flavine-adenine-dinucleotides (NAD(P)H and FADH₂) are electron carriers involved in cellular energy metabolism and in a multitude of enzymatic processes. As reduced NAD(P)H and oxidised FAD molecules are fluorescent, changes in tissue auto-fluorescence provide valuable information on the cellular redox state and energy metabolism. Since fluorescence excitation, by mercury arc lamps (HBO) is inherently coupled to photo-bleaching and photo-toxicity, microfluorimetric monitoring of energy metabolism might benefit from the replacement of HBO lamps by light emitting diodes (LEDs). Here we describe a LED-based custom-built setup for monitoring NAD(P)H and FAD fluorescence at the level of single cells (HEK293) and of brain slices. We compared NAD(P)H bleaching characteristics with two light sources (HBO lamp and LED) as well as sensitivity and signal to noise ratio of three different detector types (multi-pixel photon counter (MPPC), photomultiplier tube (PMT) and photodiode). LED excitation resulted in reduced photo-bleaching at the same fluorescence output in comparison to excitation with the HBO lamp. Transiently increasing LED power resulted in reversible bleaching of NAD(P)H fluorescence. Recovery kinetics were dependent on metabolic substrates indicating coupling of NAD(P)H fluorescence to metabolism. Electrical stimulation of brain slices induced biphasic redox changes, as indicated by NAD(P)H/FAD fluorescence transients. Increasing the gain of PMT and decreasing the LED power resulted in similar sensitivity as obtained with the MPPC and the photodiode, without worsening the signal to noise ratio. In conclusion, replacement of HBO lamp with LED might improve conventional PMT based microfluorimetry of tissue auto-fluorescence. Copyright © 2012 Elsevier B.V. All rights reserved.

A Key Enzyme of the NAD+ Salvage Pathway in Thermus thermophilus: Characterization of Nicotinamidase and the Impact of Its Gene Deletion at High Temperatures

PubMed Central

Taniguchi, Hironori; Sungwallek, Sathidaphorn; Chotchuang, Phatcharin; Okano, Kenji

2017-01-01

ABSTRACT NAD (NAD+) is a cofactor related to many cellular processes. This cofactor is known to be unstable, especially at high temperatures, where it chemically decomposes to nicotinamide and ADP-ribose. Bacteria, yeast, and higher organisms possess the salvage pathway for reconstructing NAD+ from these decomposition products; however, the importance of the salvage pathway for survival is not well elucidated, except for in pathogens lacking the NAD+ de novo synthesis pathway. Herein, we report the importance of the NAD+ salvage pathway in the thermophilic bacterium Thermus thermophilus HB8 at high temperatures. We identified the gene encoding nicotinamidase (TTHA0328), which catalyzes the first reaction of the NAD+ salvage pathway. This recombinant enzyme has a high catalytic activity against nicotinamide (Km of 17 μM, kcat of 50 s−1, kcat/Km of 3.0 × 103 s−1 · mM−1). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of T. thermophilus and disrupted the NAD+ salvage pathway in T. thermophilus. Disruption of the salvage pathway led to the severe growth retardation at a higher temperature (80°C), owing to the drastic decrease in the intracellular concentrations of NAD+ and NADH. IMPORTANCE NAD+ and other nicotinamide cofactors are essential for cell metabolism. These molecules are unstable and decompose, even under the physiological conditions in most organisms. Thermophiles can survive at high temperatures where NAD+ decomposition is, in general, more rapid. This study emphasizes that NAD+ instability and its homeostasis can be one of the important factors for thermophile survival in extreme temperatures. PMID:28630126

Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio.

PubMed

Titov, Denis V; Cracan, Valentin; Goodman, Russell P; Peng, Jun; Grabarek, Zenon; Mootha, Vamsi K

2016-04-08

A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD(+)) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD(+)/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD(+)/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state. Copyright © 2016, American Association for the Advancement of Science.

Catalases Are NAD(P)H-Dependent Tellurite Reductases

PubMed Central

Calderón, Iván L.; Arenas, Felipe A.; Pérez, José Manuel; Fuentes, Derie E.; Araya, Manuel A.; Saavedra, Claudia P.; Tantaleán, Juan C.; Pichuantes, Sergio E.; Youderian, Philip A.; Vásquez, Claudio C.

2006-01-01

Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO3 2−) to the less toxic, insoluble metal, tellurium (Te°), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical. PMID:17183702

Catalases are NAD(P)H-dependent tellurite reductases.

PubMed

Calderón, Iván L; Arenas, Felipe A; Pérez, José Manuel; Fuentes, Derie E; Araya, Manuel A; Saavedra, Claudia P; Tantaleán, Juan C; Pichuantes, Sergio E; Youderian, Philip A; Vásquez, Claudio C

2006-12-20

Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO(3)(2-)) to the less toxic, insoluble metal, tellurium (Te(o)), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical.

Malate decarboxylases: evolution and roles of NAD(P)-ME isoforms in species performing C(4) and C(3) photosynthesis.

PubMed

Maier, Alexandra; Zell, Martina B; Maurino, Veronica G

2011-05-01

In the C(4) pathway of photosynthesis two types of malate decarboxylases release CO(2) in bundle sheath cells, NADP- and NAD-dependent malic enzyme (NADP-ME and NAD-ME), located in the chloroplasts and the mitochondria of these cells, respectively. The C(4) decarboxylases involved in C(4) photosynthesis did not evolve de novo; they were recruited from existing housekeeping isoforms. NADP-ME housekeeping isoforms would function in the control of malate levels during hypoxia, pathogen defence responses, and microspore separation, while NAD-ME participates in the respiration of malate in the tricarboxylic acid cycle. Recently, the existence of three enzymatic NAD-ME entities in Arabidopsis, occurring by alternative association of two subunits, was described as a novel mechanism to regulate NAD-ME activity under changing metabolic environments. The C(4) NADP-ME is thought to have evolved from a C(3) chloroplastic ancestor, which in turn would have evolved from an ancient cytosolic enzyme. In this way, the C(4) NADP-ME would have emerged through gene duplication, acquisition of a new promoter, and neo-functionalization. In contrast, there would exist a unique NAD-ME in C(4) plants, which would have been adapted to perform a dual function through changes in the kinetic and regulatory properties of the C(3) ancestors. In addition to this, for the evolution of C(4) NAD-ME, insertion of promoters or enhancers into the single-copy genes of the C(3) ancestors would have changed the expression without gene duplication.

A Key Enzyme of the NAD+ Salvage Pathway in Thermus thermophilus: Characterization of Nicotinamidase and the Impact of Its Gene Deletion at High Temperatures.

PubMed

Taniguchi, Hironori; Sungwallek, Sathidaphorn; Chotchuang, Phatcharin; Okano, Kenji; Honda, Kohsuke

2017-09-01

NAD (NAD + ) is a cofactor related to many cellular processes. This cofactor is known to be unstable, especially at high temperatures, where it chemically decomposes to nicotinamide and ADP-ribose. Bacteria, yeast, and higher organisms possess the salvage pathway for reconstructing NAD + from these decomposition products; however, the importance of the salvage pathway for survival is not well elucidated, except for in pathogens lacking the NAD + de novo synthesis pathway. Herein, we report the importance of the NAD + salvage pathway in the thermophilic bacterium Thermus thermophilus HB8 at high temperatures. We identified the gene encoding nicotinamidase (TTHA0328), which catalyzes the first reaction of the NAD + salvage pathway. This recombinant enzyme has a high catalytic activity against nicotinamide ( K m of 17 μM, k cat of 50 s -1 , k cat / K m of 3.0 × 10 3 s -1 · mM -1 ). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of T. thermophilus and disrupted the NAD + salvage pathway in T. thermophilus Disruption of the salvage pathway led to the severe growth retardation at a higher temperature (80°C), owing to the drastic decrease in the intracellular concentrations of NAD + and NADH. IMPORTANCE NAD + and other nicotinamide cofactors are essential for cell metabolism. These molecules are unstable and decompose, even under the physiological conditions in most organisms. Thermophiles can survive at high temperatures where NAD + decomposition is, in general, more rapid. This study emphasizes that NAD + instability and its homeostasis can be one of the important factors for thermophile survival in extreme temperatures. Copyright © 2017 American Society for Microbiology.

Cofactor engineering to regulate NAD+/NADH ratio with its application to phytosterols biotransformation.

PubMed

Su, Liqiu; Shen, Yanbing; Zhang, Wenkai; Gao, Tian; Shang, Zhihua; Wang, Min

2017-10-30

Cofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD + ) metabolism, which results in a significantly altered spectrum of metabolic products. Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by Mycobacterium. Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD + -dependent process, this work utilized cofactor engineering in Mycobacterium neoaurum and investigated the effect on cofactor and phytosterols metabolism. Through the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD + /NADH ratio and the AD (D) production of M. neoaurum TCCC 11978 (MNR M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of MNR cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from Lactobacillus brevis, were successfully overexpressed and heterologously expressed in MNR M3 to improve the intracellular ratio of NAD + /NADH. After 96 h of cultivation, the expression of these two enzymes in MNR M3 resulted in the decrease in intracellular NADH level (by 51 and 67%, respectively) and the increase in NAD + /NADH ratio (by 113 and 192%, respectively). Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D

The mechanism of RNA 5' capping with NAD +, NADH and desphospho-CoA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bird, Jeremy G.; Zhang, Yu; Tian, Yuan

The chemical nature of the 5' end of RNA is a key determinant of RNA stability, processing, localization and translation efficiency and has been proposed to provide a layer of ‘epitranscriptomic’ gene regulation. Recently it has been shown that some bacterial RNA species carry a 5'-end structure reminiscent of the 5' 7-methylguanylate ‘cap’ in eukaryotic RNA. In particular, RNA species containing a 5'-end nicotinamide adenine dinucleotide (NAD+) or 3'-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH) and dpCoA caps are added to RNA after transcription initiation, inmore » a manner analogous to the addition of 7-methylguanylate caps. Here we show instead that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated ‘ab initio capping’ may occur in all organisms.« less

Purification of NAD glycohydrolase from Agkistrodon acutus venom.

PubMed

Wu, Shuang Ding; Liu, Yanli; Xu, Xiaolong; Zhu, Zhengang

2002-07-01

NAD glycohydrolase (NADase) from Agkistrodon acutus venom was purified to electrophoretic homogeneity by a fast, reproducible 3-step procedure including Q Sepharose Fast Flow, Superdex 75, and Mono S column chromatography. This new procedure gave a 15.6-fold purification with a recovery yield of 7.9% and a specific activity of 12.8 units/mg.

Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD(+) biosynthetic machinery in mammalian cells.

PubMed

Zamporlini, Federica; Ruggieri, Silverio; Mazzola, Francesca; Amici, Adolfo; Orsomando, Giuseppe; Raffaelli, Nadia

2014-11-01

The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma. © 2014 FEBS.

NMR analysis of cross strand aromatic interactions in an 8 residue hairpin and a 14 residue three stranded β-sheet peptide.

PubMed

Sonti, Rajesh; Rai, Rajkishor; Ragothama, Srinivasarao; Balaram, Padmanabhan

2012-12-13

Cross strand aromatic interactions between a facing pair of phenylalanine residues in antiparallel β-sheet structures have been probed using two structurally defined model peptides. The octapeptide Boc-LFV(D)P(L)PLFV-OMe (peptide 1) favors the β-hairpin conformation nucleated by the type II' β-turn formed by the (D)Pro-(L)Pro segment, placing Phe2 and Phe7 side chains in proximity. Two centrally positioned (D)Pro-(L)Pro segments facilitate the three stranded β-sheet formation in the 14 residue peptide Boc-LFV(D)P(L)PLFVA(D)P(L)PLFV-OMe (peptide 2) in which the Phe2/Phe7 orientations are similar to that in the octapeptide. The anticipated folded conformations of peptides 1 and 2 are established by the delineation of intramolecularly hydrogen bonded NH groups and by the observation of specific cross strand NOEs. The observation of ring current shifted aromatic protons is a diagnostic of close approach of the Phe2 and Phe7 side chains. Specific assignment of aromatic proton resonances using HSQC and HSQC-TOCSY methods allow an analysis of interproton NOEs between the spatially proximate aromatic rings. This approach facilitates specific assignments in systems containing multiple aromatic rings in spectra at natural abundance. Evidence is presented for a dynamic process which invokes a correlated conformational change about the C(α)-C(β)(χ(1)) bond for the pair of interacting Phe residues. NMR results suggest that aromatic ring orientations observed in crystals are maintained in solution. Anomalous temperature dependence of ring current induced proton chemical shifts suggests that solvophobic effects may facilitate aromatic ring clustering in apolar solvents.

NAD+ Is a Food Component That Promotes Exit from Dauer Diapause in Caenorhabditis elegans.

PubMed

Mylenko, Mykola; Boland, Sebastian; Penkov, Sider; Sampaio, Julio L; Lombardot, Benoit; Vorkel, Daniela; Verbavatz, Jean-Marc; Kurzchalia, Teymuras V

2016-01-01

The free-living soil nematode Caenorhabditis elegans adapts its development to the availability of food. When food is scarce and population density is high, worms enter a developmentally arrested non-feeding diapause stage specialized for long-term survival called the dauer larva. When food becomes available, they exit from the dauer stage, resume growth and reproduction. It has been postulated that compound(s) present in food, referred to as the "food signal", promote exit from the dauer stage. In this study, we have identified NAD+ as a component of bacterial extract that promotes dauer exit. NAD+, when dissolved in alkaline medium, causes opening of the mouth and ingestion of food. We also show that to initiate exit from the dauer stage in response to NAD+ worms require production of serotonin. Thus, C. elegans can use redox cofactors produced by dietary organisms to sense food.

Substantial conformational change mediated by charge-triad residues of the death effector domain in protein-protein interactions.

PubMed

Twomey, Edward C; Cordasco, Dana F; Kozuch, Stephen D; Wei, Yufeng

2013-01-01

Protein conformational changes are commonly associated with the formation of protein complexes. The non-catalytic death effector domains (DEDs) mediate protein-protein interactions in a variety of cellular processes, including apoptosis, proliferation and migration, and glucose metabolism. Here, using NMR residual dipolar coupling (RDC) data, we report a conformational change in the DED of the phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) protein in the complex with a mitogen-activated protein (MAP) kinase, extracellular regulated kinase 2 (ERK2), which is essential in regulating ERK2 cellular distribution and function in cell proliferation and migration. The most significant conformational change in PEA-15 happens at helices α2, α3, and α4, which also possess the highest flexibility among the six-helix bundle of the DED. This crucial conformational change is modulated by the D/E-RxDL charge-triad motif, one of the prominent structural features of DEDs, together with a number of other electrostatic and hydrogen bonding interactions on the protein surface. Charge-triad motif promotes the optimal orientation of key residues and expands the binding interface to accommodate protein-protein interactions. However, the charge-triad residues are not directly involved in the binding interface between PEA-15 and ERK2.

β-Nicotinamide Adenine Dinucleotide (β-NAD) Inhibits ATP-Dependent IL-1β Release from Human Monocytic Cells.

PubMed

Hiller, Sebastian Daniel; Heldmann, Sarah; Richter, Katrin; Jurastow, Innokentij; Küllmar, Mira; Hecker, Andreas; Wilker, Sigrid; Fuchs-Moll, Gabriele; Manzini, Ivan; Schmalzing, Günther; Kummer, Wolfgang; Padberg, Winfried; McIntosh, J Michael; Damm, Jelena; Zakrzewicz, Anna; Grau, Veronika

2018-04-10

While interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1β maturation, is released from damaged cells along with β-nicotinamide adenine dinucleotide (β-NAD). Here, we tested the hypothesis that β-NAD controls ATP-signaling and, hence, IL-1β release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2'(3')- O -(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of β-NAD. IL-1β was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca 2+ -independent phospholipase A2 (iPLA2β, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous β-NAD signaled via P2Y receptors and dose-dependently (IC 50 = 15 µM) suppressed the BzATP-induced IL-1β release. Signaling involved iPLA2β, release of a soluble mediator, and nAChR subunit α9. Patch-clamp experiments revealed that β-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular β-NAD that suppresses ATP-induced release of IL-1β by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation.

Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus.

PubMed

Pennacchio, Angela; Pucci, Biagio; Secundo, Francesco; La Cara, Francesco; Rossi, Mosè; Raia, Carlo A

2008-07-01

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh(Tt)) was heterologously overexpressed in Escherichia coli, and the protein (ADH(Tt)) was purified to homogeneity and characterized. ADH(Tt) is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to approximately 73 degrees C and a 30-min half-inactivation temperature of approximately 90 degrees C, as well as good tolerance to common organic solvents. ADH(Tt) has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and alpha-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, alpha-tetralone, and alpha-methyl and alpha-ethyl benzoylformates to (S)-(-)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-alpha-(trifluoromethyl)benzyl alcohol (93% ee), (S)-alpha-tetralol (>99% ee), methyl (R)-(-)-mandelate (92% ee), and ethyl (R)-(-)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.

NAD(P)H:Flavin Mononucleotide Oxidoreductase Inactivation during 2,4,6-Trinitrotoluene Reduction

PubMed Central

Riefler, R. Guy; Smets, Barth F.

2002-01-01

Bacteria readily transform 2,4,6-trinitrotoluene (TNT), a contaminant frequently found at military bases and munitions production facilities, by reduction of the nitro group substituents. In this work, the kinetics of nitroreduction were investigated by using a model nitroreductase, NAD(P)H:flavin mononucleotide (FMN) oxidoreductase. Under mediation by NAD(P)H:FMN oxidoreductase, TNT rapidly reacted with NADH to form 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene, whereas 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were not produced. Progressive loss of activity was observed during TNT reduction, indicating inactivation of the enzyme during transformation. It is likely that a nitrosodinitrotoluene intermediate reacted with the NAD(P)H:FMN oxidoreductase, leading to enzyme inactivation. A half-maximum constant with respect to NADH, KN, of 394 μM was measured, indicating possible NADH limitation under typical cellular conditions. A mathematical model that describes the inactivation process and NADH limitation provided a good fit to TNT reduction profiles. This work represents the first step in developing a comprehensive enzyme level understanding of nitroarene biotransformation. PMID:11916686

NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis. Isolation, characterization and biochemical properties of the enzyme.

PubMed Central

Neuhauser, W; Haltrich, D; Kulbe, K D; Nidetzky, B

1997-01-01

During growth on d-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists ofa single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in d-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 microM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuis is not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5'-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase. PMID:9307017

Increase in furfural tolerance by combinatorial overexpression of NAD salvage pathway enzymes in engineered isobutanol-producing E. coli.

PubMed

Song, Hun-Suk; Jeon, Jong-Min; Kim, Hyun-Joong; Bhatia, Shashi Kant; Sathiyanarayanan, Ganesan; Kim, Junyoung; Won Hong, Ju; Gi Hong, Yoon; Young Choi, Kwon; Kim, Yun-Gon; Kim, Wooseong; Yang, Yung-Hun

2017-12-01

To reduce the furfural toxicity for biochemical production in E. coli, a new strategy was successfully applied by supplying NAD(P)H through the nicotine amide salvage pathway. To alleviate the toxicity, nicotinamide salvage pathway genes were overexpressed in recombinant, isobutanol-producing E. coli. Gene expression of pncB and nadE respectively showed increased tolerance to furfural among these pathways. The combined expression of pncB and nadE was the most effective in increasing the tolerance of the cells to toxic aldehydes. By comparing noxE- and fdh-harbouring strains, the form of NADH, rather than NAD + , was the major effector of furfural tolerance. Overall, this study is the application of the salvage pathway to isobutanol production in the presence of furfural, and this system seems to be applicable to alleviate furfural toxicity in the production of other biochemical. Copyright © 2017 Elsevier Ltd. All rights reserved.

AMPK activation protects cells from oxidative stress-induced senescence via autophagic flux restoration and intracellular NAD(+) elevation.

PubMed

Han, Xiaojuan; Tai, Haoran; Wang, Xiaobo; Wang, Zhe; Zhou, Jiao; Wei, Xiawei; Ding, Yi; Gong, Hui; Mo, Chunfen; Zhang, Jie; Qin, Jianqiong; Ma, Yuanji; Huang, Ning; Xiang, Rong; Xiao, Hengyi

2016-06-01

AMPK activation is beneficial for cellular homeostasis and senescence prevention. However, the molecular events involved in AMPK activation are not well defined. In this study, we addressed the mechanism underlying the protective effect of AMPK on oxidative stress-induced senescence. The results showed that AMPK was inactivated in senescent cells. However, pharmacological activation of AMPK by metformin and berberine significantly prevented the development of senescence and, accordingly, inhibition of AMPK by Compound C was accelerated. Importantly, AMPK activation prevented hydrogen peroxide-induced impairment of the autophagic flux in senescent cells, evidenced by the decreased p62 degradation, GFP-RFP-LC3 cancellation, and activity of lysosomal hydrolases. We also found that AMPK activation restored the NAD(+) levels in the senescent cells via a mechanism involving mostly the salvage pathway for NAD(+) synthesis. In addition, the mechanistic relationship of autophagic flux and NAD(+) synthesis and the involvement of mTOR and Sirt1 activities were assessed. In summary, our results suggest that AMPK prevents oxidative stress-induced senescence by improving autophagic flux and NAD(+) homeostasis. This study provides a new insight for exploring the mechanisms of aging, autophagy and NAD(+) homeostasis, and it is also valuable in the development of innovative strategies to combat aging. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Computational prediction of protein hot spot residues.

PubMed

Morrow, John Kenneth; Zhang, Shuxing

2012-01-01

Most biological processes involve multiple proteins interacting with each other. It has been recently discovered that certain residues in these protein-protein interactions, which are called hot spots, contribute more significantly to binding affinity than others. Hot spot residues have unique and diverse energetic properties that make them challenging yet important targets in the modulation of protein-protein complexes. Design of therapeutic agents that interact with hot spot residues has proven to be a valid methodology in disrupting unwanted protein-protein interactions. Using biological methods to determine which residues are hot spots can be costly and time consuming. Recent advances in computational approaches to predict hot spots have incorporated a myriad of features, and have shown increasing predictive successes. Here we review the state of knowledge around protein-protein interactions, hot spots, and give an overview of multiple in silico prediction techniques of hot spot residues.

Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings

NASA Technical Reports Server (NTRS)

Warner, R. L.; Huffaker, R. C.

1989-01-01

Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.

Identification of a novel pathway of transforming growth factor-β1 regulation by extracellular NAD+ in mouse macrophages: in vitro and in silico studies.

PubMed

Zamora, Ruben; Azhar, Nabil; Namas, Rajaie; Metukuri, Mallikarjuna R; Clermont, Thierry; Gladstone, Chase; Namas, Rami A; Hermus, Linda; Megas, Cristina; Constantine, Gregory; Billiar, Timothy R; Fink, Mitchell P; Vodovotz, Yoram

2012-09-07

Extracellular β-nicotinamide adenine dinucleotide (NAD(+)) is anti-inflammatory. We hypothesized that NAD(+) would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-β1. Indeed, NAD(+) led to increases in both active and latent cell-associated TGF-β1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD(+) acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca(2+). Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca(2+) ionophores recapitulated the effects of NAD(+) on TGF-β1, whereas the cADPR antagonist 8-Br-cADPR, Ca(2+) chelation, and antagonism of L-type Ca(2+) channels suppressed these effects. The time and dose effects of NAD(+) on TGF-β1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-β1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-β1 by NAD(+). Thus, in vitro and in silico evidence points to NAD(+) as a novel modulator of TGF-β1.

Effect of charged amino acid side chain length on lateral cross-strand interactions between carboxylate- and guanidinium-containing residues in a β-hairpin.

PubMed

Kuo, Hsiou-Ting; Liu, Shing-Lung; Chiu, Wen-Chieh; Fang, Chun-Jen; Chang, Hsien-Chen; Wang, Wei-Ren; Yang, Po-An; Li, Jhe-Hao; Huang, Shing-Jong; Huang, Shou-Ling; Cheng, Richard P

2015-05-01

β-Sheet is one of the major protein secondary structures. Oppositely charged residues are frequently observed across neighboring strands in antiparallel sheets, suggesting the importance of cross-strand ion pairing interactions. The charged amino acids Asp, Glu, Arg, and Lys have different numbers of hydrophobic methylenes linking the charged functionality to the backbone. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on lateral cross-strand ion pairing interactions at non-hydrogen-bonded positions, β-hairpin peptides containing Zbb-Agx (Zbb = Asp, Glu, Aad in increasing length; Agx = Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by NMR methods. The fraction folded population and folding energy were derived from the chemical shift deviation data. Peptides with high fraction folded populations involved charged residue side chain lengths that supported high strand propensity. Double mutant cycle analysis was used to determine the interaction energy for the potential lateral ion pairs. Minimal interaction was observed between residues with short side chains, most likely due to the diffused positive charge on the guanidinium group, which weakened cross-strand electrostatic interactions with the carboxylate side chain. Only the Aad-Arg/Agh interactions with long side chains clearly exhibited stabilizing energetics, possibly relying on hydrophobics. A survey of a non-redundant protein structure database revealed that the statistical sheet pair propensity followed the trend Asp-Arg < Glu-Arg, implying the need for matching long side chains. This suggested the need for long side chains on both guanidinium-bearing and carboxylate-bearing residues to stabilize the β-hairpin motif.

Prediction of fatty acid-binding residues on protein surfaces with three-dimensional probability distributions of interacting atoms.

PubMed

Mahalingam, Rajasekaran; Peng, Hung-Pin; Yang, An-Suei

2014-08-01

Protein-fatty acid interaction is vital for many cellular processes and understanding this interaction is important for functional annotation as well as drug discovery. In this work, we present a method for predicting the fatty acid (FA)-binding residues by using three-dimensional probability density distributions of interacting atoms of FAs on protein surfaces which are derived from the known protein-FA complex structures. A machine learning algorithm was established to learn the characteristic patterns of the probability density maps specific to the FA-binding sites. The predictor was trained with five-fold cross validation on a non-redundant training set and then evaluated with an independent test set as well as on holo-apo pair's dataset. The results showed good accuracy in predicting the FA-binding residues. Further, the predictor developed in this study is implemented as an online server which is freely accessible at the following website, http://ismblab.genomics.sinica.edu.tw/. Copyright © 2014 Elsevier B.V. All rights reserved.

NNMT activation can contribute to the development of fatty liver disease by modulating the NAD + metabolism.

PubMed

Komatsu, Motoaki; Kanda, Takeshi; Urai, Hidenori; Kurokochi, Arata; Kitahama, Rina; Shigaki, Shuhei; Ono, Takashi; Yukioka, Hideo; Hasegawa, Kazuhiro; Tokuyama, Hirobumi; Kawabe, Hiroshi; Wakino, Shu; Itoh, Hiroshi

2018-06-05

Nicotinamide N-methyltransferase (NNMT) catalyses the reaction between nicotinamide (NAM) and S-adenosylmethionine to produce 1-methylnicotinamide and S-adenosylhomocysteine. Recently, this enzyme has also been reported to modulate hepatic nutrient metabolism, but its role in the liver has not been fully elucidated. We developed transgenic mice overexpressing NNMT to elucidate its role in hepatic nutrient metabolism. When fed a high fat diet containing NAM, a precursor for nicotinamide adenine dinucleotide (NAD) + , these NNMT-overexpressing mice exhibit fatty liver deterioration following increased expression of the genes mediating fatty acid uptake and decreased very low-density lipoprotein secretion. NNMT overactivation decreased the NAD + content in the liver and also decreased gene activity related to fatty acid oxidation by inhibiting NAD + -dependent deacetylase Sirt3 function. Moreover, the transgenic mice showed liver fibrosis, with the induction of inflammatory and fibrosis genes. Induced NNMT expression decreased the tissue methylation capacity, thereby reducing methylation of the connective tissue growth factor (CTGF) gene promoter, resulting in increased CTGF expression. These data indicate that NNMT links the NAD + and methionine metabolic pathways and promotes liver steatosis and fibrosis. Therefore, targeting NNMT may serve as a therapeutic strategy for treating fatty liver and fibrosis.

DNA repair deficiency sensitizes lung cancer cells to NAD+ biosynthesis blockade.

PubMed

Touat, Mehdi; Sourisseau, Tony; Dorvault, Nicolas; Chabanon, Roman M; Garrido, Marlène; Morel, Daphné; Krastev, Dragomir B; Bigot, Ludovic; Adam, Julien; Frankum, Jessica R; Durand, Sylvère; Pontoizeau, Clement; Souquère, Sylvie; Kuo, Mei-Shiue; Sauvaigo, Sylvie; Mardakheh, Faraz; Sarasin, Alain; Olaussen, Ken A; Friboulet, Luc; Bouillaud, Frédéric; Pierron, Gérard; Ashworth, Alan; Lombès, Anne; Lord, Christopher J; Soria, Jean-Charles; Postel-Vinay, Sophie

2018-04-02

Synthetic lethality is an efficient mechanism-based approach to selectively target DNA repair defects. Excision repair cross-complementation group 1 (ERCC1) deficiency is frequently found in non-small-cell lung cancer (NSCLC), making this DNA repair protein an attractive target for exploiting synthetic lethal approaches in the disease. Using unbiased proteomic and metabolic high-throughput profiling on a unique in-house-generated isogenic model of ERCC1 deficiency, we found marked metabolic rewiring of ERCC1-deficient populations, including decreased levels of the metabolite NAD+ and reduced expression of the rate-limiting NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). We also found reduced NAMPT expression in NSCLC samples with low levels of ERCC1. These metabolic alterations were a primary effect of ERCC1 deficiency, and caused selective exquisite sensitivity to small-molecule NAMPT inhibitors, both in vitro - ERCC1-deficient cells being approximately 1,000 times more sensitive than ERCC1-WT cells - and in vivo. Using transmission electronic microscopy and functional metabolic studies, we found that ERCC1-deficient cells harbor mitochondrial defects. We propose a model where NAD+ acts as a regulator of ERCC1-deficient NSCLC cell fitness. These findings open therapeutic opportunities that exploit a yet-undescribed nuclear-mitochondrial synthetic lethal relationship in NSCLC models, and highlight the potential for targeting DNA repair/metabolic crosstalks for cancer therapy.

Regulation of SIRT 1 mediated NAD dependent deacetylation: A novel role for the multifunctional enzyme CD38

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aksoy, Pinar; Escande, Carlos; Seccion Biologia Celular, Facultad de Ciencias, Universidad de la Republica, Igua 4225, Montevideo

2006-10-13

The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1more » enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes.« less

Rosuvastatin prevents angiotensin II-induced vascular changes by inhibition of NAD(P)H oxidase and COX-1

PubMed Central

Colucci, Rocchina; Fornai, Matteo; Duranti, Emiliano; Antonioli, Luca; Rugani, Ilaria; Aydinoglu, Fatma; Ippolito, Chiara; Segnani, Cristina; Bernardini, Nunzia; Taddei, Stefano; Blandizzi, Corrado; Virdis, Agostino

2013-01-01

Background and Purpose NAD(P)H oxidase and COX-1 participate in vascular damage induced by angiotensin II. We investigated the effect of rosuvastatin on endothelial dysfunction, vascular remodelling, changes in extracellular matrix components and mechanical properties of small mesenteric arteries from angiotensin II-infused rats. Experimental Approach Male rats received angiotensin II (120 ng·kg−1·min−1, subcutaneously) for 14 days with or without rosuvastatin (10 mg·kg−1·day−1, oral gavage) or vehicle. Vascular functions and morphological parameters were assessed by pressurized myography. Key Results In angiotensin II-infused rats, ACh-induced relaxation was attenuated compared with controls, less sensitive to L-NAME, enhanced by SC-560 (COX-1 inhibitor) or SQ-29548 (prostanoid TP receptor antagonist), and normalized by the antioxidant ascorbic acid or NAD(P)H oxidase inhibitors. After rosuvastatin, relaxations to ACh were normalized, fully sensitive to L-NAME, and no longer affected by SC-560, SQ-29548 or NAD(P)H oxidase inhibitors. Angiotensin II enhanced intravascular superoxide generation, eutrophic remodelling, collagen and fibronectin depositions, and decreased elastin content, resulting in increased vessel stiffness. All these changes were prevented by rosuvastatin. Angiotensin II increased phosphorylation of NAD(P)H oxidase subunit p47phox and its binding to subunit p67phox, effects inhibited by rosuvastatin. Rosuvastatin down-regulated vascular Nox4/NAD(P)H isoform and COX-1 expression, attenuated the vascular release of 6-keto-PGF1α, and enhanced copper/zinc-superoxide dismutase expression. Conclusion and Implications Rosuvastatin prevents angiotensin II-induced alterations in resistance arteries in terms of function, structure, mechanics and composition. These effects depend on restoration of NO availability, prevention of NAD(P)H oxidase-derived oxidant excess, reversal of COX-1 induction and its prostanoid production, and stimulation of

The SARM1 Toll/Interleukin-1 Receptor Domain Possesses Intrinsic NAD+ Cleavage Activity that Promotes Pathological Axonal Degeneration.

PubMed

Essuman, Kow; Summers, Daniel W; Sasaki, Yo; Mao, Xianrong; DiAntonio, Aaron; Milbrandt, Jeffrey

2017-03-22

Axonal degeneration is an early and prominent feature of many neurological disorders. SARM1 is the central executioner of the axonal degeneration pathway that culminates in depletion of axonal NAD + , yet the identity of the underlying NAD + -depleting enzyme(s) is unknown. Here, in a series of experiments using purified proteins from mammalian cells, bacteria, and a cell-free protein translation system, we show that the SARM1-TIR domain itself has intrinsic NADase activity-cleaving NAD + into ADP-ribose (ADPR), cyclic ADPR, and nicotinamide, with nicotinamide serving as a feedback inhibitor of the enzyme. Using traumatic and vincristine-induced injury models in neurons, we demonstrate that the NADase activity of full-length SARM1 is required in axons to promote axonal NAD + depletion and axonal degeneration after injury. Hence, the SARM1 enzyme represents a novel therapeutic target for axonopathies. Moreover, the widely utilized TIR domain is a protein motif that can possess enzymatic activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Coiled-coil destabilizing residues in the group A Streptococcus M1 protein are required for functional interaction.

PubMed

Stewart, Chelsea M; Buffalo, Cosmo Z; Valderrama, J Andrés; Henningham, Anna; Cole, Jason N; Nizet, Victor; Ghosh, Partho

2016-08-23

The sequences of M proteins, the major surface-associated virulence factors of the widespread bacterial pathogen group A Streptococcus, are antigenically variable but have in common a strong propensity to form coiled coils. Paradoxically, these sequences are also replete with coiled-coil destabilizing residues. These features are evident in the irregular coiled-coil structure and thermal instability of M proteins. We present an explanation for this paradox through studies of the B repeats of the medically important M1 protein. The B repeats are required for interaction of M1 with fibrinogen (Fg) and consequent proinflammatory activation. The B repeats sample multiple conformations, including intrinsically disordered, dissociated, as well as two alternate coiled-coil conformations: a Fg-nonbinding register 1 and a Fg-binding register 2. Stabilization of M1 in the Fg-nonbinding register 1 resulted in attenuation of Fg binding as expected, but counterintuitively, so did stabilization in the Fg-binding register 2. Strikingly, these register-stabilized M1 proteins gained the ability to bind Fg when they were destabilized by a chaotrope. These results indicate that M1 stability is antithetical to Fg interaction and that M1 conformational dynamics, as specified by destabilizing residues, are essential for interaction. A "capture-and-collapse" model of association accounts for these observations, in which M1 captures Fg through a dynamic conformation and then collapses into a register 2-coiled coil as a result of stabilization provided by binding energy. Our results support the general conclusion that destabilizing residues are evolutionarily conserved in M proteins to enable functional interactions necessary for pathogenesis.

Synergistic Activity between Two Antifungal Proteins, the Plant Defensin NaD1 and the Bovine Pancreatic Trypsin Inhibitor

PubMed Central

Dawson, Charlotte S.; McKenna, James A.; Quimbar, Pedro; Hayes, Brigitte M. E.; van der Weerden, Nicole L.

2017-01-01

ABSTRACT Defensins are a large family of small, cationic, cysteine-rich proteins that are part of the defense arsenal that plants use for protection against potentially damaging fungal infections. The plant defensin NaD1 from Nicotiana alata is a potent antifungal protein that inhibits growth and kills a variety of fungal pathogens that affect both plant and animal (human) hosts. Some serine protease inhibitors have also been reported to be antifungal molecules, while others have no inhibitory activity against fungi. Here we describe the synergistic activity of the plant defensin NaD1 with a selection of serine protease inhibitors against the plant pathogens Fusarium graminearum and Colletotrichum graminicola and the animal pathogen Candida albicans. The synergistic activity was not related to the protease inhibitory activity of these molecules but may arise from activation of fungal stress response pathways. The bovine pancreatic trypsin inhibitor (BPTI) displayed the most synergy with NaD1. BPTI also acted synergistically with several other antifungal molecules. The observation that NaD1 acts synergistically with protease inhibitors provides the foundation for the design of transgenic plants with improved resistance to fungal disease. It also supports the possibility of naturally occurring accessory factors that function to enhance the activity of innate immunity peptides in biological systems. IMPORTANCE This work describes the increased activity of a natural antifungal peptide in the presence of another antifungal peptide from a different family. This is termed antifungal synergy. Synergy is important for decreasing the amount of antifungal molecule needed to control the disease. Traditionally, naturally occurring antifungal molecules are assayed in isolation. Identification of synergistic interactions between antifungal peptides means that their activities in a complex biological system are likely to be different from what we observe when examining them

On tide-induced Lagrangian residual current and residual transport: 1. Lagrangian residual current

USGS Publications Warehouse

Feng, Shizuo; Cheng, Ralph T.; Pangen, Xi

1986-01-01

Residual currents in tidal estuaries and coastal embayments have been recognized as fundamental factors which affect the long-term transport processes. It has been pointed out by previous studies that it is more relevant to use a Lagrangian mean velocity than an Eulerian mean velocity to determine the movements of water masses. Under weakly nonlinear approximation, the parameter k, which is the ratio of the net displacement of a labeled water mass in one tidal cycle to the tidal excursion, is assumed to be small. Solutions for tides, tidal current, and residual current have been considered for two-dimensional, barotropic estuaries and coastal seas. Particular attention has been paid to the distinction between the Lagrangian and Eulerian residual currents. When k is small, the first-order Lagrangian residual is shown to be the sum of the Eulerian residual current and the Stokes drift. The Lagrangian residual drift velocity or the second-order Lagrangian residual current has been shown to be dependent on the phase of tidal current. The Lagrangian drift velocity is induced by nonlinear interactions between tides, tidal currents, and the first-order residual currents, and it takes the form of an ellipse on a hodograph plane. Several examples are given to further demonstrate the unique properties of the Lagrangian residual current.

Repeat dose NRPT (nicotinamide riboside and pterostilbene) increases NAD+ levels in humans safely and sustainably: a randomized, double-blind, placebo-controlled study.

PubMed

Dellinger, Ryan W; Santos, Santiago Roel; Morris, Mark; Evans, Mal; Alminana, Dan; Guarente, Leonard; Marcotulli, Eric

2017-01-01

NRPT is a combination of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD +) precursor vitamin found in milk, and pterostilbene (PT), a polyphenol found in blueberries. Here, we report this first-in-humans clinical trial designed to assess the safety and efficacy of a repeat dose of NRPT (commercially known as Basis). NRPT was evaluated in a randomized, double-blind, and placebo-controlled study in a population of 120 healthy adults between the ages of 60 and 80 years. The study consisted of three treatment arms: placebo, recommended dose of NRPT (NRPT 1X), and double dose of NRPT (NRPT 2X). All subjects took their blinded supplement daily for eight weeks. Analysis of NAD + in whole blood demonstrated that NRPT significantly increases the concentration of NAD + in a dose-dependent manner. NAD + levels increased by approximately 40% in the NRPT 1X group and approximately 90% in the NRPT 2X group after 4 weeks as compared to placebo and baseline. Furthermore, this significant increase in NAD + levels was sustained throughout the entire 8-week trial. NAD + levels did not increase for the placebo group during the trial. No serious adverse events were reported in this study. This study shows that a repeat dose of NRPT is a safe and effective way to increase NAD + levels sustainably.

Slowing ageing by design: the rise of NAD+ and sirtuin-activating compounds

PubMed Central

Bonkowski, Michael S.; Sinclair, David A.

2016-01-01

The sirtuins (SIRT1–7) are a family of nicotinamide adenine dinucleotide (NAD+)-dependent deacylases with remarkable abilities to prevent diseases and even reverse aspects of ageing. Mice engineered to express additional copies of SIRT1 or SIRT6, or treated with sirtuin-activating compounds (STACs) such as resveratrol and SRT2104 or with NAD+ precursors, have improved organ function, physical endurance, disease resistance and longevity. Trials in non-human primates and in humans have indicated that STACs may be safe and effective in treating inflammatory and metabolic disorders, among others. These advances have demonstrated that it is possible to rationally design molecules that can alleviate multiple diseases and possibly extend lifespan in humans. PMID:27552971

Protective effect of nicotinamide adenine dinucleotide (NAD+) against spinal cord ischemia-reperfusion injury via reducing oxidative stress-induced neuronal apoptosis.

PubMed

Xie, Lei; Wang, Zhenfei; Li, Changwei; Yang, Kai; Liang, Yu

2017-02-01

As previous studies demonstrate that oxidative stress and apoptosis play crucial roles in ischemic pathogenesis and nicotinamide adenine dinucleotide (NAD + ) treatment attenuates oxidative stress-induced cell death among primary neurons and astrocytes as well as significantly reduce cerebral ischemic injury in rats. We used a spinal cord ischemia injury (SCII) model in rats to verify our hypothesis that NAD + could ameliorate oxidative stress-induced neuronal apoptosis. Adult male rats were subjected to transient spinal cord ischemia for 60min, and different doses of NAD + were administered intraperitoneally immediately after the start of reperfusion. Neurological function was determined by Basso, Beattie, Bresnahan (BBB) scores. The oxidative stress level was assessed by superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. The degree of apoptosis was analyzed by deoxyuridinetriphosphate nick-end labeling (TUNEL) staining and protein levels of cleaved caspase-3 and AIF (apoptosis inducing factor). The results showed that NAD + at 50 or 100mg/kg significantly decreased the oxidative stress level and neuronal apoptosis in the spinal cord of ischemia-reperfusion rats compared with saline, as accompanied with the decreased oxidative stress, NAD + administration significantly restrained the neuronal apoptosis after ischemia injury while improved the neurological and motor function. These findings suggested that NAD + might protect against spinal cord ischemia-reperfusion via reducing oxidative stress-induced neuronal apoptosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Acute Ethanol Intake Induces NAD(P)H Oxidase Activation and Rhoa Translocation in Resistance Arteries.

PubMed

Simplicio, Janaina A; Hipólito, Ulisses Vilela; Vale, Gabriel Tavares do; Callera, Glaucia Elena; Pereira, Camila André; Touyz, Rhian M; Tostes, Rita de Cássia; Tirapelli, Carlos R

2016-11-01

The mechanism underlying the vascular dysfunction induced by ethanol is not totally understood. Identification of biochemical/molecular mechanisms that could explain such effects is warranted. To investigate whether acute ethanol intake activates the vascular RhoA/Rho kinase pathway in resistance arteries and the role of NAD(P)H oxidase-derived reactive oxygen species (ROS) on such response. We also evaluated the requirement of p47phox translocation for ethanol-induced NAD(P)H oxidase activation. Male Wistar rats were orally treated with ethanol (1g/kg, p.o. gavage) or water (control). Some rats were treated with vitamin C (250 mg/kg, p.o. gavage, 5 days) before administration of water or ethanol. The mesenteric arterial bed (MAB) was collected 30 min after ethanol administration. Vitamin C prevented ethanol-induced increase in superoxide anion (O2-) generation and lipoperoxidation in the MAB. Catalase and superoxide dismutase activities and the reduced glutathione, nitrate and hydrogen peroxide (H2O2) levels were not affected by ethanol. Vitamin C and 4-methylpyrazole prevented the increase on O2- generation induced by ethanol in cultured MAB vascular smooth muscle cells. Ethanol had no effect on phosphorylation levels of protein kinase B (Akt) and eNOS (Ser1177 or Thr495 residues) or MAB vascular reactivity. Vitamin C prevented ethanol-induced increase in the membrane: cytosol fraction ratio of p47phox and RhoA expression in the rat MAB. Acute ethanol intake induces activation of the RhoA/Rho kinase pathway by a mechanism that involves ROS generation. In resistance arteries, ethanol activates NAD(P)H oxidase by inducing p47phox translocation by a redox-sensitive mechanism. O mecanismo da disfunção vascular induzido pelo consumo de etanol não é totalmente compreendido. Justifica-se, assim a identificação de mecanismos bioquímicos e moleculares que poderiam explicar tais efeitos. Investigar se a ingestão aguda de etanol ativa a via vascular RhoA/Rho quinase

Stereospecificity of NAD+/NADH Reactions: A Project Experiment for Advanced Undergraduates.

ERIC Educational Resources Information Center

Lowrey, Jonathan S.; And Others

1981-01-01

Presents background information, materials needed, and experimental procedures to study enzymes dependent on pyridine nucleotide coenzymes (NAD/NADH). The experiments, suitable for advanced organic or biochemistry courses, require approximately 10-15 hours to complete. (SK)

Phase Variation of NadA in Invasive Neisseria meningitidis Isolates Impacts on Coverage Estimates for 4C-MenB, a MenB vaccine.

PubMed

Green, Luke R; Lucidarme, Jay; Dave, Neelam; Chan, Hannah; Clark, Stephen; Borrow, Ray; Bayliss, Christopher D

2018-06-27

A recombinant NadA protein is one of the four major protective antigens of 4C-MenB (Bexsero®), a vaccine developed for serogroup B Neisseria meningitidis (MenB). The Meningococcal Antigen Typing System (MATS) is utilised as a high throughput assay for assessing the invasive MenB strain coverage of 4C-MenB. Where present, the nadA gene is subject to phase variable changes in transcription due to a 5'TAAA repeat tract located in a regulatory region. The promoter-containing intergenic region sequences (IGR) and 5'TAAA repeat numbers were determined for 906 invasive meningococcal disease isolates possessing the nadA gene. Exclusion of the 5'TAAA repeats reduced the number of IGR alleles from 82 to 23. Repeat numbers were associated with low and high levels of NadA expression by Western blotting and ELISA. Low expression repeat numbers were present in 83% of 179 MenB isolates with NadA-2/3 or Nad-1 peptide variants and 68% of 480 MenW ST-11 complex isolates with Nad-2/3 peptide variants. For isolates with vaccine-compatible NadA variants, 93% of MATS negative isolates were associated with low expression repeat numbers whereas 63% of isolates with MATS RP scores above the 95% confidence interval for the positive bactericidal threshold had high expression repeat numbers. Analysis of the 5'TAAA repeat number has potential as a rapid, high throughput method for assessing strain coverage for the NadA-component of 4C-MenB. A key application will be assessing coverage in meningococcal disease cases where confirmation is by PCR only and MATS cannot be applied. Copyright © 2018 Green et al.

Surrogate analyte approach for quantitation of endogenous NAD(+) in human acidified blood samples using liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

PubMed

Liu, Liling; Cui, Zhiyi; Deng, Yuzhong; Dean, Brian; Hop, Cornelis E C A; Liang, Xiaorong

2016-02-01

A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantitative determination of NAD(+) in human whole blood using a surrogate analyte approach was developed and validated. Human whole blood was acidified using 0.5N perchloric acid at a ratio of 1:3 (v:v, blood:perchloric acid) during sample collection. 25μL of acidified blood was extracted using a protein precipitation method and the resulting extracts were analyzed using reverse-phase chromatography and positive electrospray ionization mass spectrometry. (13)C5-NAD(+) was used as the surrogate analyte for authentic analyte, NAD(+). The standard curve ranging from 0.250 to 25.0μg/mL in acidified human blood for (13)C5-NAD(+) was fitted to a 1/x(2) weighted linear regression model. The LC-MS/MS response between surrogate analyte and authentic analyte at the same concentration was obtained before and after the batch run. This response factor was not applied when determining the NAD(+) concentration from the (13)C5-NAD(+) standard curve since the percent difference was less than 5%. The precision and accuracy of the LC-MS/MS assay based on the five analytical QC levels were well within the acceptance criteria from both FDA and EMA guidance for bioanalytical method validation. Average extraction recovery of (13)C5-NAD(+) was 94.6% across the curve range. Matrix factor was 0.99 for both high and low QC indicating minimal ion suppression or enhancement. The validated assay was used to measure the baseline level of NAD(+) in 29 male and 21 female human subjects. This assay was also used to study the circadian effect of endogenous level of NAD(+) in 10 human subjects. Copyright © 2015 Elsevier B.V. All rights reserved.

4-Pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PyTP), a novel NAD metabolite accumulating in erythrocytes of uremic children: a biomarker for a toxic NAD analogue in other tissues?

PubMed

Synesiou, Elena; Fairbanks, Lynnette D; Simmonds, H Anne; Slominska, Ewa M; Smolenski, Ryszard T; Carrey, Elizabeth A

2011-06-01

We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP), which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD(+) metabolites (nicotinamide, N(1)-methylnicotinamide and 4Py-riboside) and the major nicotinamide metabolite N(1)-methyl-2-pyridone-5-carboxamide (2PY), with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD(+) from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD(+) analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD(+) analogue that inhibits IMP dehydrogenase in other cells.

CD73 Protein as a Source of Extracellular Precursors for Sustained NAD+ Biosynthesis in FK866-treated Tumor Cells*

PubMed Central

Grozio, Alessia; Sociali, Giovanna; Sturla, Laura; Caffa, Irene; Soncini, Debora; Salis, Annalisa; Raffaelli, Nadia; De Flora, Antonio; Nencioni, Alessio; Bruzzone, Santina

2013-01-01

NAD+ is mainly synthesized in human cells via the “salvage” pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the “salvage” pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD+ or NAD+ precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD+ precursors for NAD+ biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD+ biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors. PMID:23880765

A cation-pi interaction in the binding site of the glycine receptor is mediated by a phenylalanine residue.

PubMed

Pless, Stephan A; Millen, Kat S; Hanek, Ariele P; Lynch, Joseph W; Lester, Henry A; Lummis, Sarah C R; Dougherty, Dennis A

2008-10-22

Cys-loop receptor binding sites characteristically contain many aromatic amino acids. In nicotinic ACh and 5-HT3 receptors, a Trp residue forms a cation-pi interaction with the agonist, whereas in GABA(A) receptors, a Tyr performs this role. The glycine receptor binding site, however, contains predominantly Phe residues. Homology models suggest that two of these Phe side chains, Phe159 and Phe207, and possibly a third, Phe63, are positioned such that they could contribute to a cation-pi interaction with the primary amine of glycine. Here, we test this hypothesis by incorporation of a series of fluorinated Phe derivatives using unnatural amino acid mutagenesis. The data reveal a clear correlation between the glycine EC(50) value and the cation-pi binding ability of the fluorinated Phe derivatives at position 159, but not at positions 207 or 63, indicating a single cation-pi interaction between glycine and Phe159. The data thus provide an anchor point for locating glycine in its binding site, and demonstrate for the first time a cation-pi interaction between Phe and a neurotransmitter.

Nitrate Transport Is Independent of NADH and NAD(P)H Nitrate Reductases in Barley Seedlings 1

PubMed Central

Warner, Robert L.; Huffaker, Ray C.

1989-01-01

Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings. PMID:11537465

Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen.

PubMed

Pasqualini, Stefania; Tedeschini, Emma; Frenguelli, Giuseppe; Wopfner, Nicole; Ferreira, Fatima; D'Amato, Gennaro; Ederli, Luisa

2011-10-01

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O(3)) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O(3) fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O(3) fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O(3), determined from the mRNA levels of the major allergens. We conclude that O(3) can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. Copyright © 2011 Elsevier Ltd. All rights reserved.

Enhancement of NAD+-dependent SIRT1 deacetylase activity by methylselenocysteine resets the circadian clock in carcinogen-treated mammary epithelial cells

PubMed Central

Fang, Mingzhu; Guo, Wei-Ren; Park, Youngil; Kang, Hwan-Goo; Zarbl, Helmut

2015-01-01

We previously reported that dietary methylselenocysteine (MSC) inhibits N-methyl-N-nitrosourea (NMU)-induced mammary tumorigenesis by resetting circadian gene expression disrupted by the carcinogen at the early stage of tumorigenesis. To investigate the underlying mechanism, we developed a circadian reporter system comprised of human mammary epithelial cells with a luciferase reporter driven by the promoter of human PERIOD 2 (PER2), a core circadian gene. In this in vitro model, NMU disrupted cellular circadian rhythm in a pattern similar to that observed with SIRT1-specific inhibitors; in contrast, MSC restored the circadian rhythms disrupted by NMU and protected against SIRT1 inhibitors. Moreover, NMU inhibited intracellular NAD+/NADH ratio and reduced NAD+-dependent SIRT1 activity in a dose-dependent manner, while MSC restored NAD+/NADH and SIRT1 activity in the NMU-treated cells, indicating that the NAD+-SIRT1 pathway was targeted by NMU and MSC. In rat mammary tissue, a carcinogenic dose of NMU also disrupted NAD+/NADH oscillations and decreased SIRT1 activity; dietary MSC restored NAD+/NADH oscillations and increased SIRT1 activity in the mammary glands of NMU-treated rats. MSC-induced SIRT1 activity was correlated with decreased acetylation of BMAL1 and increased acetylation of histone 3 lysine 9 at the Per2 promoter E-Box in mammary tissue. Changes in SIRT1 activity were temporally correlated with loss or restoration of rhythmic Per2 mRNA expression in NMU-treated or MSC-rescued rat mammary glands, respectively. Together with our previous findings, these results suggest that enhancement of NAD+-dependent SIRT1 activity contributes to the chemopreventive efficacy of MSC by restoring epigenetic regulation of circadian gene expression at early stages of mammary tumorigenesis. PMID:26544624

Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans.

PubMed

Bieganowski, Pawel; Brenner, Charles

2004-05-14

NAD+ is essential for life in all organisms, both as a coenzyme for oxidoreductases and as a source of ADPribosyl groups used in various reactions, including those that retard aging in experimental systems. Nicotinic acid and nicotinamide were defined as the vitamin precursors of NAD+ in Elvehjem's classic discoveries of the 1930s. The accepted view of eukaryotic NAD+ biosynthesis, that all anabolism flows through nicotinic acid mononucleotide, was challenged experimentally and revealed that nicotinamide riboside is an unanticipated NAD+ precursor in yeast. Nicotinamide riboside kinases from yeast and humans essential for this pathway were identified and found to be highly specific for phosphorylation of nicotinamide riboside and the cancer drug tiazofurin. Nicotinamide riboside was discovered as a nutrient in milk, suggesting that nicotinamide riboside is a useful compound for elevation of NAD+ levels in humans.

[Applylication of new type combined fragments: nrDNA ITS+ nad 1-intron 2 for identification of Dendrobium species of Fengdous].

PubMed

Geng, Li-xia; Zheng, Rui; Ren, Jie; Niu, Zhi-tao; Sun, Yu-long; Xue, Qing-yun; Liu, Wei; Ding, Xiao-yu

2015-08-01

In this study, 17 kinds of Dendrobium species of Fengdous including 39 individuals were collected from 4 provinces. Mitochondrial gene sequences co I, nad 5, nad 1-intron 2 and chloroplast gene sequences rbcL, matK amd psbA-trnH were amplified from these materials, as well as nrDNA ITS. Furthermore, suitable sequences for identification of Dendrobium species of Fengdous were screened by K-2-P and P-distance. The results showed that during the mentioned 7 sequences, nrDNA ITS, nad 1-intron 2 and psbA-trnH which had a high degree of variability could be used to identify Dendrobium species of Fengdous. However, single fragment could not be used to distinguish D. moniliforme and D. huoshanense. Moreover, compared to other combined fragments, new type combined fragments nrDNA ITS+nad 1-intron 2 was more effective in identifying the original plants of Dendrobium species and could be used to identify D. huoshanense and D. moniliforme. Besides, according to the UPGMA tree constructed with nrDNA ITS+nad 1-intron 2, 3 inspected Dendrobium plants were identified as D. huoshanense, D. moniliforme and D. officinale, respectively. This study identified Dendrobium species of Fengdous by combined fragments nrDNA ITS+nad 1-intron 2 for the first time, which provided a more effective basis for identification of Dendrobium species. And this study will be helpful for regulating the market of Fengdous.

Dietary proanthocyanidins boost hepatic NAD(+) metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats.

PubMed

Aragonès, Gerard; Suárez, Manuel; Ardid-Ruiz, Andrea; Vinaixa, Maria; Rodríguez, Miguel A; Correig, Xavier; Arola, Lluís; Bladé, Cinta

2016-04-22

Proanthocyanidins (PACs) have been reported to modulate multiple targets by simultaneously controlling many pivotal metabolic pathways in the liver. However, the precise mechanism of PAC action on the regulation of the genes that control hepatic metabolism remains to be clarified. Accordingly, we used a metabolomic approach combining both nuclear magnetic resonance and mass spectrometry analysis to evaluate the changes induced by different doses of grape-seed PACs in the liver of healthy rats. Here, we report that PACs significantly increased the hepatic nicotinamide adenine dinucleotide (NAD(+)) content in a dose-dependent manner by specifically modulating the hepatic concentrations of the major NAD(+) precursors as well as the mRNA levels of the genes that encode the enzymes involved in the cellular metabolism of NAD(+). Notably, Sirtuin 1 (Sirt1) gene expression was also significantly up-regulated in a dose-response pattern. The increase in both the NAD(+) availability and Sirt1 mRNA levels, in turn, resulted in the hepatic activation of SIRT1, which was significantly associated with improved protection against hepatic triglyceride accumulation. Our data clearly indicates that PAC consumption could be a valid tool to enhance hepatic SIRT1 activity through the modulation of NAD(+) levels.

Fabrication of Flexible Arrayed Lactate Biosensor Based on Immobilizing LDH-NAD+ on NiO Film Modified by GO and MBs

PubMed Central

Yan, Siao-Jie; Liao, Yi-Hung; Lai, Chih-Hsien; Wu, You-Xiang; Wu, Cian-Yi; Chen, Hsiang-Yi; Huang, Hong-Yu; Wu, Tong-Yu

2017-01-01

We proposed the flexible arrayed lactate biosensor based on immobilizing l-lactate dehydrogenase (LDH) and nicotinamide adenine dinucleotide (NAD+) on nickel oxide (NiO) film, and which the average sensitivity could be enhanced by using graphene oxide (GO) and magnetic beads (MBs). By using GO and MBs, it exhibits excellent sensitivity (45.397 mV/mM) with a linearity of 0.992 in a range of 0.2 mM to 3 mM. According to the results of electrochemical impedance spectroscopy (EIS), the electron transfer resistance of LDH-NAD+-MBs/GPTS/GO/NiO film was smaller than those of LDH-NAD+/GPTS/GO/NiO film and LDH-NAD+/GPTS/NiO film, and it presented the outstanding electron transfer ability. After that, the limit of detection, anti-interference effect and bending test were also investigated. PMID:28704960

A computational study to identify the key residues of peroxisome proliferator-activated receptor gamma in the interactions with its antagonists.

PubMed

Sharifi, Tayebeh; Ghayeb, Yousef

2018-05-01

Peroxisome proliferator-activated receptors (PPARs) compose a family of nuclear receptors, PPARα, PPARβ, and PPARγ, which mediate the effects of lipidic ligands at the transcriptional level. Among these, the PPARγ has been known to regulate adipocyte differentiation, fatty acid storage and glucose metabolism, and is a target of antidiabetic drugs. In this work, the interactions between PPARγ and its six known antagonists were investigated using computational methods such as molecular docking, molecular dynamics (MD) simulations, and the hybrid quantum mechanics/molecular mechanics (QM/MM). The binding energies evaluated by molecular docking varied between -22.59 and -35.15 kJ mol - 1 . In addition, MD simulations were performed to investigate the binding modes and PPARγ conformational changes upon binding of antagonists. Analysis of the root-mean-square fluctuations (RMSF) of backbone atoms shows that H3 of PPARγ has a higher mobility in the absence of antagonists and moderate conformational changes were observed. The interaction energies between antagonists and each PPARγ residue involved in the interactions were studied by QM/MM calculations. These calculations reveal that antagonists with different structures show different interaction energies with the same residue of PPARγ. Therefore, it can be concluded that the key residues vary depending on the structure of the ligand, which binds to PPARγ.

Automated detection and quantification of residual brain tumor using an interactive computer-aided detection scheme

NASA Astrophysics Data System (ADS)

Gaffney, Kevin P.; Aghaei, Faranak; Battiste, James; Zheng, Bin

2017-03-01

Detection of residual brain tumor is important to evaluate efficacy of brain cancer surgery, determine optimal strategy of further radiation therapy if needed, and assess ultimate prognosis of the patients. Brain MR is a commonly used imaging modality for this task. In order to distinguish between residual tumor and surgery induced scar tissues, two sets of MRI scans are conducted pre- and post-gadolinium contrast injection. The residual tumors are only enhanced in the post-contrast injection images. However, subjective reading and quantifying this type of brain MR images faces difficulty in detecting real residual tumor regions and measuring total volume of the residual tumor. In order to help solve this clinical difficulty, we developed and tested a new interactive computer-aided detection scheme, which consists of three consecutive image processing steps namely, 1) segmentation of the intracranial region, 2) image registration and subtraction, 3) tumor segmentation and refinement. The scheme also includes a specially designed and implemented graphical user interface (GUI) platform. When using this scheme, two sets of pre- and post-contrast injection images are first automatically processed to detect and quantify residual tumor volume. Then, a user can visually examine segmentation results and conveniently guide the scheme to correct any detection or segmentation errors if needed. The scheme has been repeatedly tested using five cases. Due to the observed high performance and robustness of the testing results, the scheme is currently ready for conducting clinical studies and helping clinicians investigate the association between this quantitative image marker and outcome of patients.

Bioelectrocatalytic NAD+/NADH inter-conversion: transformation of an enzymatic fuel cell into an enzymatic redox flow battery.

PubMed

Quah, Timothy; Milton, Ross D; Abdellaoui, Sofiene; Minteer, Shelley D

2017-07-25

Diaphorase and a benzylpropylviologen redox polymer were combined to create a bioelectrode that can both oxidize NADH and reduce NAD + . We demonstrate how bioelectrocatalytic NAD + /NADH inter-conversion can transform a glucose/O 2 enzymatic fuel cell (EFC) with an open circuit potential (OCP) of 1.1 V into an enzymatic redox flow battery (ERFB), which can be rapidly recharged by operation as an EFC.

MotiveValidator: interactive web-based validation of ligand and residue structure in biomolecular complexes.

PubMed

Vařeková, Radka Svobodová; Jaiswal, Deepti; Sehnal, David; Ionescu, Crina-Maria; Geidl, Stanislav; Pravda, Lukáš; Horský, Vladimír; Wimmerová, Michaela; Koča, Jaroslav

2014-07-01

Structure validation has become a major issue in the structural biology community, and an essential step is checking the ligand structure. This paper introduces MotiveValidator, a web-based application for the validation of ligands and residues in PDB or PDBx/mmCIF format files provided by the user. Specifically, MotiveValidator is able to evaluate in a straightforward manner whether the ligand or residue being studied has a correct annotation (3-letter code), i.e. if it has the same topology and stereochemistry as the model ligand or residue with this annotation. If not, MotiveValidator explicitly describes the differences. MotiveValidator offers a user-friendly, interactive and platform-independent environment for validating structures obtained by any type of experiment. The results of the validation are presented in both tabular and graphical form, facilitating their interpretation. MotiveValidator can process thousands of ligands or residues in a single validation run that takes no more than a few minutes. MotiveValidator can be used for testing single structures, or the analysis of large sets of ligands or fragments prepared for binding site analysis, docking or virtual screening. MotiveValidator is freely available via the Internet at http://ncbr.muni.cz/MotiveValidator. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The inhibition of the mitochondrial F1FO-ATPase activity when activated by Ca2+ opens new regulatory roles for NAD.

PubMed

Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pirini, Maurizio; Pagliarani, Alessandra

2018-01-26

The mitochondrial F1FO-ATPase is uncompetitively inhibited by NAD+ only when the natural cofactor Mg2+ is replaced by Ca2+, a mode putatively involved in cell death. The Ca2+-dependent F1FO-ATPase is also inhibited when NAD+ concentration in mitochondria is raised by acetoacetate. The enzyme inhibition by NAD+ cannot be ascribed to any de-ac(et)ylation or ADP-ribosylation by sirtuines, as it is not reversed by nicotinamide. Moreover, the addition of acetyl-CoA or palmitate, which would favor the enzyme ac(et)ylation, does not affect the F1FO-ATPase activity. Consistently, NAD+ may play a new role, not associated with redox and non-redox enzymatic reactions, in the Ca2+-dependent regulation of the F1FO-ATPase activity.

Hyperthermophilic Archaeon Thermococcus kodakarensis Utilizes a Four-Step Pathway for NAD+ Salvage through Nicotinamide Deamination.

PubMed

Hachisuka, Shin-Ichi; Sato, Takaaki; Atomi, Haruyuki

2018-06-01

Many organisms possess pathways that regenerate NAD + from its degradation products, and two pathways are known to salvage NAD + from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the hyperthermophilic archaeon Thermococcus kodakarensis is supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved in de novo biosynthesis and four-step salvage of NAD + , respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD + salvage in T. kodakarensis requires deamination of Nm and proceeds through the four-step pathway. IMPORTANCE Hyperthermophiles must constantly deal with increased degradation rates of their biomolecules due to their high growth temperatures. Here, we identified the pathway that regenerates NAD + from nicotinamide (Nm) in the

Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins*

PubMed Central

Meitzler, Jennifer L.; Hinde, Sara; Bánfi, Botond; Nauseef, William M.; Ortiz de Montellano, Paul R.

2013-01-01

Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H2O2 production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1. PMID:23362256

Exogenous NAD+ decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy

PubMed Central

Zhu, Ying; Zhao, Ke-ke; Tong, Yao; Zhou, Ya-li; Wang, Yi-xiao; Zhao, Pei-quan; Wang, Zhao-yang

2016-01-01

Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD+) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD+ administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD+ administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD+ administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD+ against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD+ administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD+ administration might be potential value for the treatment of AMD. PMID:27240523

Exogenous NAD(+) decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy.

PubMed

Zhu, Ying; Zhao, Ke-Ke; Tong, Yao; Zhou, Ya-Li; Wang, Yi-Xiao; Zhao, Pei-Quan; Wang, Zhao-Yang

2016-05-31

Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD(+)) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD(+) administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD(+) administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD(+) administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD(+) against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD(+) administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD(+) administration might be potential value for the treatment of AMD.

Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa.

PubMed Central

Velasco-García, R; González-Segura, L; Muñoz-Clares, R A

2000-01-01

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)(+) to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD(+)- and NADP(+)-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)(+) and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)(+) binds first and NAD(P)H leaves last, particularly in the NADP(+)-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP(+)-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose. PMID:11104673

Melatonin protects chondrocytes from impairment induced by glucocorticoids via NAD+-dependent SIRT1.

PubMed

Yang, Wei; Kang, Xiaomin; Qin, Na; Li, Feng; Jin, Xinxin; Ma, Zhengmin; Qian, Zhuang; Wu, Shufang

2017-10-01

Intra-articular injection of glucocorticoids is used to relieve pain and inflammation in osteoarthritis patients, which is occasionally accompanied with the serious side effects of glucocorticoids in collagen-producing tissue. Melatonin is the major hormone released from the pineal gland and its beneficial effects on cartilage has been suggested. In the present study, we investigated the protective role of melatonin on matrix degeneration in chondrocytes induced by dexamethasone (Dex). The chondrocytes isolated from mice knee joint were treated with Dex, melatonin, EX527 and siRNA targeted for SIRT6, respectively. Dex treatment induced the loss of the extracellular matrix, NAD + /NADH ratio and NADPH concentration in chondrocytes. Melatonin alone have no effect on the quantity of proteoglycans and collagen type IIa1, however, the pretreatment of melatonin reversed the negative effects induced by Dex. Meanwhile, the significant decrease in NAD + /NADH ratio and NADPH concentration in Dex group were up-regulated by pretreatment of melatonin. Furthermore, it was revealed that inhibition of SIRT1 blocked the protective effects of melatonin. The enhancement of NAD + -dependent SIRT1 activity contributes to the chondroprotecfive effects of melatonin, which has a great benefit to prevent dexamethasone-induced chondrocytes impairment. Copyright © 2017 Elsevier Inc. All rights reserved.

Restoration of Mitochondrial NAD+ Levels Delays Stem Cell Senescence and Facilitates Reprogramming of Aged Somatic Cells.

PubMed

Son, Myung Jin; Kwon, Youjeong; Son, Taekwon; Cho, Yee Sook

2016-12-01

The fundamental tenet that aging is irreversible has been challenged by the development of reprogramming technology that can restore molecular and cellular age by reversing the progression of aging. The use of cells from aged individuals as sources for reprogramming or transplantation creates a major barrier in stem cell therapy with respect to cell quality and quantity. Here, we investigated the molecular features underlying senescence and rejuvenation during aged cell reprogramming and identified novel factors that can overcome age-associated barriers. Enzymes, such as nicotinamide nucleotide transhydrogenase (NNT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), that control mitochondrial NAD + levels appear to be susceptible to aging. In aged cells, mitochondrial NAD + levels decrease, accompanied by reduced SIRT3 activity; these changes severely impede cell fate transition. However, in cells collected from aged p16 knockout mice, which exhibit delayed cellular senescence, no changes in NNT or NMNAT3 expression were found. Importantly, restoring mitochondrial NAD + levels by overexpressing NNT and NMNAT3 enhanced reprogramming efficiency of aged somatic cells and extended the lifespan of human mesenchymal stem cells by delaying replicative senescence. These results demonstrate that maintenance of mitochondrial NAD + levels is critical for reversing the mechanisms of aging and ensuring that cells collected from aged individuals are of high quality. Stem Cells 2016;34:2840-2851. © 2016 AlphaMed Press.

Dietary proanthocyanidins boost hepatic NAD+ metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats

PubMed Central

Aragonès, Gerard; Suárez, Manuel; Ardid-Ruiz, Andrea; Vinaixa, Maria; Rodríguez, Miguel A.; Correig, Xavier; Arola, Lluís; Bladé, Cinta

2016-01-01

Proanthocyanidins (PACs) have been reported to modulate multiple targets by simultaneously controlling many pivotal metabolic pathways in the liver. However, the precise mechanism of PAC action on the regulation of the genes that control hepatic metabolism remains to be clarified. Accordingly, we used a metabolomic approach combining both nuclear magnetic resonance and mass spectrometry analysis to evaluate the changes induced by different doses of grape-seed PACs in the liver of healthy rats. Here, we report that PACs significantly increased the hepatic nicotinamide adenine dinucleotide (NAD+) content in a dose-dependent manner by specifically modulating the hepatic concentrations of the major NAD+ precursors as well as the mRNA levels of the genes that encode the enzymes involved in the cellular metabolism of NAD+. Notably, Sirtuin 1 (Sirt1) gene expression was also significantly up-regulated in a dose-response pattern. The increase in both the NAD+ availability and Sirt1 mRNA levels, in turn, resulted in the hepatic activation of SIRT1, which was significantly associated with improved protection against hepatic triglyceride accumulation. Our data clearly indicates that PAC consumption could be a valid tool to enhance hepatic SIRT1 activity through the modulation of NAD+ levels. PMID:27102823

Faster Rubisco Is the Key to Superior Nitrogen-Use Efficiency in NADP-Malic Enzyme Relative to NAD-Malic Enzyme C4 Grasses1

PubMed Central

Ghannoum, Oula; Evans, John R.; Chow, Wah Soon; Andrews, T. John; Conroy, Jann P.; von Caemmerer, Susanne

2005-01-01

In 27 C4 grasses grown under adequate or deficient nitrogen (N) supplies, N-use efficiency at the photosynthetic (assimilation rate per unit leaf N) and whole-plant (dry mass per total leaf N) level was greater in NADP-malic enzyme (ME) than NAD-ME species. This was due to lower N content in NADP-ME than NAD-ME leaves because neither assimilation rates nor plant dry mass differed significantly between the two C4 subtypes. Relative to NAD-ME, NADP-ME leaves had greater in vivo (assimilation rate per Rubisco catalytic sites) and in vitro Rubisco turnover rates (kcat; 3.8 versus 5.7 s−1 at 25°C). The two parameters were linearly related. In 2 NAD-ME (Panicum miliaceum and Panicum coloratum) and 2 NADP-ME (Sorghum bicolor and Cenchrus ciliaris) grasses, 30% of leaf N was allocated to thylakoids and 5% to 9% to amino acids and nitrate. Soluble protein represented a smaller fraction of leaf N in NADP-ME (41%) than in NAD-ME (53%) leaves, of which Rubisco accounted for one-seventh. Soluble protein averaged 7 and 10 g (mmol chlorophyll)−1 in NADP-ME and NAD-ME leaves, respectively. The majority (65%) of leaf N and chlorophyll was found in the mesophyll of NADP-ME and bundle sheath of NAD-ME leaves. The mesophyll-bundle sheath distribution of functional thylakoid complexes (photosystems I and II and cytochrome f) varied among species, with a tendency to be mostly located in the mesophyll. In conclusion, superior N-use efficiency of NADP-ME relative to NAD-ME grasses was achieved with less leaf N, soluble protein, and Rubisco having a faster kcat. PMID:15665246

Catalytic properties of the isolated diaphorase fragment of the NAD-reducing [NiFe]-hydrogenase from Ralstonia eutropha.

PubMed

Lauterbach, Lars; Idris, Zulkifli; Vincent, Kylie A; Lenz, Oliver

2011-01-01

The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H₂-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O₂. It comprises six subunits, HoxHYFUI₂, and incorporates a [NiFe] H+/H₂ cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with K(I) values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O₂, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis.

The Differences between NAD-ME and NADP-ME Subtypes of C4 Photosynthesis: More than Decarboxylating Enzymes.

PubMed

Rao, Xiaolan; Dixon, Richard A

2016-01-01

As an adaptation to changing climatic conditions that caused high rates of photorespiration, C 4 plants have evolved to display higher photosynthetic efficiency than C 3 plants under elevated temperature, high light intensities, and drought. The C 4 plants independently evolved more than 60 times in 19 families of angiosperms to establish similar but not uniform C 4 mechanisms to concentrate CO 2 around the carboxylating enzyme Rubisco (ribulose bisphosphate carboxylase oxygenase). C 4 photosynthesis is divided into at least two basic biochemical subtypes based on the primary decarboxylating enzymes, NAD-dependent malic enzyme (NAD-ME) and NADP-dependent malic enzyme (NADP-ME). The multiple polygenetic origins of these subtypes raise questions about the association of C 4 variation between biochemical subtypes and diverse lineages. This review addresses the differences in evolutionary scenario, leaf anatomy, and especially C 4 metabolic flow, C 4 transporters, and cell-specific function deduced from recently reported cell-specific transcriptomic, proteomic, and metabolic analyses of NAD-ME and NADP-ME subtypes. Current omic analysis has revealed the extent to which component abundances differ between the two biochemical subtypes, leading to a better understanding of C 4 photosynthetic mechanisms in NAD-ME and NADP-ME subtypes.

The Differences between NAD-ME and NADP-ME Subtypes of C4 Photosynthesis: More than Decarboxylating Enzymes

PubMed Central

Rao, Xiaolan; Dixon, Richard A.

2016-01-01

As an adaptation to changing climatic conditions that caused high rates of photorespiration, C4 plants have evolved to display higher photosynthetic efficiency than C3 plants under elevated temperature, high light intensities, and drought. The C4 plants independently evolved more than 60 times in 19 families of angiosperms to establish similar but not uniform C4 mechanisms to concentrate CO2 around the carboxylating enzyme Rubisco (ribulose bisphosphate carboxylase oxygenase). C4 photosynthesis is divided into at least two basic biochemical subtypes based on the primary decarboxylating enzymes, NAD-dependent malic enzyme (NAD-ME) and NADP-dependent malic enzyme (NADP-ME). The multiple polygenetic origins of these subtypes raise questions about the association of C4 variation between biochemical subtypes and diverse lineages. This review addresses the differences in evolutionary scenario, leaf anatomy, and especially C4 metabolic flow, C4 transporters, and cell-specific function deduced from recently reported cell-specific transcriptomic, proteomic, and metabolic analyses of NAD-ME and NADP-ME subtypes. Current omic analysis has revealed the extent to which component abundances differ between the two biochemical subtypes, leading to a better understanding of C4 photosynthetic mechanisms in NAD-ME and NADP-ME subtypes. PMID:27790235

Extracellular NAMPT/visfatin causes p53 deacetylation via NAD production and SIRT1 activation in breast cancer cells.

PubMed

Behrouzfar, Kiarash; Alaee, Mohammad; Nourbakhsh, Mitra; Gholinejad, Zafar; Golestani, Abolfazl

2017-08-01

Visfatin, which is secreted as an adipokine and cytokine, has been implicated in cancer development and progression. In this study, we investigated the NAD-producing ability of visfatin and its relationship with SIRT1 (silent information regulator 2) and p53 to clarify the role of visfatin in breast cancer. MCF-7 breast cancer cells were cultured and treated with visfatin. SIRT1 activity was assessed by measuring fluorescence intensity from fluoro-substrate peptide. To investigate the effect of visfatin on p53 acetylation, SDS-PAGE followed by western blotting was performed using specific antibodies against p53 and its acetylated form. Total NAD was measured both in cell lysate and the extracellular medium by colorimetric method. Visfatin increased both extracellular and intracellular NAD concentrations. It also induced proliferation of breast cancer cells, an effect that was abolished by inhibition of its enzymatic activity. Visfatin significantly increased SIRT1 activity, accompanied by induction of p53 deacetylation. In conclusion, the results show that extracellular visfatin produces NAD that causes upregulation of SIRT1 activity and p53 deacetylation. These findings explain the relationship between visfatin and breast cancer progression. Copyright © 2017 John Wiley & Sons, Ltd.

Environment Dictates Dependence on Mitochondrial Complex I for NAD+ and Aspartate Production and Determines Cancer Cell Sensitivity to Metformin.

PubMed

Gui, Dan Y; Sullivan, Lucas B; Luengo, Alba; Hosios, Aaron M; Bush, Lauren N; Gitego, Nadege; Davidson, Shawn M; Freinkman, Elizaveta; Thomas, Craig J; Vander Heiden, Matthew G

2016-11-08

Metformin use is associated with reduced cancer mortality, but how metformin impacts cancer outcomes is controversial. Although metformin can act on cells autonomously to inhibit tumor growth, the doses of metformin that inhibit proliferation in tissue culture are much higher than what has been described in vivo. Here, we show that the environment drastically alters sensitivity to metformin and other complex I inhibitors. We find that complex I supports proliferation by regenerating nicotinamide adenine dinucleotide (NAD)+, and metformin's anti-proliferative effect is due to loss of NAD+/NADH homeostasis and inhibition of aspartate biosynthesis. However, complex I is only one of many inputs that determines the cellular NAD+/NADH ratio, and dependency on complex I is dictated by the activity of other pathways that affect NAD+ regeneration and aspartate levels. This suggests that cancer drug sensitivity and resistance are not intrinsic properties of cancer cells, and demonstrates that the environment can dictate sensitivity to therapies that impact cell metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

ARTD1/PARP1 negatively regulates glycolysis by inhibiting hexokinase 1 independent of NAD+ depletion

PubMed Central

Fouquerel, Elise; Goellner, Eva M.; Yu, Zhongxun; Gagné, Jean-Philippe; de Moura, Michelle Barbi; Feinstein, Tim; Wheeler, David; Redpath, Philip; Li, Jianfeng; Romero, Guillermo; Migaud, Marie; Van Houten, Bennett; Poirier, Guy G.; Sobol, Robert W.

2014-01-01

Summary ARTD1 (PARP1) is a key enzyme involved in DNA repair by synthesizing poly(ADP-ribose) (PAR) in response to strand breaks and plays an important role in cell death following excessive DNA damage. ARTD1-induced cell death is associated with NAD+ depletion and ATP loss, however the molecular mechanism of ARTD1-mediated energy collapse remains elusive. Using real-time metabolic measurements, we directly compared the effects of ARTD1 activation and direct NAD+ depletion. We found that ARTD1-mediated PAR synthesis, but not direct NAD+ depletion, resulted in a block to glycolysis and ATP loss. We then established a proteomics based PAR-interactome after DNA damage and identified hexokinase 1 (HK1) as a PAR binding protein. HK1 activity is suppressed following nuclear ARTD1 activation and binding by PAR. These findings help explain how prolonged activation of ARTD1 triggers energy collapse and cell death, revealing new insight on the importance of nucleus to mitochondria communication via ARTD1 activation. PMID:25220464

Interactions between allelochemicals and the microbial community affect weed suppression following cover crop residue incorporation into soil

USDA-ARS?s Scientific Manuscript database

The objective of this study is to understand how soil microorganisms interact with cover crop-derived allelochemicals to suppress weed germination and growth following cover crop residue incorporation. We conducted a time series experiment by crossing sterilized and non-sterilized soil with four dif...

Mutational analysis of human RNA polymerase II subunit 5 (RPB5): the residues critical for interactions with TFIIF subunit RAP30 and hepatitis B virus X protein.

PubMed

Le, Thi Thu Thuy; Zhang, Shijun; Hayashi, Naoyuki; Yasukawa, Mami; Delgermaa, Luvsanjav; Murakami, Seishi

2005-09-01

RNA polymerase II (RNAPII) subunit 5 (RPB5) is positioned close to DNA downstream of the initiation site and is the site of interaction with several regulators. Hepatitis B virus X protein (HBx) binds the central part of RPB5 to modulate activated transcription, and TFIIF subunit RAP30 interacts with the same part of RPB5 that is critical for the association between TFIIF and RNAPII. However the residues necessary for these interactions remain unknown. Here we report systematic mutagenesis of the central part of RPB5 using two-step alanine scanning libraries to pinpoint critical residues for its binding to RAP30 in the TFIIF complex and/or to HBx, and identified these residues in both mammalian cells and in an in vitro binding assay. Four residues, F76, I104, T111 and S113, are critical for both TFIIF- and HBx-binding, indicating the overlapping nature of the sites of interaction. In addition, V74 and N98 are required for HBx-binding, and T56 and L58 are needed for RAP30-binding. Interestingly the residues exposed to solvent, T111 and S113, are very close to the DNA, implying that two factors may modulate the interaction between DNA and RPB5.

[Activity of liver mitochondrial NAD+-dependent dehydrogenases of the krebs cycle in rats with acetaminophen-induced hepatitis developed under conditions of alimentary protein deficiency].

PubMed

Voloshchuk, O N; Kopylchuk, G P

2016-01-01

Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage.

Differential levels of metabolic activity in isolated versus confluent/partially confluent HeLa cells are analyzed by autofluorescent NAD(P)H using multi-photon FLIM microscopy

NASA Astrophysics Data System (ADS)

Chandler, Andrea; Chandler, Aaron; Wallrabe, Horst; Periasamy, Ammasi

2017-02-01

NAD(P)H is a known biomarker for cellular metabolism; a higher ratio of enzyme-bound NAD(P)H to free/unbound NAD(P)H indicates an increase in metabolic activity. Free NADH has a shorter fluorescence lifetime (τ1), the bound version (τ2) a longer lifetime. FLIM's unique capability to establish inter alia the relative fractions of τ1 (a1%) and τ2 (a2%) in each pixel, determines the level of metabolic activity. The relative abundances of bound NAD(P)H were analyzed for single cells, confluent and partially confluent cells within 3 Fields-of-View (FoVs). A gradient of increasing a 2% levels of bound NAD(P)H from single, partially confluent to confluent cells was observed.

Few residues within an extensive binding interface drive receptor interaction and determine the specificity of arrestin proteins.

PubMed

Vishnivetskiy, Sergey A; Gimenez, Luis E; Francis, Derek J; Hanson, Susan M; Hubbell, Wayne L; Klug, Candice S; Gurevich, Vsevolod V

2011-07-08

Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements.

Few Residues within an Extensive Binding Interface Drive Receptor Interaction and Determine the Specificity of Arrestin Proteins*

PubMed Central

Vishnivetskiy, Sergey A.; Gimenez, Luis E.; Francis, Derek J.; Hanson, Susan M.; Hubbell, Wayne L.; Klug, Candice S.; Gurevich, Vsevolod V.

2011-01-01

Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements. PMID:21471193

Reduced Ssy1-Ptr3-Ssy5 (SPS) signaling extends replicative life span by enhancing NAD+ homeostasis in Saccharomyces cerevisiae.

PubMed

Tsang, Felicia; James, Christol; Kato, Michiko; Myers, Victoria; Ilyas, Irtqa; Tsang, Matthew; Lin, Su-Ju

2015-05-15

Attenuated nutrient signaling extends the life span in yeast and higher eukaryotes; however, the mechanisms are not completely understood. Here we identify the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway as a novel longevity factor. A null mutation of SSY5 (ssy5Δ) increases replicative life span (RLS) by ∼50%. Our results demonstrate that several NAD(+) homeostasis factors play key roles in this life span extension. First, expression of the putative malate-pyruvate NADH shuttle increases in ssy5Δ cells, and deleting components of this shuttle, MAE1 and OAC1, largely abolishes RLS extension. Next, we show that Stp1, a transcription factor of the SPS pathway, directly binds to the promoter of MAE1 and OAC1 to regulate their expression. Additionally, deletion of SSY5 increases nicotinamide riboside (NR) levels and phosphate-responsive (PHO) signaling activity, suggesting that ssy5Δ increases NR salvaging. This increase contributes to NAD(+) homeostasis, partially ameliorating the NAD(+) deficiency and rescuing the short life span of the npt1Δ mutant. Moreover, we observed that vacuolar phosphatase, Pho8, is partially required for ssy5Δ-mediated NR increase and RLS extension. Together, our studies present evidence that supports SPS signaling is a novel NAD(+) homeostasis factor and ssy5Δ-mediated life span extension is likely due to concomitantly increased mitochondrial and vacuolar function. Our findings may contribute to understanding the molecular basis of NAD(+) metabolism, cellular life span, and diseases associated with NAD(+) deficiency and aging. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-alpha.

PubMed

Gertzberg, Nancy; Neumann, Paul; Rizzo, Victor; Johnson, Arnold

2004-01-01

We tested the hypothesis that the NAD(P)H oxidase-dependent generation of superoxide anion (O2-*) mediates tumor necrosis factor-alpha (TNF)-induced alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The NAD(P)H oxidase subcomponents p47phox and p22phox were assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species O2-* was measured by the fluorescence of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetatedi(acetoxymethyl ester), 5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester, and dihydroethidium. TNF treatment (50 ng/ml for 4.0 h) induced 1) p47phox translocation, 2) an increase in p22phox protein, 3) increased localization of p47phox with p22phox, 4) O2-* generation, and 5) increased permeability to albumin. p22phox antisense oligonucleotide prevented the TNF-induced effect on p22phox, p47phox, O2-*, and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in O2-* and permeability to albumin was also prevented by the O2-* scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of O2-*, mediates TNF-induced barrier dysfunction in PMEM.

Streptococcus mutans NADH oxidase lies at the intersection of overlapping regulons controlled by oxygen and NAD+ levels.

PubMed

Baker, J L; Derr, A M; Karuppaiah, K; MacGilvray, M E; Kajfasz, J K; Faustoferri, R C; Rivera-Ramos, I; Bitoun, J P; Lemos, J A; Wen, Z T; Quivey, R G

2014-06-01

NADH oxidase (Nox, encoded by nox) is a flavin-containing enzyme used by the oral pathogen Streptococcus mutans to reduce diatomic oxygen to water while oxidizing NADH to NAD(+). The critical nature of Nox is 2-fold: it serves to regenerate NAD(+), a carbon cycle metabolite, and to reduce intracellular oxygen, preventing formation of destructive reactive oxygen species (ROS). As oxygen and NAD(+) have been shown to modulate the activity of the global transcription factors Spx and Rex, respectively, Nox is potentially poised at a critical junction of two stress regulons. In this study, microarray data showed that either addition of oxygen or loss of nox resulted in altered expression of genes involved in energy metabolism and transport and the upregulation of genes encoding ROS-metabolizing enzymes. Loss of nox also resulted in upregulation of several genes encoding transcription factors and signaling molecules, including the redox-sensing regulator gene rex. Characterization of the nox promoter revealed that nox was regulated by oxygen, through SpxA, and by Rex. These data suggest a regulatory loop in which the roles of nox in reduction of oxygen and regeneration of NAD(+) affect the activity levels of Spx and Rex, respectively, and their regulons, which control several genes, including nox, crucial to growth of S. mutans under conditions of oxidative stress. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Genetic interaction analysis of point mutations enables interrogation of gene function at a residue-level resolution

PubMed Central

Braberg, Hannes; Moehle, Erica A.; Shales, Michael; Guthrie, Christine; Krogan, Nevan J.

2014-01-01

We have achieved a residue-level resolution of genetic interaction mapping – a technique that measures how the function of one gene is affected by the alteration of a second gene – by analyzing point mutations. Here, we describe how to interpret point mutant genetic interactions, and outline key applications for the approach, including interrogation of protein interaction interfaces and active sites, and examination of post-translational modifications. Genetic interaction analysis has proven effective for characterizing cellular processes; however, to date, systematic high-throughput genetic interaction screens have relied on gene deletions or knockdowns, which limits the resolution of gene function analysis and poses problems for multifunctional genes. Our point mutant approach addresses these issues, and further provides a tool for in vivo structure-function analysis that complements traditional biophysical methods. We also discuss the potential for genetic interaction mapping of point mutations in human cells and its application to personalized medicine. PMID:24842270

Shade compromises the photosynthetic efficiency of NADP-ME less than that of PEP-CK and NAD-ME C4 grasses.

PubMed

Sonawane, Balasaheb V; Sharwood, Robert E; Whitney, Spencer; Ghannoum, Oula

2018-05-25

The high energy cost and apparently low plasticity of C4 photosynthesis compared with C3 photosynthesis may limit the productivity and distribution of C4 plants in low light (LL) environments. C4 photosynthesis evolved numerous times, but it remains unclear how different biochemical subtypes perform under LL. We grew eight C4 grasses belonging to three biochemical subtypes [NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEP-CK)] under shade (16% sunlight) or control (full sunlight) conditions and measured their photosynthetic characteristics at both low and high light. We show for the first time that LL (during measurement or growth) compromised the CO2-concentrating mechanism (CCM) to a greater extent in NAD-ME than in PEP-CK or NADP-ME C4 grasses by virtue of a greater increase in carbon isotope discrimination (∆P) and bundle sheath CO2 leakiness (ϕ), and a greater reduction in photosynthetic quantum yield (Φmax). These responses were partly explained by changes in the ratios of phosphoenolpyruvate carboxylase (PEPC)/initial Rubisco activity and dark respiration/photosynthesis (Rd/A). Shade induced a greater photosynthetic acclimation in NAD-ME than in NADP-ME and PEP-CK species due to a greater Rubisco deactivation. Shade also reduced plant dry mass to a greater extent in NAD-ME and PEP-CK relative to NADP-ME grasses. In conclusion, LL compromised the co-ordination of the C4 and C3 cycles and, hence, the efficiency of the CCM to a greater extent in NAD-ME than in PEP-CK species, while CCM efficiency was less impacted by LL in NADP-ME species. Consequently, NADP-ME species are more efficient at LL, which could explain their agronomic and ecological dominance relative to other C4 grasses.

Shade compromises the photosynthetic efficiency of NADP-ME less than that of PEP-CK and NAD-ME C4 grasses

PubMed Central

2018-01-01

Abstract The high energy cost and apparently low plasticity of C4 photosynthesis compared with C3 photosynthesis may limit the productivity and distribution of C4 plants in low light (LL) environments. C4 photosynthesis evolved numerous times, but it remains unclear how different biochemical subtypes perform under LL. We grew eight C4 grasses belonging to three biochemical subtypes [NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEP-CK)] under shade (16% sunlight) or control (full sunlight) conditions and measured their photosynthetic characteristics at both low and high light. We show for the first time that LL (during measurement or growth) compromised the CO2-concentrating mechanism (CCM) to a greater extent in NAD-ME than in PEP-CK or NADP-ME C4 grasses by virtue of a greater increase in carbon isotope discrimination (∆P) and bundle sheath CO2 leakiness (ϕ), and a greater reduction in photosynthetic quantum yield (Φmax). These responses were partly explained by changes in the ratios of phosphoenolpyruvate carboxylase (PEPC)/initial Rubisco activity and dark respiration/photosynthesis (Rd/A). Shade induced a greater photosynthetic acclimation in NAD-ME than in NADP-ME and PEP-CK species due to a greater Rubisco deactivation. Shade also reduced plant dry mass to a greater extent in NAD-ME and PEP-CK relative to NADP-ME grasses. In conclusion, LL compromised the co-ordination of the C4 and C3 cycles and, hence, the efficiency of the CCM to a greater extent in NAD-ME than in PEP-CK species, while CCM efficiency was less impacted by LL in NADP-ME species. Consequently, NADP-ME species are more efficient at LL, which could explain their agronomic and ecological dominance relative to other C4 grasses. PMID:29659931

Relative importance of redox buffers GSH and NAD(P)H in age-related neurodegeneration and Alzheimer disease-like mouse neurons.

PubMed

Ghosh, Debolina; Levault, Kelsey R; Brewer, Gregory J

2014-08-01

Aging, a major risk factor in Alzheimer's disease (AD), is associated with an oxidative redox shift, decreased redox buffer protection, and increased free radical reactive oxygen species (ROS) generation, probably linked to mitochondrial dysfunction. While NADH is the ultimate electron donor for many redox reactions, including oxidative phosphorylation, glutathione (GSH) is the major ROS detoxifying redox buffer in the cell. Here, we explored the relative importance of NADH and GSH to neurodegeneration in aging and AD neurons from nontransgenic and 3xTg-AD mice by inhibiting their synthesis to determine whether NADH can compensate for the GSH loss to maintain redox balance. Neurons stressed by either depleting NAD(P)H or GSH indicated that NADH redox control is upstream of GSH levels. Further, although depletion of NAD(P)H or GSH correlated linearly with neuron death, compared with GSH depletion, higher neurodegeneration was observed when NAD(P)H was extrapolated to zero, especially in old age, and in the 3xTg-AD neurons. We also observed an age-dependent loss of gene expression of key redox-dependent biosynthetic enzymes, NAMPT (nicotinamide phosphoribosyltransferase), and NNT (nicotinamide nucleotide transhydrogenase). Moreover, age-related correlations between brain NNT or NAMPT gene expression and NADPH levels suggest that these genes contribute to the age-related declines in NAD(P)H. Our data indicate that in aging and more so in AD-like neurons, NAD(P)H redox control is upstream of GSH and an oxidative redox shift that promotes neurodegeneration. Thus, NAD(P)H generation may be a more efficacious therapeutic target upstream of GSH and ROS. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

A coarse-grained model of the effective interaction for charged amino acid residues and its application to formation of GCN4-pLI tetramer

NASA Astrophysics Data System (ADS)

Kawaguchi, Kazutomo; Nakagawa, Satoshi; Kurniawan, Isman; Kodama, Koichi; Arwansyah, Muhammad Saleh; Nagao, Hidemi

2018-03-01

We present a simple coarse-grained model of the effective interaction for charged amino acid residues, such as Glu and Lys, in a water solvent. The free-energy profile as a function of the distance between two charged amino acid side-chain analogues in an explicit water solvent is calculated with all-atom molecular dynamics simulation and thermodynamic integration method. The calculated free-energy profile is applied to the coarse-grained potential of the effective interaction between two amino acid residues. The Langevin dynamics simulations with our coarse-grained potential are performed for association of a small protein complex, GCN4-pLI tetramer. The tetramer conformation reproduced by our coarse-grained model is similar to the X-ray crystallographic structure. We show that the effective interaction between charged amino acid residues stabilises association and orientation of protein complex. We also investigate the association pathways of GCN4-pLI tetramer.

Machine Learning and Network Analysis of Molecular Dynamics Trajectories Reveal Two Chains of Red/Ox-specific Residue Interactions in Human Protein Disulfide Isomerase.

PubMed

Karamzadeh, Razieh; Karimi-Jafari, Mohammad Hossein; Sharifi-Zarchi, Ali; Chitsaz, Hamidreza; Salekdeh, Ghasem Hosseini; Moosavi-Movahedi, Ali Akbar

2017-06-16

The human protein disulfide isomerase (hPDI), is an essential four-domain multifunctional enzyme. As a result of disulfide shuffling in its terminal domains, hPDI exists in two oxidation states with different conformational preferences which are important for substrate binding and functional activities. Here, we address the redox-dependent conformational dynamics of hPDI through molecular dynamics (MD) simulations. Collective domain motions are identified by the principal component analysis of MD trajectories and redox-dependent opening-closing structure variations are highlighted on projected free energy landscapes. Then, important structural features that exhibit considerable differences in dynamics of redox states are extracted by statistical machine learning methods. Mapping the structural variations to time series of residue interaction networks also provides a holistic representation of the dynamical redox differences. With emphasizing on persistent long-lasting interactions, an approach is proposed that compiled these time series networks to a single dynamic residue interaction network (DRIN). Differential comparison of DRIN in oxidized and reduced states reveals chains of residue interactions that represent potential allosteric paths between catalytic and ligand binding sites of hPDI.

An open-label, non-randomized study of the pharmacokinetics of the nutritional supplement nicotinamide riboside (NR) and its effects on blood NAD+ levels in healthy volunteers.

PubMed

Airhart, Sophia E; Shireman, Laura M; Risler, Linda J; Anderson, Gail D; Nagana Gowda, G A; Raftery, Daniel; Tian, Rong; Shen, Danny D; O'Brien, Kevin D

2017-01-01

The co-primary objectives of this study were to determine the human pharmacokinetics (PK) of oral NR and the effect of NR on whole blood nicotinamide adenine dinucleotide (NAD+) levels. Though mitochondrial dysfunction plays a critical role in the development and progression of heart failure, no mitochondria-targeted therapies have been translated into clinical practice. Recent murine studies have reported associations between imbalances in the NADH/NAD+ ratio with mitochondrial dysfunction in multiple tissues, including myocardium. Moreover, an NAD+ precursor, nicotinamide mononucleotide, improved cardiac function, while another NAD+ precursor, nicotinamide riboside (NR), improved mitochondrial function in muscle, liver and brown adipose. Thus, PK studies of NR in humans is critical for future clinical trials. In this non-randomized, open-label PK study of 8 healthy volunteers, 250 mg NR was orally administered on Days 1 and 2, then uptitrated to peak dose of 1000 mg twice daily on Days 7 and 8. On the morning of Day 9, subjects completed a 24-hour PK study after receiving 1000 mg NR at t = 0. Whole-blood levels of NR, clinical blood chemistry, and NAD+ levels were analyzed. Oral NR was well tolerated with no adverse events. Significant increases comparing baseline to mean concentrations at steady state (Cave,ss) were observed for both NR (p = 0.03) and NAD+ (p = 0.001); the latter increased by 100%. Absolute changes from baseline to Day 9 in NR and NAD+ levels correlated highly (R2 = 0.72, p = 0.008). Because NR increases circulating NAD+ in humans, NR may have potential as a therapy in patients with mitochondrial dysfunction due to genetic and/or acquired diseases.

An open-label, non-randomized study of the pharmacokinetics of the nutritional supplement nicotinamide riboside (NR) and its effects on blood NAD+ levels in healthy volunteers

PubMed Central

Airhart, Sophia E.; Shireman, Laura M.; Risler, Linda J.; Anderson, Gail D.; Nagana Gowda, G. A.; Raftery, Daniel; Tian, Rong; Shen, Danny D.; O’Brien, Kevin D.

2017-01-01

Objectives The co-primary objectives of this study were to determine the human pharmacokinetics (PK) of oral NR and the effect of NR on whole blood nicotinamide adenine dinucleotide (NAD+) levels. Background Though mitochondrial dysfunction plays a critical role in the development and progression of heart failure, no mitochondria-targeted therapies have been translated into clinical practice. Recent murine studies have reported associations between imbalances in the NADH/NAD+ ratio with mitochondrial dysfunction in multiple tissues, including myocardium. Moreover, an NAD+ precursor, nicotinamide mononucleotide, improved cardiac function, while another NAD+ precursor, nicotinamide riboside (NR), improved mitochondrial function in muscle, liver and brown adipose. Thus, PK studies of NR in humans is critical for future clinical trials. Methods In this non-randomized, open-label PK study of 8 healthy volunteers, 250 mg NR was orally administered on Days 1 and 2, then uptitrated to peak dose of 1000 mg twice daily on Days 7 and 8. On the morning of Day 9, subjects completed a 24-hour PK study after receiving 1000 mg NR at t = 0. Whole-blood levels of NR, clinical blood chemistry, and NAD+ levels were analyzed. Results Oral NR was well tolerated with no adverse events. Significant increases comparing baseline to mean concentrations at steady state (Cave,ss) were observed for both NR (p = 0.03) and NAD+ (p = 0.001); the latter increased by 100%. Absolute changes from baseline to Day 9 in NR and NAD+ levels correlated highly (R2 = 0.72, p = 0.008). Conclusions Because NR increases circulating NAD+ in humans, NR may have potential as a therapy in patients with mitochondrial dysfunction due to genetic and/or acquired diseases. PMID:29211728

Split Renilla Luciferase Protein Fragment-assisted Complementation (SRL-PFAC) to Characterize Hsp90-Cdc37 Complex and Identify Critical Residues in Protein/Protein Interactions*

PubMed Central

Jiang, Yiqun; Bernard, Denzil; Yu, Yanke; Xie, Yehua; Zhang, Tao; Li, Yanyan; Burnett, Joseph P.; Fu, Xueqi; Wang, Shaomeng; Sun, Duxin

2010-01-01

Hsp90 requires cochaperone Cdc37 to load its clients to the Hsp90 superchaperone complex. The purpose of this study was to utilize split Renilla luciferase protein fragment-assisted complementation (SRL-PFAC) bioluminescence to study the full-length human Hsp90-Cdc37 complex and to identity critical residues and their contributions for Hsp90/Cdc37 interaction in living cells. SRL-PFAC showed that full-length human Hsp90/Cdc37 interaction restored dramatically high luciferase activity through Hsp90-Cdc37-assisted complementation of the N and C termini of luciferase (compared with the set of controls). Immunoprecipitation confirmed that the expressed fusion proteins (NRL-Hsp90 and Cdc37-CRL) preserved their ability to interact with each other and also with native Hsp90 or Cdc37. Molecular dynamic simulation revealed several critical residues in the two interaction patches (hydrophobic and polar) at the interface of Hsp90/Cdc37. Mutagenesis confirmed the critical residues for Hsp90-Cdc37 complex formation. SRL-PFAC bioluminescence evaluated the contributions of these critical residues in Hsp90/Cdc37 interaction. The results showed that mutations in Hsp90 (Q133A, F134A, and A121N) and mutations in Cdc37 (M164A, R167A, L205A, and Q208A) reduced the Hsp90/Cdc37 interaction by 70–95% as measured by the resorted luciferase activity through Hsp90-Cdc37-assisted complementation. In comparison, mutations in Hsp90 (E47A and S113A) and a mutation in Cdc37 (A204E) decreased the Hsp90/Cdc37 interaction by 50%. In contrast, mutations of Hsp90 (R46A, S50A, C481A, and C598A) and mutations in Cdc37 (C54S, C57S, and C64S) did not change Hsp90/Cdc37 interactions. The data suggest that single amino acid mutation in the interface of Hsp90/Cdc37 is sufficient to disrupt its interaction, although Hsp90/Cdc37 interactions are through large regions of hydrophobic and polar interactions. These findings provides a rationale to develop inhibitors for disruption of the Hsp90/Cdc37 interaction

Split Renilla luciferase protein fragment-assisted complementation (SRL-PFAC) to characterize Hsp90-Cdc37 complex and identify critical residues in protein/protein interactions.

PubMed

Jiang, Yiqun; Bernard, Denzil; Yu, Yanke; Xie, Yehua; Zhang, Tao; Li, Yanyan; Burnett, Joseph P; Fu, Xueqi; Wang, Shaomeng; Sun, Duxin

2010-07-02

Hsp90 requires cochaperone Cdc37 to load its clients to the Hsp90 superchaperone complex. The purpose of this study was to utilize split Renilla luciferase protein fragment-assisted complementation (SRL-PFAC) bioluminescence to study the full-length human Hsp90-Cdc37 complex and to identity critical residues and their contributions for Hsp90/Cdc37 interaction in living cells. SRL-PFAC showed that full-length human Hsp90/Cdc37 interaction restored dramatically high luciferase activity through Hsp90-Cdc37-assisted complementation of the N and C termini of luciferase (compared with the set of controls). Immunoprecipitation confirmed that the expressed fusion proteins (NRL-Hsp90 and Cdc37-CRL) preserved their ability to interact with each other and also with native Hsp90 or Cdc37. Molecular dynamic simulation revealed several critical residues in the two interaction patches (hydrophobic and polar) at the interface of Hsp90/Cdc37. Mutagenesis confirmed the critical residues for Hsp90-Cdc37 complex formation. SRL-PFAC bioluminescence evaluated the contributions of these critical residues in Hsp90/Cdc37 interaction. The results showed that mutations in Hsp90 (Q133A, F134A, and A121N) and mutations in Cdc37 (M164A, R167A, L205A, and Q208A) reduced the Hsp90/Cdc37 interaction by 70-95% as measured by the resorted luciferase activity through Hsp90-Cdc37-assisted complementation. In comparison, mutations in Hsp90 (E47A and S113A) and a mutation in Cdc37 (A204E) decreased the Hsp90/Cdc37 interaction by 50%. In contrast, mutations of Hsp90 (R46A, S50A, C481A, and C598A) and mutations in Cdc37 (C54S, C57S, and C64S) did not change Hsp90/Cdc37 interactions. The data suggest that single amino acid mutation in the interface of Hsp90/Cdc37 is sufficient to disrupt its interaction, although Hsp90/Cdc37 interactions are through large regions of hydrophobic and polar interactions. These findings provides a rationale to develop inhibitors for disruption of the Hsp90/Cdc37 interaction.

Kinetic competence of the cADP-ribose-CD38 complex as an intermediate in the CD38/NAD+ glycohydrolase-catalysed reactions: implication for CD38 signalling.

PubMed Central

Cakir-Kiefer, C; Muller-Steffner, H; Oppenheimer, N; Schuber, F

2001-01-01

CD38/NAD(+) glycohydrolase is a type II transmembrane glycoprotein widely used to study T- and B-cell activation and differentiation. CD38 is endowed with two different activities: it is a signal transduction molecule and an ectoenzyme that converts NAD(+) into ADP-ribose (NAD(+) glycohydrolase activity) and small proportions of cADP-ribose (cADPR; ADP-ribosyl cyclase activity), a calcium-mobilizing metabolite, which, ultimately, can also be hydrolysed (cADPR hydrolase activity). The relationship between these two properties, and strikingly the requirement for signalling in the formation of free or enzyme-complexed cADPR, is still ill-defined. In the present study we wanted to test whether the CD38-cADPR complex is kinetically competent in the conversion of NAD(+) into the reaction product ADP-ribose. In principle, such a complex could be invoked for cross-talk, via conformational changes, with neighbouring partner(s) of CD38 thus triggering the signalling phenomena. Analysis of the kinetic parameters measured for the CD38/NAD(+) glycohydrolase-catalysed hydrolysis of 2'-deoxy-2'-aminoribo-NAD(+) and ADP-cyclo[N1,C1']-2'-deoxy-2'-aminoribose (slowly hydrolysable analogues of NAD(+) and cADPR respectively) ruled out that the CD38-cADPR complex can accumulate under steady-state conditions. This was borne out by simulation of the prevalent kinetic mechanism of CD38, which involve the partitioning of a common E.ADP-ribosyl intermediate in the formation of the enzyme-catalysed reaction products. Using this mechanism, microscopic rate conditions were found which transform a NAD(+) glycohydrolase into an ADP-ribosyl cyclase. Altogether, the present work shows that if the cross-talk with a partner depends on a conformational change of CD38, this is most probably not attributable to the formation of the CD38-cADPR complex. In line with recent results on the conformational change triggered by CD38 ligands [Berthelier, Laboureau, Boulla, Schuber and Deterre (2000) Eur. J

Reduction of Flavodoxin by Electron Bifurcation and Sodium Ion-dependent Reoxidation by NAD+ Catalyzed by Ferredoxin-NAD+ Reductase (Rnf)*

PubMed Central

Chowdhury, Nilanjan Pal; Klomann, Katharina; Seubert, Andreas; Buckel, Wolfgang

2016-01-01

Electron-transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) from Acidaminococcus fermentans catalyze the endergonic reduction of ferredoxin by NADH, which is also driven by the concomitant reduction of crotonyl-CoA by NADH, a process called electron bifurcation. Here we show that recombinant flavodoxin from A. fermentans produced in Escherichia coli can replace ferredoxin with almost equal efficiency. After complete reduction of the yellow quinone to the blue semiquinone, a second 1.4 times faster electron transfer affords the colorless hydroquinone. Mediated by a hydrogenase, protons reoxidize the fully reduced flavodoxin or ferredoxin to the semi-reduced species. In this hydrogen-generating system, both electron carriers act catalytically with apparent Km = 0.26 μm ferredoxin or 0.42 μm flavodoxin. Membrane preparations of A. fermentans contain a highly active ferredoxin/flavodoxin-NAD+ reductase (Rnf) that catalyzes the irreversible reduction of flavodoxin by NADH to the blue semiquinone. Using flavodoxin hydroquinone or reduced ferredoxin obtained by electron bifurcation, Rnf can be measured in the forward direction, whereby one NADH is recycled, resulting in the simple equation: crotonyl-CoA + NADH + H+ = butyryl-CoA + NAD+ with Km = 1.4 μm ferredoxin or 2.0 μm flavodoxin. This reaction requires Na+ (Km = 0.12 mm) or Li+ (Km = 0.25 mm) for activity, indicating that Rnf acts as a Na+ pump. The redox potential of the quinone/semiquinone couple of flavodoxin (Fld) is much higher than that of the semiquinone/hydroquinone couple. With free riboflavin, the opposite is the case. Based on this behavior, we refine our previous mechanism of electron bifurcation. PMID:27048649

Adipose tissue NAD+-homeostasis, sirtuins and poly(ADP-ribose) polymerases -important players in mitochondrial metabolism and metabolic health.

PubMed

Jokinen, Riikka; Pirnes-Karhu, Sini; Pietiläinen, Kirsi H; Pirinen, Eija

2017-08-01

Obesity, a chronic state of energy overload, is characterized by adipose tissue dysfunction that is considered to be the major driver for obesity associated metabolic complications. The reasons for adipose tissue dysfunction are incompletely understood, but one potential contributing factor is adipose tissue mitochondrial dysfunction. Derangements of adipose tissue mitochondrial biogenesis and pathways associate with obesity and metabolic diseases. Mitochondria are central organelles in energy metabolism through their role in energy derivation through catabolic oxidative reactions. The mitochondrial processes are dependent on the proper NAD + /NADH redox balance and NAD + is essential for reactions catalyzed by the key regulators of mitochondrial metabolism, sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs). Notably, obesity is associated with disturbed adipose tissue NAD + homeostasis and the balance of SIRT and PARP activities. In this review we aim to summarize existing literature on the maintenance of intracellular NAD + pools and the function of SIRTs and PARPs in adipose tissue during normal and obese conditions, with the purpose of comprehending their potential role in mitochondrial derangements and obesity associated metabolic complications. Understanding the molecular mechanisms that are the root cause of the adipose tissue mitochondrial derangements is crucial for developing new effective strategies to reverse obesity associated metabolic complications. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

De-novo NAD+ synthesis regulates SIRT1-FOXO1 apoptotic pathway in response to NQO1 substrates in lung cancer cells

PubMed Central

Cheng, Xuefang; Li, Qingran; Liu, Fang; Ye, Hui; Zhao, Min; Wang, Hong; Wang, Guangji; Hao, Haiping

2016-01-01

Tryptophan metabolism is essential in diverse kinds of tumors via regulating tumor immunology. However, the direct role of tryptophan metabolism and its signaling pathway in cancer cells remain largely elusive. Here, we establish a mechanistic link from L-type amino acid transporter 1 (LAT1) mediated transport of tryptophan and the subsequent de-novo NAD+ synthesis to SIRT1-FOXO1 regulated apoptotic signaling in A549 cells in response to NQO1 activation. In response to NQO1 activation, SIRT1 is repressed leading to the increased cellular accumulation of acetylated FOXO1 that transcriptionally activates apoptotic signaling. Decreased uptake of tryptophan due to the downregulation of LAT1 coordinates with PARP-1 hyperactivation to induce rapid depletion of NAD+ pool. Particularly, the LAT1-NAD+-SIRT1 signaling is activated in tumor tissues of patients with non-small cell lung cancer. Because NQO1 activation is characterized with oxidative challenge induced DNA damage, these results suggest that LAT1 and de-novo NAD+ synthesis in NSCLC cells may play essential roles in sensing excessive oxidative stress. PMID:27566573

De-novo NAD+ synthesis regulates SIRT1-FOXO1 apoptotic pathway in response to NQO1 substrates in lung cancer cells.

PubMed

Liu, Huiying; Xing, Rong; Cheng, Xuefang; Li, Qingran; Liu, Fang; Ye, Hui; Zhao, Min; Wang, Hong; Wang, Guangji; Hao, Haiping

2016-09-20

Tryptophan metabolism is essential in diverse kinds of tumors via regulating tumor immunology. However, the direct role of tryptophan metabolism and its signaling pathway in cancer cells remain largely elusive. Here, we establish a mechanistic link from L-type amino acid transporter 1 (LAT1) mediated transport of tryptophan and the subsequent de-novo NAD+ synthesis to SIRT1-FOXO1 regulated apoptotic signaling in A549 cells in response to NQO1 activation. In response to NQO1 activation, SIRT1 is repressed leading to the increased cellular accumulation of acetylated FOXO1 that transcriptionally activates apoptotic signaling. Decreased uptake of tryptophan due to the downregulation of LAT1 coordinates with PARP-1 hyperactivation to induce rapid depletion of NAD+ pool. Particularly, the LAT1-NAD+-SIRT1 signaling is activated in tumor tissues of patients with non-small cell lung cancer. Because NQO1 activation is characterized with oxidative challenge induced DNA damage, these results suggest that LAT1 and de-novo NAD+ synthesis in NSCLC cells may play essential roles in sensing excessive oxidative stress.

NAXE Mutations Disrupt the Cellular NAD(P)HX Repair System and Cause a Lethal Neurometabolic Disorder of Early Childhood.

PubMed

Kremer, Laura S; Danhauser, Katharina; Herebian, Diran; Petkovic Ramadža, Danijela; Piekutowska-Abramczuk, Dorota; Seibt, Annette; Müller-Felber, Wolfgang; Haack, Tobias B; Płoski, Rafał; Lohmeier, Klaus; Schneider, Dominik; Klee, Dirk; Rokicki, Dariusz; Mayatepek, Ertan; Strom, Tim M; Meitinger, Thomas; Klopstock, Thomas; Pronicka, Ewa; Mayr, Johannes A; Baric, Ivo; Distelmaier, Felix; Prokisch, Holger

2016-10-06

To safeguard the cell from the accumulation of potentially harmful metabolic intermediates, specific repair mechanisms have evolved. APOA1BP, now renamed NAXE, encodes an epimerase essential in the cellular metabolite repair for NADHX and NADPHX. The enzyme catalyzes the epimerization of NAD(P)HX, thereby avoiding the accumulation of toxic metabolites. The clinical importance of the NAD(P)HX repair system has been unknown. Exome sequencing revealed pathogenic biallelic mutations in NAXE in children from four families with (sub-) acute-onset ataxia, cerebellar edema, spinal myelopathy, and skin lesions. Lactate was elevated in cerebrospinal fluid of all affected individuals. Disease onset was during the second year of life and clinical signs as well as episodes of deterioration were triggered by febrile infections. Disease course was rapidly progressive, leading to coma, global brain atrophy, and finally to death in all affected individuals. NAXE levels were undetectable in fibroblasts from affected individuals of two families. In these fibroblasts we measured highly elevated concentrations of the toxic metabolite cyclic-NADHX, confirming a deficiency of the mitochondrial NAD(P)HX repair system. Finally, NAD or nicotinic acid (vitamin B3) supplementation might have therapeutic implications for this fatal disorder. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

NeEMO: a method using residue interaction networks to improve prediction of protein stability upon mutation.

PubMed

Giollo, Manuel; Martin, Alberto J M; Walsh, Ian; Ferrari, Carlo; Tosatto, Silvio C E

2014-01-01

The rapid growth of un-annotated missense variants poses challenges requiring novel strategies for their interpretation. From the thermodynamic point of view, amino acid changes can lead to a change in the internal energy of a protein and induce structural rearrangements. This is of great relevance for the study of diseases and protein design, justifying the development of prediction methods for variant-induced stability changes. Here we propose NeEMO, a tool for the evaluation of stability changes using an effective representation of proteins based on residue interaction networks (RINs). RINs are used to extract useful features describing interactions of the mutant amino acid with its structural environment. Benchmarking shows NeEMO to be very effective, allowing reliable predictions in different parts of the protein such as β-strands and buried residues. Validation on a previously published independent dataset shows that NeEMO has a Pearson correlation coefficient of 0.77 and a standard error of 1 Kcal/mol, outperforming nine recent methods. The NeEMO web server can be freely accessed from URL: http://protein.bio.unipd.it/neemo/. NeEMO offers an innovative and reliable tool for the annotation of amino acid changes. A key contribution are RINs, which can be used for modeling proteins and their interactions effectively. Interestingly, the approach is very general, and can motivate the development of a new family of RIN-based protein structure analyzers. NeEMO may suggest innovative strategies for bioinformatics tools beyond protein stability prediction.

Determination of the in vivo NAD:NADH ratio in Saccharomyces cerevisiae under anaerobic conditions, using alcohol dehydrogenase as sensor reaction.

PubMed

Bekers, K M; Heijnen, J J; van Gulik, W M

2015-08-01

With the current quantitative metabolomics techniques, only whole-cell concentrations of NAD and NADH can be quantified. These measurements cannot provide information on the in vivo redox state of the cells, which is determined by the ratio of the free forms only. In this work we quantified free NAD:NADH ratios in yeast under anaerobic conditions, using alcohol dehydrogenase (ADH) and the lumped reaction of glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase as sensor reactions. We showed that, with an alternative accurate acetaldehyde determination method, based on rapid sampling, instantaneous derivatization with 2,4 diaminophenol hydrazine (DNPH) and quantification with HPLC, the ADH-catalysed oxidation of ethanol to acetaldehyde can be applied as a relatively fast and simple sensor reaction to quantify the free NAD:NADH ratio under anaerobic conditions. We evaluated the applicability of ADH as a sensor reaction in the yeast Saccharomyces cerevisiae, grown in anaerobic glucose-limited chemostats under steady-state and dynamic conditions. The results found in this study showed that the cytosolic redox status (NAD:NADH ratio) of yeast is at least one order of magnitude lower, and is thus much more reduced, under anaerobic conditions compared to aerobic glucose-limited steady-state conditions. The more reduced state of the cytosol under anaerobic conditions has major implications for (central) metabolism. Accurate determination of the free NAD:NADH ratio is therefore of importance for the unravelling of in vivo enzyme kinetics and to judge accurately the thermodynamic reversibility of each redox reaction. Copyright © 2015 John Wiley & Sons, Ltd.

Renal haemodynamics and natriuretic responses to intravenous administration of diadenosine tetraphosphate (Ap4A) and nicotinamide adenine dinucleotide (NAD) in rat.

PubMed

Szczepańska-Konkel, M; Langner, G; Bednarczuk, G; Stiepanow-Trzeciak, A; Jankowski, M; Angielski, S

2003-06-01

Effects of Ap4A and NAD--precursor of adenosine, on renal plasma flow (RPF), glomerular filtration rate (GFR) and urine excretion were determined in the anaesthetised rats. Infusion of Ap4A or NAD (i.v., bolus--1 micromol/kg followed by 10 nmol/min/kg) decreased RPF and GFR (by 30 and 40%, respectively). In spite of GFR reduction during Ap4A infusion, the significant increase in sodium excretion and urine flow was noticed: fractional sodium (FENa) and urine excretion (FEurine) rose 15-fold and 2.5-fold in comparison with the control value, respectively. In contrast to Ap4A, NAD-induced decrease in GFR was associated with parallel decrease in sodium and urine excretion, thus the FENa and FEurine did not significantly change. Pretreatment with adenosine deaminase (adenosine degrading enzyme, 2 U/min/kg) or theophylline (P1-receptors antagonist, 0.2 mmol/min/kg) ceased responses to NAD, whereas Ap4A-induced changes were not affected. Pre-treatment with suramin (P2-receptors antagonist, (i.v., bolus--12 mg/kg followed by 1.2 mg/min/kg) completely abolished the renal effects of Ap4A. We conclude that Ap4A may exert specific action on renal function. It acts different from NAD that modified renal function through its hydrolysis product--adenosine. Ap4A might reduce glomerular filtration rate and evoke natriuresis and diuresis, and its effects are probably mediated through stimulation of P2-receptors.

The chemistry of nicotinamide adenine dinucleotide (NAD) analogues containing C-nucleosides related to nicotinamide riboside.

PubMed

Pankiewicz, Krzysztof W; Watanabe, Kyoichi A; Lesiak-Watanabe, Krystyna; Goldstein, Barry M; Jayaram, Hiremagalur N

2002-04-01

Oncolytic C-nucleosides, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and benzamide riboside (3-beta-D-ribofuranosylbenzamide) are converted in cell into active metabolites thiazole-4-carboxamide- and benzamide adenine dinucleotide, TAD and BAD, respectively. TAD and BAD as NAD analogues were found to bind at the nicotinamide adenine dinucleotide (cofactor NAD) site of inosine monophosphate dehydrogenase (IMPDH), an important target in cancer treatment. The synthesis and evaluation of anticancer activity of a number of C-nucleosides related to tiazofurin and nicotinamide riboside then followed and are reviewed herein. Interestingly, pyridine C-nucleosides (such as C-nicotinamide riboside) are not metabolized into the corresponding NAD analogues in cell. Their conversion by chemical methods is described. As dinucleotides these compounds show inhibition of IMPDH in low micromolar level. Also, the synthesis of BAD in metabolically stable bis(phosphonate) form is discussed indicating the usefulness of such preformed inhibitors in drug development. Among tiazofurin analogues, Franchetti and Grifantini found, that the replacement of the sulfur by oxygen (as in oxazafurin) but not the removal of nitrogen (tiophenfurin) of the thiazole ring resulted in inactive compounds. The anti cancer activity of their synthetic dinucleotide analogues indicate that inactive compounds are not only poorly metabolized in cell but also are weak inhibitors of IMPDH as dinucleotides.

Arginine 26 and aspartic acid 69 of the regulatory subunit are key residues of subunits interaction of acetohydroxyacid synthase isozyme III from E. coli.

PubMed

Zhao, Yuefang; Wen, Xin; Niu, Congwei; Xi, Zhen

2012-11-05

Acetohydroxyacid synthase (AHAS), which catalyzes the first step in the biosynthesis of branched-chain amino acids, is composed of catalytic and regulatory subunits. The enzyme exhibits full activity only when the regulatory subunit (RSU) binds to the catalytic subunit (CSU). However, the crystal structure of the holoenzyme has not been reported yet, and the molecular interaction between the CSU and RSU is also unknown. Herein, we introduced a global-surface, site-directed labeling scanning method to determine the potential interaction region of the RSU. This approach relies on the insertion of a bulky fluorescent probe at the designated site on the surface of the RSU to cause a dramatic change in holoenzyme activity by perturbing subunit interaction. Then, the key amino acid residues in the potential interaction regions were identified by site-directed mutagenesis. Compared to the wild-type, the single-point mutants R26A and D69A showed 54 and 64 % activity, respectively, whereas the double mutant (R26A+D69A) gave 14 %, thus suggesting that residues Arg26 and Asp69 are the key residues of subunit interaction with cooperative action. Additionally, the results of GST pull-down assays and pH-dependence experiments suggested that polar interaction is the main force for subunits interaction. A plausible protein-protein interaction model of the holoenzyme of Escherichia coli AHAS III is proposed, based on the mutagenesis and protein docking studies. The protocol established here should be useful for the identification of the molecular interactions between proteins. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantum oscillation signatures of spin-orbit interactions controlling the residual nodal bilayer-splitting in underdoped high-Tc cuprates

NASA Astrophysics Data System (ADS)

Harrison, Neil; Shekhter, Arkady

2015-03-01

We investigate the origin of the small residual nodal bilayer-splitting in the underdoped high-Tc superconductor YBa2Cu3O6+x using the results of recently published angle-resolved quantum oscillation data [Sebastian et al., Nature 511, 61 (2014)]. A crucial clue to the origin of the residual bilayer-splitting is found to be provided by the anomalously small Zeeman-splitting of some of the observed cyclotron orbits. We show that such an anomalously Zeeman-splitting (or small effective g-factor) for a subset of orbits can be explained by spin-orbit interactions, which become significant in the nodal regions as a result of the vanishing bilayer coupling. The primary effect of spin-orbit interactions is to cause quasiparticles traversing the nodal region of the Brillouin zone to undergo a spin flip. We suggest that the Rashba-like spin-orbit interactions, naturally present in bilayer systems, have the right symmetry and magnitude to give rise to a network of coupled orbits consistent with experimental observations in underdoped YBa2Cu3O6+x. This work is supported by the DOEm BES proposal LANLF100, while the magnet lab is supported by the NSF and Florida State.

Characterization of Frex as an NADH sensor for in vivo applications in the presence of NAD+ and at various pH values.

PubMed

Wilkening, Svea; Schmitt, Franz-Josef; Horch, Marius; Zebger, Ingo; Lenz, Oliver; Friedrich, Thomas

2017-09-01

The fluorescent biosensor Frex, recently introduced as a sensitive tool to quantify the NADH concentration in living cells, was characterized by time-integrated and time-resolved fluorescence spectroscopy regarding its applicability for in vivo measurements. Based on the purified sensor protein, it is shown that the NADH dependence of Frex fluorescence can be described by a Hill function with a concentration of half-maximal sensor response of K D  ≈ 4 µM and a Hill coefficient of n ≈ 2. Increasing concentrations of NADH have moderate effects on the fluorescence lifetime of Frex, which changes by a factor of two from about 500 ps in the absence of NADH to 1 ns under fluorescence-saturating NADH concentrations. Therefore, the observed sevenfold rise of the fluorescence intensity is primarily ascribed to amplitude changes. Notably, the dynamic range of Frex sensitivity towards NADH highly depends on the NAD + concentration, while the apparent K D for NADH is only slightly affected. We found that NAD + has a strong inhibitory effect on the fluorescence response of Frex during NADH sensing, with an apparent NAD + dissociation constant of K I  ≈ 400 µM. This finding was supported by fluorescence lifetime measurements, which showed that the addition of NAD + hardly affects the fluorescence lifetime, but rather reduces the number of Frex molecules that are able to bind NADH. Furthermore, the fluorescence responses of Frex to NADH and NAD + depend critically on pH and temperature. Thus, for in vivo applications of Frex, temperature and pH need to be strictly controlled or considered during data acquisition and analysis. If all these constraints are properly met, Frex fluorescence intensity measurements can be employed to estimate the minimum NADH concentration present within the cell at sufficiently low NAD + concentrations below 100 µM.

Mycobacterial nicotinate mononucleotide adenylyltransferase: Structure, mechanism, and implications for drug discovery

DOE PAGES

Rodionova, Irina A.; Zuccola, Harmon J.; Sorci, Leonardo; ...

2015-01-28

Nicotinate mononucleotide adenylyltransferase NadD is an essential enzyme in the biosynthesis of the NAD cofactor, which has been implicated as a target for developing new antimycobacterial therapies. Here we report the crystal structure of Mycobacterium tuberculosis NadD ( MtNadD) at a resolution of 2.4 Å. A remarkable new feature of the MtNadD structure, compared with other members of this enzyme family, is a 310 helix that locks the active site in an over-closed conformation. As a result, MtNadD is rendered inactive as it is topologically incompatible with substrate binding and catalysis. Directed mutagenesis was also used to further dissect themore » structural elements that contribute to the interactions of the two MtNadD substrates, i.e. ATP and nicotinic acid mononucleotide (NaMN). For inhibitory profiling of partially active mutants and wild type MtNadD, we used a small molecule inhibitor of MtNadD with moderate affinity ( Ki ~ 25 μM) and antimycobacterial activity (MIC 80) ~ 40-80 μM). This analysis revealed interferences with some of the residues in the NaMN binding subsite consistent with the competitive inhibition observed for the NaMN substrate (but not ATP). A detailed steady-state kinetic analysis of MtNadD suggests that ATP must first bind to allow efficient NaMN binding and catalysis. This sequential mechanism is consistent with the requirement of transition to catalytically competent (open) conformation hypothesized from structural modeling. A possible physiological significance of this mechanism is to enable the down-regulation of NAD synthesis under ATP-limiting dormancy conditions. Lastly, these findings point to a possible new strategy for designing inhibitors that lock the enzyme in the inactive over-closed conformation.« less

Genetic and molecular characterization of the guaC-nadC-aroP region of Escherichia coli K-12.

PubMed

Roberts, R E; Lienhard, C I; Gaines, C G; Smith, J M; Guest, J R

1988-01-01

The guaC (GMP reductase), nadC (quinolinate phosphoribosyltransferase), and aroP (aromatic amino acid permease) genes of Escherichia coli K-12 were located in the 2.5-min region of the chromosome (muT-guaC-nadC-aroP-aceE) by a combination of linkage analysis, deletion mapping, restriction analysis, and plasmid subcloning. The guaC locus expressed a product of Mr 37,000 with a clockwise transcriptional polarity, and the GMP reductase activities of guaC+ plasmid-containing strains were amplified 15- to 20-fold.

Simulations of the Vertical Redistribution of HNO3 by NAT or NAD PSCs: The Sensitivity to the Number of Cloud Particles Formed and the Cloud Lifetime

NASA Technical Reports Server (NTRS)

Jensen, Eric J.; Tabazadeh, Azadeh; Drdla, Katja; Toon, Owen B.; Gore, Warren J. (Technical Monitor)

2000-01-01

Recent satellite and in situ measurements have indicated that limited denitrification can occur in the Arctic stratosphere. In situ measurements from the SOLVE campaign indicate polar stratospheric clouds (PSCs) composed of small numbers (about 3 x 10^ -4 cm^-3) of 10-20 micron particles (probably NAT or NAD). These observations raise the issue of whether low number density NAT PSCs can substantially denitrify the air with reasonable cloud lifetimes. In this study, we use a one dimensional cloud model to investigate the verticle redistribution of HNO3 by NAT/NAD PSCs. The cloud formation is driven by a temperature oscillation which drops the temperature below the NAT/NAD formation threshold (about 195 K) for a few days. We assume that a small fraction of the available aerosols act as NAT nuclei when the saturation ratio of HNO3 over NAT(NAD) exceeds 10(l.5). The result is a cloud between about 16 and 20 km in the model, with NAT/NAD particle effective radii as large as about 10 microns (in agreement with the SOLVE data). We find that for typical cloud lifetimes of 2-3 days or less, the net depletion of HNO3 is no more than 1-2 ppbv, regardless of the NAT or NAD particle number density. Repeated passes of the air column through the cold pool build up the denitrification to 3-4 ppbv, and the cloud altitude steadily decreases due to the downward transport of nitric acid. Increasing the cloud lifetime results in considerably more effective denitrification, even with very low cloud particle number densities. As expected, the degree of denitrification by NAT clouds is much larger than that by NAD Clouds. Significant denitrification by NAD Clouds is only possible if the cloud lifetime is several days or more. The clouds also cause a local maximum HNO3 mixing ratio at cloud base where the cloud particles sublimate.

Binding of NAD+-Glycohydrolase to Streptolysin O Stabilizes Both Toxins and Promotes Virulence of Group A Streptococcus

PubMed Central

Velarde, Jorge J.; O’Seaghdha, Maghnus; Baddal, Buket; Bastiat-Sempe, Benedicte

2017-01-01

ABSTRACT The globally dominant, invasive M1T1 strain of group A Streptococcus (GAS) harbors polymorphisms in the promoter region of an operon that contains the genes encoding streptolysin O (SLO) and NAD+-glycohydrolase (NADase), resulting in high-level expression of these toxins. While both toxins have been shown experimentally to contribute to pathogenesis, many GAS isolates lack detectable NADase activity. DNA sequencing of such strains has revealed that reduced or absent enzymatic activity can be associated with a variety of point mutations in nga, the gene encoding NADase; a commonly observed polymorphism associated with near-complete abrogation of activity is a substitution of aspartic acid for glycine at position 330 (G330D). However, nga has not been observed to contain early termination codons or mutations that would result in a truncated protein, even when the gene product contains missense mutations that abrogate enzymatic activity. It has been suggested that NADase that lacks NAD-glycohydrolase activity retains an as-yet-unidentified inherent cytotoxicity to mammalian cells and thus is still a potent virulence factor. We now show that expression of NADase, either enzymatically active or inactive, augments SLO-mediated toxicity for keratinocytes. In culture supernatants, SLO and NADase are mutually interdependent for protein stability. We demonstrate that the two proteins interact in solution and that both the translocation domain and catalytic domain of NADase are required for maximal binding between the two toxins. We conclude that binding of NADase to SLO stabilizes both toxins, thereby enhancing GAS virulence. PMID:28900022

Ferredoxin:NAD + oxidoreductase of Thermoanaerobacterium saccharolyticum and its role in ethanol formation [Identification of a ferredoxin:NAD + oxidoreductase of Thermoanaerobacterium saccharolyticum and its role in ethanol formation

DOE PAGES

Tian, Liang; Lo, Jonathan; Shao, Xiongjun; ...

2016-09-30

Ferredoxin:NAD + oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD +. This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway in Thermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activity in vitro with a ferredoxin-based FNOR assay. To determine its role in metabolism, the tsac_1705 gene was deleted in different strains of T. saccharolyticum. In wild-type T. saccharolyticum, deletion of tsac_1705more » resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR in T. saccharolyticum. When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. As a result, we tested the effect of heterologous expression of Tsac_1705 in Clostridium thermocellum and found improvements in both the titer and the yield of ethanol.« less

Redox non-innocence permits catalytic nitrene carbonylation by (dadi)Ti=NAd (Ad = adamantyl)

DOE PAGES

Heins, Spencer P.; Wolczanski, Peter T.; Cundari, Thomas R.; ...

2017-03-06

Application of the diamide, diimine {–CH=N(1,2-C 6H 4)N(2,6- iPr 2-C 6H 3)} 2 m ((dadi) m) ligand to titanium provided adducts (dadi)TiL x (1-L x; L x = THF, PMe 2Ph, (CNMe) 2), which possess the redox formulation [(dadi) 4-]Ti(IV)L x, and 22 πe - (4n + 2). Related complexes containing titanium-ligand multiple bonds, (dadi)Ti=X (2=X; X = O, NAd), exhibit a different dadi redox state, [(dadi) 2-]Ti(IV)X, consistent with 20 πe - (4n). The Redox Non-Innocence (RNI) displayed by dadi m impedes binding by CO, and permits catalytic conversion of AdN 3 + CO to AdNCO + N 2.more » Kinetics measurements support carbonylation of 2=NAd as the rate determining step. Finally, structural and computational evidence for the observed RNI is provided.« less

Upcoming replacements for NAD83, NAVD88 and IGLD85

NASA Astrophysics Data System (ADS)

Smith, D. A.; Snay, R.

2009-05-01

The National Geodetic Survey (NGS), part of the National Oceanic and Atmospheric Administration (NOAA) is responsible for defining, maintaining and providing access to the National Spatial Reference System (NSRS) for the United States. The NSRS is the official system to which all civil federal mapping agencies should refer, and contains, amongst other things, the official geopotential (historically "vertical") datum of NAVD 88, the 3-D geometric reference system (historically "horizontal datum") of NAD 83 and great lakes datum (IGLD 85). Although part of the United States NSRS, all three of these datums have been created through international partnerships across North America. Unfortunately, time has shown both the systematic errors existent within these datums, as well as the inherent weaknesses of relying exclusively on passive monuments to define and provide access to these datums. In recognition of these issues, the National Geodetic Survey has issued a "10 year plan", available online, which outlines steps which will be taken to update NAD 83, NAVD 88 and IGLD 85 concurrently around the year 2018. The primary source of success will be in the refinement of the CORS network and the upcoming execution of the GRAV-D project (Gravity for the Re-definition of the American Vertical Datum). Conversations are ongoing with colleagues in Canada, Mexico, Central America and the Caribbean in order to coordinate all of these efforts across the entire continent. The largest changes expected to occur are the removal of over 2 meters of non-geocentricity in NAD 83; the removal of decimeters of bias and over a meter of tilt in NAVD 88; the addition of the ability to track crustal motions (subsidence, tectonics, etc) in the datums; the removal of leveling as a tool for long-line height differencing; the use of a "best" geoid as the orthometric height reference surface; the addition of datum velocities (motions of the 3-D geometric reference system origin and motions of the geoid

Method to Detect the Cellular Source of Over-Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging.

PubMed

Bremer, Daniel; Leben, Ruth; Mothes, Ronja; Radbruch, Helena; Niesner, Raluca

2017-04-03

Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme. In the case of NADPH bound to members of the NADPH oxidases family we measured a fluorescence lifetime of 3650 psec as compared to enzymes typically active in cells, in which case fluorescence lifetimes of ∼2000 psec are measured. Here we present a robust protocol based on NAD(P)H fluorescence lifetime imaging in isolated cells to distinguish between normally active enzymes and NADPH oxidases, mainly responsible for oxidative stress. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo

PubMed Central

Goody, Michelle F.; Kelly, Meghan W.; Lessard, Kevin N.; Khalil, Andre; Henry, Clarissa A.

2010-01-01

Cell-matrix adhesion complexes (CMACs) play fundamental roles during morphogenesis. Given the ubiquitous nature of CMACs and their roles in many cellular processes, one question is how specificity of CMAC function is modulated. The clearly defined cell behaviors that generate segmentally reiterated axial skeletal muscle during zebrafish development comprise an ideal system with which to investigate CMAC function during morphogenesis. We found that Nicotinamide riboside kinase 2b (Nrk2b) cell autonomously modulates the molecular composition of CMACs in vivo. Nrk2b is required for normal Laminin polymerization at the myotendinous junction (MTJ). In Nrk2b-deficient embryos, at MTJ loci where Laminin is not properly polymerized, muscle fibers elongate into adjacent myotomes and are abnormally long. In yeast and human cells, Nrk2 phosphorylates Nicotinamide Riboside and generates NAD+ through an alternative salvage pathway. Exogenous NAD+ treatment rescues MTJ development in Nrk2b-deficient embryos, but not in laminin mutant embryos. Both Nrk2b and Laminin are required for localization of Paxillin, but not β-Dystroglycan, to CMACs at the MTJ. Overexpression of Paxillin in Nrk2b-deficient embryos is sufficient to rescue MTJ integrity. Taken together, these data show that Nrk2b plays a specific role in modulating subcellular localization of discrete CMAC components that in turn play roles in musculoskeletal development. Furthermore, these data suggest that Nrk2b-mediated synthesis of NAD+ is functionally upstream of Laminin adhesion and Paxillin subcellular localization during MTJ development. These results indicate a previously unrecognized complexity to CMAC assembly in vivo and also elucidate a novel role for NAD+ during morphogenesis. PMID:20566368

Replicatively senescent human fibroblasts reveal a distinct intracellular metabolic profile with alterations in NAD+ and nicotinamide metabolism.

PubMed

James, Emma L; Lane, James A E; Michalek, Ryan D; Karoly, Edward D; Parkinson, E Kenneth

2016-12-07

Cellular senescence occurs by proliferative exhaustion (PEsen) or following multiple cellular stresses but had not previously been subject to detailed metabolomic analysis. Therefore, we compared PEsen fibroblasts with proliferating and transiently growth arrested controls using a combination of different mass spectroscopy techniques. PEsen cells showed many specific alterations in both the NAD+ de novo and salvage pathways including striking accumulations of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) in the amidated salvage pathway despite no increase in nicotinamide phosphoribosyl transferase or in the NR transport protein, CD73. Extracellular nicotinate was depleted and metabolites of the deamidated salvage pathway were reduced but intracellular NAD+ and nicotinamide were nevertheless maintained. However, sirtuin 1 was downregulated and so the accumulation of NMN and NR was best explained by reduced flux through the amidated arm of the NAD+ salvage pathway due to reduced sirtuin activity. PEsen cells also showed evidence of increased redox homeostasis and upregulated pathways used to generate energy and cellular membranes; these included nucleotide catabolism, membrane lipid breakdown and increased creatine metabolism. Thus PEsen cells upregulate several different pathways to sustain their survival which may serve as pharmacological targets for the elimination of senescent cells in age-related disease.

Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells.

PubMed

Batchuluun, Battsetseg; Inoguchi, Toyoshi; Sonoda, Noriyuki; Sasaki, Shuji; Inoue, Tomoaki; Fujimura, Yoshinori; Miura, Daisuke; Takayanagi, Ryoichi

2014-01-01

Metformin and glucagon like peptide-1 (GLP-1) prevent diabetic cardiovascular complications and atherosclerosis. However, the direct effects on hyperglycemia-induced oxidative stress in endothelial cells are not fully understood. Thus, we aimed to evaluate the effects of metformin and a GLP-1 analog, liraglutide on high glucose-induced oxidative stress. Production of reactive oxygen species (ROS), activation of protein kinase C (PKC) and NAD(P)H oxidase, and changes in signaling molecules in response to high glucose exposure were evaluated in human aortic endothelial cells with and without treatment of metformin and liraglutide, alone or in combination. PKC-NAD(P)H oxidase pathway was assessed by translocation of GFP-fused PKCβ2 isoform and GFP-fused p47phox, a regulatory subunit of NAD(P)H oxidase, in addition to endogenous PKC phosphorylation and NAD(P)H oxidase activity. High glucose-induced ROS overproduction was blunted by metformin or liraglutide treatment, with a further decrease by a combination of these drugs. Exposure to high glucose caused PKCβ2 translocation and a time-dependent phosphorylation of endogenous PKC but failed to induce its translocation and phosphorylation in the cells treated with metformin and liraglutide. Furthermore, both drugs inhibited p47phox translocation and NAD(P)H oxidase activation, and prevented the high glucose-induced changes in intracellulalr diacylglycerol (DAG) level and phosphorylation of AMP-activated protein kinase (AMPK). A combination of these drugs further enhanced all of these effects. Metformin and liraglutide ameliorate high glucose-induced oxidative stress by inhibiting PKC-NAD(P)H oxidase pathway. A combination of these two drugs provides augmented protective effects, suggesting the clinical usefulness in prevention of diabetic vascular complications. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Interaction of arginine, lysine, and guanidine with surface residues of lysozyme: implication to protein stability.

PubMed