A Novel Cofactor-binding Mode in Bacterial IMP Dehydrogenases Explains Inhibitor Selectivity*

PubMed Central

Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R.; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

2015-01-01

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5′-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. PMID:25572472

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.

NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells.

PubMed

Ratajczak, Joanna; Joffraud, Magali; Trammell, Samuel A J; Ras, Rosa; Canela, Núria; Boutant, Marie; Kulkarni, Sameer S; Rodrigues, Marcelo; Redpath, Philip; Migaud, Marie E; Auwerx, Johan; Yanes, Oscar; Brenner, Charles; Cantó, Carles

2016-10-11

NAD + is a vital redox cofactor and a substrate required for activity of various enzyme families, including sirtuins and poly(ADP-ribose) polymerases. Supplementation with NAD + precursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), protects against metabolic disease, neurodegenerative disorders and age-related physiological decline in mammals. Here we show that nicotinamide riboside kinase 1 (NRK1) is necessary and rate-limiting for the use of exogenous NR and NMN for NAD + synthesis. Using genetic gain- and loss-of-function models, we further demonstrate that the role of NRK1 in driving NAD + synthesis from other NAD + precursors, such as nicotinamide or nicotinic acid, is dispensable. Using stable isotope-labelled compounds, we confirm NMN is metabolized extracellularly to NR that is then taken up by the cell and converted into NAD + . Our results indicate that mammalian cells require conversion of extracellular NMN to NR for cellular uptake and NAD + synthesis, explaining the overlapping metabolic effects observed with the two compounds.

NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells

PubMed Central

Ratajczak, Joanna; Joffraud, Magali; Trammell, Samuel A. J.; Ras, Rosa; Canela, Núria; Boutant, Marie; Kulkarni, Sameer S.; Rodrigues, Marcelo; Redpath, Philip; Migaud, Marie E.; Auwerx, Johan; Yanes, Oscar; Brenner, Charles; Cantó, Carles

2016-01-01

NAD+ is a vital redox cofactor and a substrate required for activity of various enzyme families, including sirtuins and poly(ADP-ribose) polymerases. Supplementation with NAD+ precursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), protects against metabolic disease, neurodegenerative disorders and age-related physiological decline in mammals. Here we show that nicotinamide riboside kinase 1 (NRK1) is necessary and rate-limiting for the use of exogenous NR and NMN for NAD+ synthesis. Using genetic gain- and loss-of-function models, we further demonstrate that the role of NRK1 in driving NAD+ synthesis from other NAD+ precursors, such as nicotinamide or nicotinic acid, is dispensable. Using stable isotope-labelled compounds, we confirm NMN is metabolized extracellularly to NR that is then taken up by the cell and converted into NAD+. Our results indicate that mammalian cells require conversion of extracellular NMN to NR for cellular uptake and NAD+ synthesis, explaining the overlapping metabolic effects observed with the two compounds. PMID:27725675

Conserved water-mediated recognition and dynamics of NAD+ (carboxamide group) to hIMPDH enzyme: water mimic approach toward the design of isoform-selective inhibitor.

PubMed

Bairagya, Hridoy R; Mishra, Deepak K; Mukhopadhyay, Bishnu P; Sekar, K

2014-01-01

Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in GMP biosynthesis pathway. Type I hIMPDH is expressed at lower levels in all cells, whereas type II is especially observed in acute myelogenous leukemia, chronic myelogenous leukemia cancer cells, and 10 ns simulation of the IMP-NAD(+) complex structures (PDB ID. 1B3O and 1JCN) have revealed the presence of a few conserved hydrophilic centers near carboxamide group of NAD(+). Three conserved water molecules (W1, W, and W1') in di-nucleotide binding pocket of enzyme have played a significant role in the recognition of carboxamide group (of NAD(+)) to D274 and H93 residues. Based on H-bonding interaction of conserved hydrophilic (water molecular) centers within IMP-NAD(+)-enzyme complexes and their recognition to NAD(+), some covalent modification at carboxamide group of di-nucleotide (NAD(+)) has been made by substituting the -CONH2group by -CONHNH2 (carboxyl hydrazide group) using water mimic inhibitor design protocol. The modeled structure of modified ligand may, though, be useful for the development of antileukemic agent or it could be act as better inhibitor for hIMPDH-II.

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

PubMed

Brickman, Timothy J; Suhadolc, Ryan J; McKelvey, Pamela J; Armstrong, Sandra K

2017-02-01

Nicotinamide adenine dinucleotide (NAD) is produced via de novo biosynthesis pathways and by salvage or recycling routes. The classical Bordetella bacterial species are known to be auxotrophic for nicotinamide or nicotinic acid. This study confirmed that Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis have the recycling/salvage pathway genes pncA and pncB, for use of nicotinamide or nicotinic acid, respectively, for NAD synthesis. Although these Bordetellae lack the nadA and nadB genes needed for de novo NAD biosynthesis, remarkably, they have one de novo pathway gene, nadC, encoding quinolinate phosphoribosyltransferase. Genomic analyses of taxonomically related Bordetella and Achromobacter species also indicated the presence of an 'orphan' nadC and the absence of nadA and nadB. When supplied as the sole NAD precursor, quinolinate promoted B. bronchiseptica growth, and the ability to use it required nadC. Co-expression of Bordetella nadC with the nadB and nadA genes of Paraburkholderia phytofirmans allowed B. bronchiseptica to grow in the absence of supplied pyridines, indicative of de novo NAD synthesis and functional confirmation of Bordetella NadC activity. Expression of nadC in B. bronchiseptica was influenced by nicotinic acid and by a NadQ family transcriptional repressor, indicating that these organisms prioritize their use of pyridines for NAD biosynthesis. © 2016 John Wiley & Sons Ltd.

Identification of Novel Triazole-Based Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors Endowed with Antiproliferative and Antiinflammatory Activity.

PubMed

Travelli, Cristina; Aprile, Silvio; Rahimian, Reza; Grolla, Ambra A; Rogati, Federica; Bertolotti, Mattia; Malagnino, Floriana; di Paola, Rosanna; Impellizzeri, Daniela; Fusco, Roberta; Mercalli, Valentina; Massarotti, Alberto; Stortini, Giorgio; Terrazzino, Salvatore; Del Grosso, Erika; Fakhfouri, Gohar; Troiani, Maria Pia; Alisi, Maria Alessandra; Grosa, Giorgio; Sorba, Giovanni; Canonico, Pier Luigi; Orsomando, Giuseppe; Cuzzocrea, Salvatore; Genazzani, Armando A; Galli, Ubaldina; Tron, Gian Cesare

2017-03-09

Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme involved in the recycling of nicotinamide to maintain adequate NAD levels inside the cells. It has been postulated to be a pharmacological target, as it is overexpressed in cancer cells as well as in inflammatory diseases. We describe the synthesis and characterization of a novel class of one-digit nanomolar NAMPT inhibitors based on in vitro characterization. The most active compound tested, 30c, displayed activity in xenograft and allograft models, strengthening the potential of NAMPT inhibitors as antitumoral drugs. Furthermore, in the present contribution we describe the ability of 30c to significantly improve the outcome of colitis in mice. Given that this is the first report of an effect of NAMPT inhibitors in colitis, this result paves the way for novel applications for this class of compounds.

A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity

DOE PAGES

Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; ...

2015-01-09

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD +, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexesmore » with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD + and XMP/NAD +. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD +-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD +-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. As a result, these findings offer a potential strategy for further ligand optimization.« less

A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity.

PubMed

Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

2015-02-27

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+), which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD(+) and XMP/NAD(+). In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD(+) adenosine moiety. More importantly, this new NAD(+)-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD(+)-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Role of Nicotinamide Adenine Dinucleotide and Related Precursors as Therapeutic Targets for Age-Related Degenerative Diseases: Rationale, Biochemistry, Pharmacokinetics, and Outcomes.

PubMed

Braidy, Nady; Berg, Jade; Clement, James; Khorshidi, Fatemeh; Poljak, Anne; Jayasena, Tharusha; Grant, Ross; Sachdev, Perminder

2018-05-11

Nicotinamide adenine dinucleotide (NAD + ) is an essential pyridine nucleotide that serves as an essential cofactor and substrate for a number of critical cellular processes involved in oxidative phosphorylation and ATP production, DNA repair, epigenetically modulated gene expression, intracellular calcium signaling, and immunological functions. NAD + depletion may occur in response to either excessive DNA damage due to free radical or ultraviolet attack, resulting in significant poly(ADP-ribose) polymerase (PARP) activation and a high turnover and subsequent depletion of NAD + , and/or chronic immune activation and inflammatory cytokine production resulting in accelerated CD38 activity and decline in NAD + levels. Recent studies have shown that enhancing NAD + levels can profoundly reduce oxidative cell damage in catabolic tissue, including the brain. Therefore, promotion of intracellular NAD + anabolism represents a promising therapeutic strategy for age-associated degenerative diseases in general, and is essential to the effective realization of multiple benefits of healthy sirtuin activity. The kynurenine pathway represents the de novo NAD + synthesis pathway in mammalian cells. NAD + can also be produced by the NAD + salvage pathway. Recent Advances: In this review, we describe and discuss recent insights regarding the efficacy and benefits of the NAD + precursors, nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN), in attenuating NAD + decline in degenerative disease states and physiological aging. Results obtained in recent years have shown that NAD + precursors can play important protective roles in several diseases. However, in some cases, these precursors may vary in their ability to enhance NAD + synthesis via their location in the NAD + anabolic pathway. Increased synthesis of NAD + promotes protective cell responses, further demonstrating that NAD + is a regulatory molecule associated with several biochemical pathways. In the next few years, the refinement of personalized therapy for the use of NAD + precursors and improved detection methodologies allowing the administration of specific NAD + precursors in the context of patients' NAD + levels will lead to a better understanding of the therapeutic role of NAD + precursors in human diseases. Antioxid. Redox Signal. 00, 000-000.

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

Synthesis, conformational analysis, and biological activity of new analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) as IMP dehydrogenase inhibitors.

PubMed

Franchetti, Palmarisa; Cappellacci, Loredana; Pasqualini, Michela; Petrelli, Riccardo; Jayaprakasan, Vetrichelvan; Jayaram, Hiremagalur N; Boyd, Donald B; Jain, Manojkumar D; Grifantini, Mario

2005-03-15

Thiazole-4-carboxamide adenine dinucleotide (TAD) analogues T-2'-MeAD (1) and T-3'-MeAD (2) containing, respectively, a methyl group at the ribose 2'-C-, and 3'-C-position of the adenosine moiety, were prepared as potential selective human inosine monophosphate dehydrogenase (IMPDH) type II inhibitors. The synthesis of heterodinucleotides was carried out by CDI-catalyzed coupling reaction of unprotected 2'-C-methyl- or 3'-C-methyl-adenosine 5'-monophosphate with 2',3'-O-isopropylidene-tiazofurin 5'-monophosphate, and then deisopropylidenation. Biological evaluation of dinucleotides 1 and 2 as inhibitors of recombinant human IMPDH type I and type II resulted in a good activity. Inhibition of both isoenzymes by T-2'-MeAD and T-3'-MeAD was noncompetitive with respect to NAD substrate. Binding of T-3'-MeAD was comparable to that of parent compound TAD, while T-2'-MeAD proved to be a weaker inhibitor. However, no significant difference was found in inhibition of the IMPDH isoenzymes. T-2'-MeAD and T-3'-MeAD were found to inhibit the growth of K562 cells (IC(50) 30.7 and 65.0muM, respectively).

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.

Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways

PubMed Central

Ma, Menggen; Song, Mingzhou

2010-01-01

Lignocellulosic biomass conversion inhibitors, furfural and HMF, inhibit microbial growth and interfere with subsequent fermentation of ethanol, posing significant challenges for a sustainable cellulosic ethanol conversion industry. Numerous yeast genes were found to be associated with the inhibitor tolerance. However, limited knowledge is available about mechanisms of the tolerance and the detoxification of the biomass conversion inhibitors. Using a robust standard for absolute mRNA quantification assay and a recently developed tolerant ethanologenic yeast Saccharomyces cerevisiae NRRL Y-50049, we investigate pathway-based transcription profiles relevant to the yeast tolerance and the inhibitor detoxification. Under the synergistic inhibitory challenges by furfural and HMF, Y-50049 was able to withstand the inhibitor stress, in situ detoxify furfural and HMF, and produce ethanol, while its parental control Y-12632 failed to function till 65 h after incubation. The tolerant strain Y-50049 displayed enriched genetic background with significantly higher abundant of transcripts for at least 16 genes than a non-tolerant parental strain Y-12632. The enhanced expression of ZWF1 appeared to drive glucose metabolism in favor of pentose phosphate pathway over glycolysis at earlier steps of glucose metabolisms. Cofactor NAD(P)H generation steps were likely accelerated by enzymes encoded by ZWF1, GND1, GND2, TDH1, and ALD4. NAD(P)H-dependent aldehyde reductions including conversion of furfural and HMF, in return, provided sufficient NAD(P)+ for NAD(P)H regeneration in the yeast detoxification pathways. Enriched genetic background and a well maintained redox balance through reprogrammed expression responses of Y-50049 were accountable for the acquired tolerance and detoxification of furfural to furan methanol and HMF to furan dimethanol. We present significant gene interactions and regulatory networks involved in NAD(P)H regenerations and functional aldehyde reductions under the inhibitor stress. PMID:19517136

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 derangement. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Discovery and Characterization of Novel Nonsubstrate and Substrate NAMPT Inhibitors

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

Wilsbacher, Julie L.; Cheng, Min; Cheng, Dong

Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell deathmore » when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236–45.« less

Reciprocal regulation by hypoxia-inducible factor-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in osteoarthritis.

PubMed

Oh, H; Kwak, J-S; Yang, S; Gong, M-K; Kim, J-H; Rhee, J; Kim, S K; Kim, H-E; Ryu, J-H; Chun, J-S

2015-12-01

Hypoxia-inducible factor-2α (HIF-2α) transcriptionally upregulates Nampt in articular chondrocytes. NAMPT, which exhibits nicotinamide phosphoribosyltransferase activity, in turn causes osteoarthritis (OA) in mice by stimulating the expression of matrix-degrading enzymes. Here, we sought to elucidate whether HIF-2α activates the NAMPT-NAD(+)-SIRT axis in chondrocytes and thereby contributes to the pathogenesis of OA. Assays of NAD levels, SIRT activity, reporter gene activity, mRNA, and protein levels were conducted in primary cultured mouse articular chondrocytes. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2α (Ad-Epas1) or NAMPT (Ad-Nampt). The functions of SIRT in OA were examined by IA co-injection of SIRT inhibitors or adenovirus expressing individual SIRT isoforms or shRNA targeting specific SIRT isoforms. HIF-2α activated the NAMPT-NAD(+)-SIRT axis in chondrocytes by upregulating NAMPT, which stimulated NAD(+) synthesis and thereby activated SIRT family members. The activated NAMPT-SIRT pathway, in turn, promoted HIF-2α protein stability by negatively regulating its hydroxylation and 26S proteasome-mediated degradation, resulting in increased HIF-2α transcriptional activity. Among SIRT family members (SIRT1-7), SIRT2 and SIRT4 were positively associated with HIF-2α stability and transcriptional activity in chondrocytes. This reciprocal regulation was required for the expression of catabolic matrix metalloproteinases (MMP3, MMP12, and MMP13) and OA cartilage destruction caused by IA injection of Ad-Epas1 Ad-Nampt. The reciprocal regulation of HIF-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in OA cartilage destruction caused by HIF-2α or NAMPT. Copyright © 2015 The Authors. Published by Elsevier Ltd.. 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

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

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.

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.

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

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.

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

Fahrer, Joerg, E-mail: joerg.fahrer@uni-ulm.de; Wagner, Silvia; Buerkle, Alexander

Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin didmore » not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.« less

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

Vasoactivity of rucaparib, a PARP-1 inhibitor, is a complex process that involves myosin light chain kinase, P2 receptors, and PARP itself.

PubMed

McCrudden, Cian M; O'Rourke, Martin G; Cherry, Kim E; Yuen, Hiu-Fung; O'Rourke, Declan; Babur, Muhammad; Telfer, Brian A; Thomas, Huw D; Keane, Patrick; Nambirajan, Thiagarajan; Hagan, Chris; O'Sullivan, Joe M; Shaw, Chris; Williams, Kaye J; Curtin, Nicola J; Hirst, David G; Robson, Tracy

2015-01-01

Therapeutic inhibition of poly(ADP-ribose) polymerase (PARP), as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK) 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation.

Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice.

PubMed

Neelakantan, Harshini; Vance, Virginia; Wetzel, Michael D; Wang, Hua-Yu Leo; McHardy, Stanton F; Finnerty, Celeste C; Hommel, Jonathan D; Watowich, Stanley J

2018-01-01

There is a critical need for new mechanism-of-action drugs that reduce the burden of obesity and associated chronic metabolic comorbidities. A potentially novel target to treat obesity and type 2 diabetes is nicotinamide-N-methyltransferase (NNMT), a cytosolic enzyme with newly identified roles in cellular metabolism and energy homeostasis. To validate NNMT as an anti-obesity drug target, we investigated the permeability, selectivity, mechanistic, and physiological properties of a series of small molecule NNMT inhibitors. Membrane permeability of NNMT inhibitors was characterized using parallel artificial membrane permeability and Caco-2 cell assays. Selectivity was tested against structurally-related methyltransferases and nicotinamide adenine dinucleotide (NAD + ) salvage pathway enzymes. Effects of NNMT inhibitors on lipogenesis and intracellular levels of metabolites, including NNMT reaction product 1-methylnicotianamide (1-MNA) were evaluated in cultured adipocytes. Effects of a potent NNMT inhibitor on obesity measures and plasma lipid were assessed in diet-induced obese mice fed a high-fat diet. Methylquinolinium scaffolds with primary amine substitutions displayed high permeability from passive and active transport across membranes. Importantly, methylquinolinium analogues displayed high selectivity, not inhibiting related SAM-dependent methyltransferases or enzymes in the NAD + salvage pathway. NNMT inhibitors reduced intracellular 1-MNA, increased intracellular NAD + and S-(5'-adenosyl)-l-methionine (SAM), and suppressed lipogenesis in adipocytes. Treatment of diet-induced obese mice systemically with a potent NNMT inhibitor significantly reduced body weight and white adipose mass, decreased adipocyte size, and lowered plasma total cholesterol levels. Notably, administration of NNMT inhibitors did not impact total food intake nor produce any observable adverse effects. These results support development of small molecule NNMT inhibitors as therapeutics to reverse diet-induced obesity and validate NNMT as a viable target to treat obesity and related metabolic conditions. Increased flux of key cellular energy regulators, including NAD + and SAM, may potentially define the therapeutic mechanism-of-action of NNMT inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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

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

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.

A [32P]-NAD+-based method to identify and quantitate long residence time enoyl-ACP reductase inhibitors

PubMed Central

Yu, Weixuan; Neckles, Carla; Chang, Andrew; Bommineni, Gopal Reddy; Spagnuolo, Lauren; Zhang, Zhuo; Liu, Nina; Lai, Christina; Truglio, James; Tonge, Peter J.

2015-01-01

The classical methods for quantifying drug-target residence time (tR) use loss or regain of enzyme activity in progress curve kinetic assays. However, such methods become imprecise at very long residence times, mitigating the use of alternative strategies. Using the NAD(P)H-dependent FabI enoyl-ACP reductase as a model system, we developed a Penefsky column-based method for direct measurement of tR, where the off-rate of the drug was determined with radiolabeled [adenylate-32P] NAD(P+) cofactor. Twenty-three FabI inhibitors were analyzed and a mathematical model was used to estimate limits to the tR values of each inhibitor based on percent drug-target complex recovery following gel filtration. In general, this method showed good agreement with the classical steady state kinetic methods for compounds with tR values of 10-100 min. In addition, we were able to identify seven long tR inhibitors (100-1500 min) and to accurately determine their tR values. The method was then used to measure tR as a function of temperature, an analysis not previously possible using the standard kinetic approach due to decreased NAD(P)H stability at elevated temperatures. In general, a 4-fold difference in tR was observed when the temperature was increased from 25 °C to 37 °C . PMID:25684450

Role of the epidermal growth factor receptor in signaling strain-dependent activation of the brain natriuretic peptide gene.

PubMed

Anderson, Hope D I; Wang, Feng; Gardner, David G

2004-03-05

The epidermal growth factor receptor (EGFR) and ectoshedding of heparin-binding epidermal growth factor (HBEGF), an EGFR ligand, have been linked to the development of cardiac myocyte hypertrophy. However, the precise role that the liganded EGFR plays in the transcriptional activation of the gene program that accompanies hypertrophy remains undefined. Utilizing the human (h) BNP gene as a model of hypertrophy-dependent gene activation, we show that activation of the EGFR plays an important role in mediating mechanical strain-dependent stimulation of the hBNP promoter. Strain promotes endothelin (ET) generation through NAD(P)H oxidase-dependent production of reactive oxygen species. ET in turn induces metalloproteinase-mediated cleavage of pro-HBEGF and ectoshedding of HBEGF, which activates the EGFR and stimulates hBNP promoter activity. HBEGF also stimulates other phenotypic markers of hypertrophy including protein synthesis and sarcomeric assembly. The antioxidant N-acetylcysteine or the NAD(P)H oxidase inhibitor, apocynin, inhibited strain-dependent activation of the ET-1 promoter, HBEGF shedding, and hBNP promoter activation. The metalloproteinase inhibitor, GM-6001, prevented the induction of HBEGF ectoshedding and the hBNP promoter response to strain, suggesting a critical role for the metalloproteinase-dependent cleavage event in signaling the strain response. These findings suggest that metalloproteinase activity as an essential step in this pathway may prove to be a relevant therapeutic target in the management of cardiac hypertrophy.

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 endogenous vascular antioxidant defences. PMID:22817606

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.

Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration

PubMed Central

Mukherjee, Sarmistha; Chellappa, Karthikeyani; Moffitt, Andrea; Ndungu, Joan; Dellinger, Ryan W.; Davis, James G.; Agarwal, Beamon; Baur, Joseph A.

2016-01-01

The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing Nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nampt overexpressing mice were mildly hyperglycemic at baseline and, similarly to the mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking Nampt in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR. Conclusion NAD availability is limiting during liver regeneration and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. PMID:27809334

Reverse electron transport effects on NADH formation and metmyoglobin reduction.

PubMed

Belskie, K M; Van Buiten, C B; Ramanathan, R; Mancini, R A

2015-07-01

The objective was to determine if NADH generated via reverse electron flow in beef mitochondria can be used for electron transport-mediated reduction and metmyoglobin reductase pathways. Beef mitochondria were isolated from bovine hearts (n=5) and reacted with combinations of succinate, NAD, and mitochondrial inhibitors to measure oxygen consumption and NADH formation. Mitochondria and metmyoglobin were reacted with succinate, NAD, and mitochondrial inhibitors to measure electron transport-mediated metmyoglobin reduction and metmyoglobin reductase activity. Addition of succinate and NAD increased oxygen consumption, NADH formation, electron transport-mediated metmyoglobin reduction, and reductase activity (p<0.05). Addition of antimycin A prevented electron flow beyond complex III, therefore, decreasing oxygen consumption and electron transport-mediated metmyoglobin reduction. Addition of rotenone prevented reverse electron flow, increased oxygen consumption, increased electron transport-mediated metmyoglobin reduction, and decreased NADH formation. Succinate and NAD can generate NADH in bovine tissue postmortem via reverse electron flow and this NADH can be used by both electron transport-mediated and metmyoglobin reductase pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

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.

Exploring NAD+ metabolism in host-pathogen interactions.

PubMed

Mesquita, Inês; Varela, Patrícia; Belinha, Ana; Gaifem, Joana; Laforge, Mireille; Vergnes, Baptiste; Estaquier, Jérôme; Silvestre, Ricardo

2016-03-01

Nicotinamide adenine dinucleotide (NAD(+)) is a vital molecule found in all living cells. NAD(+) intracellular levels are dictated by its synthesis, using the de novo and/or salvage pathway, and through its catabolic use as co-enzyme or co-substrate. The regulation of NAD(+) metabolism has proven to be an adequate drug target for several diseases, including cancer, neurodegenerative or inflammatory diseases. Increasing interest has been given to NAD(+) metabolism during innate and adaptive immune responses suggesting that its modulation could also be relevant during host-pathogen interactions. While the maintenance of NAD(+) homeostatic levels assures an adequate environment for host cell survival and proliferation, fluctuations in NAD(+) or biosynthetic precursors bioavailability have been described during host-pathogen interactions, which will interfere with pathogen persistence or clearance. Here, we review the double-edged sword of NAD(+) metabolism during host-pathogen interactions emphasizing its potential for treatment of infectious diseases.

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.

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

Inhibiting poly ADP-ribosylation increases fatty acid oxidation and protects against fatty liver disease.

PubMed

Gariani, Karim; Ryu, Dongryeol; Menzies, Keir J; Yi, Hyon-Seung; Stein, Sokrates; Zhang, Hongbo; Perino, Alessia; Lemos, Vera; Katsyuba, Elena; Jha, Pooja; Vijgen, Sandrine; Rubbia-Brandt, Laura; Kim, Yong Kyung; Kim, Jung Tae; Kim, Koon Soon; Shong, Minho; Schoonjans, Kristina; Auwerx, Johan

2017-01-01

To date, no pharmacological therapy has been approved for non-alcoholic fatty liver disease (NAFLD). The aim of the present study was to evaluate the therapeutic potential of poly ADP-ribose polymerase (PARP) inhibitors in mouse models of NAFLD. As poly ADP-ribosylation (PARylation) of proteins by PARPs consumes nicotinamide adenine dinucleotide (NAD + ), we hypothesized that overactivation of PARPs drives NAD + depletion in NAFLD. Therefore, we assessed the effectiveness of PARP inhibition to replenish NAD + and activate NAD + -dependent sirtuins, hence improving hepatic fatty acid oxidation. To do this, we examined the preventive and therapeutic benefits of the PARP inhibitor (PARPi), olaparib, in different models of NAFLD. The induction of NAFLD in C57BL/6J mice using a high-fat high-sucrose (HFHS)-diet increased PARylation of proteins by PARPs. As such, increased PARylation was associated with reduced NAD + levels and mitochondrial function and content, which was concurrent with elevated hepatic lipid content. HFHS diet supplemented with PARPi reversed NAFLD through repletion of NAD + , increasing mitochondrial biogenesis and β-oxidation in liver. Furthermore, PARPi reduced reactive oxygen species, endoplasmic reticulum stress and fibrosis. The benefits of PARPi treatment were confirmed in mice fed with a methionine- and choline-deficient diet and in mice with lipopolysaccharide-induced hepatitis; PARP activation was attenuated and the development of hepatic injury was delayed in both models. Using Sirt1 hep-/- mice, the beneficial effects of a PARPi-supplemented HFHS diet were found to be Sirt1-dependent. Our study provides a novel and practical pharmacological approach for treating NAFLD, fueling optimism for potential clinical studies. Non-alcoholic fatty liver disease (NAFLD) is now considered to be the most common liver disease in the Western world and has no approved pharmacological therapy. PARP inhibitors given as a treatment in two different mouse models of NAFLD confer a protection against its development. PARP inhibitors may therefore represent a novel and practical pharmacological approach for treating NAFLD. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration.

PubMed

Mukherjee, Sarmistha; Chellappa, Karthikeyani; Moffitt, Andrea; Ndungu, Joan; Dellinger, Ryan W; Davis, James G; Agarwal, Beamon; Baur, Joseph A

2017-02-01

The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nicotinamide phosphoribosyltransferase overexpressing mice were mildly hyperglycemic at baseline and, similar to mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking nicotinamide phosphoribosyltransferase in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR. NAD availability is limiting during liver regeneration, and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. (Hepatology 2017;65:616-630). © 2016 by the American Association for the Study of Liver Diseases.

A Nampt inhibitor FK866 mimics vitamin B3 deficiency by causing senescence of human fibroblastic Hs68 cells via attenuation of NAD(+)-SIRT1 signaling.

PubMed

Song, Tuzz-Ying; Yeh, Shu-Lan; Hu, Miao-Lin; Chen, Mei-Yau; Yang, Nae-Cherng

2015-12-01

Vitamin B3 (niacin) deficiency can cause pellagra with symptoms of dermatitis, diarrhea and dementia. However, it is unclear whether the vitamin B3 deficiency causes human aging. FK866 (a Nampt inhibitor) can reduce intracellular NAD(+) level and induce senescence of human Hs68 cells. However, the mechanisms underlying FK866-induced senescence of Hs68 cells are unclear. In this study, we used FK866 to mimic the effects of vitamin B3 deficiency to reduce the NAD(+) level and investigated the mechanisms of FK866-induced senescence of Hs68 cells. We hypothesized that FK866 induced the senescence of Hs68 cells via an attenuation of NAD(+)-silent information regulator T1 (SIRT1) signaling. We found that FK866 induced cell senescence and diminished cellular NAD(+) levels and SIRT1 activity (detected by acetylation of p53), and these effects were dramatically antagonized by co-treatment with nicotinic acid, nicotinamide, or NAD(+). In contrast, the protein expression of SIRT1, AMP-activated protein kinase, mammalian target of rapamycin, and nicotinamide phosphoribosyltransferase (Nampt) was not affected by FK866. In addition, the role of GSH in the FK866-induced cells senescence may be limited, as N-acetylcysteine did not antagonize FK866-induced cell senescence. These results suggest that FK866 induces cell senescence via attenuation of NAD(+)-SIRT1 signaling. The effects of vitamin B3 deficiency on human aging warrant further investigation.

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, the proapoptotic action of palmitate seems not to be mediated by decreased NAD levels.

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

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.

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 individually. This study identified synergy between an antifungal peptide and a group of peptides that do not affect fungal growth in vitro. This provides the foundation for generation of transgenic plants with increased resistance to fungal disease and identification of antifungal accessory factors that enhance the activity of innate immune molecules but do not have an antifungal effect on their own. PMID:29062897

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.

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.

Molecular evolution of the nicotinic acid requirement within the Shigella/EIEC pathotype.

PubMed

Di Martino, Maria Letizia; Fioravanti, Rosa; Barbabella, Giada; Prosseda, Gianni; Colonna, Bianca; Casalino, Mariassunta

2013-12-01

Nicotinamide adenine dinucleotide (NAD) is a crucial cofactor in several anabolic and catabolic reactions. NAD derives from quinolinic acid (QUIN) which in Escherichia coli is obtained through a pyridine salvage pathway or a de novo synthesis pathway. In the latter case, two enzymes, L-aspartate oxidase (NadB) and quinolinate synthase (NadA), are required for the synthesis of QUIN. In contrast to its E. coli ancestor, Shigella spp., the causative agent of bacillary dissentery, lacks the de novo pathway and strictly requires nicotinic acid for growth (Nic⁻ phenotype). This phenotype depends on the silencing of the nadB and nadA genes and its pathoadaptive nature is suggested by the observation that QUIN attenuates the Shigella invasive process. Shigella shares the pathogenicity mechanism with enteronvasive E. coli (EIEC), a group of pathogenic E. coli. On the basis of this similarity EIEC and Shigella have been grouped into a single E. coli pathotype. However EIEC strains do not constitute a homogeneous group and do not possess the complete set of characters that define Shigella strains. In this work we have analysed thirteen EIEC strains belonging to different serotypes and originating from different geographic areas. We show that, in contrast to Shigella, only some EIEC strains require nicotinic acid for growth in minimal medium. Moreover, by studying the emergence of the Nic⁻ phenotype in all serotypes of S. flexneri, as well as in S. sonnei and S. dysenteriae, we describe which molecular rearrangements occurred and which mutations are responsible for the inactivation of the nadA and nadB genes. Our data confirm that the genome of Shigella is extremely dynamic and support the hypothesis that EIEC might reflect an earlier stage of the pathoadaptation process undergone by Shigella. Copyright © 2013. Published by Elsevier GmbH.

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

Computational optimization of AG18051 inhibitor for amyloid-beta binding alcohol dehydrogenase enzyme

NASA Astrophysics Data System (ADS)

Marques, Alexandra T.; Antunes, Agostinho; Fernandes, Pedro A.; Ramos, Maria J.

Amyloid-beta (Abeta) binding alcohol dehydrogenase (ABAD) is a multifunctional enzyme involved in maintaining the homeostasis. The enzyme can also mediate some diseases, including genetic diseases, Alzheimer's disease, and possibly some prostate cancers. Potent inhibitors of ABAD might facilitate a better clarification of the functions of the enzyme under normal and pathogenic conditions and might also be used for therapeutic intervention in disease conditions mediated by the enzyme. The AG18051 is the only presently available inhibitor of ABAD. It binds in the active-site cavity of the enzyme and reacts with the NAD+ cofactor to form a covalent adduct. In this work, we use computational methods to perform a rational optimization of the AG18051 inhibitor, through the introduction of chemical substitutions directed to improve the affinity of the inhibitor to the enzyme. The molecular mechanics-Poisson-Boltzmann surface area methodology was used to predict the relative free binding energy of the different modified inhibitor-NAD-enzyme complexes. We show that it is possible to increase significantly the affinity of the inhibitor to the enzyme with small modifications, without changing the overall structure and ADME (absorption, distribution, metabolism, and excretion) properties of the original inhibitor.

Chronic alcohol binging injures the liver and other organs by reducing NAD⁺ levels required for sirtuin's deacetylase activity.

PubMed

French, Samuel W

2016-04-01

NAD(+) levels are markedly reduced when blood alcohol levels are high during binge drinking. This causes liver injury to occur because the enzymes that require NAD(+) as a cofactor such as the sirtuin de-acetylases cannot de-acetylate acetylated proteins such as acetylated histones. This prevents the epigenetic changes that regulate metabolic processes and which prevent organ injury such as fatty liver in response to alcohol abuse. Hyper acetylation of numerous regulatory proteins develops. Systemic multi-organ injury occurs when NAD(+) is reduced. For instance the Circadian clock is altered if NAD(+) is not available. Cell cycle arrest occurs due to up regulation of cell cycle inhibitors leading to DNA damage, mutations, apoptosis and tumorigenesis. NAD(+) is linked to aging in the regulation of telomere stability. NAD(+) is required for mitochondrial renewal. Alcohol dehydrogenase is present in every visceral organ in the body so that there is a systemic reduction of NAD(+) levels in all of these organs during binge drinking. Copyright © 2016. Published by Elsevier Inc.

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.

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.

NAD(+)- dependent deacetylase SIRT3 regulates mitochondrial protein synthesis by deacetylation of the ribosomal protein MRPL10

USDA-ARS?s Scientific Manuscript database

A member of the sirtuin family of NAD (+)-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated ...

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.

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.

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

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.

Structure-function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa.

PubMed

Yates, Susan P; Taylor, Patricia L; Jørgensen, René; Ferraris, Dana; Zhang, Jie; Andersen, Gregers R; Merrill, A Rod

2005-02-01

The mono-ADPRT (mono-ADP-ribosyltransferase), Pseudomonas aeruginosa ETA (exotoxin A), catalyses the transfer of ADP-ribose from NAD+ to its protein substrate. A series of water-soluble compounds that structurally mimic the nicotinamide moiety of NAD+ was investigated for their inhibition of the catalytic domain of ETA. The importance of an amide locked into a hetero-ring structure and a core hetero-ring system that is planar was a trend evident by the IC50 values. Also, the weaker inhibitors have core ring structures that are less planar and thus more flexible. One of the most potent inhibitors, PJ34, was further characterized and shown to exhibit competitive inhibition with an inhibition constant K(i) of 140 nM. We also report the crystal structure of the catalytic domain of ETA in complex with PJ34, the first example of a mono-ADPRT in complex with an inhibitor. The 2.1 A (1 A=0.1 nm) resolution structure revealed that PJ34 is bound within the nicotinamide-binding pocket and forms stabilizing hydrogen bonds with the main chain of Gly-441 and to the side-chain oxygen of Gln-485, a member of a proposed catalytic loop. Structural comparison of this inhibitor complex with diphtheria toxin (a mono-ADPRT) and with PARPs [poly(ADP-ribose) polymerases] shows similarity of the catalytic residues; however, a loop similar to that found in ETA is present in diphtheria toxin but not in PARP. The present study provides insight into the important features required for inhibitors that mimic NAD+ and their binding to the mono-ADPRT family of toxins.

Expression patterns of nicotinamide phosphoribosyltransferase and nicotinic acid phosphoribosyltransferase in human malignant lymphomas.

PubMed

Olesen, Uffe Høgh; Hastrup, Nina; Sehested, Maxwell

2011-04-01

The purpose of the study was to determine in human malignant lymphomas the expression patterns of nicotinamide phosphoribosyltransferase (NAMPT) and nicotinic acid phosphoribosyltransferase (NAPRT), the primary, rate-limiting enzymes in the synthesis of NAD+. NAMPT is a potential biomarker for sensitivity to NAMPT inhibitors and NAPRT is a biomarker for the use of nicotinic acid as a chemoprotectant in treatment with NAMPT inhibitors. The NAMPT inhibitor, APO866, is currently in clinical phase II trials in lymphomas. The expression of NAMPT and NAPRT was investigated in 53 samples of malignant lymphomas (diffuse large B-cell lymphoma, follicular B-cell lymphoma, Hodgkin's lymphoma and peripheral T-cell lymphoma). The expression of NAMPT was generally high in the more aggressive malignant lymphomas, with >80% strong expression, whereas the expression in the more indolent follicular lymphoma (FL) was significantly lower (>75% moderate or low expression, p = 0.0002). NAMPT was very highly expressed in Hodgkin Reed-Sternberg cells in Hodgkin's lymphoma. NAPRT expression was more varied (p > 0.0001) with 30-50% low expression except for Hodgkin's lymphoma where 85% displayed low expression (p = 0.0024). In conclusion, FL are a promising target for NAMPT inhibitors whereas substantial subsets of malignant lymphomas especially in Hodgkin lymphoma may be suitable for a combination treatment with nicotinic acid and NAMPT inhibitors. © 2011 The Authors. APMIS © 2011 APMIS.

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.

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.

Acute Mitochondrial Inhibition by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK) 1/2 Inhibitors Regulates Proliferation*

PubMed Central

Ripple, Maureen O.; Kim, Namjoon; Springett, Roger

2013-01-01

The Ras-MEK1/2-ERK1/2 kinase signaling pathway regulates proliferation, survival, and differentiation and, because it is often aberrant in tumors, is a popular target for small molecule inhibition. A novel metabolic analysis that measures the real-time oxidation state of NAD(H) and the hemes of the electron transport chain and oxygen consumption within intact, living cells found that structurally distinct MEK1/2 inhibitors had an immediate, dose-dependent effect on mitochondrial metabolism. The inhibitors U0126, MIIC and PD98059 caused NAD(H) reduction, heme oxidation, and decreased oxygen consumption, characteristic of complex I inhibition. PD198306, an orally active MEK1/2 inhibitor, acted as an uncoupler. Each MEK1/2 inhibitor depleted phosphorylated ERK1/2 and inhibited proliferation, but the most robust antiproliferative effects always correlated with the metabolic failure which followed mitochondrial inhibition rather than inhibition of MEK1/2. This warrants rethinking the role of ERK1/2 in proliferation and emphasizes the importance of mitochondrial function in this process. PMID:23235157

Profiling the role of mammalian target of rapamycin in the vascular smooth muscle metabolome in pulmonary arterial hypertension

PubMed Central

Kudryashova, Tatiana V.; Goncharov, Dmitry A.; Pena, Andressa; Ihida-Stansbury, Kaori; DeLisser, Horace; Kawut, Steven M.

2015-01-01

Abstract Increased proliferation and resistance to apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMCs), coupled with metabolic reprogramming, are key components of pulmonary vascular remodeling, a major and currently irreversible pathophysiological feature of pulmonary arterial hypertension (PAH). We recently reported that activation of mammalian target of rapamycin (mTOR) plays a key role in increased energy generation and maintenance of the proliferative, apoptosis-resistant PAVSMC phenotype in human PAH, but the downstream effects of mTOR activation on PAH PAVSMC metabolism are not clear. Using liquid and gas chromatography–based mass spectrometry, we performed pilot metabolomic profiling of human microvascular PAVSMCs from idiopathic-PAH subjects before and after treatment with the selective adenosine triphosphate–competitive mTOR inhibitor PP242 and from nondiseased lungs. We have shown that PAH PAVSMCs have a distinct metabolomic signature of altered metabolites—components of fatty acid synthesis, deficiency of sugars, amino sugars, and nucleotide sugars—intermediates of protein and lipid glycosylation, and downregulation of key biochemicals involved in glutathione and nicotinamide adenine dinucleotide (NAD) metabolism. We also report that mTOR inhibition attenuated or reversed the majority of the PAH-specific abnormalities in lipogenesis, glycosylation, glutathione, and NAD metabolism without affecting altered polyunsaturated fatty acid metabolism. Collectively, our data demonstrate a critical role of mTOR in major PAH PAVSMC metabolic abnormalities and suggest the existence of de novo lipid synthesis in PAVSMCs in human PAH that may represent a new, important component of disease pathogenesis worthy of future investigation. PMID:26697174

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

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.

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.

Acetylcholine promotes the emergence and elongation of lateral roots of Raphanus sativus

PubMed Central

Sugiyama, Kou-ichi

2011-01-01

Radish (Raphanus sativus L.) was grown on four layers of paper towel moistened with distilled water with and without acetylcholine (ACh) for five days in the dark after sowing. ACh at 1 nM promoted the growth (emergence and elongation) of lateral roots of radish plants, but had no effect on the stems and main roots. Moreover, ACh enhanced the dry weight of roots [main (primary) + lateral roots]. Neostigmine, an inhibitor of acetylcholinesterase (AChE) also promoted the emergence and elongation of lateral roots, and atropine, a competitive inhibitor of ACh receptor, suppressed the emergence and elongation. ACh promoted the activities of glyceraldehyde-3-phosephate dehydrogenase (G-3-PD), nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase (NAD-ICDH), succinate dehydrogenase (SDH) and cytochrome-c oxidase (Cyt-c OD) in seedlings. Moreover, ACh suppressed the activity of AChE and increased the amount of proteins and pyridine nucleotides (NAD and NADH) in the roots of the seedlings. It also increased the activities of NAD-forming enzymes [NAD synthetase and ATP-nicotinamide mononucleotide (ATP-NMN) adenyltransferase], and enhanced the amount of DNA in the roots of the seedlings. The relationship between ACh and the emergence and growth of lateral roots was discussed from a biochemical viewpoint. PMID:21900743

Pro-inflammatory effects of interleukin-17A on vascular smooth muscle cells involve NAD(P)H- oxidase derived reactive oxygen species.

PubMed

Pietrowski, Eweline; Bender, Bianca; Huppert, Jula; White, Robin; Luhmann, Heiko J; Kuhlmann, Christoph R W

2011-01-01

T cells are known for their contribution to the inflammatory element of atherosclerosis. Recently, it has been demonstrated that the Th17 derived cytokine IL-17 is involved in the pro-inflammatory response of vascular smooth muscle cells (VSMC). The aim of the present study was to examine whether reactive oxygen species (ROS) might be involved in this context. The effect of IL-17A on ROS generation was examined using the fluorescent dye 2'7'-dichlorodihydrofluorescein (H(2)DCF) in primary murine VSMC. IL-17A induced an increase in H(2)DCF fluorescence in VSMC, and this effect was blocked by the NAD(P)H-oxidase inhibitor apocynin and siRNA targeting Nox2. The p38-MAPK inhibitors SB203580 and SB202190 dose-dependently reduced the IL-17A induced ROS production. The IL-17A induced release of the pro-inflammatory cytokines IL-6, G-CSF, GM-CSF and MCP-1 from VSMC, as detected by the Luminex technology, was completely abolished by NAD(P)H-oxidase inhibition. Taken together, our data indicate that IL-17A causes the NAD(P)H-oxidase dependent generation of ROS leading to a pro-inflammatory activation of VSMC. Copyright © 2010 S. Karger AG, Basel.

Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

PubMed

Roessler, Claudia; Nowak, Theresa; Pannek, Martin; Gertz, Melanie; Nguyen, Giang T T; Scharfe, Michael; Born, Ilona; Sippl, Wolfgang; Steegborn, Clemens; Schutkowski, Mike

2014-09-26

Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Discovery of potent and selective sirtuin 2 (SIRT2) inhibitors using a fragment-based approach.

PubMed

Cui, Huaqing; Kamal, Zeeshan; Ai, Teng; Xu, Yanli; More, Swati S; Wilson, Daniel J; Chen, Liqiang

2014-10-23

Sirtuin 2 (SIRT2) is one of the sirtuins, a family of NAD(+)-dependent deacetylases that act on a variety of histone and non-histone substrates. Accumulating biological functions and potential therapeutic applications have drawn interest in the discovery and development of SIRT2 inhibitors. Herein we report our discovery of novel SIRT2 inhibitors using a fragment-based approach. Inspired by the purported close binding proximity of suramin and nicotinamide, we prepared two sets of fragments, namely, the naphthylamide sulfonic acids and the naphthalene-benzamides and -nicotinamides. Biochemical evaluation of these two series provided structure-activity relationship (SAR) information, which led to the design of (5-benzamidonaphthalen-1/2-yloxy)nicotinamide derivatives. Among these inhibitors, one compound exhibited high anti-SIRT2 activity (48 nM) and excellent selectivity for SIRT2 over SIRT1 and SIRT3. In vitro, it also increased the acetylation level of α-tubulin, a well-established SIRT2 substrate, in both concentration- and time-dependent manners. Further kinetic studies revealed that this compound behaves as a competitive inhibitor against the peptide substrate and most likely as a noncompetitive inhibitor against NAD(+). Taken together, these results indicate that we have discovered a potent and selective SIRT2 inhibitor whose novel structure merits further exploration.

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.

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.

Antitumor effect of combined NAMPT and CD73 inhibition in an ovarian cancer model

PubMed Central

Magnone, Mirko; Zamporlini, Federica; Emionite, Laura; Sturla, Laura; Bianchi, Giovanna; Vigliarolo, Tiziana; Nahimana, Aimable; Nencioni, Alessio; Raffaelli, Nadia; Bruzzone, Santina

2016-01-01

Nicotinamide phosphoribosyltransferase (NAMPT) is a crucial enzyme in the biosynthesis of intracellular NAD+. NAMPT inhibitors have potent anticancer activity in several preclinical models by depleting NAD+ and ATP levels. Recently, we demonstrated that CD73 enables the utilization of extracellular NAD+/nicotinamide mononucleotide (NMN) by converting them to Nicotinamide riboside (NR), which can cross the plasmamembrane and fuel intracellular NAD+ biosynthesis in human cells. These processes are herein confirmed to also occur in a human ovarian carcinoma cell line (OVCAR-3), by means of CD73 or NRK1 specific silencing. Next, we investigated the anti-tumor activity of the simultaneous inhibition of NAMPT (with FK866) and CD73 (with α, β-methylene adenosine 5′-diphosphate, APCP), in an in vivo human ovarian carcinoma model. Interestingly, the combined therapy was found to significantly decrease intratumor NAD+, NMN and ATP levels, compared with single treatments. In addition, the concentration of these nucleotides in ascitic exudates was more remarkably reduced in animals treated with both FK866 and APCP compared with single treatments. Importantly, tumors treated with FK866 in combination with APCP contained a statistically significant lower proportion of Ki67 positive proliferating cells and a higher percentage of necrotic area. Finally, a slight but significant increase in animal survival in response to the combined therapy, compared to the single agents, could be demonstrated. Our results indicate that the pharmacological inhibition of CD73 enzymatic activity could be considered as a means to potentiate the anti-cancer effects of NAMPT inhibitors. PMID:26658104

Antitumor effect of combined NAMPT and CD73 inhibition in an ovarian cancer model.

PubMed

Sociali, Giovanna; Raffaghello, Lizzia; Magnone, Mirko; Zamporlini, Federica; Emionite, Laura; Sturla, Laura; Bianchi, Giovanna; Vigliarolo, Tiziana; Nahimana, Aimable; Nencioni, Alessio; Raffaelli, Nadia; Bruzzone, Santina

2016-01-19

Nicotinamide phosphoribosyltransferase (NAMPT) is a crucial enzyme in the biosynthesis of intracellular NAD+. NAMPT inhibitors have potent anticancer activity in several preclinical models by depleting NAD+ and ATP levels. Recently, we demonstrated that CD73 enables the utilization of extracellular NAD+/nicotinamide mononucleotide (NMN) by converting them to Nicotinamide riboside (NR), which can cross the plasmamembrane and fuel intracellular NAD+ biosynthesis in human cells. These processes are herein confirmed to also occur in a human ovarian carcinoma cell line (OVCAR-3), by means of CD73 or NRK1 specific silencing. Next, we investigated the anti-tumor activity of the simultaneous inhibition of NAMPT (with FK866) and CD73 (with α, β-methylene adenosine 5'-diphosphate, APCP), in an in vivo human ovarian carcinoma model. Interestingly, the combined therapy was found to significantly decrease intratumor NAD+, NMN and ATP levels, compared with single treatments. In addition, the concentration of these nucleotides in ascitic exudates was more remarkably reduced in animals treated with both FK866 and APCP compared with single treatments. Importantly, tumors treated with FK866 in combination with APCP contained a statistically significant lower proportion of Ki67 positive proliferating cells and a higher percentage of necrotic area. Finally, a slight but significant increase in animal survival in response to the combined therapy, compared to the single agents, could be demonstrated. Our results indicate that the pharmacological inhibition of CD73 enzymatic activity could be considered as a means to potentiate the anti-cancer effects of NAMPT inhibitors.

Mechanisms of direct inhibition of the respiratory sulfate-reduction pathway by (per)chlorate and nitrate.

PubMed

Carlson, Hans K; Kuehl, Jennifer V; Hazra, Amrita B; Justice, Nicholas B; Stoeva, Magdalena K; Sczesnak, Andrew; Mullan, Mark R; Iavarone, Anthony T; Engelbrektson, Anna; Price, Morgan N; Deutschbauer, Adam M; Arkin, Adam P; Coates, John D

2015-06-01

We investigated perchlorate (ClO(4)(-)) and chlorate (ClO(3)(-)) (collectively (per)chlorate) in comparison with nitrate as potential inhibitors of sulfide (H(2)S) production by mesophilic sulfate-reducing microorganisms (SRMs). We demonstrate the specificity and potency of (per)chlorate as direct SRM inhibitors in both pure cultures and undefined sulfidogenic communities. We demonstrate that (per)chlorate and nitrate are antagonistic inhibitors and resistance is cross-inducible implying that these compounds share at least one common mechanism of resistance. Using tagged-transposon pools we identified genes responsible for sensitivity and resistance in Desulfovibrio alaskensis G20. We found that mutants in Dde_2702 (Rex), a repressor of the central sulfate-reduction pathway were resistant to both (per)chlorate and nitrate. In general, Rex derepresses its regulon in response to increasing intracellular NADH:NAD(+) ratios. In cells in which respiratory sulfate reduction is inhibited, NADH:NAD(+) ratios should increase leading to derepression of the sulfate-reduction pathway. In support of this, in (per)chlorate or nitrate-stressed wild-type G20 we observed higher NADH:NAD(+) ratios, increased transcripts and increased peptide counts for genes in the core Rex regulon. We conclude that one mode of (per)chlorate and nitrate toxicity is as direct inhibitors of the central sulfate-reduction pathway. Our results demonstrate that (per)chlorate are more potent inhibitors than nitrate in both pure cultures and communities, implying that they represent an attractive alternative for controlling sulfidogenesis in industrial ecosystems. Of these, perchlorate offers better application logistics because of its inhibitory potency, solubility, relative chemical stability, low affinity for mineral cations and high mobility in environmental systems.

Mitochondrial electron transport and glycolysis are coupled in articular cartilage.

PubMed

Martin, J A; Martini, A; Molinari, A; Morgan, W; Ramalingam, W; Buckwalter, J A; McKinley, T O

2012-04-01

Although the majority of the adenosine triphosphate (ATP) in chondrocytes is made by glycolysis rather than by oxidative phosphorylation in mitochondria there is evidence to suggest that reactive oxygen species produced by mitochondrial electron transport (ET) help to maintain cellular redox balance in favor of glycolysis. The objective of this study was to test this hypothesis by determining if rotenone, which inhibits ET and blocks oxidant production inhibits glycolytic ATP synthesis. Bovine osteochondral explants were treated with rotenone, an ET inhibitor; or oligomycin an ATP synthase inhibitor; or 2-fluoro-2-deoxy-D-glucose, a glycolysis inhibiter; or peroxide, an exogenous oxidant; or mitoquinone (MitoQ), a mitochondria-targeted anti-oxidant. Cartilage extracts were assayed for ATP, nicotine adenine dinucleotide (NAD+/H), and culture medium was assayed for pyruvate and lactate after 24 h of treatment. Imaging studies were used to measure superoxide production in cartilage. Rotenone and 2-FG caused a significant decline in cartilage ATP (P < 0.001). In contrast, ATP levels were not affected by oligomycin. Peroxide treatment blocked rotenone effects on ATP, while treatment with MitoQ significantly suppressed ATP levels. Rotenone and 2-FG caused a significant decline in pyruvate, but not in lactate production. NADH:NAD+ ratios decreased significantly in both rotenone and 2-FG-treated explants (P < 0.05). Rotenone also significantly reduced superoxide production. These findings showing a link between glycolysis and ET are consistent with previous reports on the critical need for oxidants to support normal chondrocyte metabolism. They suggest a novel role for mitochondria in cartilage homeostasis that is independent of oxidative phosphorylation. Copyright © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Oxidant injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide.

PubMed Central

Schraufstatter, I U; Hinshaw, D B; Hyslop, P A; Spragg, R G; Cochrane, C G

1986-01-01

To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure of P388D1 murine macrophages to hydrogen peroxide. Total intracellular NAD+ levels in P388D1 cells decreased with H2O2 concentrations of 40 microM or higher. Doses of H2O2 between 0.1 and 2.5 mM led to an 80% depletion of NAD within 20 min. With doses of H2O2 of 250 microM or lower, the fall in NAD and, as shown previously, ATP, was reversible. Higher doses of H2O2 that cause ultimate lysis of the cells, induced an irreversible depletion of NAD and ATP. Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 microM H2O2 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H2O2. H2O2 at 40 microM or higher inhibited DNA synthesis, and induced SSB within less than 30 s. These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase, and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis. PMID:2937805

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

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.

Pathways of nitrogen assimilation in cowpea nodules studied using /sup 15/N/sub 2/ and allopurinol. [Vigna unguiculata L. Walp. cv Vita

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

Atkins, C.A.; Storer, P.J.; Pate, J.S.

1988-01-01

In the presence of 0.5 millimolar allopurinol (4-hydroxypyrazolo (3,4-d)pyrimidine), an inhibitor of NAD:xanthine oxidoreductase (EC 1.2.3.2), intact attached nodules of cowpea (vigna unguiculata L. Walp. cv Vita 3) formed (/sup 15/N)xanthine from /sup 15/N/sub 2/ at rates equivalent to those of ureide synthesis, confirming the direct assimilation of fixed nitrogen into purines. Xanthine accumulated in nodules and was exported in increasing amounts in xylem of allopurinol-treated plants. Other intermediates of purine oxidation, de novo purine synthesis, and ammonia assimilation did not increase and, over the time course of experiments (4 hours), allopurinol had no effect on nitrogenase (EC 1.87.99.2) activity.more » Negligible /sup 15/N -labeling of asparagine from /sup 15/N/sub 2/ was observed, suggesting that the significant pool (up to 14 micromoles per gram of nodule fresh weight) of this amide in cowpea nodules was not formed directly from fixation but may have accumulated as a consequence of phloem delivery.« less

Crystallization of human nicotinamide phosphoribosyltransferase

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

Takahashi, Ryo; Nakamura, Shota; Yoshida, Takuya

2007-05-01

Human nicotinamide phosphoribosyltransferase has been crystallized using microseeding methods and X-ray diffraction data have been collected at 2.0 Å resolution. In the NAD biosynthetic pathway, nicotinamide phosphoribosyltransferase (NMPRTase; EC 2.4.2.12) plays an important role in catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide and 5′-phosphoribosyl-1′-pyrophosphate. Because the diffraction pattern of the initally obtained crystals was not suitable for structure analysis, the crystal quality was improved by successive use of the microseeding technique. The resultant crystals diffracted to 2.0 Å resolution. These crystals belonged to space group P21, with unit-cell parameters a = 60.56, b = 106.40, c = 82.78 Å.more » Here, the crystallization of human NMPRTase is reported in the free form; the crystals should be useful for inhibitor-soaking experiments on the enzyme.« less

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.

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

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.

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.

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

Pleiotropic effects of the sirtuin inhibitor sirtinol involves concentration-dependent modulation of multiple nuclear receptor-mediated pathways in the androgen-responsive prostate cancer cell LNCaP

USDA-ARS?s Scientific Manuscript database

Sirtinol, a purported specific inhibitor of the nicotinamide adenine dinucleotide (NAD)-dependent type III histone deacetylase (also known as sirtuin), has been used extensively to identify chemopreventive/chemotherapeutic agents that modulate the activity of this group of enzymes. However, the mole...

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.

Activation of Akt by Advanced Glycation End Products (AGEs): Involvement of IGF-1 Receptor and Caveolin-1

PubMed Central

Yang, Su-Jung; Chen, Chen-Yu; Chang, Geen-Dong; Wen, Hui-Chin; Chen, Ching-Yu; Chang, Shi-Chuan; Liao, Jyh-Fei; Chang, Chung-Ho

2013-01-01

Diabetes is characterized by chronic hyperglycemia, which in turn facilitates the formation of advanced glycation end products (AGEs). AGEs activate signaling proteins such as Src, Akt and ERK1/2. However, the mechanisms by which AGEs activate these kinases remain unclear. We examined the effect of AGEs on Akt activation in 3T3-L1 preadipocytes. Addition of AGEs to 3T3-L1 cells activated Akt in a dose- and time-dependent manner. The AGEs-stimulated Akt activation was blocked by a PI3-kinase inhibitor LY 294002, Src inhibitor PP2, an antioxidant NAC, superoxide scavenger Tiron, or nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase inhibitor DPI, suggesting the involvement of Src and NAD(P)H oxidase in the activation of PI3-kinase-Akt pathway by AGEs. AGEs-stimulated Src tyrosine phosphorylation was inhibited by NAC, suggesting that Src is downstream of NAD(P)H oxidase. The AGEs-stimulated Akt activity was sensitive to Insulin-like growth factor 1 receptor (IGF-1R) kinase inhibitor AG1024. Furthermore, AGEs induced phosphorylation of IGF-1 receptorβsubunit (IGF-1Rβ) on Tyr1135/1136, which was sensitive to PP2, indicating that AGEs stimulate Akt activity by transactivating IGF-1 receptor. In addition, the AGEs-stimulated Akt activation was attenuated by β-methylcyclodextrin that abolishes the structure of caveolae, and by lowering caveolin-1 (Cav-1) levels with siRNAs. Furthermore, addition of AGEs enhanced the interaction of phospho-Cav-1 with IGF-1Rβ and transfection of 3T3-L1 cells with Cav-1 Y14F mutants inhibited the activation of Akt by AGEs. These results suggest that AGEs activate NAD(P)H oxidase and Src which in turn phosphorylates IGF-1 receptor and Cav-1 leading to activation of IGF-1 receptor and the downstream Akt in 3T3-L1 cells. AGEs treatment promoted the differentiation of 3T3-L1 preadipocytes and addition of AG1024, LY 294002 or Akt inhibitor attenuated the promoting effect of AGEs on adipogenesis, suggesting that IGF-1 receptor, PI3-Kinase and Akt are involved in the facilitation of adipogenesis by AGEs. PMID:23472139

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

Oligo-carrageenan kappa increases NADPH, ascorbate and glutathione syntheses and TRR/TRX activities enhancing photosynthesis, basal metabolism, and growth in Eucalyptus trees.

PubMed

González, Alberto; Moenne, Fabiola; Gómez, Melissa; Sáez, Claudio A; Contreras, Rodrigo A; Moenne, Alejandra

2014-01-01

In order to analyze the effect of OC kappa in redox status, photosynthesis, basal metabolism and growth in Eucalyptus globulus, trees were treated with water (control), with OC kappa at 1 mg mL(-1), or treated with inhibitors of NAD(P)H, ascorbate (ASC), and glutathione (GSH) syntheses and thioredoxin reductase (TRR) activity, CHS-828, lycorine, buthionine sulfoximine (BSO), and auranofin, respectively, and with OC kappa, and cultivated for 4 months. Treatment with OC kappa induced an increase in NADPH, ASC, and GSH syntheses, TRR and thioredoxin (TRX) activities, photosynthesis, growth and activities of basal metabolism enzymes such as rubisco, glutamine synthetase (GlnS), adenosine 5'-phosphosulfate reductase (APR), involved in C, N, and S assimilation, respectively, Krebs cycle and purine/pyrimidine synthesis enzymes. Treatment with inhibitors and OC kappa showed that increases in ASC, GSH, and TRR/TRX enhanced NADPH synthesis, increases in NADPH and TRR/TRX enhanced ASC and GSH syntheses, and only the increase in NADPH enhanced TRR/TRX activities. In addition, the increase in NADPH, ASC, GSH, and TRR/TRX enhanced photosynthesis and growth. Moreover, the increase in NADPH, ASC and TRR/TRX enhanced activities of rubisco, Krebs cycle, and purine/pyrimidine synthesis enzymes, the increase in GSH, NADPH, and TRR/TRX enhanced APR activity, and the increase in NADPH and TRR/TRX enhanced GlnS activity. Thus, OC kappa increases NADPH, ASC, and GSH syntheses leading to a more reducing redox status, the increase in NADPH, ASC, GSH syntheses, and TRR/TRX activities are cross-talking events leading to activation of photosynthesis, basal metabolism, and growth in Eucalyptus trees.

Methylene blue stimulates substrate-level phosphorylation catalysed by succinyl-CoA ligase in the citric acid cycle.

PubMed

Komlódi, T; Tretter, L

2017-09-01

Methylene blue (MB), a potential neuroprotective agent, is efficient in various neurodegenerative disease models. Beneficial effects of MB have been attributed to improvements in mitochondrial functions. Substrate-level phosphorylation (SLP) results in the production of ATP independent from the ATP synthase (ATP-ase). In energetically compromised mitochondria, ATP produced by SLP can prevent the reversal of the adenine nucleotide translocase and thus the hydrolysis of glycolytic ATP. The aim of the present study was to investigate the effect of MB on mitochondrial SLP catalysed by succinyl-CoA ligase. Measurements were carried out on isolated guinea pig cortical mitochondria respiring on α-ketoglutarate, glutamate, malate or succinate. The mitochondrial functions and parameters like ATP synthesis, oxygen consumption, membrane potential, and NAD(P)H level were followed online, in parallel with the redox state of MB. SLP-mediated ATP synthesis was measured in the presence of inhibitors for ATP-ase and adenylate kinase. In the presence of the ATP-ase inhibitor oligomycin MB stimulated respiration with all of the respiratory substrates. However, the rate of ATP synthesis increased only with substrates α-ketoglutarate and glutamate (forming succinyl-CoA). MB efficiently stimulated SLP and restored the membrane potential in mitochondria also with the combined inhibition of Complex I and ATP synthase. ATP formed by SLP alleviated the energetic insufficiency generated by the lack of oxidative phosphorylation. Thus, the MB-mediated stimulation of SLP might be important in maintaining the energetic competence of mitochondria and in preventing the mitochondrial hydrolysis of glycolytic ATP. The mitochondrial effects of MB are explained by the ability to accept electrons from reducing equivalents and transfer them to cytochrome c bypassing the respiratory Complexes I and III. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

A molecular model for the active site of S-adenosyl- l-homocysteine hydrolase

NASA Astrophysics Data System (ADS)

Yeh, Jerry C.; Borchardt, Ronald T.; Vedani, Angelo

1991-06-01

S-adenosyl- l-homocysteine hydrolase (AdoHcy hydrolase, EC 3.3.1.1.), a specific target for antiviral drug design, catalyzes the hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy) as well as the synthesis of AdoHcy from Ado and Hcy. The enzyme isolated from different sources has been shown to contain tightly bound NAD+. Based on the 2.0 Å-resolution X-ray crystal structure of dogfish lactate dehydrogenase (LDH), which is functionally homologous to AdoHcy hydrolase, and the primary sequence of rat liver AdoHcy hydrolase, we have derived a molecular model of an extended active site for AdoHcy hydrolase. The computational mutation was performed using the software MUTAR (Yeh et al., University of Kansas, Lawrence), followed by molecular mechanics optimizations using the programs AMBER (Singh et al., University of California, San Francisco) and YETI (Vedani, University of Kansas). Solvation of the model structure was achieved by use of the program SOLVGEN (Jacober, University of Kansas); 56 water molecules were explicitly included in all refinements. Some of these may be involved in the catalytic reaction. We also studied a model of the complex of AdoHcy hydrolase with NAD+, as well as the ternary complexes of the redox reaction catalyzed by AdoHcy hydrolase and has been used to differentiate the relative binding strength of inhibitors.

The nuclear protein PH5P of the inter-alpha-inhibitor superfamily: a missing link between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and a novel actor of DNA repair?

PubMed

Jean, L; Risler, J L; Nagase, T; Coulouarn, C; Nomura, N; Salier, J P

1999-03-05

Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick sensor involved in cellular processes where nicking and rejoining of DNA strands are required. The inter-alpha-inhibitor family is comprized of several plasma proteins that all harbor one or more so-called heavy chains designated H1-H4. The latter originate from precursor polypeptides H1P-H4P whose upper two thirds are highly homologous. We now describe a novel protein that includes (i) a so-called BRCT domain found in many proteins involved in DNA repair, (ii) an area that is homologous to the NAD-dependent catalytic domain of poly(ADP-ribose)polymerase, (iii) an area that is homologous to the upper two thirds of precursor polypeptides H1P-H4P and (iv) a proline-rich region with a potential nuclear localization signal. This protein now designated PH5P points to as yet unsuspected links between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and is likely to be involved in DNA repair.

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.

Critical role of PI3-kinase/Akt activation in the PARP inhibitor induced heart function recovery during ischemia-reperfusion.

PubMed

Kovacs, Krisztina; Toth, Ambrus; Deres, Peter; Kalai, Tamas; Hideg, Kalman; Gallyas, Ferenc; Sumegi, Balazs

2006-02-14

Poly(ADP-ribose) polymerase (PARP) inhibitors protect hearts from ischemia-reperfusion (IR)-induced damages by limiting nicotinamide adenine dinucleotide (NAD+) and ATP depletion, and by other, not yet elucidated mechanisms. Our preliminary data suggested that PARP catalyzed ADP-ribosylations may affect signaling pathways in cardiomyocytes. To clarify this possibility, we studied the effect of a well-characterized (4-hydroxyquinazoline) and a novel (carboxaminobenzimidazol-derivative) PARP inhibitor on the activation of phosphatidylinositol-3-kinase (PI3-kinase)/Akt pathway in Langendorff-perfused hearts. PARP inhibitors promoted the restoration of myocardial energy metabolism (assessed by 31P nuclear magnetic resonance spectroscopy) and cardiac function compared to untreated hearts. PARP inhibitors also attenuated the infarct size and reduced the IR-induced lipid peroxidation, protein oxidation and total peroxide concentration. Moreover, PARP inhibitors facilitated Akt phosphorylation and activation, as well as the phosphorylation of its downstream target glycogen synthase kinase-3beta (GSK-3beta) in normoxia and, more robustly, during IR. Blocking PI3-kinase by wortmannin or LY294002 reduced the PARP inhibitor-elicited robust Akt and GSK-3beta phosphorylation upon ischemia-reperfusion, and significantly diminished the recovery of ATP and creatine phosphate showing the importance of Akt activation in the recovery of energy metabolism. In addition, inhibition of PI3-kinase/Akt pathway decreased the protective effect of PARP inhibitors on infarct size and the recovery of heart functions. All these data suggest that contrary to the original view, which considered preservation of NAD+ and consequently ATP pools as the exclusive underlying mechanism for the cytoprotective effect of PARP inhibitors, the activation of PI3-kinase/Akt pathway and related processes are at least equally important in the cardioprotective effects of PARP inhibitors during ischemia-reperfusion.

Profiles of the biosynthesis and metabolism of pyridine nucleotides in potatoes (Solanum tuberosum L.).

PubMed

Katahira, Riko; Ashihara, Hiroshi

2009-12-01

As part of a research program on nucleotide metabolism in potato tubers (Solanum tuberosum L.), profiles of pyridine (nicotinamide) metabolism were examined based on the in situ metabolic fate of radio-labelled precursors and the in vitro activities of enzymes. In potato tubers, [(3)H]quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [(14)C]nicotinamide, a catabolite of NAD, were utilised for pyridine nucleotide synthesis. The in situ tracer experiments and in vitro enzyme assays suggest the operation of multiple pyridine nucleotide cycles. In addition to the previously proposed cycle consisting of seven metabolites, we found a new cycle that includes newly discovered nicotinamide riboside deaminase which is also functional in potato tubers. This cycle bypasses nicotinamide and nicotinic acid; it is NAD --> nicotinamide mononucleotide --> nicotinamide riboside --> nicotinic acid riboside --> nicotinic acid mononucleotide --> nicotinic acid adenine dinucleotide --> NAD. Degradation of the pyridine ring was extremely low in potato tubers. Nicotinic acid glucoside is formed from nicotinic acid in potato tubers. Comparative studies of [carboxyl-(14)C]nicotinic acid metabolism indicate that nicotinic acid is converted to nicotinic acid glucoside in all organs of potato plants. Trigonelline synthesis from [carboxyl-(14)C]nicotinic acid was also found. Conversion was greater in green parts of plants, such as leaves and stem, than in underground parts of potato plants. Nicotinic acid utilised for the biosynthesis of these conjugates seems to be derived not only from the pyridine nucleotide cycle, but also from the de novo synthesis of nicotinic acid mononucleotide.

Discovery of a Highly Selective NAMPT Inhibitor That Demonstrates Robust Efficacy and Improved Retinal Toxicity with Nicotinic Acid Coadministration.

PubMed

Zhao, Genshi; Green, Colin F; Hui, Yu-Hua; Prieto, Lourdes; Shepard, Robert; Dong, Sucai; Wang, Tao; Tan, Bo; Gong, Xueqian; Kays, Lisa; Johnson, Robert L; Wu, Wenjuan; Bhattachar, Shobha; Del Prado, Miriam; Gillig, James R; Fernandez, Maria-Carmen; Roth, Ken D; Buchanan, Sean; Kuo, Ming-Shang; Geeganage, Sandaruwan; Burkholder, Timothy P

2017-12-01

NAMPT, an enzyme essential for NAD + biosynthesis, has been extensively studied as an anticancer target for developing potential novel therapeutics. Several NAMPT inhibitors have been discovered, some of which have been subjected to clinical investigations. Yet, the on-target hematological and retinal toxicities have hampered their clinical development. In this study, we report the discovery of a unique NAMPT inhibitor, LSN3154567. This molecule is highly selective and has a potent and broad spectrum of anticancer activity. Its inhibitory activity can be rescued with nicotinic acid (NA) against the cell lines proficient, but not those deficient in NAPRT1, essential for converting NA to NAD + LSN3154567 also exhibits robust efficacy in multiple tumor models deficient in NAPRT1. Importantly, this molecule when coadministered with NA does not cause observable retinal and hematological toxicities in the rodents, yet still retains robust efficacy. Thus, LSN3154567 has the potential to be further developed clinically into a novel cancer therapeutic. Mol Cancer Ther; 16(12); 2677-88. ©2017 AACR . ©2017 American Association for Cancer Research.

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.

Synthesis of β-Nicotinamide Riboside Using an Efficient Two-Step Methodology.

PubMed

Zhang, Ning; Sauve, Anthony A

2017-12-24

A two-step chemical method for the synthesis of β-nicotinamide riboside (NR) is described. NR has achieved wide use as an NAD + precursor (vitamin B3) and can significantly increase central metabolite NAD + concentrations in mammalian cells. β-NR can be prepared with an efficient two-step procedure. The synthesis is initiated via coupling of commercially available 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose with ethyl nicotinate in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf). 1 H NMR showed that the product was formed with complete stereoselectivity to produce only the β-isomer in high yield (>90% versus starting sugar). The clean stereochemical result suggests that the coupling proceeds via a cationic cis-1,2-acyloxonium-sugar intermediate, which controls addition by nucleophiles to generate predominantly β-stereochemistry. The subsequent deprotection of esters in methanolic ammonia generates the desired product in 85% overall yield versus sugar. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

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

Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions.

PubMed

Hasegawa, Satoshi; Suda, Masako; Uematsu, Kimio; Natsuma, Yumi; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

2013-02-01

We previously demonstrated efficient L-valine production by metabolically engineered Corynebacterium glutamicum under oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis of l-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase from Lysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gene ldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress the L-valine yield. Eliminating these by-products therefore was deemed key to improving theL-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase gene ppc, succinate production was effectively suppressed, but both glucose consumption and L-valine production dropped considerably due to the severely elevated intracellular NADH/NAD(+) ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, including gapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higher L-valine yield and productivity. Deleting the alanine transaminase gene avtA suppressed alanine production. The resultant strain produced 1,280 mM L-valine at a yield of 88% mol mol of glucose(-1) after 24 h under oxygen deprivation, a vastly improved yield over our previous best.

Engineering of Corynebacterium glutamicum for High-Yield l-Valine Production under Oxygen Deprivation Conditions

PubMed Central

Hasegawa, Satoshi; Suda, Masako; Uematsu, Kimio; Natsuma, Yumi; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki

2013-01-01

We previously demonstrated efficient l-valine production by metabolically engineered Corynebacterium glutamicum under oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis of l-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase from Lysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gene ldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress the l-valine yield. Eliminating these by-products therefore was deemed key to improving the l-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase gene ppc, succinate production was effectively suppressed, but both glucose consumption and l-valine production dropped considerably due to the severely elevated intracellular NADH/NAD+ ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, including gapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higher l-valine yield and productivity. Deleting the alanine transaminase gene avtA suppressed alanine production. The resultant strain produced 1,280 mM l-valine at a yield of 88% mol mol of glucose−1 after 24 h under oxygen deprivation, a vastly improved yield over our previous best. PMID:23241971

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 + .

Improved synthesis of chiral alcohols with Escherichia coli cells co-expressing pyridine nucleotide transhydrogenase, NADP+-dependent alcohol dehydrogenase and NAD+-dependent formate dehydrogenase.

PubMed

Weckbecker, Andrea; Hummel, Werner

2004-11-01

Recombinant pyridine nucleotide transhydrogenase (PNT) from Escherichia coli has been used to regenerate NAD+ and NADPH. The pnta and pntb genes encoding for the alpha- and beta-subunits were cloned and co-expressed with NADP+-dependent alcohol dehydrogenase (ADH) from Lactobacillus kefir and NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii. Using this whole-cell biocatalyst, efficient conversion of prochiral ketones to chiral alcohols was achieved: 66% acetophenone was reduced to (R)-phenylethanol over 12 h, whereas only 19% (R)-phenylethanol was formed under the same conditions with cells containing ADH and FDH genes but without PNT genes. Cells that were permeabilized with toluene showed ketone reduction only if both cofactors were present.

Proinflammatory Actions of Visfatin/Nicotinamide Phosphoribosyltransferase (Nampt) Involve Regulation of Insulin Signaling Pathway and Nampt Enzymatic Activity*

PubMed Central

Jacques, Claire; Holzenberger, Martin; Mladenovic, Zvezdana; Salvat, Colette; Pecchi, Emilie; Berenbaum, Francis; Gosset, Marjolaine

2012-01-01

Visfatin (also termed pre-B-cell colony-enhancing factor (PBEF) or nicotinamide phosphoribosyltransferase (Nampt)) is a pleiotropic mediator acting on many inflammatory processes including osteoarthritis. Visfatin exhibits both an intracellular enzymatic activity (nicotinamide phosphoribosyltransferase, Nampt) leading to NAD synthesis and a cytokine function via the binding to its hypothetical receptor. We recently reported the role of visfatin in prostaglandin E2 (PGE2) synthesis in chondrocytes. Here, our aim was to characterize the signaling pathways involved in this response in exploring both the insulin receptor (IR) signaling pathway and Nampt activity. IR was expressed in human and murine chondrocytes, and visfatin triggered Akt phosphorylation in murine chondrocytes. Blocking IR expression with siRNA or activity using the hydroxy-2-naphthalenyl methyl phosphonic acid tris acetoxymethyl ester (HNMPA-(AM)3) inhibitor diminished visfatin-induced PGE2 release in chondrocytes. Moreover, visfatin-induced IGF-1R−/− chondrocytes released higher concentration of PGE2 than IGF-1R+/+ cells, a finding confirmed with an antibody that blocked IGF-1R. Using RT-PCR, we found that visfatin did not regulate IR expression and that an increased insulin release was also unlikely to be involved because insulin was unable to increase PGE2 release. Inhibition of Nampt activity using the APO866 inhibitor gradually decreased PGE2 release, whereas the addition of exogenous nicotinamide increased it. We conclude that the proinflammatory actions of visfatin in chondrocytes involve regulation of IR signaling pathways, possibly through the control of Nampt enzymatic activity. PMID:22399297

Tomato Fruit Chromoplasts Behave as Respiratory Bioenergetic Organelles during Ripening1[W][OPEN

PubMed Central

Renato, Marta; Pateraki, Irini; Boronat, Albert; Azcón-Bieto, Joaquín

2014-01-01

During tomato (Solanum lycopersicum) fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts. It was recently reported that tomato chromoplasts can synthesize ATP through a respiratory process called chromorespiration. Here we show that chromoplast oxygen consumption is stimulated by the electron donors NADH and NADPH and is sensitive to octyl gallate (Ogal), a plastidial terminal oxidase inhibitor. The ATP synthesis rate of isolated chromoplasts was dependent on the supply of NAD(P)H and was fully inhibited by Ogal. It was also inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting the involvement of a chemiosmotic gradient. In addition, ATP synthesis was sensitive to 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a cytochrome b6f complex inhibitor. The possible participation of this complex in chromorespiration was supported by the detection of one of its components (cytochrome f) in chromoplasts using immunoblot and immunocytochemical techniques. The observed increased expression of cytochrome c6 during ripening suggests that it could act as electron acceptor of the cytochrome b6f complex in chromorespiration. The effects of Ogal on respiration and ATP levels were also studied in tissue samples. Oxygen uptake of mature green fruit and leaf tissues was not affected by Ogal, but was inhibited increasingly in fruit pericarp throughout ripening (up to 26% in red fruit). Similarly, Ogal caused a significant decrease in ATP content of red fruit pericarp. The number of energized mitochondria, as determined by confocal microscopy, strongly decreased in fruit tissue during ripening. Therefore, the contribution of chromoplasts to total fruit respiration appears to increase in late ripening stages. PMID:25125503

Isoproterenol-stimulated labelling of particulate proteins by using [adenylate-32P]NAD+ independent on a cAMP-dependent protein kinase in parotid acinar cells.

PubMed

Sugiya, H; Hara-Yokoyama, M; Furuyama, S

1992-03-30

When saponin-permeabilized rat parotid acinar cells were incubated with [adenylate-32P]NAD+, labelling of proteins (33, 27 and 23 kDa) in particulate fractions of the cells was stimulated by isoproterenol. The effect of isoproterenol was completely blocked by a beta-antagonist. Both forskolin or cAMP mimicked the effect of isoproterenol on the labelling. However, an inhibitor of cAMPdPK failed to induce complete inhibition of the effects of isoproterenol, forskolin and cAMP. When the labelled proteins were treated with snake venom phosphodiesterase, neither [32P]5'-AMP nor [32P]phosphoribosyladenosine was released. These results suggest that covalent modification of proteins with NAD+, which is distinct from ADP-ribosylation and cAMPdPK-dependent phosphorylation, is coupled to beta-receptor-cAMP signalling system in rat parotid acinar cells.

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.

NAMPT/PBEF1 enzymatic activity is indispensable for myeloma cell growth and osteoclast activity

PubMed Central

Venkateshaiah, Sathisha Upparahalli; Khan, Sharmin; Ling, Wen; Bam, Rakesh; Li, Xin; van Rhee, Frits; Usmani, Saad; Barlogie, Bart; Epstein, Joshua; Yaccoby, Shmuel

2015-01-01

Multiple myeloma (MM) cells typically grow in focal lesions, stimulating osteoclasts that destroy bone and support MM. Osteoclasts and MM cells are hypermetabolic. The coenzyme nicotinamide adenine dinucleotide (NAD+) is not only essential for cellular metabolism; it also affects activity of NAD-dependent enzymes, such as PARP-1 and SIRT-1. Nicotinamide phos-phoribosyltransferase (NAMPT/PBEF/visfatin, encoded by PBEF1) is a rate-limiting enzyme in NAD+ biosynthesis from nicotinamide. Coculture of primary MM cells with osteoclasts induced PBEF1 upregulation in both cell types. PBEF1 expression was higher in experimental myelomatous bones than in nonmyelomatous bone and higher in MM patients’ plasma cells than in healthy donors’ counterparts. APO866 is a specific PBEF1 inhibitor known to deplete cellular NAD+, APO866 at low nanomolar concentrations inhibited growth of primary MM cells or MM cell lines cultured alone or cocultured with osteoclasts and induced apoptosis in these cells. PBEF1 activity and NAD+ content were reduced in MM cells by APO866, resulting in lower activity of PARP-1 and SIRT-1. The inhibitory effect of APO866 on MM cell growth was abrogated by supplementation of extracellular NAD+ or NAM. APO866 inhibited NF-κB activity in osteoclast precursors and suppressed osteoclast formation and activity. PBEF1 knockdown similarly inhibited MM cell growth and osteoclast formation. In the SCID-rab model, APO866 inhibited growth of primary MM and H929 cells and prevented bone disease. These findings indicate that MM cells and osteoclasts are highly sensitive to NAD+ depletion and that PBEF1 inhibition represents a novel approach to target cellular metabolism and inhibit PARP-1 and bone disease in MM. PMID:23435312

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 + for ADP-ribosylation of proteins involved in transcribing and translating the phage genome. We show here that phage KVP40 encodes a functional pyridine nucleotide scavenging pathway that is expressed during the metabolic period of the infection cycle. The pathway is conserved in other large, dsDNA phages in which the two genes, nadV and natV , share an evolutionary history in their respective phage-host group. Copyright © 2017 American Society for Microbiology.

Hydrogen peroxide-induced injury of cells and its prevention by inhibitors of poly(ADP-ribose) polymerase.

PubMed Central

Schraufstatter, I U; Hyslop, P A; Hinshaw, D B; Spragg, R G; Sklar, L A; Cochrane, C G

1986-01-01

H2O2, in concentrations achieved in the proximity of stimulated leukocytes, induces injury and lysis of target cells. This may be an important aspect of inflammatory injury of tissues. Cell lysis in two target cells, the murine macrophage-like tumor cell line P388D1 and human peripheral lymphocytes, was found to be associated with activation of poly(ADP-ribose) polymerase (EC 2.4.2.30), a nuclear enzyme. This enzyme is activated under various conditions of DNA damage. Poly(ADP-ribose) polymerase utilizes nicotinamide adenine dinucleotide (NAD) as substrate and has been previously shown to consume NAD during exposure of cells to oxidants that was associated with inhibition of glycolysis, a decrease in cellular ATP, and cell death. In the current studies, inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide, nicotinamide, or theophylline in cells exposed to lethal concentrations of H2O2 prevented the sequence of events that eventually led to cell lysis--i.e., the decrease in NAD, followed by depletion of ATP, influx of extracellular Ca2+, actin polymerization and, finally, cell death. DNA damage, the initial stimulus for poly(ADP-ribose) polymerase activation, occurred despite the inhibition of this enzyme. Cells exposed to oxidant in the presence of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide failed to demonstrate repair of DNA strand breaks. PMID:2941760

Multiple turnovers of the nicotino-enzyme PdxB require α-keto acids as co-substrates

PubMed Central

Rudolph, Johannes; Kim, Juhan; Copley, Shelley D.

2012-01-01

PdxB catalyzes the second step in the biosynthesis of pyridoxal phosphate by oxidizing 4-phospho-D-erythronate (4PE) to 2-oxo-3-hydroxy-4-phospho-butanoate (OHPB) with concomitant reduction of NAD+ to NADH. PdxB is a nicotino-enzyme wherein the NAD(H) cofactor remains tightly bound to PdxB. It has been a mystery how PdxB performs multiple turnovers since addition of free NAD+ does not re-oxidize the enzyme-bound NADH following conversion of 4PE to OHPB. We have solved this mystery by demonstrating that a variety of physiologically available α-ketoacids serve as oxidants of PdxB to sustain multiple turnovers. In a coupled assay using the next two enzymes of the biosynthetic pathway for pyridoxal phosphate (SerC and PdxA), we have found that α-ketoglutarate, oxaloacetic acid, and pyruvate are equally good substrates for PdxB (kcat/Km values ~ 1 × 104 M-1s-1). The kinetic parameters for the substrate 4PE include a kcat of 1.4 s-1, a Km of 2.9 μM, and a kcat/Km of 6.7 × 106 M-1s-1. Additionally, we have characterized the stereochemistry of α-ketoglutarate reduction by showing that D-2-HGA, but not L-2-HGA, is a competitive inhibitor vs. 4PE and a noncompetitive inhibitor vs. α-ketoglutarate. PMID:20831184

Glucose metabolism in batch and continuous cultures of Gluconacetobacter diazotrophicus PAL 3.

PubMed

Luna, María F; Bernardelli, Cecilia E; Galar, María L; Boiardi, José L

2006-03-01

Periplasmic glucose oxidation (by way of a pyrrolo-quinoline-quinone [PQQ]-linked glucose dehydrogenase [GDH]) was observed in continuous cultures of Gluconacetobacter diazotrophicus regardless of the carbon source (glucose or gluconate) and the nitrogen source (N(2) or NH(3)). Its synthesis was stimulated by conditions of high energetic demand (i.e., N(2)-fixation) and/or C-limitation. Under C-excess conditions, PQQ-GDH synthesis increased with the glucose concentration in the culture medium. In batch cultures, PQQ-GDH was actively expressed in very early stages with higher activities under conditions of N(2)-fixation. Hexokinase activity was almost absent under any culture condition. Cytoplasmic nicotinamide adenine dinucleotide (NAD)-linked glucose dehydrogenase (GDH) was expressed in continuous cultures under all tested conditions, and its synthesis increased with the glucose concentration. In contrast, low activities of this enzyme were detected in batch cultures. Periplasmic oxidation, by way of PQQ-GDH, seems to be the principal pathway for metabolism of glucose in G. Diazotrophicus, and NAD-GDH is an alternative route under certain environmental conditions.

Use of NAD(P)H fluorescence measurement for on-line monitoring of metabolic state of Azohydromonas australica in poly(3-hydroxybutyrate) production.

PubMed

Gahlawat, Geeta; Srivastava, Ashok K

2013-02-01

Culture fluorescence measurement is an indirect and non-invasive method of biomass estimation to assess the metabolic state of the microorganism in a fermentation process. In the present investigation, NAD(P)H fluorescence has been used for on-line in situ characterization of metabolic changes occurring during different phases of batch cultivation of Azohydromonas australica in growth associated poly(3-hydroxybutyrate) or PHB production. A linear correlation between biomass concentration and net NAD(P)H fluorescence was obtained during early log phase (3-12 h) and late log phase (24-39 h) of PHB fermentation. After 12 h (mid log phase) cultivation PHB accumulation shot up and a drop in culture fluorescence was observed which synchronously exhibited continuous utilization of NAD(P)H for the synthesis of biomass and PHB formation simultaneously. A decrease in the observed net fluorescence value was observed again towards the end of fermentation (at 39 h) which corresponded very well with the culture starvation and substrate depletion towards the end of cultivation inside the bioreactor. It was therefore concluded that NAD(P)H fluorescence measurements could be used for indication of the time of fresh nutrient (substrate) feed during substrate limitation to further enhance the PHB production.

Pyridine nucleotides in regulation of cell death and survival by redox and non-redox reactions.

PubMed

Novak Kujundžić, Renata; Žarković, Neven; Gall Trošelj, Koraljka

2014-01-01

Changes of the level and ratios of pyridine nucleotides determine metabolism- dependent cellular redox status and the activity of poly(ADP-ribose) polymerases (PARPs) and sirtuins, thereby influencing several processes closely related to cell survival and death. Pyridine nucleotides participate in numerous metabolic reactions whereby their net cellular level remains constant, but the ratios of NAD+/NADP+ and NADH/NADPH oscillate according to metabolic changes in response to diverse stress signals. In non-redox reactions, NAD+ is degraded and quickly, afterward, resynthesized in the NAD+ salvage pathway, unless overwhelming activation of PARP-1 consumes NAD+ to the point of no return, when the cell can no longer generate enough ATP to accommodate NAD+ resynthesis. The activity of PARP-1 is mandatory for the onset of cytoprotective autophagy on sublethal stress signals. It has become increasingly clear that redox status, largely influenced by the metabolism-dependent composition of the pyridine nucleotides pool, plays an important role in the synthesis of pro-apoptotic and anti-apoptotic sphingolipids. Awareness of the involvement of the prosurvival sphingolipid, sphingosine-1-phosphate, in transition from inflammation to malignant transformation has recently emerged. Here, the participation of pyridine nucleotides in redox and non-redox reactions, sphingolipid metabolism, and their role in cell fate decisions is reviewed.

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

PnuC and the utilization of the nicotinamide riboside analog 3-aminopyridine in Haemophilus influenzae.

PubMed

Sauer, Elizabeta; Merdanovic, Melisa; Mortimer, Anne Price; Bringmann, Gerhard; Reidl, Joachim

2004-12-01

The utilization pathway for the uptake of NAD and nicotinamide riboside was previously characterized for Haemophilus influenzae. We now report on the cellular location, topology, and substrate specificity of PnuC. pnuC of H. influenzae is only distantly related to pnuC of Escherichia coli and Salmonella enterica serovar Typhimurium. When E. coli PnuC was expressed in an H. influenzae pnuC mutant, it was able to take up only nicotinamide riboside and not nicotinamide mononucleotide. Therefore, we postulated that PnuC transporters in general possess specificity for nicotinamide riboside. Earlier studies showed that 3-aminopyridine derivatives (e.g., 3-aminopyridine adenine dinucleotide) are inhibitory for H. influenzae growth. By testing characterized strains with mutations in the NAD utilization pathway, we show that 3-aminopyridine riboside is inhibitory to H. influenzae and is taken up by the NAD-processing and nicotinamide riboside route. 3-Aminopyridine riboside is utilized effectively in a pnuC+ background. In addition, we demonstrate that 3-aminopyridine adenine dinucleotide resynthesis is produced by NadR. 3-Aminopyridine riboside-resistant H. influenzae isolates were characterized, and mutations in nadR could be detected. We also tested other species of the family Pasteurellaceae, Pasteurella multocida and Actinobacillus actinomycetemcomitans, and found that 3-aminopyridine riboside does not act as a growth inhibitor; hence, 3-aminopyridine riboside represents an anti-infective agent with a very narrow host range.

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

Effect of Polyhydroxybutyrate (PHB) storage on L-arginine production in recombinant Corynebacterium crenatum using coenzyme regulation.

PubMed

Xu, Meijuan; Qin, Jingru; Rao, Zhiming; You, Hengyi; Zhang, Xian; Yang, Taowei; Wang, Xiaoyuan; Xu, Zhenghong

2016-01-19

Corynebacterium crenatum SYPA 5 is the industrial strain for L-arginine production. Poly-β-hydroxybutyrate (PHB) is a kind of biopolymer stored as bacterial reserve materials for carbon and energy. The introduction of the PHB synthesis pathway into several strains can regulate the global metabolic pathway. In addition, both the pathways of PHB and L-arginine biosynthesis in the cells are NADPH-dependent. NAD kinase could upregulate the NADPH concentration in the bacteria. Thus, it is interesting to investigate how both PHB and NAD kinase affect the L-arginine biosynthesis in C. crenatum SYPA 5. C. crenatum P1 containing PHB synthesis pathway was constructed and cultivated in batch fermentation for 96 h. The enzyme activities of the key enzymes were enhanced comparing to the control strain C. crenatum SYPA 5. More PHB was found in C. crenatum P1, up to 12.7 % of the dry cell weight. Higher growth level and enhanced glucose consumptions were also observed in C. crenatum P1. With respect to the yield of L-arginine, it was 38.54 ± 0.81 g/L, increasing by 20.6 %, comparing to the control under the influence of PHB accumulation. For more NADPH supply, C. crenatum P2 was constructed with overexpression of NAD kinase based on C. crenatum P1. The NADPH concentration was increased in C. crenatum P2 comparing to the control. PHB content reached 15.7 % and 41.11 ± 1.21 g/L L-arginine was obtained in C. crenatum P2, increased by 28.6 %. The transcription levels of key L-arginine synthesis genes, argB, argC, argD and argJ in recombinant C. crenatum increased 1.9-3.0 times compared with the parent strain. Accumulation of PHB by introducing PHB synthesis pathway, together with up-regulation of coenzyme level by overexpressing NAD kinase, enables the recombinant C. crenatum to serve as high-efficiency cell factories in the long-time L-arginine fermentation. Furthermore, batch cultivation of the engineered C. crenatum revealed that it could accumulate both extracellular L-arginine and intracellular PHB simultaneously. All of these have a potential biotechnological application as a strategy for high-yield L-arginine.

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

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.).

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.

NAD⁺ repletion improves mitochondrial and stem cell function and enhances life span in mice.

PubMed

Zhang, Hongbo; Ryu, Dongryeol; Wu, Yibo; Gariani, Karim; Wang, Xu; Luan, Peiling; D'Amico, Davide; Ropelle, Eduardo R; Lutolf, Matthias P; Aebersold, Ruedi; Schoonjans, Kristina; Menzies, Keir J; Auwerx, Johan

2016-06-17

Adult stem cells (SCs) are essential for tissue maintenance and regeneration yet are susceptible to senescence during aging. We demonstrate the importance of the amount of the oxidized form of cellular nicotinamide adenine dinucleotide (NAD(+)) and its effect on mitochondrial activity as a pivotal switch to modulate muscle SC (MuSC) senescence. Treatment with the NAD(+) precursor nicotinamide riboside (NR) induced the mitochondrial unfolded protein response and synthesis of prohibitin proteins, and this rejuvenated MuSCs in aged mice. NR also prevented MuSC senescence in the mdx (C57BL/10ScSn-Dmd(mdx)/J) mouse model of muscular dystrophy. We furthermore demonstrate that NR delays senescence of neural SCs and melanocyte SCs and increases mouse life span. Strategies that conserve cellular NAD(+) may reprogram dysfunctional SCs and improve life span in mammals. Copyright © 2016, American Association for the Advancement of Science.

Oligo-carrageenan kappa-induced reducing redox status and increase in TRR/TRX activities promote activation and reprogramming of terpenoid metabolism in Eucalyptus trees.

PubMed

González, Alberto; Gutiérrez-Cutiño, Marlen; Moenne, Alejandra

2014-06-05

In order to analyze whether the reducing redox status and activation of thioredoxin reductase (TRR)/thioredoxin(TRX) system induced by oligo-carrageenan (OC) kappa in Eucalyptus globulus activate secondary metabolism increasing terpenoid synthesis, trees were sprayed on the leaves with water, with OC kappa, or with inhibitors of NAD(P)H, ascorbate (ASC) and (GSH) synthesis and TRR activity, CHS-828, lycorine, buthionine sulfoximine (BSO) and auranofine, respectively, and with OC kappa and cultivated for four months. The main terpenoids in control Eucalyptus trees were eucalyptol (76%), α-pinene (7.4%), aromadendrene (3.6%), silvestrene (2.8%), sabinene (2%) and α-terpineol (0.9%). Treated trees showed a 22% increase in total essential oils as well as a decrease in eucalyptol (65%) and sabinene (0.8%) and an increase in aromadendrene (5%), silvestrene (7.8%) and other ten terpenoids. In addition, treated Eucalyptus showed seven de novo synthesized terpenoids corresponding to carene, α-terpinene, α-fenchene, γ-maaliene, spathulenol and α-camphenolic aldehyde. Most increased and de novo synthesized terpenoids have potential insecticidal and antimicrobial activities. Trees treated with CHS-828, lycorine, BSO and auranofine and with OC kappa showed an inhibition of increased and de novo synthesized terpenoids. Thus, OC kappa-induced reducing redox status and activation of TRR/TRX system enhance secondary metabolism increasing the synthesis of terpenoids and reprogramming of terpenoid metabolism in Eucalyptus trees.

Tomato fruit chromoplasts behave as respiratory bioenergetic organelles during ripening.

PubMed

Renato, Marta; Pateraki, Irini; Boronat, Albert; Azcón-Bieto, Joaquín

2014-10-01

During tomato (Solanum lycopersicum) fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts. It was recently reported that tomato chromoplasts can synthesize ATP through a respiratory process called chromorespiration. Here we show that chromoplast oxygen consumption is stimulated by the electron donors NADH and NADPH and is sensitive to octyl gallate (Ogal), a plastidial terminal oxidase inhibitor. The ATP synthesis rate of isolated chromoplasts was dependent on the supply of NAD(P)H and was fully inhibited by Ogal. It was also inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting the involvement of a chemiosmotic gradient. In addition, ATP synthesis was sensitive to 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a cytochrome b6f complex inhibitor. The possible participation of this complex in chromorespiration was supported by the detection of one of its components (cytochrome f) in chromoplasts using immunoblot and immunocytochemical techniques. The observed increased expression of cytochrome c6 during ripening suggests that it could act as electron acceptor of the cytochrome b6f complex in chromorespiration. The effects of Ogal on respiration and ATP levels were also studied in tissue samples. Oxygen uptake of mature green fruit and leaf tissues was not affected by Ogal, but was inhibited increasingly in fruit pericarp throughout ripening (up to 26% in red fruit). Similarly, Ogal caused a significant decrease in ATP content of red fruit pericarp. The number of energized mitochondria, as determined by confocal microscopy, strongly decreased in fruit tissue during ripening. Therefore, the contribution of chromoplasts to total fruit respiration appears to increase in late ripening stages. © 2014 American Society of Plant Biologists. All Rights Reserved.

Reaching for mechanistic consensus across life kingdoms: structure and insights into catalysis of the myo-inositol-1-phosphate synthase (mIPS) from Archaeoglobus fulgidus.

PubMed

Stieglitz, Kimberly A; Yang, Hongying; Roberts, Mary F; Stec, Boguslaw

2005-01-11

myo-Inositol-1-phosphate synthase (mIPS) catalyzes the first step in the synthesis of l-myo-inositol-1-phosphate. We have solved and refined the structure of the mIPS from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus at 1.9 A resolution. The enzyme crystallized from poly(ethylene glycol) in the P1 space group with one tetramer in the asymmetric unit and provided a view of the entire biologically active oligomer. Despite significant changes in sequence length and amino acid composition, the general architecture of the archaeal enzyme is similar to that of the eukaryotic mIPS from Saccharomyces cerevisiae and bacterial mIPS from Mycobacterium tuberculosis. The enhanced thermostability of the archaeal enzyme as compared to that from yeast is consistent with deletion of a number of surface loops that results in a significantly smaller protein. In the structure of the A. fulgidus mIPS, the active sites of all four subunits were fully ordered and contained NAD(+) and inorganic phosphate. The structure also contained a single metal ion (identified as K(+)) in two of the four subunits. The analysis of the electrostatic potential maps of the protein suggested the presence of a second metal-ion-binding site in close proximity to the first metal ion and NAD(+). The modeling of the substrate and known inhibitors suggests a critical role for the second metal ion in catalysis and provides insights into the common elements of the catalytic cycle in enzymes from different life kingdoms.

Structural Basis for Nicotinamide Inhibition and Base Exchange in Sir2 Enzymes

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

Sanders, B.; Zhao, K; Slama, J

2007-01-01

The Sir2 family of proteins consists of broadly conserved NAD+-dependent deacetylases that are implicated in diverse biological processes, including DNA regulation, metabolism, and longevity. Sir2 proteins are regulated in part by the cellular concentrations of a noncompetitive inhibitor, nicotinamide, that reacts with a Sir2 reaction intermediate via a base-exchange reaction to reform NAD+ at the expense of deacetylation. To gain a mechanistic understanding of nicotinamide inhibition in Sir2 enzymes, we captured the structure of nicotinamide bound to a Sir2 homolog, yeast Hst2, in complex with its acetyl-lysine 16 histone H4 substrate and a reaction intermediate analog, ADP-HPD. Together with relatedmore » biochemical studies and structures, we identify a nicotinamide inhibition and base-exchange site that is distinct from the so-called 'C pocket' binding site for the nicotinamide group of NAD+. These results provide insights into the Sir2 mechanism of nicotinamide inhibition and have important implications for the development of Sir2-specific effectors.« less

EFFECTS OF X IRRADIATION ON ENZYME SYNTHESIS DURING LIVER REGENERATION

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

Myers, D.K.

1962-05-01

Twenty-four different enzymes or enzyme systems were assayed in regenerating rat liver from control and irradiated animals at various times after partial hepatectomy. X irradiation, either of the whole liver region or of an exteriorized liver lobule, interfered with the accumulation of only three of these enzymes: deoxycytidylate deaminase, thymidine phosphorylase, and NAD pyrophosphorylase. Irradiation did not affect the synthesis of related enzymes such as adenosine and guanine deaminases, and inosine and uridine phosphorylases. The effects of irradiation on enzyme synthesis in regenerating liver would appear to be highly selective. (auth)

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.

Partial reactions of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase

PubMed Central

Barnett, J. E. G.; Rasheed, A.; Corina, D. L.

1973-01-01

After removal of tightly bound NAD+ by using charcoal, a preparation of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase catalysed the reduction of 5-keto-d-glucitol 6-phosphate and 5-keto-d-glucose 6-phosphate by [4-3H]NADH to give [5-3H]-glucitol 6-phosphate and [5-3H]glucose 6-phosphate respectively. The position of the tritium atom in the latter was shown by degradation. Both enzyme-catalysed reductions were strongly inhibited by 2-deoxy-d-glucose 6-phosphate, a powerful competitive inhibitor of inositol cyclase. The charcoal-treated enzyme preparation also converted 5-keto-d-glucose 6-phosphate into [3H]myoinositol 1-phosphate in the presence of [4-3H]NADH, but less effectively. These partial reactions of inositol cyclase are interpreted as providing strong evidence for the formation of 5-keto-d-glucose 6-phosphate as an enzyme-bound intermediate in the conversion of d-glucose 6-phosphate into 1 l-myoinositol 1-phosphate. The enzyme was partially inactivated by NaBH4 in the presence of NAD+. Glucose 6-phosphate did not increase the inactivation, and there was no inactivation in the absence of NAD+. There was no evidence for Schiff base formation during the cyclization. d-Glucitol 6-phosphate (l-sorbitol 1-phosphate) was a good inhibitor of the overall reaction. It did not inactivate the enzyme. The apparent molecular weight of inositol cyclase as determined by Sephadex chromatography was 2.15×105. PMID:4352864

SIRT1 inhibition during the hypoinflammatory phenotype of sepsis enhances immunity and improves outcome

PubMed Central

Vachharajani, Vidula T.; Liu, Tiefu; Brown, Candice M.; Wang, Xianfeng; Buechler, Nancy L.; Wells, Jonathan David; Yoza, Barbara K.; McCall, Charles E.

2014-01-01

Mechanism-based sepsis treatments are unavailable, and their incidence is rising worldwide. Deaths occur during the early acute phase of hyperinflammation or subsequent postacute hypoinflammatory phase with sustained organ failure. The acute sepsis phase shifts rapidly, and multiple attempts to treat early excessive inflammation have uniformly failed. We reported in a sepsis cell model and human sepsis blood leukocytes that nuclear NAD+ sensor SIRT1 deacetylase remodels chromatin at specific gene sets to switch the acute-phase proinflammatory response to hypoinflammatory. Importantly, SIRT1 chromatin reprogramming is reversible, suggesting that inhibition of SIRT1 might reverse postacute-phase hypoinflammation. We tested this concept in septic mice, using the highly specific SIRT1 inhibitor EX-527, a small molecule that closes the NAD+ binding site of SIRT1. Strikingly, when administered 24 h after sepsis, all treated animals survived, whereas only 40% of untreated mice survived. EX-527 treatment reversed the inability of leukocytes to adhere at the small intestine MVI, reversed in vivo endotoxin tolerance, increased leukocyte accumulation in peritoneum, and improved peritoneal bacterial clearance. Mechanistically, the SIRT1 inhibitor restored repressed endothelial E-selectin and ICAM-1 expression and PSGL-1 expression on the neutrophils. Systemic benefits of EX-527 treatment included stabilized blood pressure, improved microvascular blood flow, and a shift toward proimmune macrophages in spleen and bone marrow. Our findings reveal that modifying the SIRT1 NAD+ axis may provide a novel way to treat sepsis in its hypoinflammatory phase. PMID:25001863

Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H2-driven NAD+-reduction in the presence of O2.

PubMed

Preissler, Janina; Wahlefeld, Stefan; Lorent, Christian; Teutloff, Christian; Horch, Marius; Lauterbach, Lars; Cramer, Stephen P; Zebger, Ingo; Lenz, Oliver

2018-01-01

Biocatalysts that mediate the H 2 -dependent reduction of NAD + to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD + -reducing [NiFe]‑hydrogenase that sustains catalytic activity at high temperatures and in the presence of O 2 , which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD + -reducing [NiFe]‑hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1 T (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H 2 -oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H 2 -mediated NAD + reduction activity was observed at 80°C and pH6.5, and catalytic activity was found to be sustained at low O 2 concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]‑hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD + -reducing [NiFe]‑hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H 2 -driven cofactor recycling under oxic conditions at elevated temperatures. Copyright © 2017 Elsevier B.V. All rights reserved.

Aerobic denitration of 2,4,6-trinitrotoluene in the presence of phenazine compounds and reduced pyridine nucleotides.

PubMed

Stenuit, Ben; Lamblin, Guillaume; Cornelis, Pierre; Agathos, Spiros N

2012-10-02

Phenazine-containing spent culture supernatants of Pseudomonas aeruginosa concentrated with a C18 solid-phase extraction cartridge initiate NAD(P)H-dependent denitration of 2,4,6-trinitrotoluene (TNT). In this study, TNT denitration was investigated under aerobic conditions using two phenazine secondary metabolites excreted by P. aeruginosa, pyocyanin (Py) and its precursor phenazine-1- carboxylic acid (PCA), and two chemically synthesized pyocyanin analogs, phenazine methosulfate (PMS+) and phenazine ethosulfate (PES+). The biomimetic Py/NAD(P)H/O2 system was characterized and found to extensively denitrate TNT in unbuffered aqueous solution with minor production of toxic amino aromatic derivatives. To a much lesser extent, TNT denitration was also observed with PMS+ and PES+ in the presence of NAD(P)H. No TNT denitration was detected with the biomimetic PCA/NAD(P)H/O2 system. Electron paramagnetic resonance (EPR) spectroscopy analysis of the biomimetic Py/NAD(P)H/O2 system revealed the generation of superoxide radical anions (O2 •−). In vitro TNT degradation experiments in the presence of specific inhibitors of reactive oxygen species suggest a nucleophilic attack of superoxide radical anion followed by TNT denitration through an as yet unknown mechanism. The results of this research confirm the high functional versatility of the redox-active metabolite pyocyanin and the susceptibility of aromatic compounds bearing electron withdrawing substituents, such as nitro groups, to superoxide-driven nucleophilic attack.

Poly(ADP-ribose) polymerase-dependent energy depletion occurs through inhibition of glycolysis.

PubMed

Andrabi, Shaida A; Umanah, George K E; Chang, Calvin; Stevens, Daniel A; Karuppagounder, Senthilkumar S; Gagné, Jean-Philippe; Poirier, Guy G; Dawson, Valina L; Dawson, Ted M

2014-07-15

Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated "parthanatos" in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD(+) and energetic collapse, which have been thought to be caused by the consumption of cellular NAD(+) by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD(+) depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD(+) depletion in N-methyl-N-nitroso-N-nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1-mediated mitochondrial dysfunction. Depleting neurons of NAD(+) with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase.

A theoretical study of the molecular mechanism of the GAPDH Trypanosoma cruzi enzyme involving iodoacetate inhibitor

NASA Astrophysics Data System (ADS)

Carneiro, Agnaldo Silva; Lameira, Jerônimo; Alves, Cláudio Nahum

2011-10-01

The glyceraldehyde-3-phosphate dehydrogenase enzyme (GAPDH) is an important biological target for the development of new chemotherapeutic agents against Chagas disease. In this Letter, the inhibition mechanism of GAPDH involving iodoacetate (IAA) inhibitor was studied using the hybrid quantum mechanical/molecular mechanical (QM/MM) approach and molecular dynamic simulations. Analysis of the potential energy surface and potential of mean force show that the covalent attachment of IAA inhibitor to the active site of the enzyme occurs as a concerted process. In addition, the energy terms decomposition shows that NAD+ plays an important role in stabilization of the reagents and transition state.

Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells.

PubMed

Yang, Tianle; Chan, Noel Yan-Ki; Sauve, Anthony A

2007-12-27

A new two-step methodology achieves stereoselective synthesis of beta-nicotinamide riboside and a series of related amide, ester, and acid nucleosides. Compounds were prepared through a triacetylated-nicotinate ester nucleoside, via coupling of either ethylnicotinate or phenylnicotinate with 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose. Nicotinamide riboside, nicotinic acid riboside, O-ethylnicotinate riboside, O-methylnicotinate riboside, and several N-alkyl derivatives increased NAD+ concentrations from 1.2-2.7-fold in several mammalian cell lines. These findings establish bioavailability and potent effects of these nucleosides in stimulating the increase of NAD+ concentrations in mammalian cells.

Role of Gts1p in regulation of energy-metabolism oscillation in continuous cultures of the yeast Saccharomyces cerevisiae.

PubMed

Xu, Zhaojun; Tsurugi, Kunio

2007-03-01

Energy-metabolism oscillation (EMO) in an aerobic chemostat culture of yeast is basically regulated by a feedback loop of redox reactions in energy metabolism and modulated by metabolism of storage carbohydrates. In this study, we investigated the role of Gts1p in the stabilization of EMO, using the GTS1-deleted transformant gts1Delta. We found that fluctuations in the redox state of the NAD co-factor and levels of redox-regulated metabolites in glycolysis, especially of ethanol, are markedly reduced in amplitude during EMO of gts1Delta, while respiration indicated by the oxygen uptake rate (OUR) and energy charge is not so affected throughout EMO in gts1Delta. Further, the transitions of the levels of OUR, NAD(+) : NADH ratio and intracellular pH between the two phases were apparently retarded compared with those in the wild-type, suggesting attenuation of EMO in gts1Delta. Furthermore, the mRNA levels of genes encoding enzymes for the synthesis of trehalose and glycogen are fairly reduced in gts1Delta, consistent with the decreased synthesis of storage carbohydrates. In addition, the level of inorganic phosphate, which is required for the reduction of NAD(+) and mainly supplied from trehalose synthesis, was decreased in the early respiro-fermentative phase in gts1Delta. Thus, we suggested that the deletion of GTS1 as a transcriptional co-activator for these genes inhibited the metabolism of storage carbohydrates, which causes attenuation of the feedback loop of dehydrogenase reactions in glycolysis with the restricted fluctuation of ethanol as a main synchronizing agent for EMO in a cell population.

WldS but not Nmnat1 protects dopaminergic neurites from MPP+ neurotoxicity.

PubMed

Antenor-Dorsey, Jo Ann V; O'Malley, Karen L

2012-02-08

The WldS mouse mutant ("Wallerian degeneration-slow") delays axonal degeneration in a variety of disorders including in vivo models of Parkinson's disease. The mechanisms underlying WldS -mediated axonal protection are unclear, although many studies have attributed WldS neuroprotection to the NAD+-synthesizing Nmnat1 portion of the fusion protein. Here, we used dissociated dopaminergic cultures to test the hypothesis that catalytically active Nmnat1 protects dopaminergic neurons from toxin-mediated axonal injury. Using mutant mice and lentiviral transduction of dopaminergic neurons, the present findings demonstrate that WldS but not Nmnat1, Nmnat3, or cytoplasmically-targeted Nmnat1 protects dopamine axons from the parkinsonian mimetic N-methyl-4-phenylpyridinium (MPP+). Moreover, NAD+ synthesis is not required since enzymatically-inactive WldS still protects. In addition, NAD+ by itself is axonally protective and together with WldS is additive in the MPP+ model. Our data suggest that NAD+ and WldS act through separate and possibly parallel mechanisms to protect dopamine axons. As MPP+ is thought to impair mitochondrial function, these results suggest that WldS might be involved in preserving mitochondrial health or maintaining cellular metabolism.

Nicotinamide Riboside Preserves Cardiac Function in a Mouse Model of Dilated Cardiomyopathy.

PubMed

Diguet, Nicolas; Trammell, Samuel A J; Tannous, Cynthia; Deloux, Robin; Piquereau, Jérôme; Mougenot, Nathalie; Gouge, Anne; Gressette, Mélanie; Manoury, Boris; Blanc, Jocelyne; Breton, Marie; Decaux, Jean-François; Lavery, Gareth G; Baczkó, István; Zoll, Joffrey; Garnier, Anne; Li, Zhenlin; Brenner, Charles; Mericskay, Mathias

2018-05-22

Myocardial metabolic impairment is a major feature in chronic heart failure. As the major coenzyme in fuel oxidation and oxidative phosphorylation and a substrate for enzymes signaling energy stress and oxidative stress response, nicotinamide adenine dinucleotide (NAD + ) is emerging as a metabolic target in a number of diseases including heart failure. Little is known on the mechanisms regulating homeostasis of NAD + in the failing heart. To explore possible alterations of NAD + homeostasis in the failing heart, we quantified the expression of NAD + biosynthetic enzymes in the human failing heart and in the heart of a mouse model of dilated cardiomyopathy (DCM) triggered by Serum Response Factor transcription factor depletion in the heart (SRF HKO ) or of cardiac hypertrophy triggered by transverse aorta constriction. We studied the impact of NAD + precursor supplementation on cardiac function in both mouse models. We observed a 30% loss in levels of NAD + in the murine failing heart of both DCM and transverse aorta constriction mice that was accompanied by a decrease in expression of the nicotinamide phosphoribosyltransferase enzyme that recycles the nicotinamide precursor, whereas the nicotinamide riboside kinase 2 (NMRK2) that phosphorylates the nicotinamide riboside precursor is increased, to a higher level in the DCM (40-fold) than in transverse aorta constriction (4-fold). This shift was also observed in human failing heart biopsies in comparison with nonfailing controls. We show that the Nmrk2 gene is an AMP-activated protein kinase and peroxisome proliferator-activated receptor α responsive gene that is activated by energy stress and NAD + depletion in isolated rat cardiomyocytes. Nicotinamide riboside efficiently rescues NAD + synthesis in response to FK866-mediated inhibition of nicotinamide phosphoribosyltransferase and stimulates glycolysis in cardiomyocytes. Accordingly, we show that nicotinamide riboside supplementation in food attenuates the development of heart failure in mice, more robustly in DCM, and partially after transverse aorta constriction, by stabilizing myocardial NAD + levels in the failing heart. Nicotinamide riboside treatment also robustly increases the myocardial levels of 3 metabolites, nicotinic acid adenine dinucleotide, methylnicotinamide, and N1-methyl-4-pyridone-5-carboxamide, that can be used as validation biomarkers for the treatment. The data show that nicotinamide riboside, the most energy-efficient among NAD precursors, could be useful for treatment of heart failure, notably in the context of DCM, a disease with few therapeutic options. © 2017 American Heart Association, Inc.

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

Multiphoton fluorescence lifetime imaging of metabolic status in mesenchymal stem cell during adipogenic differentiation

NASA Astrophysics Data System (ADS)

Meleshina, A. V.; Dudenkova, V. V.; Shirmanova, M. V.; Bystrova, A. S.; Zagaynova, E. V.

2016-03-01

Non-invasive imaging of cell metabolism is a valuable approach to assess the efficacy of stem cell therapy and understand the tissue development. In this study we analyzed metabolic trajectory of the mesenchymal stem cells (MCSs) during differentiation into adipocytes by measuring fluorescence lifetimes of free and bound forms of the reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD). Undifferentiated MSCs and MSCs on the 5, 12, 19, 26 days of differentiation were imaged on a Zeiss 710 microscope with fluorescence lifetime imaging (FLIM) system B&H (Germany). Fluorescence of NAD(P)H and FAD was excited at 750 nm and 900 nm, respectively, by a femtosecond Ti:sapphire laser and detected in a range 455-500 nm and 500-550 nm, correspondingly. We observed the changes in the NAD(P)H and FAD fluorescence lifetimes and their relative contributions in the differentiated adipocytes compare to undifferentiated MSCs. Increase of fluorescence lifetimes of the free and bound forms of NAD(P)H and the contribution of protein-bound NAD(P)H was registered, that can be associated with a metabolic switch from glycolysis to oxidative phosphorylation and/or synthesis of lipids in adipogenically differentiated MSCs. We also found that the contribution of protein-bound FAD decreased during differentiation. After carrying out appropriate biochemical measurements, the observed changes in cellular metabolism can potentially serve to monitor stem cell differentiation by FLIM.

H2O2 accelerates cellular senescence by accumulation of acetylated p53 via decrease in the function of SIRT1 by NAD+ depletion.

PubMed

Furukawa, Ayako; Tada-Oikawa, Saeko; Kawanishi, Shosuke; Oikawa, Shinji

2007-01-01

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD(+)-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H(2)O(2) and examined DNA cleavage, depletion of intracellular NAD(+), expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated beta-galactosidase (SA-beta-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H(2)O(2), though no cell death was observed. NAD(+) levels in TIG-3 cells treated with H(2)O(2) were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD(+) levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H(2)O(2) treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H(2)O(2) induced cell cycle arrest at 24h and increased SA-beta-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.

Streptozotocin Diabetes CORRELATION WITH EXTENT OF DEPRESSION OF PANCREATIC ISLET NICOTINAMIDE ADENINE DINUCLEOTIDE

PubMed Central

Anderson, Tom; Schein, Philip S.; McMenamin, Mary G.; Cooney, David A.

1974-01-01

The diabetogenic activity of streptozotocin has been correlated with a reduction in pyridine nucleotide synthesis in the mouse pancreatic islet. To determine the specificity of this reduction for diabetogenicity, a comparative study of streptozotocin, its cytotoxic moiety, 1-methyl-1-nitrosourea, and alloxan was performed. Streptozotocin administered intraperitoneally (i.p.) producd a dose-related reduction in islet NAD which was proportional to the degree of diabetogenicity. A diabetogenic dose, 200 mg/kg, attained a peak plasma N-nitroso intact streptozotocin concentration of 0.224 μmol/ml and reduced the mean islet NAD from a control of 0.78 to 0.15 pmol. At borderline, 150 mg/kg, and nondiabetogenic, 100 mg/kg, doses, plasma concentrations reached 0.161 and 0.136 μmol/ml, and NAD was 0.36 and 0.86 pmol/islet, respectively. 1-Methyl-1-nitrosourea, 100 mg/kg, attained a maximum N-nitroso intact 1-methyl-1-nitrosourea concentration of 0.162 μmol/ml and reduced the mean NAD to 0.58 pmol/islet, and was nondiabetogenic; 200 mg/kg attained a peak plasma concentration of 0.344 μmol/ml and depressed NAD to 0.38 pmol/islet, and was inconsistently diabetogenic. Islet NAD of 0.4 pmol/islet or greater is required for integrity of the beta cell. A diabetogenic dose of alloxan, 500 mg/kg, did not depress NAD, 0.85 pmol/islet, therefore confirming that its mechanism of diabetogenicity differs from that of streptozotocin. In vivo uptake of [methyl-14C]streptozotocin by islets was 3.8 times that of [methyl-14C]-1-methyl-1-nitrosourea, whereas uptake by the exocrine pancreas favored 1-methyl-1-nitrosourea over streptozotocin 2.4:1. The decreased islet uptake of 1-methyl-1-nitrosourea correlates with the 3.5 times increased molar dosage required to produce islet NAD depression comparable to that of streptozotocin, 150 mg/kg. These studies indicate that the glucose carrier of streptozotocin facilitates uptake of its cytotoxic group, 1-methyl-1-nitrosourea, into islets. PMID:4369217

Paper-Based Device for Rapid Visualization of NADH Based on Dissolution of Gold Nanoparticles.

PubMed

Liang, Pingping; Yu, Haixiang; Guntupalli, Bhargav; Xiao, Yi

2015-07-15

We describe a paper-based device that enables rapid and sensitive room-temperature detection of dihydronicotinamide adenine dinucleotide (NADH) via a colorimetric readout and demonstrate its value for monitoring NAD+-driven enzymatic reactions. Our system is based on NADH-mediated inhibition of gold nanoparticle (AuNPs) dissolution in a Au3+-cetyltrimethylammonium bromide (CTAB) solution. We fabricated a device consisting of a mixed cellulose ester paper featuring a wax-encircled, AuNP-coated film atop a cotton absorbent layer sandwiched between two plastic cover layers. In the absence of NADH, the Au3+-CTAB complex dissolves the AuNP layer completely, generating a white color in the test zone. In the presence of NADH, Au3+ is rapidly reduced to Au+, greatly decreasing the dissolution of AuNPs and yielding a red color that becomes stronger at increasing concentrations of NADH. This device exploits capillary force-assisted vertical diffusion, allowing us to apply a 25 μL sample to a surface-confined test zone to achieve a detection limit of 12.5 μM NADH. We used the enzyme glucose dehydrogenase as a model to demonstrate that our paper-based device can monitor NAD+-driven biochemical processes with and without selective dehydrogenase inhibitors by naked-eye observation within 4 min at room temperature in a small sample volume. We believe that our paper-based device could offer a valuable and low-cost analytical tool for monitoring NAD+-associated enzymatic reactions and screening for dehydrogenase inhibitors in a variety of testing contexts.

Role of malate dehydrogenase in facilitating lactate dehydrogenase to support the glycolysis pathway in tumors.

PubMed

Mansouri, Siavash; Shahriari, Ali; Kalantar, Hadi; Moini Zanjani, Taraneh; Haghi Karamallah, Mojtaba

2017-04-01

High aerobic glycolysis, as one of the hallmarks of cancer cells, requires nicotinamide adenine dinucleotide (NAD + ) as a vital co-factor, to guarantee the flow of glycolysis. Malate dehydrogenase (MDH), as an important enzyme in cancer metabolism, is a source of NAD + additional to lactate dehydrogenase (LDH). The current study aimed to elucidate the kinetic parameters of MDH in human breast cancer and evaluate its supportive role in the glycolysis pathway. The Michaelis-Menten constant (K m ) and maximum velocity (V max ) of MDH were determined in the crude extracts of human breast tumors and healthy tissue samples, which were obtained directly from the operating theatre. To assess the potential role of MDH in supporting glycolysis, the MDH activity was measured when the LDH activity was inhibited by different concentrations of oxamate, an inhibitor of LDH in breast cancer cell lines. The K m of cancerous MDH (C-MDH) was the same as the healthy MDH, although the V max of C-MDH was higher relative to the healthy MDH. Notably, the MDH activity was increased in the MDA-MB-231 cell line, which was treated with the LDH inhibitor (oxamate), but not in the MCF-7 cell line (P<0.05). The higher tendency of C-MDH for NAD + and malate generation in cancer cells is an effective approach for supporting glycolysis. Increasing MDH activity in the absence of LDH demonstrates the supportive role of MDH in glycolysis. Therefore, decreasing MDH activity and expression in a forward reaction may present as a valid molecular target to abolish its potential effect on tumor metabolism.

The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro

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

Skardelly, Marco, E-mail: Marco.Skardelly@med.uni-tuebingen.de; Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig; Glien, Anja

In allogenic and xenogenic transplantation, adequate immunosuppression plays a major role in graft survival, especially over the long term. The effect of immunosuppressive drugs on neural stem/progenitor cell fate has not been sufficiently explored. The focus of this study is to systematically investigate the effects of the following four different immunotherapeutic strategies on human neural progenitor cell survival/death, proliferation, metabolic activity, differentiation and migration in vitro: (1) cyclosporine A (CsA), a calcineurin inhibitor; (2) everolimus (RAD001), an mTOR-inhibitor; (3) mycophenolic acid (MPA, mycophenolate), an inhibitor of inosine monophosphate dehydrogenase and (4) prednisolone, a steroid. At the minimum effective concentration (MEC),more » we found a prominent decrease in hNPCs' proliferative capacity (BrdU incorporation), especially for CsA and MPA, and an alteration of the NAD(P)H-dependent metabolic activity. Cell death rate, neurogenesis, gliogenesis and cell migration remained mostly unaffected under these conditions for all four immunosuppressants, except for apoptotic cell death, which was significantly increased by MPA treatment. - Highlights: • Four immunosuppresants (ISs) were tested in human neural progenitor cells in vitro. • Cyclosporine A and mycophenolic acid showed a prominent anti-proliferative activity • Mycophenolic acid exhibited a significant pro-apoptotic effect. • NAD(P)H-dependent metabolic activity was occasionally induced by ISs. • Neuronal differentiation and migration potential remained unaffected by ISs treatment.« less

Which interventions are cost-effective for the management of whiplash-associated and neck pain-associated disorders? A systematic review of the health economic literature by the Ontario Protocol for Traffic Injury Management (OPTIMa) Collaboration.

PubMed

van der Velde, Gabrielle; Yu, Hainan; Paulden, Mike; Côté, Pierre; Varatharajan, Sharanya; Shearer, Heather M; Wong, Jessica J; Randhawa, Kristi; Southerst, Danielle; Mior, Silvano; Sutton, Deborah; Jacobs, Craig; Taylor-Vaisey, Anne

2016-12-01

Whiplash-associated disorders (WAD) and neck pain and associated disorders (NAD) are prevalent conditions that impact society and impose a significant economic burden on health-care systems. Health economic evidence on WAD and NAD interventions has been sparse: only three economic evaluations of interventions for NAD were identified by the Bone and Joint Decade 2000-2010 Task Force on Neck Pain and Its Associated Disorders (NPTF). An updated overview is needed to inform health-care policy and guidelines. This study aimed to determine the cost-effectiveness of interventions for grades I-III WAD and NAD in children and adults. Systematic review of health economic literature, best-evidence synthesis. We systematically searched CINAHL, the Cochrane economic databases (Health Technology Assessment, NHS Economic Evaluation Database), EconLit, EMBASE, MEDLINE, PsycINFO, and Tufts CEA Registry from 2000 to 2015 for economic evaluations of WAD and NAD interventions. We appraised relevant evaluations using the Scottish Intercollegiate Guidelines Network Methodology Criteria for Economic Evaluations. We extracted data, including mean costs (standardized to 2013 Canadian dollars [CAD]) and quality-adjusted life years (QALYs), from studies with adequate methodological quality. We recalculated cost-effectiveness statistics based on the standardized currency using a willingness-to-pay of CAD $50,000 per additional QALY. Funding was provided by the Ministry of Finance. Our search identified 1,616 citations. Six studies fulfilled our selection criteria, including three studies previously reviewed by the NPTF. Structured education appears cost-effective for adults with WAD. For adults with NAD, acupuncture added to routine medical care; manual therapy; multimodal care that includes manual therapy; advice and exercise; and psychological care using cognitive-behavioral therapy appear cost-effective. In contrast, adding manual therapy or diathermy to advice and exercise; multimodal care by a physiotherapist or physician; and behavioral-graded activity do not appear cost-effective for adults with NAD. Our review adds to the findings of the NPTF. Recent evidence suggests that structured education is cost-effective for WAD, whereas advice and exercise and multimodal care that include manual therapy are cost-effective for NAD. Obtaining more robust health economic evidence for non-invasive interventions for WAD and NAD in children and adults remains an essential research priority. Copyright © 2015 Elsevier Inc. All rights reserved.

Ikkepsilon regulates viral-induced interferon regulatory factor-3 activation via a redox-sensitive pathway

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

Indukuri, Hemalatha; Castro, Shawn M.; Liao, S.-M.

2006-09-15

Respiratory syncytial virus (RSV)-induced chemokine gene expression occurs through the activation of a subset of transcription factors, including Interferon Regulatory Factor (IRF)-3. In this study, we have investigated the signaling pathway leading to RSV-induced IRF-3 activation and whether it is mediated by intracellular reactive oxygen species (ROS) generation. Our results show that RSV infection induces expression and catalytic activity of IKK{epsilon}, a noncanonical IKK-like kinase. Expression of a kinase-inactive IKK{epsilon} blocks RSV-induced IRF-3 serine phosphorylation, nuclear translocation and DNA-binding, leading to inhibition of RANTES gene transcription, mRNA expression and protein synthesis. Treatment of alveolar epithelial cells with antioxidants or withmore » NAD(P)H oxidase inhibitors abrogates RSV-induced chemokine secretion, IRF-3 phosphorylation and IKK{epsilon} induction, indicating that ROS generation plays a fundamental role in the signaling pathway leading to IRF-3 activation, therefore, identifying a novel molecular target for the development of strategies aimed to modify the inflammatory response associated with RSV infection of the lung.« less

[Cyclosporin A causes oxidative stress and mitochondrial dysfunction in renal tubular cells].

PubMed

Pérez de Hornedo, J; de Arriba, G; Calvino, M; Benito, S; Parra, T

2007-01-01

Reactive oxygen species (ROS) have been implicated in cyclosporin A (CsA) nephrotoxicity. As mitochondria are one of the main sources of ROS in cells, we evaluated the role of CsA in mitochondrial structure and function in LLC-PK1 cells. We incubated cells with CsA 1 microM for 24 hours and studies were performed with flow citometry and confocal microscopy. We studied mitochondrial NAD(P)H content, superoxide anion (O2.-) production (MitoSOX Red), oxidation of cardiolipin of inner mitochondrial membrane (NAO) and mitochondrial membrane potential (DIOC2(3)). Also we analyzed the intracellular ROS synthesis (H2DCF-DA) and reduced glutation (GSH) of cells. Our results showed that CsA decreased NAD(P)H and membrane potential, and increased O2.- in mitochondria. CsA also provoked oxidation of cardiolipin. Furthermore, CsA increased intracellular ROS production and decreased GSH content. These results suggest that CsA has crucial effects in mitochondria. CsA modified mitochondrial physiology through the decrease of antioxidant mitochondrial compounds as NAD(P)H and the dissipation of mitochondrial membrane potential and increase of oxidants as O2.-. Also, CsA alters lipidic structure of inner mitochondrial membrane through the oxidation of cardiolipin. These effects trigger a chain of events that favour intracellular synthesis of ROS and depletion of GSH that can compromise cellular viability. Nephrotoxic cellular effects of CsA can be explained, at least in part, through its influence on mitochondrial functionalism.

Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism.

PubMed

Belenky, Peter; Christensen, Kathryn C; Gazzaniga, Francesca; Pletnev, Alexandre A; Brenner, Charles

2009-01-02

NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification.

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

Fluorometric Measurement of Pyridine Nucleotide Reduction in the Giant Axon of the Squid

PubMed Central

Doane, Marshall G.

1967-01-01

By monitoring the fluorescence of the isolated giant axon of the squid Loligo pealei, it was possible to follow changes in its oxidation-reduction state as caused by the action of anoxia, cyanide, Amytal, and azide. The response to oxygen depletion was very rapid, the NAD within the axon being 90% reduced within 1–2 min. Cyanide and Amytal gave essentially similar results, although somewhat longer periods of time elapsed during their onset and washout periods. The extent of NAD reduction was essentially the same under conditions of anoxia and treatment with cyanide and Amytal. Azide was less effective in this respect, and at comparatively high levels of concentration (25–50 mM) gave values of 40% or less of the reduction observed with the other inhibitors. The application of ouabain and strophanthidin gave no observable NAD reduction. Variations in the time required to consume given quantities of dissolved oxygen before and after stimulation indicated an increase of 10–20% in oxygen uptake rate associated with activity, although this figure appeared to be a function of the surface-to-volume ratio of the axon. A biochemical analysis of axoplasm for oxidized and reduced pyridine nucleotide was made. Fluorometric examination of centrifuged axoplasm indicated that the NAD-NADH was largely confined to the mitochondria of the axon. PMID:4384698

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.

Are manual therapies, passive physical modalities, or acupuncture effective for the management of patients with whiplash-associated disorders or neck pain and associated disorders? An update of the Bone and Joint Decade Task Force on Neck Pain and Its Associated Disorders by the OPTIMa collaboration.

PubMed

Wong, Jessica J; Shearer, Heather M; Mior, Silvano; Jacobs, Craig; Côté, Pierre; Randhawa, Kristi; Yu, Hainan; Southerst, Danielle; Varatharajan, Sharanya; Sutton, Deborah; van der Velde, Gabrielle; Carroll, Linda J; Ameis, Arthur; Ammendolia, Carlo; Brison, Robert; Nordin, Margareta; Stupar, Maja; Taylor-Vaisey, Anne

2016-12-01

In 2008, the Bone and Joint Decade 2000-2010 Task Force on Neck Pain and Its Associated Disorders (Neck Pain Task Force) found limited evidence on the effectiveness of manual therapies, passive physical modalities, or acupuncture for the management of whiplash-associated disorders (WAD) or neck pain and associated disorders (NAD). This review aimed to update the findings of the Neck Pain Task Force, which examined the effectiveness of manual therapies, passive physical modalities, and acupuncture for the management of WAD or NAD. This is a systematic review and best evidence synthesis. The sample includes randomized controlled trials, cohort studies, and case-control studies comparing manual therapies, passive physical modalities, or acupuncture with other interventions, placebo or sham, or no intervention. The outcome measures were self-rated or functional recovery, pain intensity, health-related quality of life, psychological outcomes, or adverse events. We systematically searched five databases from 2000 to 2014. Random pairs of independent reviewers critically appraised eligible studies using the Scottish Intercollegiate Guidelines Network criteria. Studies with a low risk of bias were stratified by the intervention's stage of development (exploratory vs. evaluation) and synthesized following best evidence synthesis principles. Funding was provided by the Ministry of Finance. We screened 8,551 citations, and 38 studies were relevant and 22 had a low risk of bias. Evidence from seven exploratory studies suggests that (1) for recent but not persistent NAD grades I-II, thoracic manipulation offers short-term benefits; (2) for persistent NAD grades I-II, technical parameters of cervical mobilization (eg, direction or site of manual contact) do not impact outcomes, whereas one session of cervical manipulation is similar to Kinesio Taping; and (3) for NAD grades I-II, strain-counterstrain treatment is no better than placebo. Evidence from 15 evaluation studies suggests that (1) for recent NAD grades I-II, cervical and thoracic manipulation provides no additional benefit to high-dose supervised exercises, and Swedish or clinical massage adds benefit to self-care advice; (2) for persistent NAD grades I-II, home-based cupping massage has similar outcomes to home-based muscle relaxation, low-level laser therapy (LLLT) does not offer benefits, Western acupuncture provides similar outcomes to non-penetrating placebo electroacupuncture, and needle acupuncture provides similar outcomes to sham-penetrating acupuncture; (3) for WAD grades I-II, needle electroacupuncture offers similar outcomes as simulated electroacupuncture; and (4) for recent NAD grades III, a semi-rigid cervical collar with rest and graded strengthening exercises lead to similar outcomes, and LLLT does not offer benefits. Our review adds new evidence to the Neck Pain Task Force and suggests that mobilization, manipulation, and clinical massage are effective interventions for the management of neck pain. It also suggests that electroacupuncture, strain-counterstrain, relaxation massage, and some passive physical modalities (heat, cold, diathermy, hydrotherapy, and ultrasound) are not effective and should not be used to manage neck pain. Copyright © 2015 Elsevier Inc. All rights reserved.

Regulation of the Biosynthesis of Amino Acids of the Aspartate Family in Coliform Bacteria and Pseudomonads

PubMed Central

Cohen, G. N.; Stanier, R. Y.; Bras, Gisele Le

1969-01-01

The control of aspartokinase and homoserine dehydrogenase activities was compared in aerobic and fermentative pseudomonads (genera Pseudomonas and Aeromonas), and in coliform bacteria representative of the principal genera of the Enterobacteriaceae. Isofunctional aspartokinases subject to independent end-product control occur in the Enterobacteriaceae and in Aeromonas. In Pseudomonas, there appears to be a single aspartokinase, subject to concerted feedback inhibition by lysine and threonine. Within this genus, the sensitivity of aspartokinase to the single allosteric inhibitors varies considerably: the aspartokinase of the acidovorans group is little affected by the single inhibitors, whereas that of the fluorescent group is severely inhibited by either amino acid at high concentration. In all bacteria examined, homoserine dehydrogenase activity is inhibited by threonine; inhibition is more severe in aerobic pseudomonads than in the other groups. In most of the bacteria examined, either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate can serve as a cofactor for this enzyme, though the relative activity with the two pyridine nucleotides varies widely. Aerobic pseudomonads of the acidovorans group contain a homoserine dehydrogenase that is absolutely specific for NAD. The taxonomic implications of these findings are discussed. PMID:4391829

The Nrf2-antioxidant response element pathway: a target for regulating energy metabolism

USDA-ARS?s Scientific Manuscript database

The nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that responds to oxidative stress by binding to the antioxidant response element (ARE) in the promoter of genes coding for antioxidant enzymes like NAD(P)H:quinone oxidoreductase 1 (NQO1) and proteins for glutathione synthesis. ...

DNA synthesis inhibitors for the treatment of gastrointestinal cancer.

PubMed

Yasui, Hiroshi; Tsurita, Giichiro; Imai, Kohzoh

2014-11-01

Intensive laboratory, preclinical and clinical studies have identified and validated molecular targets in cancers, leading to a shift toward the development of novel, rationally designed and specific therapeutic agents. However, gastrointestinal cancers continue to have a poor prognosis, largely due to drug resistance. Here, we discuss the current understanding of DNA synthesis inhibitors and their mechanisms of action for the treatment of gastrointestinal malignancies. Conventional agents, including DNA synthesis inhibitors such as fluoropyrimidines and platinum analogs, remain the most effective therapeutics and are the standards against which new drugs are compared. Novel DNA synthesis inhibitors for the treatment of gastrointestinal malignancies include a combination of the antimetabolite TAS-102, which consists of trifluorothymidine with a thymidine phosphorylase inhibitor, and a novel micellar formulation of cisplatin NC-6004 that uses a nanotechnology-based drug delivery system. The challenges of translational cancer research using DNA synthesis inhibitors include the identification of drugs that are specific to tumor cells to reduce toxicity and increase antitumor efficacy, biomarkers to predict pharmacological responses to chemotherapeutic drugs, identification of ways to overcome drug resistance and development of novel combination therapies with DNA synthesis inhibitors and other cancer therapies, such as targeted molecular therapeutics. Here, we discuss the current understanding of DNA synthesis inhibitors and their mechanisms of action for the treatment of gastrointestinal malignancies.

Allopurinol-mediated lignocellulose-derived microbial inhibitor tolerance by Clostridium beijerinckii during acetone-butanol-ethanol (ABE) fermentation.

PubMed

Ujor, Victor; Agu, Chidozie Victor; Gopalan, Venkat; Ezeji, Thaddeus Chukwuemeka

2015-04-01

In addition to glucans, xylans, and arabinans, lignocellulosic biomass hydrolysates contain significant levels of nonsugar components that are toxic to the microbes that are typically used to convert biomass to biofuels and chemicals. To enhance the tolerance of acetone-butanol-ethanol (ABE)-generating Clostridium beijerinckii NCIMB 8052 to these lignocellulose-derived microbial inhibitory compounds (LDMICs; e.g., furfural), we have been examining different metabolic perturbation strategies to increase the cellular reductant pools and thereby facilitate detoxification of LDMICs. As part of these efforts, we evaluated the effect of allopurinol, an inhibitor of NAD(P)H-generating xanthine dehydrogenase (XDH), on C. beijerinckii grown in furfural-supplemented medium and found that it unexpectedly increased the rate of detoxification of furfural by 1.4-fold and promoted growth, butanol, and ABE production by 1.2-, 2.5-, and 2-fold, respectively. Since NAD(P)H/NAD(P)(+) levels in C. beijerinckii were largely unchanged upon allopurinol treatment, we postulated and validated a possible basis in DNA repair to account for the solventogenic gains with allopurinol. Following the observation that supplementation of allopurinol in the C. beijerinckii growth media mitigates the toxic effects of nalidixic acid, a DNA-damaging antibiotic, we found that allopurinol elicited 2.4- and 6.7-fold increase in the messenger RNA (mRNA) levels of xanthine and hypoxanthine phosphoribosyltransferases, key purine-salvage enzymes. Consistent with this finding, addition of inosine (a precursor of hypoxanthine) and xanthine led to 1.4- and 1.7-fold increase in butanol production in furfural-challenged cultures of C. beijerinckii. Taken together, our results provide a purine salvage-based rationale for the unanticipated effect of allopurinol in improving furfural tolerance of the ABE-fermenting C. beijerinckii.

Mitochondrial complex I and NAD(P)H oxidase are major sources of exacerbated oxidative stress in pressure-overloaded ischemic-reperfused hearts.

PubMed

Mozaffari, Mahmood S; Baban, Babak; Liu, Jun Yao; Abebe, Worku; Sullivan, Jennifer C; El-Marakby, Ahmed

2011-03-01

We tested the hypothesis that pressure overload exacerbates oxidative stress associated with augmented mitochondrial permeability transition (MPT) pore opening and cell death in ischemic-reperfused hearts. Pressure overload decreased the level of reduced glutathione but increased nitrotyrosine and 8-hydroxydeoxyguanosine levels in ischemic-reperfused hearts. The activity of catalase, but not superoxide dismutase (SOD), was lower in ischemic-reperfused hearts perfused at higher pressure. Mitochondria from ischemic-reperfused hearts subjected to higher perfusion pressure displayed significantly greater [³H]-2-deoxyglucose-6-P entrapment suggestive of greater MPT pore opening and consistent with greater necrosis and apoptosis. Tempol (SOD mimetic) reduced infarct size in both groups but it remained greater in the higher pressure group. By contrast, uric acid (peroxynitrite scavenger) markedly reduced infarct size at higher pressure, effectively eliminating the differential between the two groups. Inhibition of xanthine oxidase, with allopurinol, reduced infarct size but did not eliminate the differential between the two groups. However, amobarbital (inhibitor of mitochondrial complex I) or apocynin [inhibitor of NAD(P)H oxidase] reduced infarct size at both pressures and also abrogated the differential between the two groups. Consistent with the effect of apocynin, pressure-overloaded hearts displayed significantly higher NAD(P)H oxidase activity. Furthermore, pressure-overloaded hearts displayed increased nitric oxide synthase activity which, along with increased propensity to superoxide generation, may underlie uric acid-induced cardioprotection. In conclusion, increased oxidative and nitrosative stress, coupled with lack of augmented SOD and catalase activities, contributes importantly to the exacerbating impact of pressure overload on MPT pore opening and cell death in ischemic-reperfused hearts.

SimC7 Is a Novel NAD(P)H-Dependent Ketoreductase Essential for the Antibiotic Activity of the DNA Gyrase Inhibitor Simocyclinone.

PubMed

Schäfer, Martin; Le, Tung B K; Hearnshaw, Stephen J; Maxwell, Anthony; Challis, Gregory L; Wilkinson, Barrie; Buttner, Mark J

2015-06-19

Simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü6040. The simocyclinone (sim) biosynthetic gene cluster has been sequenced and a hypothetical biosynthetic pathway has been proposed. The tetraene linker in SD8 was suggested to be the product of a modular type I polyketide synthase working in trans with two monofunctional enzymes. One of these monofunctional enzymes, SimC7, was proposed to supply a dehydratase activity missing from two modules of the polyketide synthase. In this study, we report the function of SimC7. We isolated the entire ~72-kb sim cluster on a single phage artificial chromosome clone and produced simocyclinone heterologously in a Streptomyces coelicolor strain engineered for improved antibiotic production. Deletion of simC7 resulted in the production of a novel simocyclinone, 7-oxo-SD8, which unexpectedly carried a normal tetraene linker but was altered in the angucyclinone moiety. We demonstrate that SimC7 is an NAD(P)H-dependent ketoreductase that catalyzes the conversion of 7-oxo-SD8 into SD8. 7-oxo-SD8 was essentially inactive as a DNA gyrase inhibitor, and the reduction of the keto group by SimC7 was shown to be crucial for high-affinity binding to the enzyme. Thus, SimC7 is an angucyclinone ketoreductase that is essential for the biological activity of simocyclinone. Copyright © 2015. Published by Elsevier Ltd.

Towards an integrative model of C4 photosynthetic subtypes: insights from comparative transcriptome analysis of NAD-ME, NADP-ME, and PEP-CK C4 species

PubMed Central

Bräutigam, Andrea; Schliesky, Simon; Külahoglu, Canan; Osborne, Colin P.; Weber, Andreas P.M.

2014-01-01

C4 photosynthesis affords higher photosynthetic carbon conversion efficiency than C3 photosynthesis and it therefore represents an attractive target for engineering efforts aiming to improve crop productivity. To this end, blueprints are required that reflect C4 metabolism as closely as possible. Such blueprints have been derived from comparative transcriptome analyses of C3 species with related C4 species belonging to the NAD-malic enzyme (NAD-ME) and NADP-ME subgroups of C4 photosynthesis. However, a comparison between C3 and the phosphoenolpyruvate carboxykinase (PEP-CK) subtype of C4 photosynthesis is still missing. An integrative analysis of all three C4 subtypes has also not been possible to date, since no comparison has been available for closely related C3 and PEP-CK C4 species. To generate the data, the guinea grass Megathyrsus maximus, which represents a PEP-CK species, was analysed in comparison with a closely related C3 sister species, Dichanthelium clandestinum, and with publicly available sets of RNA-Seq data from C4 species belonging to the NAD-ME and NADP-ME subgroups. The data indicate that the core C4 cycle of the PEP-CK grass M. maximus is quite similar to that of NAD-ME species with only a few exceptions, such as the subcellular location of transfer acid production and the degree and pattern of up-regulation of genes encoding C4 enzymes. One additional mitochondrial transporter protein was associated with the core cycle. The broad comparison identified sucrose and starch synthesis, as well as the prevention of leakage of C4 cycle intermediates to other metabolic pathways, as critical components of C4 metabolism. Estimation of intercellular transport fluxes indicated that flux between cells is increased by at least two orders of magnitude in C4 species compared with C3 species. In contrast to NAD-ME and NADP-ME species, the transcription of photosynthetic electron transfer proteins was unchanged in PEP-CK. In summary, the PEP-CK blueprint of M. maximus appears to be simpler than those of NAD-ME and NADP-ME plants. PMID:24642845

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.

Lactate is oxidized outside of the mitochondrial matrix in rodent brain.

PubMed

Herbst, Eric A F; George, Mitchell A J; Brebner, Karen; Holloway, Graham P; Kane, Daniel A

2018-05-01

The nature and existence of mitochondrial lactate oxidation is debated in the literature. Obscuring the issue are disparate findings in isolated mitochondria, as well as relatively low rates of lactate oxidation observed in permeabilized muscle fibres. However, respiration with lactate has yet to be directly assessed in brain tissue with the mitochondrial reticulum intact. To determine if lactate is oxidized in the matrix of brain mitochondria, oxygen consumption was measured in saponin-permeabilized mouse brain cortex samples, and rat prefrontal cortex and hippocampus (dorsal) subregions. While respiration in the presence of ADP and malate increased with the addition of lactate, respiration was maximized following the addition of exogenous NAD + , suggesting maximal lactate metabolism involves extra-matrix lactate dehydrogenase. This was further supported when NAD + -dependent lactate oxidation was significantly decreased with the addition of either low-concentration α-cyano-4-hydroxycinnamate or UK-5099, inhibitors of mitochondrial pyruvate transport. Mitochondrial respiration was comparable between glutamate, pyruvate, and NAD + -dependent lactate oxidation. Results from the current study demonstrate that permeabilized brain is a feasible model for assessing lactate oxidation, and support the interpretation that lactate oxidation occurs outside the mitochondrial matrix in rodent brain.

NADP/sup +/ enhances cholera and pertussis toxin-catalyzed ADP-ribosylation of membrane proteins

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

Kawai, Y.; Whitsel, C.; Arinze, I.J.

1986-05-01

Cholera or pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation is frequently used to estimate the concentration of the stimulatory (Ns) or inhibitory (Ni) guanine nucleotide regulatory proteins which modulate the activity of adenylate cyclase. With this assay, however, the degradation of the substrate, NAD/sup +/, by endogenous enzymes such as NAD/sup +/-glycohydrolase (NADase) present in the test membranes can influence the results. In this study the authors show that both cholera and pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation of liver membrane proteins is markedly enhanced by NADP/sup +/. The effect is concentration dependent; with 20 ..mu..M (/sup 32/P)NAD/sup +/ as substrate maximal enhancement is obtainedmore » at 0.5-1.0 mM NADP/sup +/. The enhancement of (/sup 32/P)ADP-ribosylation by NADP/sup +/ was much greater than that by other known effectors such as Mg/sup 2 +/, phosphate or isoniazid. The effect of NADP/sup +/ on ADP-ribosylation may occur by inhibition of the degradation of NAD/sup +/ probably by acting as an alternate substrate for NADase. Among inhibitors tested (NADP/sup +/, isoniazid, imidazole, nicotinamide, L-Arg-methyl-ester and HgCl/sub 2/) to suppress NADase activity, NADP/sup +/ was the most effective and, 10 mM, inhibited activity of the enzyme by about 90%. In membranes which contain substantial activities of NADase the inclusion of NADP/sup +/ in the assay is necessary to obtain maximal ADP-ribosylation.« less

The TosMIC approach to 3-(oxazol-5-yl) indoles: application to the synthesis of indole-based IMPDH inhibitors.

PubMed

Dhar, T G Murali; Shen, Zhongqi; Fleener, Catherine A; Rouleau, Katherine A; Barrish, Joel C; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2002-11-18

A modified approach to the synthesis of 3-(oxazolyl-5-yl) indoles is reported. This method was applied to the synthesis of series of novel indole based inhibitors of inosine monophosphate dehydrogenase (IMPDH). The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.

5-((3-Amidobenzyl)oxy)nicotinamides as Sirtuin 2 Inhibitors.

PubMed

Ai, Teng; Wilson, Daniel J; More, Swati S; Xie, Jiashu; Chen, Liqiang

2016-04-14

Derived from our previously reported human sirtuin 2 (SIRT2) inhibitors that were based on a 5-aminonaphthalen-1-yloxy nicotinamide core structure, 5-((3-amidobenzyl)oxy)nicotinamides offered excellent activity against SIRT2 and high isozyme selectivity over SIRT1 and SIRT3. Selected compounds also exhibited generally favorable in vitro absorption, distribution, metabolism, and excretion properties. Kinetic studies revealed that a representative SIRT2 inhibitor acted competitively against both NAD(+) and the peptide substrate, an inhibitory modality that was supported by our computational study. More importantly, two selected compounds exhibited significant protection against α-synuclein aggregation-induced cytotoxicity in SH-SY5Y cells. Therefore, 5-((3-amidobenzyl)oxy)nicotinamides represent a new class of SIRT2 inhibitors that are attractive candidates for further lead optimization in our continued effort to explore selective inhibition of SIRT2 as a potential therapy for Parkinson's disease.

Reversible, partial inactivation of plant betaine aldehyde dehydrogenase by betaine aldehyde: mechanism and possible physiological implications.

PubMed

Zárate-Romero, Andrés; Murillo-Melo, Darío S; Mújica-Jiménez, Carlos; Montiel, Carmina; Muñoz-Clares, Rosario A

2016-04-01

In plants, the last step in the biosynthesis of the osmoprotectant glycine betaine (GB) is the NAD(+)-dependent oxidation of betaine aldehyde (BAL) catalysed by some aldehyde dehydrogenase (ALDH) 10 enzymes that exhibit betaine aldehyde dehydrogenase (BADH) activity. Given the irreversibility of the reaction, the short-term regulation of these enzymes is of great physiological relevance to avoid adverse decreases in the NAD(+):NADH ratio. In the present study, we report that the Spinacia oleracea BADH (SoBADH) is reversibly and partially inactivated by BAL in the absence of NAD(+)in a time- and concentration-dependent mode. Crystallographic evidence indicates that the non-essential Cys(450)(SoBADH numbering) forms a thiohemiacetal with BAL, totally blocking the productive binding of the aldehyde. It is of interest that, in contrast to Cys(450), the catalytic cysteine (Cys(291)) did not react with BAL in the absence of NAD(+) The trimethylammonium group of BAL binds in the same position in the inactivating or productive modes. Accordingly, BAL does not inactivate the C(450)SSoBADH mutant and the degree of inactivation of the A(441)I and A(441)C mutants corresponds to their very different abilities to bind the trimethylammonium group. Cys(450)and the neighbouring residues that participate in stabilizing the thiohemiacetal are strictly conserved in plant ALDH10 enzymes with proven or predicted BADH activity, suggesting that inactivation by BAL is their common feature. Under osmotic stress conditions, this novel partial and reversible covalent regulatory mechanism may contribute to preventing NAD(+)exhaustion, while still permitting the synthesis of high amounts of GB and avoiding the accumulation of the toxic BAL. © 2016 Authors; published by Portland Press Limited.

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

Primary deuterium and tritium isotope effects upon V/K in the liver alcohol dehydrogenase reaction with ethanol.

PubMed

Damgaard, S E

1981-09-29

The primary isotope effect upon V/K when ethanol stereospecifically labeled with deuterium or tritium is oxidized by liver alcohol dehydrogenase has been measured between pH 6 and 9. The deuterium isotope effect was obtained with high reproducibility by the use of two different radioactive tracers, viz. 14C and 3H, to follow the rate of acetaldehyde formation from deuterium-labeled ethanol and normal ethanol, respectively. Synthesis of the necessary labeled compounds is described in this and earlier work referred to. V/K isotope effects for both tritium and deuterium have been measured with three different coenzymes, NAD+, thio-NAD+, and acetyl-NAD+. With NAD+ at pH 7, D(V/K) was 3.0 and T(V/K) was 6.5. With increasing pH, these values decreased to 1.5 and 2.5 at pH 9. The intrinsic isotope effect evaluated by the method of Northrop [Northrop, D.B. (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M, H., & Northrop, D. B., Eds.) pp 112-152, University Park Press, Baltimore] varies little with pH. It amounts to about 10 with NAD+ and about 5 with the coenzyme analogues. Commitment functions and their dependence upon pH calculated in this connection appear to be in agreement with known kinetic parameters of liver alcohol dehydrogenase. This assay method was also applied in vivo in the rat. Being a noninvasive method because only trace amounts of isotopes are needed, it may yield information about alternative routes of ethanol oxidation in vivo. In naive rats at low concentrations of ethanol, it confirms the discrete role of the non alcohol dehydrogenase systems.

Pulsed addition of HMF and furfural to batch-grown xylose-utilizing Saccharomyces cerevisiae results in different physiological responses in glucose and xylose consumption phase

PubMed Central

2013-01-01

Background Pretreatment of lignocellulosic biomass generates a number of undesired degradation products that can inhibit microbial metabolism. Two of these compounds, the furan aldehydes 5-hydroxymethylfurfural (HMF) and 2-furaldehyde (furfural), have been shown to be an impediment for viable ethanol production. In the present study, HMF and furfural were pulse-added during either the glucose or the xylose consumption phase in order to dissect the effects of these inhibitors on energy state, redox metabolism, and gene expression of xylose-consuming Saccharomyces cerevisiae. Results Pulsed addition of 3.9 g L-1 HMF and 1.2 g L-1 furfural during either the glucose or the xylose consumption phase resulted in distinct physiological responses. Addition of furan aldehydes in the glucose consumption phase was followed by a decrease in the specific growth rate and the glycerol yield, whereas the acetate yield increased 7.3-fold, suggesting that NAD(P)H for furan aldehyde conversion was generated by acetate synthesis. No change in the intracellular levels of NAD(P)H was observed 1 hour after pulsing, whereas the intracellular concentration of ATP increased by 58%. An investigation of the response at transcriptional level revealed changes known to be correlated with perturbations in the specific growth rate, such as protein and nucleotide biosynthesis. Addition of furan aldehydes during the xylose consumption phase brought about an increase in the glycerol and acetate yields, whereas the xylitol yield was severely reduced. The intracellular concentrations of NADH and NADPH decreased by 58 and 85%, respectively, hence suggesting that HMF and furfural drained the cells of reducing power. The intracellular concentration of ATP was reduced by 42% 1 hour after pulsing of inhibitors, suggesting that energy-requiring repair or maintenance processes were activated. Transcriptome profiling showed that NADPH-requiring processes such as amino acid biosynthesis and sulfate and nitrogen assimilation were induced 1 hour after pulsing. Conclusions The redox and energy metabolism were found to be more severely affected after pulsing of furan aldehydes during the xylose consumption phase than during glucose consumption. Conceivably, this discrepancy resulted from the low xylose utilization rate, hence suggesting that xylose metabolism is a feasible target for metabolic engineering of more robust xylose-utilizing yeast strains. PMID:24341320

Concurrent targeting of nitrosative stress-PARP pathway corrects functional, behavioral and biochemical deficits in experimental diabetic neuropathy

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

Negi, Geeta; Kumar, Ashutosh; Sharma, Shyam S., E-mail: sssharma@niper.ac.in

2010-01-01

Peroxynitrite mediated nitrosative stress, an indisputable initiator of DNA damage and overactivation of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated after sensing DNA damage, are two crucial pathogenetic mechanisms in diabetic neuropathy. The intent of the present study was to investigate the effect of combination of a peroxynitrite decomposition catalyst (PDC), FeTMPyP and a PARP inhibitor, 4-ANI against diabetic peripheral neuropathy. The end points of evaluation of the study included motor nerve conduction velocity (MNCV) and nerve blood flow (NBF) for evaluating nerve functions; thermal hyperalgesia and mechanical allodynia for assessing nociceptive alterations, malondialdehyde and peroxynitrite levels to detect oxidativemore » stress-nitrosative stress; NAD concentration in sciatic nerve to assess overactivation of PARP. Additionally immunohistochemical studies for nitrotyrosine and Poly(ADP-ribose) (PAR) was also performed. Treatment with the combination of FeTMPyP and 4-ANI led to significant improvement in nerve functions and pain parameters and also attenuated the oxidative-nitrosative stress markers. Further, the combination also reduced the overactivation of PARP as evident from increased NAD levels and decreased PAR immunopositivity in sciatic nerve microsections. Thus, it can be concluded that treatment with the combination of a PDC and PARP inhibitor attenuates alteration in peripheral nerves in diabetic neuropathy (DN).« less

[Assessment of mitochondrial metabolic oxidative state in living cardiomyocytes with spectrally-resolved fluorescence lifetime spectroscopy of NAD(P)H].

PubMed

Cheng, Ying; Ren, Mingming; Niu, Yanyan; Qiao, Jianhua; Aneba, S; Chorvat, D; Chorvatova, A

2009-12-01

The primary function of cardiac mitochondria is the production of ATP to support heart contraction. Examination of the mitochondrial redox state is therefore crucially important to sensitively detect early signs of mitochondrial function in pathophysiological conditions, such as ischemia, diabetes and heart failure. We study fingerprinting of mitochondrial metabolic oxidative state in living cardiomyocytes with spectrally-resolved fluorescence lifetime spectroscopy of NAD(P)H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply. Here NAD(P)H is studied as a marker for non-invasive fluorescent probing of the mitochondrial function. NAD(P) H fluorescence is recorded in cardiac cells following excitation with 375nm UV-light and detection by spectrally-resolved time-correlated single photon counting (TCSPC), based on the simultaneous measurement of the fluorescence spectra and fluorescence lifetimes. Modulation of NADH production and/or mitochondrial respiration is tested to study dynamic characteristics of NAD(P) H fluorescence decay. Our results show that at least a 3-exponential decay model, with 0.4-0.7ns, 1.2-1.9ns and 8.0-13. Ons lifetime pools is necessary to describe cardiomyocyte autofluorescence (AF) within 420-560nm spectral range. Increased mitochondrial NADH production by ketone bodies enhanced the fluorescence intensity, without significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF intensity and shortened the average fluorescence lifetime. Dinitrophenol (DNP), an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF intensity, broadened the spectral shoulder at 520 nm and increased the average fluorescence lifetime. These effects are comparable to the study of NADH fluorescence decay in vitro. In the present contribution we demonstrated that spectrally-resolved fluorescence lifetime technique provides promising new tool for analysis of mitochondrial NAD(P) H fluorescence with good reproducibility in living cardiomyocytes. This approach will enhance our knowledge about cardiomyocyte oxidative metabolism and/or its dysfunction at a cellular level. In the future, this approach can prove helpful in the clinical diagnosis and treatment of mitochondrial disorder.

ENZYME ACTIVITIES DURING THE ASEXUAL CYCLE OF NEUROSPORA CRASSA

PubMed Central

Stine, G. J.

1968-01-01

Three enzymes, (a) nicotinamide adenine diphosphate-dependent glutamic dehydrogenase (NAD enzyme), (b) nictoinamide adenine triphosphate-dependent glutamic dehydrogenase (NADP enzyme), and (c) nicotinamide-adenine dinucleotidase (NADase), were measured in separate extracts of Neurospora crassa grown in Vogel's medium N and medium N + glutamate. Specific activities and total units per culture of each enzyme were determined at nine separate intervals phased throughout the asexual cycle. The separate dehydrogenases were lowest in the conidia, increased slowly during germination, and increased rapidly during logarithmic mycelial growth. The amounts of these enzymes present during germination were small when compared with those found later during the production of the conidiophores. The NAD enzyme may be necessary for pregermination synthesis. The NADP-enzyme synthesis was associated with the appearance of the germ tube. Although higher levels of the dehydrogenases in the conidiophores resulted in more enzyme being found in the differentiated conidia, the rate of germination was uneffected. The greatest activity for the NADase enzyme was associated with the conidia, early phases of germination, and later production of new conidia. NADase decreased significantly with the onset of logarithmic growth, remained low during the differentiation of conidiophores, and increased considerably as the conidiophores aged. PMID:4384627

Are psychological interventions effective for the management of neck pain and whiplash-associated disorders? A systematic review by the Ontario Protocol for Traffic Injury Management (OPTIMa) Collaboration.

PubMed

Shearer, Heather M; Carroll, Linda J; Wong, Jessica J; Côté, Pierre; Varatharajan, Sharanya; Southerst, Danielle; Sutton, Deborah A; Randhawa, Kristi A; Yu, Hainan; Mior, Silvano A; van der Velde, Gabrielle M; Nordin, Margareta C; Stupar, Maja; Taylor-Vaisey, Anne L

2016-12-01

In 2008, the lack of published evidence prevented the Bone and Joint Decade 2000-2010 Task Force on Neck Pain and Its Associated Disorders (Neck Pain Task Force [NPTF]) from commenting on the effectiveness of psychological interventions for the management of neck pain. This study aimed to update findings of the NPTF and evaluate the effectiveness of psychological interventions for the management of neck pain and associated disorders (NAD) or whiplash-associated disorders (WAD). This study used systematic review and best-evidence synthesis. Randomized controlled trials, cohort studies, and case-control studies comparing psychological interventions to other non-invasive interventions or no intervention were the samples used in this study. The outcome measures are (1) self-rated recovery; (2) functional recovery; (3) clinical outcomes; (4) administrative outcomes; and (5) adverse effects. We searched six databases from 1990 to 2015. Randomized controlled trials, cohort studies, and case-control studies meeting our selection criteria were eligible for critical appraisal. Random pairs of independent reviewers used the Scottish Intercollegiate Guidelines Network criteria to critically appraise eligible studies. Studies with a low risk of bias were synthesized following best evidence synthesis principles. This study was funded by the Ontario Ministry of Finance. We screened 1,919 articles, 19 were eligible for critical appraisal and 10 were judged to have low risk of bias. We found no clear evidence supporting relaxation training or cognitive behavioral therapy (CBT) for persistent grades I-III NAD for reducing pain intensity or disability. Similarly, we did not find evidence to support the effectiveness of biofeedback or relaxation training for persistent grade II WAD, and there is conflicting evidence for the use of CBT in this population. However, adding a progressive goal attainment program to functional restoration physiotherapy may benefit patients with persistent grades I-III WAD. Furthermore, Jyoti meditation may help reduce neck pain intensity and bothersomeness in patients with persistent NAD. We did not find evidence for or against the use of psychological interventions in patients with recent onset NAD or WAD. We found evidence that a progressive goal attainment program may be helpful for the management of persistent WAD and that Jyoti meditation may benefit patients with persistent NAD. The limited evidence of effectiveness for psychological interventions may be due to several factors, such as interventions that are ineffective, poorly conceptualized, or poorly implemented. Further methodologically rigorous research is needed. Copyright © 2015 Elsevier Inc. All rights reserved.

Kinesin spindle protein (KSP) inhibitors. Part 7: Design and synthesis of 3,3-disubstituted dihydropyrazolobenzoxazines as potent inhibitors of the mitotic kinesin KSP.

PubMed

Garbaccio, Robert M; Tasber, Edward S; Neilson, Lou Anne; Coleman, Paul J; Fraley, Mark E; Olson, Christy; Bergman, Jeff; Torrent, Maricel; Buser, Carolyn A; Rickert, Keith; Walsh, Eileen S; Hamilton, Kelly; Lobell, Robert B; Tao, Weikang; South, Vicki J; Diehl, Ronald E; Davide, Joseph P; Yan, Youwei; Kuo, Lawrence C; Li, Chunze; Prueksaritanont, Thomayant; Fernandez-Metzler, Carmen; Mahan, Elizabeth A; Slaughter, Donald E; Salata, Joseph J; Kohl, Nancy E; Huber, Hans E; Hartman, George D

2007-10-15

Observations from two structurally related series of KSP inhibitors led to the proposal and discovery of dihydropyrazolobenzoxazines that possess ideal properties for cancer drug development. The synthesis and characterization of this class of inhibitors along with relevant pharmacokinetic and in vivo data are presented. The synthesis is highlighted by a key [3+2] cycloaddition to form the pyrazolobenzoxazine core followed by diastereospecific installation of a quaternary center.

Regulation of synthesis and activity of NAD(+)-dependent 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) by dexamethasone and phorbol ester in human erythroleukemia (HEL) cells

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

Xun, C.Q.; Ensor, C.M.; Tai, H.H.

1991-06-28

Dexamethasone stimulated 15-PGDH activity in HEL cells in a time and concentration dependent manner. Increase in 15-PGDH activity by dexamethasone was found to be accompanied by an increase in enzyme synthesis as revealed by Western blot and (35S)methionine labeling studies. In addition to dexamethasone, other anti-inflammatory steroids also increased 15-PGDH activity in the order of their glucocorticoid activity. Among sex steroids only progesterone increased significantly 15-PGDH activity. 12-0-Tetradecanoylphorbol-13-acetate (TPA) also induced the synthesis of 15-PGDH but inhibited the enzyme activity. It appears that TPA caused a time dependent inactivation of 15-PGDH by a protein kinase C mediated mechanism.

Synthesis and Evaluation of Novel RSK Inhibitors in a Living Human Breast Model

DTIC Science & Technology

2015-09-01

the n- propyl carbamate analogue 9, and the sec-butyl carbamate analogue 10. The modified synthetic route could also be applied to the synthesis of...AWARD NUMBER: W81XWH-11-1-0068 TITLE: Synthesis and Evaluation of Novel RSK Inhibitors in a Living Human Breast Model PRINCIPAL INVESTIGATOR...DATES COVERED 01 Jul 2011 – 30 Jun 2015 4. TITLE AND SUBTITLE Synthesis and Evaluation of Novel RSK Inhibitors in a Living Human Breast Model 5a

Generation of reactive oxygen species in the reaction catalyzed by alpha-ketoglutarate dehydrogenase.

PubMed

Tretter, Laszlo; Adam-Vizi, Vera

2004-09-08

Alpha-ketoglutarate dehydrogenase (alpha-KGDH), a key enzyme in the Krebs' cycle, is a crucial early target of oxidative stress (Tretter and Adam-Vizi, 2000). The present study demonstrates that alpha-KGDH is able to generate H(2)O(2) and, thus, could also be a source of reactive oxygen species (ROS) in mitochondria. Isolated alpha-KGDH with coenzyme A (HS-CoA) and thiamine pyrophosphate started to produce H(2)O(2) after addition of alpha-ketoglutarate in the absence of nicotinamide adenine dinucleotide-oxidized (NAD(+)). NAD(+), which proved to be a powerful inhibitor of alpha-KGDH-mediated H(2)O(2) formation, switched the H(2)O(2) forming mode of the enzyme to the catalytic [nicotinamide adenine dinucleotide-reduced (NADH) forming] mode. In contrast, NADH stimulated H(2)O(2) formation by alpha-KGDH, and for this, neither alpha-ketoglutarate nor HS-CoA were required. When all of the substrates and cofactors of the enzyme were present, the NADH/NAD(+) ratio determined the rate of H(2)O(2) production. The higher the NADH/NAD(+) ratio the higher the rate of H(2)O(2) production. H(2)O(2) production as well as the catalytic function of the enzyme was activated by Ca(2+). In synaptosomes, using alpha-ketoglutarate as respiratory substrate, the rate of H(2)O(2) production increased by 2.5-fold, and aconitase activity decreased, indicating that alpha-KGDH can generate H(2)O(2) in in situ mitochondria. Given the NADH/NAD(+) ratio as a key regulator of H(2)O(2) production by alpha-KGDH, it is suggested that production of ROS could be significant not only in the respiratory chain but also in the Krebs' cycle when oxidation of NADH is impaired. Thus alpha-KGDH is not only a target of ROS but could significantly contribute to generation of oxidative stress in the mitochondria.

Towards an integrative model of C4 photosynthetic subtypes: insights from comparative transcriptome analysis of NAD-ME, NADP-ME, and PEP-CK C4 species.

PubMed

Bräutigam, Andrea; Schliesky, Simon; Külahoglu, Canan; Osborne, Colin P; Weber, Andreas P M

2014-07-01

C4 photosynthesis affords higher photosynthetic carbon conversion efficiency than C3 photosynthesis and it therefore represents an attractive target for engineering efforts aiming to improve crop productivity. To this end, blueprints are required that reflect C4 metabolism as closely as possible. Such blueprints have been derived from comparative transcriptome analyses of C3 species with related C4 species belonging to the NAD-malic enzyme (NAD-ME) and NADP-ME subgroups of C4 photosynthesis. However, a comparison between C3 and the phosphoenolpyruvate carboxykinase (PEP-CK) subtype of C4 photosynthesis is still missing. An integrative analysis of all three C4 subtypes has also not been possible to date, since no comparison has been available for closely related C3 and PEP-CK C4 species. To generate the data, the guinea grass Megathyrsus maximus, which represents a PEP-CK species, was analysed in comparison with a closely related C3 sister species, Dichanthelium clandestinum, and with publicly available sets of RNA-Seq data from C4 species belonging to the NAD-ME and NADP-ME subgroups. The data indicate that the core C4 cycle of the PEP-CK grass M. maximus is quite similar to that of NAD-ME species with only a few exceptions, such as the subcellular location of transfer acid production and the degree and pattern of up-regulation of genes encoding C4 enzymes. One additional mitochondrial transporter protein was associated with the core cycle. The broad comparison identified sucrose and starch synthesis, as well as the prevention of leakage of C4 cycle intermediates to other metabolic pathways, as critical components of C4 metabolism. Estimation of intercellular transport fluxes indicated that flux between cells is increased by at least two orders of magnitude in C4 species compared with C3 species. In contrast to NAD-ME and NADP-ME species, the transcription of photosynthetic electron transfer proteins was unchanged in PEP-CK. In summary, the PEP-CK blueprint of M. maximus appears to be simpler than those of NAD-ME and NADP-ME plants. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

beta-1,2,3-Triazolyl-nucleosides as nicotinamide riboside mimics.

PubMed

Amigues, E J; Armstrong, E; Dvorakova, M; Migaud, M E; Huang, M

2009-03-01

The synthesis of a series of pyridine- and piperidine-substituted 1,2,3-triazolides linked to a riboside moiety is described. The presence of a triazolide substituent on the pyridine moiety permitted the facile reduction of the latter under mild hydrogenation conditions. These analogues were modelled as to define their similarity to nicotinamide riboside and quantify their ability to bind NAD-dependent protein deacetylases.

Differential signatures of bacterial and mammalian IMP dehydrogenase enzymes.

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

Zhang, R.; Evans, G.; Rotella, F.

1999-06-01

IMP dehydrogenase (IMPDH) is an essential enzyme of de novo guanine nucleotide synthesis. IMPDH inhibitors have clinical utility as antiviral, anticancer or immunosuppressive agents. The essential nature of this enzyme suggests its therapeutic applications may be extended to the development of antimicrobial agents. Bacterial IMPDH enzymes show bio- chemical and kinetic characteristics that are different than the mammalian IMPDH enzymes, suggesting IMPDH may be an attractive target for the development of antimicrobial agents. We suggest that the biochemical and kinetic differences between bacterial and mammalian enzymes are a consequence of the variance of specific, identifiable amino acid residues. Identification ofmore » these residues or combination of residues that impart this mammalian or bacterial enzyme signature is a prerequisite for the rational identification of agents that specifically target the bacterial enzyme. We used sequence alignments of IMPDH proteins to identify sequence signatures associated with bacterial or eukaryotic IMPDH enzymes. These selections were further refined to discern those likely to have a role in catalysis using information derived from the bacterial and mammalian IMPDH crystal structures and site-specific mutagenesis. Candidate bacterial sequence signatures identified by this process include regions involved in subunit interactions, the active site flap and the NAD binding region. Analysis of sequence alignments in these regions indicates a pattern of catalytic residues conserved in all enzymes and a secondary pattern of amino acid conservation associated with the major phylogenetic groups. Elucidation of the basis for this mammalian/bacterial IMPDH signature will provide insight into the catalytic mechanism of this enzyme and the foundation for the development of highly specific inhibitors.« less

Oxidized LDL activates blood platelets through CD36/NOX2–mediated inhibition of the cGMP/protein kinase G signaling cascade

PubMed Central

Magwenzi, Simbarashe; Woodward, Casey; Wraith, Katie S.; Aburima, Ahmed; Raslan, Zaher; Jones, Huw; McNeil, Catriona; Wheatcroft, Stephen; Yuldasheva, Nadira; Febbriao, Maria; Kearney, Mark

2015-01-01

Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in dyslipidemic disorders. Although oxLDL stimulates activatory signaling, it is unclear how these events drive accelerated thrombosis. Here, we describe a mechanism for oxLDL-mediated platelet hyperactivity that requires generation of reactive oxygen species (ROS). Under arterial flow, oxLDL triggered sustained generation of platelet intracellular ROS, which was blocked by CD36 inhibitors, mimicked by CD36-specific oxidized phospholipids, and ablated in CD36−/− murine platelets. oxLDL-induced ROS generation was blocked by the reduced NAD phosphate oxidase 2 (NOX2) inhibitor, gp91ds-tat, and absent in NOX2−/− mice. The synthesis of ROS by oxLDL/CD36 required Src-family kinases and protein kinase C (PKC)-dependent phosphorylation and activation of NOX2. In functional assays, oxLDL abolished guanosine 3′,5′-cyclic monophosphate (cGMP)-mediated signaling and inhibited platelet aggregation and arrest under flow. This was prevented by either pharmacologic inhibition of NOX2 in human platelets or genetic ablation of NOX2 in murine platelets. Platelets from hyperlipidemic mice were also found to have a diminished sensitivity to cGMP when tested ex vivo, a phenotype that was corrected by infusion of gp91ds-tat into the mice. This study demonstrates that oxLDL and hyperlipidemia stimulate the generation of NOX2-derived ROS through a CD36-PKC pathway and may promote platelet hyperactivity through modulation of cGMP signaling. PMID:25710879

Resveratrol improves left ventricular diastolic relaxation in type 2 diabetes by inhibiting oxidative/nitrative stress: in vivo demonstration with magnetic resonance imaging

PubMed Central

Zhang, Hanrui; Morgan, Brandon; Potter, Barry J.; Ma, Lixin; Dellsperger, Kevin C.; Ungvari, Zoltan

2010-01-01

Resveratrol is a natural phytophenol that exhibits cardioprotective effects. This study was designed to elucidate the mechanisms by which resveratrol protects against diabetes-induced cardiac dysfunction. Normal control (m-Leprdb) mice and type 2 diabetic (Leprdb) mice were treated with resveratrol orally for 4 wk. In vivo MRI showed that resveratrol improved cardiac function by increasing the left ventricular diastolic peak filling rate in Leprdb mice. This protective role is partially explained by resveratrol's effects in improving nitric oxide (NO) production and inhibiting oxidative/nitrative stress in cardiac tissue. Resveratrol increased NO production by enhancing endothelial NO synthase (eNOS) expression and reduced O2·− production by inhibiting NAD(P)H oxidase activity and gp91phox mRNA and protein expression. The increased nitrotyrosine (N-Tyr) protein expression in Leprdb mice was prevented by the inducible NO synthase (iNOS) inhibitor 1400W. Resveratrol reduced both N-Tyr and iNOS expression in Leprdb mice. Furthermore, TNF-α mRNA and protein expression, as well as NF-κB activation, were reduced in resveratrol-treated Leprdb mice. Both Leprdb mice null for TNF-α (dbTNF−/dbTNF− mice) and Leprdb mice treated with the NF-κB inhibitor MG-132 showed decreased NAD(P)H oxidase activity and iNOS expression as well as elevated eNOS expression, whereas m-Leprdb mice treated with TNF-α showed the opposite effects. Thus, resveratrol protects against cardiac dysfunction by inhibiting oxidative/nitrative stress and improving NO availability. This improvement is due to the role of resveratrol in inhibiting TNF-α-induced NF-κB activation, therefore subsequently inhibiting the expression and activation of NAD(P)H oxidase and iNOS as well as increasing eNOS expression in type 2 diabetes. PMID:20675566

5-HT1A and 5-HT7 receptor crosstalk in the regulation of emotional memory: implications for effects of selective serotonin reuptake inhibitors.

PubMed

Eriksson, Therese M; Holst, Sarah; Stan, Tiberiu L; Hager, Torben; Sjögren, Benita; Ogren, Sven Öve; Svenningsson, Per; Stiedl, Oliver

2012-11-01

This study utilized pharmacological manipulations to analyze the role of direct and indirect activation of 5-HT(7) receptors (5-HT(7)Rs) in passive avoidance learning by assessing emotional memory in male C57BL/6J mice. Additionally, 5-HT(7)R binding affinity and 5-HT(7)R-mediated protein phosphorylation of downstream signaling targets were determined. Elevation of 5-HT by the selective serotonin reuptake inhibitor (SSRI) fluoxetine had no effect by itself, but facilitated emotional memory performance when combined with the 5-HT(1A)R antagonist NAD-299. This facilitation was blocked by the selective 5-HT(7)R antagonist SB269970, revealing excitatory effects of the SSRI via 5-HT(7)Rs. The enhanced memory retention by NAD-299 was blocked by SB269970, indicating that reduced activation of 5-HT(1A)Rs results in enhanced 5-HT stimulation of 5-HT(7)Rs. The putative 5-HT(7)R agonists LP-44 when administered systemically and AS19 when administered both systemically and into the dorsal hippocampus failed to facilitate memory. This finding is consistent with the low efficacy of LP-44 and AS19 to stimulate protein phosphorylation of 5-HT(7)R-activated signaling cascades. In contrast, increasing doses of the dual 5-HT(1A)R/5-HT(7)R agonist 8-OH-DPAT impaired memory, while co-administration with NAD-299 facilitated of emotional memory in a dose-dependent manner. This facilitation was blocked by SB269970 indicating 5-HT(7)R activation by 8-OH-DPAT. Dorsohippocampal infusion of 8-OH-DPAT impaired passive avoidance retention through hippocampal 5-HT(1A)R activation, while 5-HT(7)Rs appear to facilitate memory processes in a broader cortico-limbic network and not the hippocampus alone. Copyright © 2012 Elsevier Ltd. All rights reserved.

NAD Kinases: Metabolic Targets Controlling Redox Co-enzymes and Reducing Power Partitioning in Plant Stress and Development

PubMed Central

Li, Bin-Bin; Wang, Xiang; Tai, Li; Ma, Tian-Tian; Shalmani, Abdullah; Liu, Wen-Ting; Li, Wen-Qiang; Chen, Kun-Ming

2018-01-01

NAD(H) and NADP(H) are essential co-enzymes which dominantly control a number of fundamental biological processes by acting as reducing power and maintaining the intracellular redox balance of all life kingdoms. As the only enzymes that catalyze NAD(H) and ATP to synthesize NADP(H), NAD Kinases (NADKs) participate in many essential metabolic reactions, redox sensitive regulation, photosynthetic performance and also reactive oxygen species (ROS) homeostasis of cells and therefore, play crucial roles in both development and stress responses of plants. NADKs are highly conserved enzymes in amino acid sequences but have multiple subcellular localization and diverse functions. They may function as monomers, dimers or multimers in cells but the enzymatic properties in plants are not well elucidated yet. The activity of plant NADK is regulated by calcium/calmodulin and plays crucial roles in photosynthesis and redox co-enzyme control. NADK genes are expressed in almost all tissues and developmental stages of plants with specificity for different members. Their transcripts can be greatly stimulated by a number of environmental factors such as pathogenic attack, irritant applications and abiotic stress treatments. Using transgenic approaches, several studies have shown that NADKs are involved in chlorophyll synthesis, photosynthetic efficiency, oxidative stress protection, hormone metabolism and signaling regulation, and therefore contribute to the growth regulation and stress tolerance of plants. In this review, the enzymatic properties and functional mechanisms of plant NADKs are thoroughly investigated based on literature and databases. The results obtained here are greatly advantageous for further exploration of NADK function in plants. PMID:29662499

A conjugated fatty acid present at high levels in bitter melon seed favorably affects lipid metabolism in hepatocytes by increasing NAD(+)/NADH ratio and activating PPARα, AMPK and SIRT1 signaling pathway.

PubMed

Chen, Gou-Chun; Su, Hui-Min; Lin, Yu-Shun; Tsou, Po-Yen; Chyuan, Jong-Ho; Chao, Pei-Min

2016-07-01

α-Eleostearic acid (α-ESA), or the cis-9, trans-11, trans-13 isomer of conjugated linolenic acid, is a special fatty acid present at high levels in bitter melon seed oil. The aim of this study was to examine the effect of α-ESA on hepatic lipid metabolism. Using H4IIEC3 hepatoma cell line, we showed that α-ESA significantly lowered intracellular triglyceride accumulation compared to α-linolenic acid (LN), used as a fatty acid control, in a dose- and time-dependent manner. The effects of α-ESA on enzyme activities and mRNA profiles in H4IIEC3 cells suggested that enhanced fatty acid oxidation and lowered lipogenesis were involved in α-ESA-mediated triglyceride lowering effects. In addition, α-ESA triggered AMP-activated protein kinase (AMPK) activation without altering sirtuin 1 (SIRT1) protein levels. When cells were treated with vehicle control (VC), LN alone (LN; 100μmol/L) or in combination with α-ESA (LN+α-ESA; 75+25μmol/L) for 24h, acetylation of forkhead box protein O1 was decreased, while the NAD(+)/NADH ratio, mRNA levels of NAMPT and PTGR1 and enzyme activity of nicotinamide phosphoribosyltransferase were increased by LN+α-ESA treatment compared to treatment with LN alone, suggesting that α-ESA activates SIRT1 by increasing NAD(+) synthesis and NAD(P)H consumption. The antisteatosis effect of α-ESA was confirmed in mice treated with a high-sucrose diet supplemented with 1% α-ESA for 5weeks. We conclude that α-ESA favorably affects hepatic lipid metabolism by increasing cellular NAD(+)/NADH ratio and activating PPARα, AMPK and SIRT1 signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Photosystem I from plants as a bacterial cytochrome P450 surrogate electron donor: terminal hydroxylation of branched hydrocarbon chains.

PubMed

Jensen, Kenneth; Johnston, Jonathan B; de Montellano, Paul R Ortiz; Møller, Birger Lindberg

2012-02-01

The ability of cytochrome P450 enzymes to catalyze highly regio- and stereospecific hydroxylations makes them attractive alternatives to approaches based on chemical synthesis but they require expensive cofactors, e.g. NAD(P)H, which limits their commercial potential. Ferredoxin (Fdx) is a multifunctional electron carrier that in plants accepts electrons from photosystem I (PSI) and facilitates photoreduction of NADP(+) to NADPH mediated by ferredoxin-NAD(P)H oxidoreductase (FdR). In bacteria, the electron flow is reversed and Fdx accepts electrons from NADPH via FdR and serves as the direct electron donor to bacterial P450s. By combining the two systems, we demonstrate that irradiation of PSI can drive the activity of a bacterial P450, CYP124 from Mycobacterium tuberculosis. The substitution of the costly cofactor NADPH with sunlight illustrates the potential of the light-driven hydroxylation system for biotechnology applications.

The use of nicotinamide in the prevention of type 1 diabetes.

PubMed

Elliott, R B; Pilcher, C C; Stewart, A; Fergusson, D; McGregor, M A

1993-11-30

Nicotinamide can protect the NOD mouse from diabetes if given early enough and in sufficient dose. The effect partly wanes with time. There is reduced islet inflammation. Similar protective effects can be demonstrated in quasi-experimental interventions in humans--both diabetes related and unrelated deemed at risk of developing diabetes by reason of having islet cell antibodies. Nicotinamide protects isolated islets in vitro from the toxicity of a number of agents, but only in doses that produce significant PARP inhibition, and increased intracellular levels of NAD. It is unlikely that the protective effect demonstrated in humans is due to significant PARP inhibition, as the levels of nicotinamide achieved with the doses used are too low. Other effects of the vitamin are more likely, e.g., increase in NAD pool size by de novo synthesis, or inhibition of free radical generation. The drug appears to be safe in the doses employed in humans.

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

PubMed

Zepeda, S; Monasterio, O; Ureta, T

1990-03-15

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.

Studies of Toxoplasma gondii and Plasmodium falciparum enoyl acyl carrier protein reductase and implications for the development of antiparasitic agents

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

Muench, Stephen P.; Prigge, Sean T.; McLeod, Rima

2007-03-01

The crystal structures of T. gondii and P. falciparum ENR in complex with NAD{sup +} and triclosan and of T. gondii ENR in an apo form have been solved to 2.6, 2.2 and 2.8 Å, respectively. Recent studies have demonstrated that submicromolar concentrations of the biocide triclosan arrest the growth of the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii and inhibit the activity of the apicomplexan enoyl acyl carrier protein reductase (ENR). The crystal structures of T. gondii and P. falciparum ENR in complex with NAD{sup +} and triclosan and of T. gondii ENR in an apo form have beenmore » solved to 2.6, 2.2 and 2.8 Å, respectively. The structures of T. gondii ENR have revealed that, as in its bacterial and plant homologues, a loop region which flanks the active site becomes ordered upon inhibitor binding, resulting in the slow tight binding of triclosan. In addition, the T. gondii ENR–triclosan complex reveals the folding of a hydrophilic insert common to the apicomplexan family that flanks the substrate-binding domain and is disordered in all other reported apicomplexan ENR structures. Structural comparison of the apicomplexan ENR structures with their bacterial and plant counterparts has revealed that although the active sites of the parasite enzymes are broadly similar to those of their bacterial counterparts, there are a number of important differences within the drug-binding pocket that reduce the packing interactions formed with several inhibitors in the apicomplexan ENR enzymes. Together with other significant structural differences, this provides a possible explanation of the lower affinity of the parasite ENR enzyme family for aminopyridine-based inhibitors, suggesting that an effective antiparasitic agent may well be distinct from equivalent antimicrobials.« less

Loss of the RNA polymerase III repressor MAF1 confers obesity resistance

PubMed Central

Bonhoure, Nicolas; Byrnes, Ashlee; Moir, Robyn D.; Hodroj, Wassim; Preitner, Frédéric; Praz, Viviane; Marcelin, Genevieve; Chua, Streamson C.; Martinez-Lopez, Nuria; Singh, Rajat; Moullan, Norman; Auwerx, Johan; Willemin, Gilles; Shah, Hardik; Hartil, Kirsten; Vaitheesvaran, Bhavapriya; Kurland, Irwin

2015-01-01

MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1−/− mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1−/− mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD+, and is associated with obesity resistance. Consistent with this, NAD+ levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences. PMID:25934505

Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

NASA Technical Reports Server (NTRS)

McNally, J. Scott; Davis, Michael E.; Giddens, Don P.; Saha, Aniket; Hwang, Jinah; Dikalov, Sergey; Jo, Hanjoong; Harrison, David G.

2003-01-01

Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

High-Resolution Crystal Structures of Streptococcus pneumoniae Nicotinamidase with Trapped Intermediates Provide Insights into the Catalytic Mechanism and Inhibition by Aldehydes

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

French, Jarrod B.; Cen, Yana; Sauve, Anthony A.

2010-11-11

Nicotinamidases are salvage enzymes that convert nicotinamide to nicotinic acid. These enzymes are essential for the recycling of nicotinamide into NAD{sup +} in most prokaryotes and most single-cell and multicellular eukaryotes, but not in mammals. The significance of these enzymes for nicotinamide salvage and for NAD{sup +} homeostasis has stimulated interest in nicotinamidases as possible antibiotic targets. Nicotinamidases are also regulators of intracellular nicotinamide concentrations, thereby regulating signaling of downstream NAD{sup +}-consuming enzymes, such as the NAD{sup +}-dependent deacetylases (sirtuins). Here, we report several high-resolution crystal structures of the nicotinamidase from Streptococcus pneumoniae (SpNic) in unliganded and ligand-bound forms. Themore » structure of the C136S mutant in complex with nicotinamide provides details about substrate binding, while a trapped nicotinoyl thioester in a complex with SpNic reveals the structure of the proposed thioester reaction intermediate. Examination of the active site of SpNic reveals several important features, including a metal ion that coordinates the substrate and the catalytically relevant water molecule and an oxyanion hole that both orients the substrate and offsets the negative charge that builds up during catalysis. Structures of this enzyme with bound nicotinaldehyde inhibitors elucidate the mechanism of inhibition and provide further details about the catalytic mechanism. In addition, we provide a biochemical analysis of the identity and role of the metal ion that orients the ligand in the active site and activates the water molecule responsible for hydrolysis of the substrate. These data provide structural evidence of several proposed reaction intermediates and allow for a more complete understanding of the catalytic mechanism of this enzyme.« less

An intact eight-membered water chain in drosophilid alcohol dehydrogenases is essential for optimal enzyme activity.

PubMed

Wuxiuer, Yimingjiang; Morgunova, Ekaterina; Cols, Neus; Popov, Alexander; Karshikoff, Andrey; Sylte, Ingebrigt; Gonzàlez-Duarte, Roser; Ladenstein, Rudolf; Winberg, Jan-Olof

2012-08-01

All drosophilid alcohol dehydrogenases contain an eight-member water chain connecting the active site with the solvent at the dimer interface. A similar water chain has also been shown to exist in other short-chain dehydrogenase/reductase (SDR) enzymes, including therapeutically important SDRs. The role of this water chain in the enzymatic reaction is unknown, but it has been proposed to be involved in a proton relay system. In the present study, a connecting link in the water chain was removed by mutating Thr114 to Val114 in Scaptodrosophila lebanonensis alcohol dehydrogenase (SlADH). This threonine is conserved in all drosophilid alcohol dehydrogenases but not in other SDRs. X-ray crystallography of the SlADH(T114V) mutant revealed a broken water chain, the overall 3D structure of the binary enzyme-NAD(+) complex was almost identical to the wild-type enzyme (SlADH(wt) ). As for the SlADH(wt) , steady-state kinetic studies revealed that catalysis by the SlADH(T114V) mutant was consistent with a compulsory ordered reaction mechanism where the co-enzyme binds to the free enzyme. The mutation caused a reduction of the k(on) velocity for NAD(+) and its binding strength to the enzyme, as well as the rate of hydride transfer (k) in the ternary enzyme-NAD(+) -alcohol complex. Furthermore, it increased the pK(a) value of the group in the binary enzyme-NAD(+) complex that regulates the k(on) velocity of alcohol and alcohol-competitive inhibitors. Overall, the results indicate that an intact water chain is essential for optimal enzyme activity and participates in a proton relay system during catalysis. © 2012 The Authors Journal compilation © 2012 FEBS.

Hypercholesterolemia-induced erectile dysfunction: endothelial nitric oxide synthase (eNOS) uncoupling in the mouse penis by NAD(P)H oxidase

PubMed Central

Musicki, Biljana; Liu, Tongyun; Lagoda, Gwen A.; Strong, Travis D.; Sezen, Sena F.; Johnson, Justin M.; Burnett, Arthur L.

2010-01-01

INTRODUCTION Hypercholesterolemia induces erectile dysfunction (ED) mostly by increasing oxidative stress and impairing endothelial function in the penis, but the mechanisms regulating reactive oxygen species (ROS) production in the penis are not understood. AIMS We evaluated whether hypercholesterolemia activates nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase in the penis, providing an initial source of ROS to induce endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction resulting in ED. METHODS Low-density-lipoprotein receptor (LDLR)–null mice were fed Western diet for 4 weeks to induce early-stage hyperlipidemia. Wild type (WT) mice fed regular chow served as controls. Mice received NAD(P)H oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Erectile function was assessed in response to cavernous nerve electrical stimulation. Markers of endothelial function (phospho [P]-vasodilator-stimulated-protein [VASP]-Ser-239), oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NAD[P]H oxidase subunits p67phox, p47phox, and gp91phox), P-eNOS-Ser-1177, and eNOS were measured by Western blot in penes. MAIN OUTCOME MEASURES Molecular mechanisms of ROS generation and endothelial dysfunction in hypercholesterolemia-induced ED. RESULTS Erectile response was significantly (P<0.05) reduced in hypercholesterolemic LDLR-null mice compared to WT mice. Relative to WT mice, hypercholesterolemia increased (P<0.05) protein expressions of NAD(P)H oxidase subunits p67phox, p47phox and gp91phox, eNOS uncoupling, and 4-HNE-modified proteins, and reduced (P<0.05) P-VASP-Ser-239 expression in the penis. Apocynin treatment of LDLR-null mice preserved (P<0.05) maximal intracavernosal pressure, and reversed (P < 0.05) the abnormalities in protein expressions of gp67phox and gp47phox, 4-HNE, P-VASP-Ser-239, and eNOS uncoupling in the penis. Apocynin treatment of WT mice did not affect any of these parameters. Protein expressions of P-eNOS-Ser-1177 and total eNOS were unaffected by hypercholesterolemia. CONCLUSION Activated NAD(P)H oxidase in the penis is an initial source of oxidative stress resulting in eNOS uncoupling, thus providing a mechanism of eNOS uncoupling and endothelial dysfunction in hypercholesterolemia-induced ED. PMID:20626609

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

Oral Treatment of Rodents with Insecticides for Control of Sand Flies (Diptera: Psychodidae) and the Fluorescent Tracer Technique (FTT) as a Tool to Evaluate Potential Sand Fly Control Methods

DTIC Science & Technology

2011-03-01

chitin synthesis inhibitors diflubenzuron and novaluron evaluated in these studies were...effective against sand fly larvae and palatable to hamsters. In contrast to the chitin synthesis inhibitors and juvenile hormone analogs...concentrations (mg/kg) Effects Chitin synthesis inhibitor Diflubenzuron 8.97, 89.7, 897 Mortality at larva-to-pupa molt Novaluron 9.88, 98.8,

Nicotinamide mononucleotide (NMN) supplementation ameliorates the impact of maternal obesity in mice: comparison with exercise.

PubMed

Uddin, Golam Mezbah; Youngson, Neil A; Doyle, Bronte M; Sinclair, David A; Morris, Margaret J

2017-11-08

Maternal overnutrition increases the risk of long-term metabolic dysfunction in offspring. Exercise improves metabolism partly by upregulating mitochondrial biogenesis or function, via increased levels of nicotinamide adenine dinucleotide (NAD + ). We have shown that the NAD + precursor, nicotinamide mononucleotide (NMN) can reverse some of the negative consequences of high fat diet (HFD) consumption. To investigate whether NMN can impact developmentally-set metabolic deficits, we compared treadmill exercise and NMN injection in offspring of obese mothers. Five week old lean and obese female C57BL6/J mice were mated with chow fed males. Female offspring weaned onto HFD were given treadmill exercise for 9 weeks, or NMN injection daily for 18 days. Maternal obesity programmed increased adiposity and liver triglycerides, with decreased glucose tolerance, liver NAD + levels and citrate synthase activity in offspring. Both interventions reduced adiposity, and showed a modest improvement in glucose tolerance and improved markers of mitochondrial function. NMN appeared to have stronger effects on liver fat catabolism (Hadh) and synthesis (Fasn) than exercise. The interventions appeared to exert the most global benefit in mice that were most metabolically challenged (HFD-consuming offspring of obese mothers). This work encourages further study to confirm the suitability of NMN for use in reversing metabolic dysfunction linked to programming by maternal obesity.

Nicotinamide: a vitamin able to shift macrophage differentiation toward macrophages with restricted inflammatory features.

PubMed

Weiss, Ronald; Schilling, Erik; Grahnert, Anja; Kölling, Valeen; Dorow, Juliane; Ceglarek, Uta; Sack, Ulrich; Hauschildt, Sunna

2015-11-01

The differentiation of human monocytes into macrophages is influenced by environmental signals. Here we asked in how far nicotinamide (NAM), a vitamin B3 derivative known to play a major role in nicotinamide adenine dinucleotide (NAD)-mediated signaling events, is able to modulate monocyte differentiation into macrophages developed in the presence of granulocyte macrophage colony-stimulating factor (GM-MØ) or macrophage colony-stimulating factor (M-MØ). We found that GM-MØ undergo biochemical, morphological and functional modifications in response to NAM, whereas M-MØ were hardly affected. GM-MØ exposed to NAM acquired an M-MØ-like structure while the LPS-induced production of pro-inflammatory cytokines and COX-derived eicosanoids were down-regulated. In contrast, NAM had no effect on the production of IL-10 or the cytochrome P450-derived eicosanoids. Administration of NAM enhanced intracellular NAD concentrations; however, it did not prevent the LPS-mediated drain on NAD pools. In search of intracellular molecular targets of NAM known to be involved in LPS-induced cytokine and eicosanoid synthesis, we found NF-κB activity to be diminished. In conclusion, our data show that vitamin B3, when present during the differentiation of monocytes into GM-MØ, interferes with biochemical pathways resulting in strongly reduced pro-inflammatory features. © The Author(s) 2015.

Nicotinamide Riboside and Nicotinic Acid Riboside Salvage in Fungi and Mammals

PubMed Central

Belenky, Peter; Christensen, Kathryn C.; Gazzaniga, Francesca; Pletnev, Alexandre A.; Brenner, Charles

2009-01-01

NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification. PMID:19001417

Restructuring of the dinucleotide-binding fold in an NADP(H) sensor protein

PubMed Central

Zheng, Xiaofeng; Dai, Xueyu; Zhao, Yanmei; Chen, Qiang; Lu, Fei; Yao, Deqiang; Yu, Quan; Liu, Xinping; Zhang, Chuanmao; Gu, Xiaocheng; Luo, Ming

2007-01-01

NAD(P) has long been known as an essential energy-carrying molecule in cells. Recent data, however, indicate that NAD(P) also plays critical signaling roles in regulating cellular functions. The crystal structure of a human protein, HSCARG, with functions previously unknown, has been determined to 2.4-Å resolution. The structure reveals that HSCARG can form an asymmetrical dimer with one subunit occupied by one NADP molecule and the other empty. Restructuring of its NAD(P)-binding Rossmann fold upon NADP binding changes an extended loop to an α-helix to restore the integrity of the Rossmann fold. The previously unobserved restructuring suggests that HSCARG may assume a resting state when the level of NADP(H) is normal within the cell. When the NADP(H) level passes a threshold, an extensive restructuring of HSCARG would result in the activation of its regulatory functions. Immunofluorescent imaging shows that HSCARG redistributes from being associated with intermediate filaments in the resting state to being dispersed in the nucleus and the cytoplasm. The structural change of HSCARG upon NADP(H) binding could be a new regulatory mechanism that responds only to a significant change of NADP(H) levels. One of the functions regulated by HSCARG may be argininosuccinate synthetase that is involved in NO synthesis. PMID:17496144

Design, Synthesis, Biological Evaluation, and X-ray Studies of HIV-1 Protease Inhibitors with Modified P2′ Ligands of Darunavir

PubMed Central

Fyvie, W. Sean; Brindisi, Margherita; Steffey, Melinda; Agniswamy, Johnson; Wang, Yuan-Fang; Aoki, Manabu; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

2018-01-01

The structure-based design, synthesis, and biological evaluation of a series of nonpeptidic HIV-1 protease inhibitors with rationally designed P2′ ligands are described. The inhibitors are designed to enhance backbone binding interactions, particularly at the S2′ subsite. Synthesis of inhibitors was carried out efficiently. The stereochemistry of alcohol functionalities of the P2′ ligands was set by asymmetric reduction of the corresponding ketone using (R,R)- or (S,S)-Noyori catalysts. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 3g and 3h showed enzyme Ki values of 27.9 and 49.7 pM and antiviral activity of 6.2 and 3.9 nM, respectively. These inhibitors also remained quite potent against darunavir-resistant HIV-1 variants. An X-ray structure of inhibitor 3g in complex with HIV-1 protease revealed key interactions in the S2′ subsite. PMID:29110408

A complex effect of arsenite on the formation of alpha-ketoglutarate in rat liver mitochondria.

PubMed

Lenartowicz, E

1990-12-01

This investigation presents disturbances of the mitochondrial metabolism by arsenite, a hydrophilic dithiol reagent known as an inhibitor of mitochondrial alpha-keto acid dehydrogenases. Arsenite at concentrations of 0.1-1.0 mM was shown to induce a considerable oxidation of intramitochondrial NADPH, NADH, and glutathione without decreasing the mitochondrial membrane potential. The oxidation of NAD(P)H required the presence of phosphate and was sensitive to ruthenium red, but occurred without the addition of calcium salts. Mitochondrial reactions producing alpha-ketoglutarate from glutamate and isocitrate were modulated by arsenite through various mechanisms: (i) both glutamate transaminations, with oxaloacetate and with pyruvate, were inhibited by accumulating alpha-ketoglutarate; however, at low concentrations of alpha-ketoglutarate the aspartate aminotransferase reaction was stimulated due to the increase of NAD+ content; (ii) the oxidation of isocitrate was stimulated at its low concentration only, due to the oxidation of NADPH and NADH; this oxidation was prevented by concentrations of citrate or isocitrate greater than 1 mM; (iii) the conversion of isocitrate to citrate was suppressed, presumably as a result of the decrease of Mg2+ concentration in mitochondria. Thus the depletion of mitochondrial vicinal thiol groups in hydrophilic domains disturbs the mitochondrial metabolism not only by the inhibition of alpha-keto acid dehydrogenases but also by the oxidation of NAD(P)H and, possibly, by the change in the ion concentrations.

Global Kinetic Analysis of Mammalian E3 Reveals pH-dependent NAD+/NADH Regulation, Physiological Kinetic Reversibility, and Catalytic Optimum*

PubMed Central

Moxley, Michael A.; Beard, Daniel A.; Bazil, Jason N.

2016-01-01

Mammalian E3 is an essential mitochondrial enzyme responsible for catalyzing the terminal reaction in the oxidative catabolism of several metabolites. E3 is a key regulator of metabolic fuel selection as a component of the pyruvate dehydrogenase complex (PDHc). E3 regulates PDHc activity by altering the affinity of pyruvate dehydrogenase kinase, an inhibitor of the enzyme complex, through changes in reduction and acetylation state of lipoamide moieties set by the NAD+/NADH ratio. Thus, an accurate kinetic model of E3 is needed to predict overall mammalian PDHc activity. Here, we have combined numerous literature data sets and new equilibrium spectroscopic experiments with a multitude of independently collected forward and reverse steady-state kinetic assays using pig heart E3. The latter kinetic assays demonstrate a pH-dependent transition of NAD+ activation to inhibition, shown here, to our knowledge, for the first time in a single consistent data set. Experimental data were analyzed to yield a thermodynamically constrained four-redox-state model of E3 that simulates pH-dependent activation/inhibition and active site redox states for various conditions. The developed model was used to determine substrate/product conditions that give maximal E3 rates and show that, due to non-Michaelis-Menten behavior, the maximal flux is different compared with the classically defined kcat. PMID:26644471

The requirement for bivalent cations in formation of nicotinamide–adenine dinucleotide by nicotinamide mononucleotide adenylyltransferase of pig-liver nuclei

PubMed Central

Jackson, J. F.; Atkinson, M. R.

1966-01-01

1. The requirement for bivalent cations in catalysis of NAD formation from ATP and NMN in the presence of NMN adenylyltransferase of pig-liver nuclei was studied. Rates of NAD formation in the presence of the activating cations Cd2+, Mn2+, Mg2+, Zn2+, Co2+ and Ni2+ were approximately a linear function of heats of hydration of the corresponding ions. Ba2+, Sr2+, Ca2+, Cu2+ and Be2+ did not activate the enzyme; Be2+ inhibited the reaction in the presence of Mg2+ and, to a greater extent, in the presence of Ni2+. 2. Michaelis constants for NAD formation, measured in a coupled assay with NMN adenylyltransferase and alcohol dehydrogenase at pH8·0 and 25°, in the presence of 3mm concentrations of the unvaried reactants, were 88±7μm-ATP, 42±4μm-NMN and 85±4μm-Mg2+. The results at this pH and at pH7·5 were consistent with mechanisms in which Mg2+–ATP complex is a reactant and free ATP a competitive inhibitor. 3. Formation of nicotinamide–hypoxanthine dinucleotide from NMN and ITP in the presence of the transferase was also more rapid with Ni2+ and Co2+ than with Mg2+. PMID:4291356

Monitoring Mitochondrial Electron Fluxes Using NAD(P)H-Flavoprotein Fluorometry Reveals Complex Action of Isoflurane on Cardiomyocytes

PubMed Central

Sedlic, Filip; Pravdic, Danijel; Hirata, Naoyuki; Mio, Yasushi; Sepac, Ana; Camara, Amadou K.; Wakatsuki, Tetsuro; Bosnjak, Zeljko J.; Bienengraeber, Martin

2010-01-01

Mitochondrial bioenergetic studies mostly rely on isolated mitochondria thus excluding the regulatory role of other cellular compartments important for the overall mitochondrial function. In intact cardiomyocytes, we followed the dynamics of electron fluxes along specific sites of the electron transport chain (ETC) by simultaneous detection of NAD(H)P and flavoprotein (FP) fluorescence intensities using a laser-scanning confocal microscope. This method was used to delineate the effects of isoflurane, a volatile anesthetic and cardioprotective agent, on the ETC. Comparison to the effects of well-characterized ETC inhibitors and uncoupling agent revealed two distinct effects of isoflurane: uncoupling-induced mitochondrial depolarization and inhibition of ETC at the level of complex I. In correlation, oxygen consumption measurements in cardiomyocytes confirmed a dose-dependent, dual effect of isoflurane, and in isolated mitochondria an obstruction of the ETC primarily at the level of complex I. These effects are likely responsible for the reported mild stimulation of mitochondrial reactive oxygen species (ROS) production required for the cardioprotective effects of isoflurane. In conclusion, isoflurane exhibits complex effects on the ETC in intact cardiomyocytes, altering its electron fluxes, and thereby enhancing ROS production. The NAD(P)H-FP fluorometry is a useful method for exploring the effect of drugs on mitochondria and identifying their specific sites of action within the ETC of intact cardiomyocytes. PMID:20646994

Separation of cordycepin from Cordyceps militaris fermentation supernatant using preparative HPLC and evaluation of its antibacterial activity as an NAD+-dependent DNA ligase inhibitor

PubMed Central

Zhou, Xiaofeng; Cai, Guoqiang; He, Yi; Tong, Guotong

2016-01-01

Cordycepin exhibits various bio-activities, including anticancer, antibacterial, antiviral and immune regulation activities, and is a significant focus of research. However, the preparation of high-purity cordycepin remains challenging. Also, the molecular target with which cordycepin interacts to cause an antibacterial effect remains unknown. In the present study, cordycepin was prepared by preparative high-performance liquid chromatography (prep-HPLC) and the purity obtained was 99.6%, indicating that this technique may be useful for the large-scale isolation of cordycepin in the future. The results of computational molecular docking analysis indicated that the interaction energy between cordycepin and NAD+-dependent DNA ligase (LigA) was lower than that between cordycepin and other common antibacterial targets. The highly pure cordycepin obtained by prep-HPLC demonstrated inhibitory activity against LigA from various bacteria in vitro. In conclusion, cordycepin may be useful as a broad-spectrum antibiotic targeting LigA in various bacteria. PMID:27588098

The multifaceted influence of histone deacetylases on DNA damage signalling and DNA repair

PubMed Central

Roos, Wynand Paul; Krumm, Andrea

2016-01-01

Histone/protein deacetylases play multiple roles in regulating gene expression and protein activation and stability. Their deregulation during cancer initiation and progression cause resistance to therapy. Here, we review the role of histone deacetylases (HDACs) and the NAD+ dependent sirtuins (SIRTs) in the DNA damage response (DDR). These lysine deacetylases contribute to DNA repair by base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), homologous recombination (HR) and interstrand crosslink (ICL) repair. Furthermore, we discuss possible mechanisms whereby these histone/protein deacetylases facilitate the switch between DNA double-strand break (DSB) repair pathways, how SIRTs play a central role in the crosstalk between DNA repair and cell death pathways due to their dependence on NAD+, and the influence of small molecule HDAC inhibitors (HDACi) on cancer cell resistance to genotoxin based therapies. Throughout the review, we endeavor to identify the specific HDAC targeted by HDACi leading to therapy sensitization. PMID:27738139

Erasers of Histone Acetylation: The Histone Deacetylase Enzymes

PubMed Central

Seto, Edward; Yoshida, Minoru

2014-01-01

Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl functional groups from the lysine residues of both histone and nonhistone proteins. In humans, there are 18 HDAC enzymes that use either zinc- or NAD+-dependent mechanisms to deacetylate acetyl lysine substrates. Although removal of histone acetyl epigenetic modification by HDACs regulates chromatin structure and transcription, deacetylation of nonhistones controls diverse cellular processes. HDAC inhibitors are already known potential anticancer agents and show promise for the treatment of many diseases. PMID:24691964

SCD1 inhibition causes cancer cell death by depleting mono-unsaturated fatty acids.

PubMed

Mason, Paul; Liang, Beirong; Li, Lingyun; Fremgen, Trisha; Murphy, Erin; Quinn, Angela; Madden, Stephen L; Biemann, Hans-Peter; Wang, Bing; Cohen, Aharon; Komarnitsky, Svetlana; Jancsics, Kate; Hirth, Brad; Cooper, Christopher G F; Lee, Edward; Wilson, Sean; Krumbholz, Roy; Schmid, Steven; Xiang, Yibin; Booker, Michael; Lillie, James; Carter, Kara

2012-01-01

Increased metabolism is a requirement for tumor cell proliferation. To understand the dependence of tumor cells on fatty acid metabolism, we evaluated various nodes of the fatty acid synthesis pathway. Using RNAi we have demonstrated that depletion of fatty-acid synthesis pathway enzymes SCD1, FASN, or ACC1 in HCT116 colon cancer cells results in cytotoxicity that is reversible by addition of exogenous fatty acids. This conditional phenotype is most pronounced when SCD1 is depleted. We used this fatty-acid rescue strategy to characterize several small-molecule inhibitors of fatty acid synthesis, including identification of TOFA as a potent SCD1 inhibitor, representing a previously undescribed activity for this compound. Reference FASN and ACC inhibitors show cytotoxicity that is less pronounced than that of TOFA, and fatty-acid rescue profiles consistent with their proposed enzyme targets. Two reference SCD1 inhibitors show low-nanomolar cytotoxicity that is offset by at least two orders of magnitude by exogenous oleate. One of these inhibitors slows growth of HCT116 xenograft tumors. Our data outline an effective strategy for interrogation of on-mechanism potency and pathway-node-specificity of fatty acid synthesis inhibitors, establish an unambiguous link between fatty acid synthesis and cancer cell survival, and point toward SCD1 as a key target in this pathway.

Visible light-driven NADH regeneration sensitized by proflavine for biocatalysis.

PubMed

Nam, Dong Heon; Park, Chan Beum

2012-06-18

Harvest time: Proflavine drives the reduction of NAD(+) in the presence of a Rh-based electron mediator. Photoregenerated NADH was enzymatically active for oxidation by NADH-dependent L-glutamate dehydrogenase for the synthesis of L-glutamate. This work suggests that proflavine has the potential to become an efficient light-harvesting component in biocatalytic photosynthesis driven by solar energy. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phytochelatin synthesis and glutathione levels in response to heavy metals in tomato cells.

PubMed

Scheller, H V; Huang, B; Hatch, E; Goldsbrough, P B

1987-12-01

Cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, produce phytochelatins (poly[gamma-glutamylcysteinyl]glycines) when exposed to cadmium. The synthesis of these peptides is accompanied by a decline in cellular levels of glutathione. Buthionine sulfoximine, an inhibitor of glutathione synthesis, inhibits the sustained production of phytochelatins. However, phytochelatin synthesis can occur in the presence of buthionine sulfoximine provided that sufficient glutathione is available. These results indicate that glutathione is a substrate for phytochelatin synthesis. The protein synthesis inhibitor cycloheximide does not affect the initial production of phytochelatin.

A modified approach to 2-(N-aryl)-1,3-oxazoles: application to the synthesis of the IMPDH inhibitor BMS-337197 and analogues.

PubMed

Dhar, T G Murali; Guo, Junqing; Shen, Zhongqi; Pitts, William J; Gu, Henry H; Chen, Bang-Chi; Zhao, Rulin; Bednarz, Mark S; Iwanowicz, Edwin J

2002-06-13

[structure: see text] A modified approach to the synthesis of 2-(N-aryl)-1,3-oxazoles, employing an optimized iminophosphorane/heterocumulene-mediated methodology, and its application to the synthesis of BMS-337197, a potent inhibitor of IMPDH, are described.

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.

Talc

MedlinePlus

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In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

PubMed

Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

2016-05-01

Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

Rapid synthesis of triazine inhibitors of inosine monophosphate dehydrogenase.

PubMed

Pitts, William J; Guo, Junqing; Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Watterson, Scott H; Bednarz, Mark S; Chen, Bang Chi; Barrish, Joel C; Bassolino, Donna; Cheney, Daniel; Fleener, Catherine A; Rouleau, Katherine A; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2002-08-19

A series of novel triazine-based small molecule inhibitors (IV) of inosine monophosphate dehydrogenase was prepared. The synthesis and the structure-activity relationships (SAR) derived from in vitro studies are described.

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.

Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways.

PubMed

Martínez, Irene; Zhu, Jiangfeng; Lin, Henry; Bennett, George N; San, Ka-Yiu

2008-11-01

Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.

Predicted Role of NAD Utilization in the Control of Circadian Rhythms during DNA Damage Response

PubMed Central

Luna, Augustin; McFadden, Geoffrey B.; Aladjem, Mirit I.; Kohn, Kurt W.

2015-01-01

The circadian clock is a set of regulatory steps that oscillate with a period of approximately 24 hours influencing many biological processes. These oscillations are robust to external stresses, and in the case of genotoxic stress (i.e. DNA damage), the circadian clock responds through phase shifting with primarily phase advancements. The effect of DNA damage on the circadian clock and the mechanism through which this effect operates remains to be thoroughly investigated. Here we build an in silico model to examine damage-induced circadian phase shifts by investigating a possible mechanism linking circadian rhythms to metabolism. The proposed model involves two DNA damage response proteins, SIRT1 and PARP1, that are each consumers of nicotinamide adenine dinucleotide (NAD), a metabolite involved in oxidation-reduction reactions and in ATP synthesis. This model builds on two key findings: 1) that SIRT1 (a protein deacetylase) is involved in both the positive (i.e. transcriptional activation) and negative (i.e. transcriptional repression) arms of the circadian regulation and 2) that PARP1 is a major consumer of NAD during the DNA damage response. In our simulations, we observe that increased PARP1 activity may be able to trigger SIRT1-induced circadian phase advancements by decreasing SIRT1 activity through competition for NAD supplies. We show how this competitive inhibition may operate through protein acetylation in conjunction with phosphorylation, consistent with reported observations. These findings suggest a possible mechanism through which multiple perturbations, each dominant during different points of the circadian cycle, may result in the phase advancement of the circadian clock seen during DNA damage. PMID:26020938

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

Development of Pyrazolone and Isoxazol-5-one Cambinol Analogues as Sirtuin Inhibitors

PubMed Central

2015-01-01

Sirtuins are a family of NAD+-dependent protein deacetylases that play critical roles in epigenetic regulation, stress responses, and cellular aging in eukaryotic cells. In an effort to identify small molecule inhibitors of sirtuins for potential use as chemotherapeutics as well as tools to modulate sirtuin activity, we previously identified a nonselective sirtuin inhibitor called cambinol (IC50 ≈ 50 μM for SIRT1 and SIRT2) with in vitro and in vivo antilymphoma activity. In the current study, we used saturation transfer difference (STD) NMR experiments with recombinant SIRT1 and 20 to map parts of the inhibitor that interacted with the protein. Our ongoing efforts to optimize cambinol analogues for potency and selectivity have resulted in the identification of isoform selective analogues: 17 with >7.8-fold selectivity for SIRT1, 24 with >15.4-fold selectivity for SIRT2, and 8 with 6.8- and 5.3-fold selectivity for SIRT3 versus SIRT1 and SIRT2, respectively. In vitro cytotoxicity studies with these compounds as well as EX527, a potent and selective SIRT1 inhibitor, suggest that antilymphoma activity of this compound class may be predominantly due to SIRT2 inhibition. PMID:24697269

Discovery of Novel Inhibitors and Fluorescent Probe Targeting NAMPT.

PubMed

Wang, Xia; Xu, Tian-Ying; Liu, Xin-Zhu; Zhang, Sai-Long; Wang, Pei; Li, Zhi-Yong; Guan, Yun-Feng; Wang, Shu-Na; Dong, Guo-Qiang; Zhuo, Shu; Le, Ying-Ying; Sheng, Chun-Quan; Miao, Chao-Yu

2015-07-31

Nicotinamide phosphoribosyltransferase (NAMPT) is a promising antitumor target. Novel NAMPT inhibitors with diverse chemotypes are highly desirable for development of antitumor agents. Using high throughput screening system targeting NAMPT on a chemical library of 30000 small-molecules, we found a non-fluorescent compound F671-0003 and a fluorescent compound M049-0244 with excellent in vitro activity (IC50: 85 nM and 170 nM respectively) and anti-proliferative activity against HepG2 cells. These two compounds significantly depleted cellular NAD levels. Exogenous NMN rescued their anti-proliferative activity against HepG2 cells. Structure-activity relationship study proposed a binding mode for NAMPT inhibitor F671-0003 and highlighted the importance of hydrogen bonding, hydrophobic and π-π interactions in inhibitor binding. Imaging study provided the evidence that fluorescent compound M049-0244 (3 μM) significantly stained living HepG2 cells. Cellular fluorescence was further verified to be NAMPT dependent by using RNA interference and NAMPT over expression transgenic mice. Our findings provide novel antitumor lead compounds and a "first-in-class" fluorescent probe for imaging NAMPT.

Lactate oxidation coupled to energy production in mitochondria like particles from Setaria digitata, a filarial parasite.

PubMed

Sivan, V M; Raj, R K

1994-10-14

In the filarial parasite, Setaria digitata, the mitochondria like particles (MLP) show NAD reduction with sodium lactate. The MLP also reduces dye and ferricyanide with lactate. The ferricyanide reduction by lactate is found to be sensitive to the cytochrome o inhibitor orthohydroxy diphenyl (OHD) and complex I inhibitor rotenone, modulated by ADP (+) and ATP (-) and inhibited by pyruvate and oxaloacetate. MLP shows lactate oxidation sensitive to OHD, rotenone and sodium malonate. Thus, the lactate utilizing complex system, consisting of an NADH generating MLP bound lactate dehydrogenase and a lactate flavocytochrome reductase tightly linked to complex I and cytochrome o, produces ATP in functional association with fumarate reductase complex and other enzyme systems. Hence, this study provides new dimensions to the study of metabolism in filarial parasites.

Comparative Proteomic Analysis of Tolerance and Adaptation of Ethanologenic Saccharomyces cerevisiae to Furfural, a Lignocellulosic Inhibitory Compound▿ †

PubMed Central

Lin, Feng-Ming; Qiao, Bin; Yuan, Ying-Jin

2009-01-01

The molecular mechanism involved in tolerance and adaptation of ethanologenic Saccharomyces cerevisiae to inhibitors (such as furfural, acetic acid, and phenol) represented in lignocellulosic hydrolysate is still unclear. Here, 18O-labeling-aided shotgun comparative proteome analysis was applied to study the global protein expression profiles of S. cerevisiae under conditions of treatment of furfural compared with furfural-free fermentation profiles. Proteins involved in glucose fermentation and/or the tricarboxylic acid cycle were upregulated in cells treated with furfural compared with the control cells, while proteins involved in glycerol biosynthesis were downregulated. Differential levels of expression of alcohol dehydrogenases were observed. On the other hand, the levels of NADH, NAD+, and NADH/NAD+ were reduced whereas the levels of ATP and ADP were increased. These observations indicate that central carbon metabolism, levels of alcohol dehydrogenases, and the redox balance may be related to tolerance of ethanologenic yeast for and adaptation to furfural. Furthermore, proteins involved in stress response, including the unfolded protein response, oxidative stress, osmotic and salt stress, DNA damage and nutrient starvation, were differentially expressed, a finding that was validated by quantitative real-time reverse transcription-PCR to further confirm that the general stress responses are essential for cellular defense against furfural. These insights into the response of yeast to the presence of furfural will benefit the design and development of inhibitor-tolerant ethanologenic yeast by metabolic engineering or synthetic biology. PMID:19363068

Certhrax Toxin, an Anthrax-related ADP-ribosyltransferase from Bacillus cereus*

PubMed Central

Visschedyk, Danielle; Rochon, Amanda; Tempel, Wolfram; Dimov, Svetoslav; Park, Hee-Won; Merrill, A. Rod

2012-01-01

We identified Certhrax, the first anthrax-like mART toxin from the pathogenic G9241 strain of Bacillus cereus. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we have shown that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. Like anthrax lethal factor, Certhrax was found to require protective antigen for host cell entry. This two-domain enzyme was shown to be 60-fold more toxic to mammalian cells than anthrax lethal factor. Certhrax localizes to distinct regions within mouse RAW264.7 cells by 10 min postinfection and is extranuclear in its cellular location. Substitution of catalytic residues shows that the mART function is responsible for the toxicity, and it binds NAD+ with high affinity (KD = 52.3 ± 12.2 μm). We report the 2.2 Å Certhrax structure, highlighting its structural similarities and differences with anthrax lethal factor. We also determined the crystal structures of two good inhibitors (P6 (KD = 1.7 ± 0.2 μm, Ki = 1.8 ± 0.4 μm) and PJ34 (KD = 5.8 ± 2.6 μm, Ki = 9.6 ± 0.3 μm)) in complex with Certhrax. As with other toxins in this family, the phosphate-nicotinamide loop moves toward the NAD+ binding site with bound inhibitor. These results indicate that Certhrax may be important in the pathogenesis of B. cereus. PMID:22992735

Initial-rate kinetics of human NMN-adenylyltransferases: substrate and metal ion specificity, inhibition by products and multisubstrate analogues, and isozyme contributions to NAD+ biosynthesis.

PubMed

Sorci, Leonardo; Cimadamore, Flavio; Scotti, Stefania; Petrelli, Riccardo; Cappellacci, Loredana; Franchetti, Palmarisa; Orsomando, Giuseppe; Magni, Giulio

2007-04-24

Initial-rate and product inhibition studies revealed distinctive ordered ternary complex kinetic mechanisms, substrate specificities, and metal ion preferences for the three isozymes of human nicotinamide mononucleotide adenylyl-transferase (NMNAT, EC 2.7.7.1). ATP binds before NMN with nuclear isozyme NMNAT1 and Golgi apparatus NMNAT2, but the opposite order is observed with the mitochondrial isozyme NMNAT3. Only the latter utilizes ITP efficiently in place of ATP, and while NMNH conversion to NADH by NMNAT1 and NMNAT3 occurs at similar rates, conversion by NMNAT2 is much slower. These isozymes can also be discriminated by their action on tiazofurin monophosphate (TrMP), a metabolite of the antineoplastic prodrug tiazofurin. Our finding that TrMP is only a substrate with NMNAT1 and NMNAT3 reveals for the first time an organelle selectivity in the metabolism of this important drug. In search of additional ways to discriminate these isozymes, we synthesized and tested the P1-(nicotinamide/nicotinate-riboside-5')-Pn-(adenosine-5') dinucleotides Np3AD, Np4AD, and Nap4AD. In addition to being highly effective inhibitors, these multisubstrate geometric inhibitors gave inhibition patterns that are consistent with the aforementioned isozyme differences in substrate binding order. Distinctive differences in their substrate specificity and metal ion selectivity also permitted us to quantify individual isozyme contributions to NAD+ formation in human cell extracts.

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

SCD1 Inhibition Causes Cancer Cell Death by Depleting Mono-Unsaturated Fatty Acids

PubMed Central

Mason, Paul; Liang, Beirong; Li, Lingyun; Fremgen, Trisha; Murphy, Erin; Quinn, Angela; Madden, Stephen L.; Biemann, Hans-Peter; Wang, Bing; Cohen, Aharon; Komarnitsky, Svetlana; Jancsics, Kate; Hirth, Brad; Cooper, Christopher G. F.; Lee, Edward; Wilson, Sean; Krumbholz, Roy; Schmid, Steven; Xiang, Yibin; Booker, Michael; Lillie, James; Carter, Kara

2012-01-01

Increased metabolism is a requirement for tumor cell proliferation. To understand the dependence of tumor cells on fatty acid metabolism, we evaluated various nodes of the fatty acid synthesis pathway. Using RNAi we have demonstrated that depletion of fatty-acid synthesis pathway enzymes SCD1, FASN, or ACC1 in HCT116 colon cancer cells results in cytotoxicity that is reversible by addition of exogenous fatty acids. This conditional phenotype is most pronounced when SCD1 is depleted. We used this fatty-acid rescue strategy to characterize several small-molecule inhibitors of fatty acid synthesis, including identification of TOFA as a potent SCD1 inhibitor, representing a previously undescribed activity for this compound. Reference FASN and ACC inhibitors show cytotoxicity that is less pronounced than that of TOFA, and fatty-acid rescue profiles consistent with their proposed enzyme targets. Two reference SCD1 inhibitors show low-nanomolar cytotoxicity that is offset by at least two orders of magnitude by exogenous oleate. One of these inhibitors slows growth of HCT116 xenograft tumors. Our data outline an effective strategy for interrogation of on-mechanism potency and pathway-node-specificity of fatty acid synthesis inhibitors, establish an unambiguous link between fatty acid synthesis and cancer cell survival, and point toward SCD1 as a key target in this pathway. PMID:22457791

Synthesis and optimization of thiadiazole derivatives as a novel class of substrate competitive c-Jun N-terminal kinase inhibitors

PubMed Central

De, Surya K.; Chen, Vida; Stebbins, John L.; Chen, Li-Hsing; Cellitti, Jason F.; Machleidt, Thomas; Barile, Elisa; Riel-Mehan, Megan; Dahl, Russell; Yang, Li; Emdadi, Aras; Murphy, Ria; Pellecchia, Maurizio

2009-01-01

A series of thiadiazole derivatives has been designed as potential allosteric, substrate competitive inhibitors of the protein kinase JNK. We report on the synthesis, characterization and evaluation of a series of compounds that resulted in the identification of potent and selective JNK inhibitors targeting its JIP-1 docking site. PMID:20045647

Loss of the RNA polymerase III repressor MAF1 confers obesity resistance.

PubMed

Bonhoure, Nicolas; Byrnes, Ashlee; Moir, Robyn D; Hodroj, Wassim; Preitner, Frédéric; Praz, Viviane; Marcelin, Genevieve; Chua, Streamson C; Martinez-Lopez, Nuria; Singh, Rajat; Moullan, Norman; Auwerx, Johan; Willemin, Gilles; Shah, Hardik; Hartil, Kirsten; Vaitheesvaran, Bhavapriya; Kurland, Irwin; Hernandez, Nouria; Willis, Ian M

2015-05-01

MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences. © 2015 Bonhoure et al.; Published by Cold Spring Harbor Laboratory Press.

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

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

Kolodkin-Gal, I; Elsholz, AKW; Muth, C

2013-04-29

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa(3) and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showedmore » that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD(+))/NADH ratio via binding of NAD(+) to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.« less

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

PubMed Central

Kolodkin-Gal, Ilana; Elsholz, Alexander K.W.; Muth, Christine; Girguis, Peter R.; Kolter, Roberto; Losick, Richard

2013-01-01

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio via binding of NAD+ to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration. PMID:23599347

Bacterial fatty acid metabolism in modern antibiotic discovery.

PubMed

Yao, Jiangwei; Rock, Charles O

2017-11-01

Bacterial fatty acid synthesis is essential for many pathogens and different from the mammalian counterpart. These features make bacterial fatty acid synthesis a desirable target for antibiotic discovery. The structural divergence of the conserved enzymes and the presence of different isozymes catalyzing the same reactions in the pathway make bacterial fatty acid synthesis a narrow spectrum target rather than the traditional broad spectrum target. Furthermore, bacterial fatty acid synthesis inhibitors are single-targeting, rather than multi-targeting like traditional monotherapeutic, broad-spectrum antibiotics. The single-targeting nature of bacterial fatty acid synthesis inhibitors makes overcoming fast-developing, target-based resistance a necessary consideration for antibiotic development. Target-based resistance can be overcome through multi-targeting inhibitors, a cocktail of single-targeting inhibitors, or by making the single targeting inhibitor sufficiently high affinity through a pathogen selective approach such that target-based mutants are still susceptible to therapeutic concentrations of drug. Many of the pathogens requiring new antibiotic treatment options encode for essential bacterial fatty acid synthesis enzymes. This review will evaluate the most promising targets in bacterial fatty acid metabolism for antibiotic therapeutics development and review the potential and challenges in advancing each of these targets to the clinic and circumventing target-based resistance. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016 Elsevier B.V. All rights reserved.

The roles of the cyclo-oxygenases types one and two in prostaglandin synthesis in human fetal membranes at term.

PubMed

Sawdy, R J; Slater, D M; Dennes, W J; Sullivan, M H; Bennett, P R

2000-01-01

The aim of this study was to determine the relative contributions of cyclo-oxygenase (COX) types 1 and 2 to prostaglandin synthesis at term. Fetal membranes were collected from 6 pregnancies after elective caesarean section at term, prior to labour. The presence of COX-1 and COX-2 protein was determined using Western analysis. The relative contributions of the two isoforms of COX to prostaglandin synthesis were determined by incubation of fetal membrane discs with either a COX-2 selective inhibitor, SC236, or a COX-1 selective inhibitor, SC560, and measurement of prostaglandin release during 24 h using enzyme-linked immuno-sorbent assay (ELISA). Both COX-1 and COX-2 protein were demonstrated in amnion and chorion-decidua. The COX-2 selective inhibitor, SC-236, significantly reduced prostaglandin synthesis, both in its COX-2 specific and higher, non-specific concentration ranges. The COX-1 selective inhibitor, SC-560, had no effect upon prostaglandin synthesis in its COX-1 specific concentration range, but did significantly reduce prostaglandin synthesis at higher, non-selective concentrations. Fetal membranes contain both COX-1 and COX-2 at term, but only COX-2 contributes towards prostaglandin synthesis. COX-2 selective NSAI drugs will be as effective as non-selective agents in inhibition of fetal membrane prostaglandin synthesis and may represent a new strategy for tocolysis. Copyright 2000 Harcourt Publishers Ltd.

Role of Sirt1 during the ageing process: relevance to protection of synapses in the brain.

PubMed

Godoy, Juan A; Zolezzi, Juan M; Braidy, Nady; Inestrosa, Nibaldo C

2014-12-01

Ageing is a stochastic process associated with a progressive decline in physiological functions which predispose to the pathogenesis of several neurodegenerative diseases. The intrinsic complexity of ageing remains a significant challenge to understand the cause of this natural phenomenon. At the molecular level, ageing is thought to be characterized by the accumulation of chronic oxidative damage to lipids, proteins and nucleic acids caused by free radicals. Increased oxidative stress and misfolded protein formations, combined with impaired compensatory mechanisms, may promote neurodegenerative disorders with age. Nutritional modulation through calorie restriction has been shown to be effective as an anti-ageing factor, promoting longevity and protecting against neurodegenerative pathology in yeast, nematodes and murine models. Calorie restriction increases the intracellular levels of the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD(+)), a co-substrate for the sirtuin 1 (Sirt1, silent mating-type information regulator 2 homolog 1) activity and a cofactor for oxidative phosphorylation and ATP synthesis. Promotion of intracellular NAD(+) anabolism is speculated to induce neuroprotective effects against amyloid-β-peptide (Aβ) toxicity in some models for Alzheimer's disease (AD). The NAD(+)-dependent histone deacetylase, Sirt1, has been implicated in the ageing process. Sirt1 serves as a deacetylase for numerous proteins involved in several cellular pathways, including stress response and apoptosis, and plays a protective role in neurodegenerative disorders, such as AD.

Nuclear CD38 in retinoic acid-induced HL-60 cells

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

Yalcintepe, Leman; Albeniz, Isil; Adin-Cinar, Suzan

2005-02-01

The cell surface antigen, CD38, is a 45-kDa transmembrane protein which is predominantly expressed on hematopoietic cells during differentiation. As a bifunctional ectoenzyme, it catalyzes the synthesis of cyclic ADP-ribose (cADPR) from NAD{sup +} and hydrolysis of either NAD{sup +} or cADPR to ADP-ribose. All-trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that the nuclei of RA-treated human HL-60 myeloblastic cells reveal enzymatic activities inherent to CD38. Thus, GDP-ribosyl cyclase and NAD{sup +} glycohydrolase activities in the nuclear fraction increased very significantly in response to incubation with RA. Withmore » Western blotting, we detected in the nuclear protein fraction from RA-treated cells a {approx}43-kDa protein band which was reactive with the CD38-specific monoclonal antibody OKT10. The expression of CD38 in HL-60 nuclei was also shown with FACScan analysis. RA treatment gave rise to an increase in in vitro ADP ribosylation of the {approx}43-kDa nuclear protein. Moreover, nuclei isolated from RA-treated HL-60 cells revealed calcium release in response to cADPR, whereas a similar response was not observed in control nuclei. These results suggest that CD38 is expressed in HL-60 cell nuclei during RA-induced differentiation.« less

Identification of an anti-MRSA dihydrofolate reductase inhibitor from a diversity-oriented synthesis.

PubMed

Wyatt, Emma E; Galloway, Warren R J D; Thomas, Gemma L; Welch, Martin; Loiseleur, Olivier; Plowright, Alleyn T; Spring, David R

2008-10-28

The screening of a diversity-oriented synthesis library followed by structure-activity relationship investigations have led to the discovery of an anti-MRSA agent which operates as an inhibitor of Staphylococcus aureus dihydrofolate reductase.

Dynamical Systems in Neuropharmacology

DTIC Science & Technology

1992-04-15

induced hyperactivity with the selective serotonin uptake blockers fluoxetine , sertraline, or zimelidine or with the serotonin synthesis inhibitor PCPA. By...Conversely, the catecholamine synthesis inhibitor AMPT, which blocks the effects of amphetamine, did not affect the response to MDMA. We applied our scaling

Selective inhibition of influenza virus protein synthesis by inhibitors of DNA function. [UV radiation

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

Minor, P.D.; Dimmock, N.J.

1977-05-15

Various known inhibitors of cellular DNA function were shown to inhibit cellular RNA synthesis and influenza (fowl plague) virus multiplication. The drugs were investigated for their effect upon the synthesis of influenza virus proteins. According to this effect they could be classified with previously studied compounds as follows: Group I (ethidium bromide, proflavine, and N-nitroquinoline-N-oxide) inhibited both viral and cellular protein synthesis; Group II (nogalomycin, daunomycin and ..cap alpha..-amanitin) inhibited viral but not cellular protein synthesis, and all viral proteins were inhibited coordinately; Group III (mithramycin, echinomycin, and actinomycin D) inhibited all viral but not cellular protein synthesis at highmore » concentrations, but at a lower critical concentration inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein preferentially; Group IV(uv irradiation and camptothecin) inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein, but not other viral proteins, even at high doses. The mode of action of these inhibitors is discussed in relation to the mechanism of the nuclear events upon which influenza virus multiplication is dependent.« less

Metabolism of organic acids, nitrogen and amino acids in chlorotic leaves of 'Honeycrisp' apple (Malus domestica Borkh) with excessive accumulation of carbohydrates.

PubMed

Wang, Huicong; Ma, Fangfang; Cheng, Lailiang

2010-07-01

Metabolite profiles and activities of key enzymes in the metabolism of organic acids, nitrogen and amino acids were compared between chlorotic leaves and normal leaves of 'Honeycrisp' apple to understand how accumulation of non-structural carbohydrates affects the metabolism of organic acids, nitrogen and amino acids. Excessive accumulation of non-structural carbohydrates and much lower CO(2) assimilation were found in chlorotic leaves than in normal leaves, confirming feedback inhibition of photosynthesis in chlorotic leaves. Dark respiration and activities of several key enzymes in glycolysis and tricarboxylic acid (TCA) cycle, ATP-phosphofructokinase, pyruvate kinase, citrate synthase, aconitase and isocitrate dehydrogenase were significantly higher in chlorotic leaves than in normal leaves. However, concentrations of most organic acids including phosphoenolpyruvate (PEP), pyruvate, oxaloacetate, 2-oxoglutarate, malate and fumarate, and activities of key enzymes involved in the anapleurotic pathway including PEP carboxylase, NAD-malate dehydrogenase and NAD-malic enzyme were significantly lower in chlorotic leaves than in normal leaves. Concentrations of soluble proteins and most free amino acids were significantly lower in chlorotic leaves than in normal leaves. Activities of key enzymes in nitrogen assimilation and amino acid synthesis, including nitrate reductase, glutamine synthetase, ferredoxin and NADH-dependent glutamate synthase, and glutamate pyruvate transaminase were significantly lower in chlorotic leaves than in normal leaves. It was concluded that, in response to excessive accumulation of non-structural carbohydrates, glycolysis and TCA cycle were up-regulated to "consume" the excess carbon available, whereas the anapleurotic pathway, nitrogen assimilation and amino acid synthesis were down-regulated to reduce the overall rate of amino acid and protein synthesis.

Lysine Deacetylase Inhibitors in Parasites: Past, Present, and Future Perspectives.

PubMed

Hailu, Gebremedhin S; Robaa, Dina; Forgione, Mariantonietta; Sippl, Wolfgang; Rotili, Dante; Mai, Antonello

2017-06-22

Current therapies for human parasite infections rely on a few drugs, most of which have severe side effects, and their helpfulness is being seriously compromised by the drug resistance problem. Globally, this is pushing discovery research of antiparasitic drugs toward new agents endowed with new mechanisms of action. By using a "drug repurposing" strategy, histone deacetylase inhibitors (HDACi), which are presently clinically approved for cancer use, are now under investigation for various parasite infections. Because parasitic Zn 2+ - and NAD + -dependent HDACs play crucial roles in the modulation of parasite gene expression and many of them are pro-survival for several parasites under various conditions, they are now emerging as novel potential antiparasitic targets. This Perspective summarizes the state of knowledge of HDACi (both class I/II HDACi and sirtuin inhibitors) targeted to the main human parasitic diseases (schistosomiasis, malaria, trypanosomiasis, leishmaniasis, and toxoplasmosis) and provides visions into the main issues that challenge their development as antiparasitic agents.

Structure-based design and profiling of novel 17β-HSD14 inhibitors.

PubMed

Braun, Florian; Bertoletti, Nicole; Möller, Gabriele; Adamski, Jerzy; Frotscher, Martin; Guragossian, Nathalie; Madeira Gírio, Patrícia Alexandra; Le Borgne, Marc; Ettouati, Laurent; Falson, Pierre; Müller, Sebastian; Vollmer, Günther; Heine, Andreas; Klebe, Gerhard; Marchais-Oberwinkler, Sandrine

2018-05-22

The human enzyme 17β-hydroxysteroid dehydrogenase 14 (17β-HSD14) oxidizes the hydroxyl group at position 17 of estradiol and 5-androstenediol using NAD + as cofactor. However, the physiological role of the enzyme remains unclear. We recently described the first class of nonsteroidal inhibitors for this enzyme with compound 1 showing a high 17β-HSD14 inhibitory activity. Its crystal structure was used as starting point for a structure-based optimization in this study. The goal was to develop a promising chemical probe to further investigate the enzyme. The newly designed compounds revealed mostly very high inhibition of the enzyme and for seven of them the crystal structures of the corresponding inhibitor-enzyme complexes were resolved. The crystal structures disclosed that a small change in the substitution pattern of the compounds resulted in an alternative binding mode for one inhibitor. The profiling of a set of the most potent inhibitors identified 13 (K i  = 9 nM) with a good selectivity profile toward three 17β-HSDs and the estrogen receptor alpha. This inhibitor displayed no cytotoxicity, good solubility, and auspicious predicted bioavailability. Overall, 13 is a highly interesting 17β-HSD14 inhibitor, which might be used as chemical probe for further investigation of the target enzyme. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Inhibitor effects during the cell cycle in Chlamydomonas reinhardtii. Determination of transition points in asynchronous cultures

PubMed Central

1975-01-01

A wide variety of inhibitors (drugs, antibiotics, and antimetabolites) will block cell division within an ongoing cell cycle in autotrophic cultures of Chlamydomonas reinhardtii. To determine when during the cell cycle a given inhibitor is effective in preventing cell division, a technique is described which does not rely on the use of synchronous cultures. The technique permits the measurement of transition points, the cell cycle stage at which the subsequent cell division becomes insensitive to the effects of an inhibitor. A map of transition points in the cell cycle reveals that they are grouped into two broad periods, the second and fourth quarters. In general, inhibitors which block organellar DNA, RNA, and protein synthesis have second-quarter transition points, while those which inhibit nuclear cytoplasmic macromolecular synthesis have fourth-quarter transition points. The specific grouping of these transition points into two periods suggests that the synthesis of organellar components is completed midway through the cell cycle and that the synthesis of nonorganellar components required for cell division is not completed until late in the cell cycle. PMID:1176526

Family-wide analysis of poly(ADP-ribose) polymerase activity

PubMed Central

Uchima, Lilen; Rood, Jenny; Zaja, Roko; Hay, Ronald T.; Ahel, Ivan; Chang, Paul

2014-01-01

The poly(ADP-ribose) polymerase (PARP) protein family generates ADP-ribose (ADPr) modifications onto target proteins using NAD+ as substrate. Based on the composition of three NAD+ coordinating amino acids, the H-Y-E motif, each PARP is predicted to generate either poly(ADP-ribose) (PAR) or mono(ADP-ribose) (MAR). However, the reaction product of each PARP has not been clearly defined, and is an important priority since PAR and MAR function via distinct mechanisms. Here we show that the majority of PARPs generate MAR, not PAR, and demonstrate that the H-Y-E motif is not the sole indicator of PARP activity. We identify automodification sites on seven PARPs, and demonstrate that MAR and PAR generating PARPs modify similar amino acids, suggesting that the sequence and structural constraints limiting PARPs to MAR synthesis do not limit their ability to modify canonical amino acid targets. In addition, we identify cysteine as a novel amino acid target for ADP-ribosylation on PARPs. PMID:25043379

Metabolic engineering of Synechocystis sp. PCC 6803 for enhanced ethanol production based on flux balance analysis.

PubMed

Yoshikawa, Katsunori; Toya, Yoshihiro; Shimizu, Hiroshi

2017-05-01

Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production due to its ability to directly convert atmospheric carbon dioxide into ethanol using photosystems. To enhance ethanol production in Synechocystis sp. PCC 6803, metabolic engineering was performed based on in silico simulations, using the genome-scale metabolic model. Comprehensive reaction knockout simulations by flux balance analysis predicted that the knockout of NAD(P)H dehydrogenase enhanced ethanol production under photoautotrophic conditions, where ammonium is the nitrogen source. This deletion inhibits the re-oxidation of NAD(P)H, which is generated by ferredoxin-NADP + reductase and imposes re-oxidation in the ethanol synthesis pathway. The effect of deleting the ndhF1 gene, which encodes NADH dehydrogenase subunit 5, on ethanol production was experimentally evaluated using ethanol-producing strains of Synechocystis sp. PCC 6803. The ethanol titer of the ethanol-producing ∆ndhF1 strain increased by 145%, compared with that of the control strain.

Divergent Pathways Involving 1,3-Dipolar Addition and N-N Bond Splitting of an Organic Azide across a Zirconium Methylidene.

PubMed

Kurogi, Takashi; Mane, Manoj V; Zheng, Shuai; Carroll, Patrick J; Baik, Mu-Hyun; Mindiola, Daniel J

2018-02-12

The zirconium methylidene (PNP)Zr=CH 2 (OAr) (1) reacts with N 3 Ad to give two products (PNP)Zr=NAd(OAr) (2) and (PNP)Zr(η 2 -N=NAd)(N=CH 2 )(OAr) (3), both resulting from a common cycloaddition intermediate (PNP)Zr(CH 2 N 3 Ad)(OAr) (A). Using a series of control experiments in combination with DFT calculations, it was found that 2 results from a nitrene by a carbene metathesis reaction in which N 2 acts as a delivery vehicle and forms N 2 CH 2 as a side product. In the case of 3, N-N bond splitting of the azide at the α-position allowed the isolation of a rare example of a parent ketimide complex of zirconium. Isotopic labeling studies and solid-state X-ray analysis are presented for 2 and 3, in addition to an independent synthesis for the former. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced biosynthesis of chiral phenyllactic acid from L-phenylalanine through a new whole-cell biocatalyst.

PubMed

Zheng, Zhaojuan; Xia, Meijuan; Fang, Xuchao; Jiang, Ting; Ouyang, Jia

2018-06-22

Phenyllactic acid (PLA) is a high-value compound, which was usually produced by lactic acid bacteria (LAB) as biocatalysts and glucose or phenylpyruvic acid (PPA) as starting materials for PLA synthesis in previous studies. However, the PLA produced using LAB is a racemic mixture. Besides, both glucose and PPA were unsatisfactory substrates, as the former could not produce high concentrations of PLA while the latter is not a renewable and green substrate. To overcome these drawbacks, in this study, a new biotransformation process was developed for chiral PLA production from L-phenylalanine via the intermediate PPA using recombinant Escherichia coli co-expressing L-amino acid deaminase, NAD-dependent L-lactate dehydrogenase or NAD-dependent D-lactate dehydrogenase, and formate dehydrogenase. After optimization, the recombinant E. coli produced L- and D-PLA at concentrations of 59.9 and 60.3 mM in 6 h, respectively. Hence, this process provides an effective and promising alternative method for chiral PLA production.

Respiratory processes in non-photosynthetic plastids

PubMed Central

Renato, Marta; Boronat, Albert; Azcón-Bieto, Joaquín

2015-01-01

Chlororespiration is a respiratory process located in chloroplast thylakoids which consists in an electron transport chain from NAD(P)H to oxygen. This respiratory chain involves the NAD(P)H dehydrogenase complex, the plastoquinone pool and the plastid terminal oxidase (PTOX), and it probably acts as a safety valve to prevent the over-reduction of the photosynthetic machinery in stress conditions. The existence of a similar respiratory activity in non-photosynthetic plastids has been less studied. Recently, it has been reported that tomato fruit chromoplasts present an oxygen consumption activity linked to ATP synthesis. Etioplasts and amyloplasts contain several electron carriers and some subunits of the ATP synthase, so they could harbor a similar respiratory process. This review provides an update on the study about respiratory processes in chromoplasts, identifying the major gaps that need to be addressed in future research. It also reviews the proteomic data of etioplasts and amyloplasts, which suggest the presence of a respiratory electron transport chain in these plastids. PMID:26236317

The inhibitory effect of beta-lapachone on RANKL-induced osteoclastogenesis

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

Gu, Dong Ryun; Center for Metabolic Function Regulation; Lee, Joon No

β-lapachone (β-L) is a substrate of reduced nicotinamide adenine dinucleotide (NADH): quinone oxidoreductase 1 (NQO1). NQO1 reduces quinones to hydroquinones using NADH as an electron donor and consequently increases the intracellular NAD+/NADH ratio. The activation of NQO1 by β-L has beneficial effects on several metabolic syndromes, such as obesity, hypertension, and renal injury. However, the effect of β-L on bone metabolism remains unclear. Here, we show that β-L might be a potent inhibitor of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. β-L inhibited osteoclast formation in a dose-dependent manner and also reduced the expression of osteoclast differentiation marker genes,more » such as tartrate-resistant acid phosphatase (Acp5 or TRAP), cathepsin K (CtsK), the d2 isoform of vacuolar ATPase V0 domain (Atp6v0d2), osteoclast-associated receptor (Oscar), and dendritic cell-specific transmembrane protein (Dc-stamp). β-L treatment of RANKL-induced osteoclastogenesis significantly increased the cellular NAD+/NADH ratio and resulted in the activation of 5′ AMP-activated protein kinase (AMPK), a negative regulator of osteoclast differentiation. In addition, β-L treatment led to significant suppression of the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and peroxisome proliferator-activated receptor gamma coactivator 1β (PGC1β), which can stimulate osteoclastogenesis. β-L treatment downregulated c-Fos and nuclear factor of activated T-cells 1 (NFATc1), which are master transcription factors for osteoclastogenesis. Taken together, the results demonstrated that β-L inhibits RANKL-induced osteoclastogenesis and could be considered a potent inhibitor of RANKL-mediated bone diseases, such as postmenopausal osteoporosis, rheumatoid arthritis, and periodontitis. - Highlights: • β-lapachone (β-L) inhibits RANKL-mediated osteoclastogenesis. • β-L increases the intracellular NAD+/NADH ratio, which is followed by activation of AMPK in osteoclasts. • The activation of AMPK by β-L inhibits c-Fos and NFATc1 expression in RANKL-induced osteoclastogenesis. • β-L also suppresses c-Fos and NFATc1 expression via downregulation of PPARγ and PGC1β expression.« less

Contribution of NAD 2D-NMR in liquid crystals to the determination of hydrogen isotope profile of methyl groups in miliacin

NASA Astrophysics Data System (ADS)

Berdagué, Philippe; Lesot, Philippe; Jacob, Jérémy; Terwilliger, Valery J.; Le Milbeau, Claude

2016-01-01

The hydrogen isotopic composition (δD or (D/H) value) of molecular biomarkers preserved in sedimentary archives is increasingly used to provide clues about the evolution of past climatic conditions. The rationale is that intact biomarkers retain isotopic information related to the climatic conditions that prevailed at the time of their synthesis. Some of these biomarkers may be degraded during diagenesis, however. The extent to which these degradations alter the original δD value of the source biomarker is presently debated and the capacity to resolve this question by determination of compound-specific δD values alone is limited. The ;bulk; or ;global; δD value of any molecule is in fact a composite of δD values at each site within this molecule (δDi or (D/H)i with i = number of hydrogen/deuterium atoms in the considered molecule). Determination of this site-specific δDi value in biomarkers could not only yield outstanding paleoenvironmental information but also help forecast the impacts of diagenesis and define essential steps in biosynthetic pathways. This task is analytically challenging. Here, we examined the capabilities of natural abundance deuterium 2D-NMR (NAD 2D-NMR) using homopolypeptide liquid crystals as an NMR solvent to: (i) analyze the NAD spectra of biomakers; (ii) determine the site-specific distribution of hydrogen in the nine methyl groups (δDMei with i = 23-31) of miliacin, a pentacyclic triterpene of the amyrin family and key biomarker for broomcorn millet in sedimentary archives. Relative (D/H)Mei values were established by anisotropic NAD 2D-NMR. Then absolute δDMei values were obtained by determining δDMei value of the methoxy group of miliacin using two independent approaches: isotropic NAD NMR (SNIF-NMR™) and GC-irMS. The resulting isotope profile for miliacin shows, for the first time, large variations in δDMei values that can directly be explained by biosynthetic processes. This approach has also the potential to permit predicting fractionations during pentacyclic triterpene diagenesis.

3-cyanoindole-based inhibitors of inosine monophosphate dehydrogenase: synthesis and initial structure-activity relationships.

PubMed

Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Chen, Ping; Norris, Derek; Watterson, Scott H; Ballentine, Shelley K; Fleener, Catherine A; Rouleau, Katherine A; Barrish, Joel C; Townsend, Robert; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2003-10-20

A series of novel small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH), based upon a 3-cyanoindole core, were explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SAR), derived from in vitro studies, for this new series of inhibitors is given.

An Ancient Pathway Combining Carbon Dioxide Fixation with the Generation and Utilization of a Sodium Ion Gradient for ATP Synthesis

PubMed Central

Poehlein, Anja; Schmidt, Silke; Kaster, Anne-Kristin; Goenrich, Meike; Vollmers, John; Thürmer, Andrea; Bertsch, Johannes; Schuchmann, Kai; Voigt, Birgit; Hecker, Michael; Daniel, Rolf; Thauer, Rudolf K.; Gottschalk, Gerhard; Müller, Volker

2012-01-01

Synthesis of acetate from carbon dioxide and molecular hydrogen is considered to be the first carbon assimilation pathway on earth. It combines carbon dioxide fixation into acetyl-CoA with the production of ATP via an energized cell membrane. How the pathway is coupled with the net synthesis of ATP has been an enigma. The anaerobic, acetogenic bacterium Acetobacterium woodii uses an ancient version of this pathway without cytochromes and quinones. It generates a sodium ion potential across the cell membrane by the sodium-motive ferredoxin:NAD oxidoreductase (Rnf). The genome sequence of A. woodii solves the enigma: it uncovers Rnf as the only ion-motive enzyme coupled to the pathway and unravels a metabolism designed to produce reduced ferredoxin and overcome energetic barriers by virtue of electron-bifurcating, soluble enzymes. PMID:22479398

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

Acute effects of moclobemide and deprenyl on 5-HT synthesis rates in the rat brain: An autoradiographic study.

PubMed

Nishi, Kyoko; Mück-Seler, Dorotea; Hasegawa, Shu; Watanabe, Arata; Diksic, Mirko

2006-10-16

Serotonin (5-HT), norepinephrine (NE) and dopamine (DA) released from nerve terminals in the brain are primarily removed from the synaptic cleft by a reuptake mechanism. In part, the homeostasis is maintained by monoamine oxidase (MAO) deamination achieved primarily intracellularly. The present study's aim was to examine the effect of the acute administration of the MAO inhibitors, moclobemide (a MAO-A inhibitor) and deprenyl (a MAO-B inhibitor), on 5-HT synthesis rates, measured in discrete regions of the rat brain by an autoradiographic method, using alpha-[14C]methyl-l-tryptophan as a tracer. MAO inhibitors have different effects on 5-HT synthesis rates in the cell bodies and areas of the nerve terminals. Moclobemide (10 mg/kg, i.p. 30 min before the tracer injection) and deprenyl (3 mg/kg, i.p. 2 h before the tracer injection) decreased the 5-HT synthesis rates in the dorsal (-18% and -22%) and median (-22% and -33%) raphe, respectively. Moclobemide also significantly decreased 5-HT synthesis in the entire nerve terminal areas investigated. The reductions were between 23% (cingulate cortex) and 50% (locus coeruleus). Deprenyl did not significantly affect 5-HT synthesis in the nerve terminals. The present results suggest that MAO-A, and to a lesser extent, MAO-B, are involved in the regulation of 5-HT synthesis in the rat brain. The mechanism(s) of MAO inhibitors' action on 5-HT synthesis in the raphe nuclei are probably related to an increase in the extraneuronal 5-HT concentration and also to the interaction between the serotonergic and catecholaminergic neurons. The reduction of 5-HT synthesis in the raphe nuclei likely occurs by an action of extracellular 5-HT via the dendritic autoreceptors with a possible contribution from the action of extracellular DA and NE. In the terminal regions, the most likely mechanism is via the presynaptic autoreceptors through which elevated extraneuronal 5-HT acts on synthesis control. However, there is also a possibility that the elevation in intraneuronal 5-HT directly inhibits its synthesis, especially following deprenyl treatment. A great influence of moclobemide on 5-HT synthesis could be related to its antidepressant action.

Microgravity

NASA Image and Video Library

2004-04-15

The Commercial Vapor Diffusion Apparatus will be used to perform 128 individual crystal growth investigations for commercial and science research. These experiments will grow crystals of several different proteins, including HIV-1 Protease Inhibitor, Glycogen Phosphorylase A, and NAD Synthetase. The Commercial Vapor Diffusion Apparatus supports multiple commercial investigations within a controlled environment. The goal of the Commercial Protein Crystal Growth payload on STS-95 is to grow large, high-quality crystals of several different proteins of interest to industry, and to continue to refine the technology and procedures used in microgravity for this important commercial research.

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

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.

Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification

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

Keating, David H.; Zhang, Yaoping; Ong, Irene M.

2014-08-13

Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and proteinmore » levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts.« less

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.

Lupin nad9 and nad6 genes and their expression: 5' termini of the nad9 gene transcripts differentiate lupin species.

PubMed

Rurek, Michał; Nuc, Katarzyna; Raczyńska, Katarzyna Dorota; Augustyniak, Halina

2003-10-02

The mitochondrial nad9 and nad6 genes were analyzed in four lupin species: Lupinus luteus, Lupinus angustifolius, Lupinus albus and Lupinus mutabilis. The nucleotide sequence of these genes confirmed their high conservation, however, higher number of nucleotide substitution was observed in the L. albus genes. Southern hybridizations confirmed the presence of single copy number of these genes in L. luteus, L. albus and L. angustifolius. The expression of nad9 and nad6 genes was analyzed by Northern in different tissue types of analyzed lupin species. Transcription analyses of the two nad genes displayed single predominant mRNA species of about 0.6 kb in L. luteus and L. angustifolius. The L. albus transcripts were larger in size. The nad9 and nad6 transcripts were modified by RNA editing at 8 and 11 positions, in L. luteus and L. angustifolius, respectively. The gene order, rps3-rpl16-nad9, found in Arabidopsis thaliana is also conserved in L. luteus and L. angustifolius mitochondria. L. luteus and L. angustifolius showed some variability in the sequence of the nad9 promoter region. The last feature along with the differences observed in nad9 mRNA 5' termini of two lupins differentiate L. luteus and L. angustifolius species.

Nicotinamide adenine dinucleotide is transported into mammalian mitochondria.

PubMed

Davila, Antonio; Liu, Ling; Chellappa, Karthikeyani; Redpath, Philip; Nakamaru-Ogiso, Eiko; Paolella, Lauren M; Zhang, Zhigang; Migaud, Marie E; Rabinowitz, Joshua D; Baur, Joseph A

2018-06-12

Mitochondrial NAD levels influence fuel selection, circadian rhythms, and cell survival under stress. It has alternately been argued that NAD in mammalian mitochondria arises from import of cytosolic nicotinamide (NAM), nicotinamide mononucleotide (NMN), or NAD itself. We provide evidence that murine and human mitochondria take up intact NAD. Isolated mitochondria preparations cannot make NAD from NAM, and while NAD is synthesized from NMN, it does not localize to the mitochondrial matrix or effectively support oxidative phosphorylation. Treating cells with nicotinamide riboside that is isotopically labeled on the nicotinamide and ribose moieties results in the appearance of doubly labeled NAD within mitochondria. Analogous experiments with doubly labeled nicotinic acid riboside (labeling cytosolic NAD without labeling NMN) demonstrate that NAD(H) is the imported species. Our results challenge the long-held view that the mitochondrial inner membrane is impermeable to pyridine nucleotides and suggest the existence of an unrecognized mammalian NAD (or NADH) transporter. © 2018, Davila et al.

The design and synthesis of novel N-hydroxyformamide inhibitors of ADAM-TS4 for the treatment of osteoarthritis.

PubMed

De Savi, Chris; Pape, Andrew; Cumming, John G; Ting, Attilla; Smith, Peter D; Burrows, Jeremy N; Mills, Mark; Davies, Chris; Lamont, Scott; Milne, David; Cook, Calum; Moore, Peter; Sawyer, Yvonne; Gerhardt, Stefan

2011-03-01

Two series of N-hydroxyformamide inhibitors of ADAM-TS4 were identified from screening compounds previously synthesised as inhibitors of matrix metalloproteinase-13 (collagenase-3). Understanding of the binding mode of this class of compound using ADAM-TS1 as a structural surrogate has led to the discovery of potent and very selective inhibitors with favourable DMPK properties. Synthesis, structure-activity relationships, and strategies to improve selectivity and lower in vivo metabolic clearance are described. Copyright © 2011 Elsevier Ltd. All rights reserved.

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.

Tracing regulatory routes in metabolism using generalised supply-demand analysis.

PubMed

Christensen, Carl D; Hofmeyr, Jan-Hendrik S; Rohwer, Johann M

2015-12-03

Generalised supply-demand analysis is a conceptual framework that views metabolism as a molecular economy. Metabolic pathways are partitioned into so-called supply and demand blocks that produce and consume a particular intermediate metabolite. By studying the response of these reaction blocks to perturbations in the concentration of the linking metabolite, different regulatory routes of interaction between the metabolite and its supply and demand blocks can be identified and their contribution quantified. These responses are mediated not only through direct substrate/product interactions, but also through allosteric effects. Here we subject previously published kinetic models of pyruvate metabolism in Lactococcus lactis and aspartate-derived amino acid synthesis in Arabidopsis thaliana to generalised supply-demand analysis. Multiple routes of regulation are brought about by different mechanisms in each model, leading to behavioural and regulatory patterns that are generally difficult to predict from simple inspection of the reaction networks depicting the models. In the pyruvate model the moiety-conserved cycles of ATP/ADP and NADH/NAD(+) allow otherwise independent metabolic branches to communicate. This causes the flux of one ATP-producing reaction block to increase in response to an increasing ATP/ADP ratio, while an NADH-consuming block flux decreases in response to an increasing NADH/NAD(+) ratio for certain ratio value ranges. In the aspartate model, aspartate semialdehyde can inhibit its supply block directly or by increasing the concentration of two amino acids (Lys and Thr) that occur as intermediates in demand blocks and act as allosteric inhibitors of isoenzymes in the supply block. These different routes of interaction from aspartate semialdehyde are each seen to contribute differently to the regulation of the aspartate semialdehyde supply block. Indirect routes of regulation between a metabolic intermediate and a reaction block that either produces or consumes this intermediate can play a much larger regulatory role than routes mediated through direct interactions. These indirect routes of regulation can also result in counter-intuitive metabolic behaviour. Performing generalised supply-demand analysis on two previously published models demonstrated the utility of this method as an entry point in the analysis of metabolic behaviour and the potential for obtaining novel results from previously analysed models by using new approaches.

SIRT1 deacetylates RFX5 and antagonizes repression of collagen type I (COL1A2) transcription in smooth muscle cells

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

Xia, Jun; Department of Respiratory Medicine, Jiangsu Provincial Hospital of Chinese Traditional Medicine; Wu, Xiaoyan

Highlights: Black-Right-Pointing-Pointer SIRT1 interacts with and deacetylates RFX5. Black-Right-Pointing-Pointer SIRT1 activation attenuates whereas SIRT1 inhibition enhances collagen repression by RFX5 in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 promotes cytoplasmic localization and proteasomal degradation of RFX5 and cripples promoter recruitment of RFX5. Black-Right-Pointing-Pointer IFN-{gamma} represses SIRT1 expression in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 agonist alleviates collagen repression by IFN-{gamma} in vascular smooth muscle cells. -- Abstract: Decreased expression of collagen by vascular smooth muscle cells (SMCs) within the atherosclerotic plaque contributes to the thinning of the fibrous cap and poses a great threat to plaque rupture. Elucidation of the mechanismmore » underlying repressed collagen type I (COL1A2) gene would potentially provide novel solutions that can prevent rupture-induced complications. We have previously shown that regulatory factor for X-box (RFX5) binds to the COL1A2 transcription start site and represses its transcription. Here we report that SIRT1, an NAD-dependent, class III deacetylase, forms a complex with RFX5. Over-expression of SIRT1 or NAMPT, which synthesizes NAD+ to activate SIRT1, or treatment with the SIRT1 agonist resveratrol decreases RFX5 acetylation and disrupts repression of the COL1A2 promoter activity by RFX5. On the contrary, knockdown of SIRT1 or treatment with SIRT1 inhibitors induces RFX5 acetylation and enhances the repression of collagen transcription. SIRT1 antagonizes RFX5 activity by promoting its nuclear expulsion and proteasomal degradation hence dampening its binding to the COL1A2 promoter. The pro-inflammatory cytokine IFN-{gamma} represses COL1A2 transcription by down-regulating SIRT1 expression in SMCs. Therefore, our data have identified as novel pathway whereby SIRT1 maintains collagen synthesis in SMCs by modulating RFX5 activity.« less

A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana

PubMed Central

Wang, Chenggang; Zhou, Mingqi; Zhang, Xudong; Yao, Jin; Zhang, Yanping; Mou, Zhonglin

2017-01-01

Nicotinamide adenine dinucleotide (NAD+) participates in intracellular and extracellular signaling events unrelated to metabolism. In animals, purinergic receptors are required for extracellular NAD+ (eNAD+) to evoke biological responses, indicating that eNAD+ may be sensed by cell-surface receptors. However, the identity of eNAD+-binding receptors still remains elusive. Here, we identify a lectin receptor kinase (LecRK), LecRK-I.8, as a potential eNAD+ receptor in Arabidopsis. The extracellular lectin domain of LecRK-I.8 binds NAD+ with a dissociation constant of 436.5 ± 104.8 nM, although much higher concentrations are needed to trigger in vivo responses. Mutations in LecRK-I.8 inhibit NAD+-induced immune responses, whereas overexpression of LecRK-I.8 enhances the Arabidopsis response to NAD+. Furthermore, LecRK-I.8 is required for basal resistance against bacterial pathogens, substantiating a role for eNAD+ in plant immunity. Our results demonstrate that lectin receptors can potentially function as eNAD+-binding receptors and provide direct evidence for eNAD+ being an endogenous signaling molecule in plants. DOI: http://dx.doi.org/10.7554/eLife.25474.001 PMID:28722654

The mitochondrial SIR2 related protein 2 (SIR2RP2) impacts Leishmania donovani growth and infectivity

PubMed Central

Mittal, Nimisha; Muthuswami, Rohini

2017-01-01

Background Leishmania donovani, a protozoan parasite is the major causative agent of visceral leishmaniasis. Increased toxicity and resistance to the existing repertoire of drugs has been reported. Hence, an urgent need exists for identifying newer drugs and drug targets. Previous reports have shown sirtuins (Silent Information Regulator) from kinetoplastids as promising drug targets. Leishmania species code for three SIR2 (Silent Information Regulator) related proteins. Here, we for the first time report the functional characterization of SIR2 related protein 2 (SIR2RP2) of L. donovani. Methodology Recombinant L. donovani SIR2RP2 was expressed in E. coli and purified. The enzymatic functions of SIR2RP2 were determined. The subcellular localization of LdSIR2RP2 was done by constructing C-terminal GFP-tagged full-length LdSIR2RP2. Deletion mutants of LdSIR2RP2 were generated in Leishmania by double targeted gene replacement methodology. These null mutants were tested for their proliferation, virulence, cell cycle defects, mitochondrial functioning and sensitivity to known SIR2 inhibitors. Conclusion Our data suggests that LdSIR2RP2 possesses NAD+-dependent ADP-ribosyltransferase activity. However, NAD+-dependent deacetylase and desuccinylase activities were not detected. The protein localises to the mitochondrion of the promastigotes. Gene deletion studies showed that ΔLdSIR2RP2 null mutants had restrictive growth phenotype associated with accumulation of cells in the G2/M phase and compromised mitochondrial functioning. The null mutants had attenuated infectivity. Deletion of LdSIR2RP2 resulted in increased sensitivity of the parasites to the known SIR2 inhibitors. The sirtuin inhibitors inhibited the ADP-ribosyltransferase activity of recombinant LdSIR2RP2. In conclusion, sirtuins could be used as potential new drug targets for visceral leishmaniasis. PMID:28493888

Synthesis of Lysine Methyltransferase Inhibitors

NASA Astrophysics Data System (ADS)

Ye, Tao; Hui, Chunngai

2015-07-01

Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

Nicotinamide riboside kinases display redundancy in mediating nicotinamide mononucleotide and nicotinamide riboside metabolism in skeletal muscle cells.

PubMed

Fletcher, Rachel S; Ratajczak, Joanna; Doig, Craig L; Oakey, Lucy A; Callingham, Rebecca; Da Silva Xavier, Gabriella; Garten, Antje; Elhassan, Yasir S; Redpath, Philip; Migaud, Marie E; Philp, Andrew; Brenner, Charles; Canto, Carles; Lavery, Gareth G

2017-08-01

Augmenting nicotinamide adenine dinucleotide (NAD + ) availability may protect skeletal muscle from age-related metabolic decline. Dietary supplementation of NAD + precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) appear efficacious in elevating muscle NAD + . Here we sought to identify the pathways skeletal muscle cells utilize to synthesize NAD + from NMN and NR and provide insight into mechanisms of muscle metabolic homeostasis. We exploited expression profiling of muscle NAD + biosynthetic pathways, single and double nicotinamide riboside kinase 1/2 (NRK1/2) loss-of-function mice, and pharmacological inhibition of muscle NAD + recycling to evaluate NMN and NR utilization. Skeletal muscle cells primarily rely on nicotinamide phosphoribosyltransferase (NAMPT), NRK1, and NRK2 for salvage biosynthesis of NAD + . NAMPT inhibition depletes muscle NAD + availability and can be rescued by NR and NMN as the preferred precursors for elevating muscle cell NAD + in a pathway that depends on NRK1 and NRK2. Nrk2 knockout mice develop normally and show subtle alterations to their NAD+ metabolome and expression of related genes. NRK1, NRK2, and double KO myotubes revealed redundancy in the NRK dependent metabolism of NR to NAD + . Significantly, these models revealed that NMN supplementation is also dependent upon NRK activity to enhance NAD + availability. These results identify skeletal muscle cells as requiring NAMPT to maintain NAD + availability and reveal that NRK1 and 2 display overlapping function in salvage of exogenous NR and NMN to augment intracellular NAD + availability.

Design and synthesis of potent, orally-active DGAT-1 inhibitors containing a dioxino[2,3-d]pyrimidine core.

PubMed

Dow, Robert L; Andrews, Melissa; Aspnes, Gary E; Balan, Gayatri; Michael Gibbs, E; Guzman-Perez, Angel; Karki, Kapil; Laperle, Jennifer L; Li, Jian-Cheng; Litchfield, John; Munchhof, Michael J; Perreault, Christian; Patel, Leena

2011-10-15

A novel series of potent DGAT-1 inhibitors was developed originating from the lactam-based clinical candidate PF-04620110. Incorporation of a dioxino[2,3-d]pyrimidine-based core afforded good alignment of pharmacophore features and resulted in improved passive permeability. Development of an efficient, homochiral synthesis of these targets facilitated confirmation of predictions regarding the stereochemical-dependence of DGAT-1 inhibition for this series. Compound 10 was shown to be a potent inhibitor of human DGAT-1 (10 nM) and to suppress triglyceride synthesis at oral doses of <3mg/kg. Copyright © 2011 Elsevier Ltd. All rights reserved.

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.

PROTEASOME INHIBITOR TREATMENT REDUCED FATTY ACID, TRIACYLGLYCEROL AND CHOLESTEROL SYNTHESIS

PubMed Central

Oliva, Joan; French, Samuel W.; Li, Jun; Bardag-Gorce, Fawzia

2014-01-01

In the present study, the beneficial effects of proteasome inhibitor treatment in reducing ethanol-induced steatosis were investigated. A microarray analysis was performed on the liver of rats injected with PS-341 (Bortezomib, Velcade®), and the results showed that proteasome inhibitor treatment significantly reduced the mRNA expression of SREBP-1c, and the downstream lipogenic enzymes, such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis. ELOVL6, which is responsible for fatty acids long chain elongation, was also significantly down regulated by proteasome inhibitor treatment. Moreover, PS-341 administration significantly reduced the expression of acyl-glycerol-3-phosphate acyltransferase (AGPAT), and diacylglycerol acyltransferase (DGAT), enzyme involved in triacylglycerol (TAG) synthesis. Finally, PS-341 was found to down regulate the enzymes 3-hydroxy-3-methylglutaryl-CoenzymeA synthase (HMG-CoA synthase) that is responsible for cholesterol synthesis. Proteasome inhibitor was also found to play a role in intestinal lipid adsorption because apolipoproteins A (apoA-I, apoAII, apoA-IV and ApoCIII) were down regulated by proteasome inhibitor treatment, especially ApoA-II that is known to be a marker of alcohol consumption. Proteasome inhibitor treatment also decreased apobec-1 complementation factor (ACF) leading to lower level of editing and production of ApoB protein. Moreover apolipoprotein C-III, a major component of chylomicrons was significantly down regulated. However, lipoprotein lipase (Lpl) and High density lipoprotein binding protein (Hdlbp) mRNA levels were increased by proteasome inhibitor treatment. These results suggested that proteasome inhibitor treatment could be used to reduce the alcohol-enhanced lipogenesis and alcohol-induced liver steatosis. A morphologic analysis, performed on the liver of rats fed ethanol for one month and treated with PS-341, showed that proteasome inhibitor treatment significantly decreased ethanol-induced liver steatosis. SREBP-1c, FAS and ACC were increased by ethanol feeding alone, but were significantly decreased when proteasome inhibitor was administered to rats fed ethanol. Our results also show that both mRNA and protein levels of these lipogenic enzymes, up regulated by ethanol, were then down regulated when proteasome inhibitor was administered to rats fed ethanol. It was also confirmed that alcohol feeding caused an increase in AGPAT and DGAT, which was prevented by proteasome inhibitor treatment of the animal fed ethanol. Chronic alcohol feeding did not affect the gene expression of HMG-CoA synthase. However, PS341 administration significantly reduced the HMG-CoA synthase mRNA levels, confirming the results obtained with the microarray analysis. C/EBP transcription factors alpha (CCAAT/enhancer-binding protein alpha) has been shown to positively regulate SREBP-1c mRNA expression, thus regulating lipogenesis. Proteasome inhibition caused a decrease in C/EBP alpha mRNA expression, indicating that C/EBP down regulation may be the mechanism by which proteasome inhibitor treatment reduced lipogenesis. In conclusion, our results indicate that proteasome activity is not only involved in down regulating fatty acid synthesis and triacylglycerol synthesis, but also cholesterol synthesis and intestinal lipid adsorption. Proteasome inhibitor, administrated at a non-toxic low dose, played a beneficial role in reducing lipogenesis caused by chronic ethanol feeding and these beneficial effects are obtained because of the specificity and reversibility of the proteasome inhibitor used. PMID:22445925

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 our understanding of NAD biosynthesis and are valuable for manipulation of NAD homeostasis for metabolic engineering. Copyright © 2017 American Society for Microbiology.

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.

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

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.V. All rights reserved.

Biosensor reveals multiple sources for mitochondrial NAD⁺.

PubMed

Cambronne, Xiaolu A; Stewart, Melissa L; Kim, DongHo; Jones-Brunette, Amber M; Morgan, Rory K; Farrens, David L; Cohen, Michael S; Goodman, Richard H

2016-06-17

Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments. We found that the concentrations of free NAD(+) in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD(+) biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD(+) concentrations. Copyright © 2016, American Association for the Advancement of Science.

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.

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

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.

Recent advances in the study of 11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2)Inhibitors.

PubMed

Zhou, Chunchun; Ye, Fan; Wu, He; Ye, Hui; Chen, Quanxu

2017-06-01

11β-Hydroxysteroid dehydrogenase (11β-HSD), which interconverts hormonally active cortisol and inactive cortisone in multiple human tissues, has two distinct isoforms named 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) and 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2). 11β-HSD2 is an NAD + -dependent oxidase which lowers cortisol by converting it to cortisone while 11β-HSD1 mainly catalyzes the reduction which converts cortisone into cortisol. Selective inhibition of 11β-HSD2 is generally detrimental to health because the accumulation of cortisol can cause metabolic symptoms such as apparent mineralocorticoid excess (AME), fetal developmental defects and lower testosterone levels in males. There has been some advances on the study of 11β-HSD2 inhibitors and we think it necessary to make a summary of the characteristics and inhibiting properties of latest 11β-HSD2 inhibitors. As another review on 11β-HSD2 inhibitors has been issued on 2011 (see review (Ma et al., 2011)), this mini-review concerns advances during the last 5 years. Copyright © 2017 Elsevier B.V. All rights reserved.

Dihydroquinazolines as a novel class of Trypanosoma brucei trypanothione reductase inhibitors: discovery, synthesis, and characterization of their binding mode by protein crystallography.

PubMed

Patterson, Stephen; Alphey, Magnus S; Jones, Deuan C; Shanks, Emma J; Street, Ian P; Frearson, Julie A; Wyatt, Paul G; Gilbert, Ian H; Fairlamb, Alan H

2011-10-13

Trypanothione reductase (TryR) is a genetically validated drug target in the parasite Trypanosoma brucei , the causative agent of human African trypanosomiasis. Here we report the discovery, synthesis, and development of a novel series of TryR inhibitors based on a 3,4-dihydroquinazoline scaffold. In addition, a high resolution crystal structure of TryR, alone and in complex with substrates and inhibitors from this series, is presented. This represents the first report of a high resolution complex between a noncovalent ligand and this enzyme. Structural studies revealed that upon ligand binding the enzyme undergoes a conformational change to create a new subpocket which is occupied by an aryl group on the ligand. Therefore, the inhibitor, in effect, creates its own small binding pocket within the otherwise large, solvent exposed active site. The TryR-ligand structure was subsequently used to guide the synthesis of inhibitors, including analogues that challenged the induced subpocket. This resulted in the development of inhibitors with improved potency against both TryR and T. brucei parasites in a whole cell assay.

Cell proliferation and progesterone synthesis depend on lipid metabolism in bovine granulosa cells.

PubMed

Elis, Sebastien; Desmarchais, Alice; Maillard, Virginie; Uzbekova, Svetlana; Monget, Philippe; Dupont, Joëlle

2015-03-15

In dairy cows, lipids are essential to support energy supplies for all biological functions, especially during early lactation. Lipid metabolism is crucial for sustaining proper reproductive function. Alteration of lipid metabolism impacts follicular development and affects oocyte developmental competence. Indeed, nonesterified fatty acids are able to decrease granulosa cell (GC) proliferation and affect estradiol synthesis, thus potentially affecting follicular growth and viability. The objective of this study was to assess the impact of lipid metabolism on bovine GCs, through the use of the lipid metabolism inhibitors etomoxir, an inhibitor of fatty acid (FA) oxidation through inhibition of carnitine palmitoyl transferase 1 (CPT1), and C75, an inhibitor of FA synthesis through inhibition of fatty acid synthase. We showed that etomoxir and C75 significantly inhibited DNA synthesis in vitro; C75 also significantly decreased progesterone synthesis. Both inhibitors significantly reduced AMPK (5' adenosine monophosphate-activated protein kinase) and acetyl-CoA carboxylase phosphorylation. Etomoxir also affected the AKT (protein kinase B) signaling pathway. Combined, these data suggest that both FA oxidation and synthesis are important for the bovine GCs to express a proliferative and steroidogenic phenotype and, thus, for sustaining follicular growth. Despite these findings, it is important to note that the changes caused by the inhibitors of FA metabolism on GCs in vitro are globally mild, suggesting that lipid metabolism is not as critical in GCs as was observed in the oocyte-cumulus complex. Further studies are needed to investigate the detailed mechanisms by which lipid metabolism interacts with GC functions. Copyright © 2015 Elsevier Inc. All rights reserved.

Discovery of novel selenium derivatives as Pin1 inhibitors by high-throughput screening

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

Subedi, Amit; Graduate School of Science and Engineering, Saitama University, Saitama, 338-8570; Shimizu, Takeshi

2016-06-03

Peptidyl prolyl cis/trans isomerization by Pin1 regulates various oncogenic signals during cancer progression, and its inhibition through multiple approaches has established Pin1 as a therapeutic target. However, lack of simplified screening systems has limited the discovery of potent Pin1 inhibitors. We utilized phosphorylation-dependent binding of Pin1 to its specific substrate to develop a screening system for Pin1 inhibitors. Using this system, we screened a chemical library, and identified a novel selenium derivative as Pin1 inhibitor. Based on structure-activity guided chemical synthesis, we developed more potent Pin1 inhibitors that inhibited cancer cell proliferation. -- Highlights: •Novel screening for Pin1 inhibitors basedmore » on Pin1 binding is developed. •A novel selenium compound is discovered as Pin1 inhibitor. •Activity guided chemical synthesis of selenium derivatives resulted potent Pin1 inhibitors.« less

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

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

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

Inhibition of cereal rust fungi by both class I and II defensins derived from the flowers of Nicotiana alata.

PubMed

Dracatos, Peter M; van der Weerden, Nicole L; Carroll, Kate T; Johnson, Elizabeth D; Plummer, Kim M; Anderson, Marilyn A

2014-01-01

Defensins are a large family of small, cysteine-rich, basic proteins, produced by most plants and plant tissues. They have a primary function in defence against fungal disease, although other functions have been described. This study reports the isolation and characterization of a class I secreted defensin (NaD2) from the flowers of Nicotiana alata, and compares its antifungal activity with the class II defensin (NaD1) from N. alata flowers, which is stored in the vacuole. NaD2, like all other class I defensins, lacks the C-terminal pro-peptide (CTPP) characteristic of class II defensins. NaD2 is most closely related to Nt-thionin from N. tabacum (96% identical) and shares 81% identity with MtDef4 from alfalfa. The concentration required to inhibit in vitro fungal growth by 50% (IC50 ) was assessed for both NaD1 and NaD2 for the biotrophic basidiomycete fungi Puccinia coronata f. sp. avenae (Pca) and P. sorghi (Ps), the necrotrophic pathogenic ascomycetes Fusarium oxysporum f. sp. vasinfectum (Fov), F. graminearum (Fgr), Verticillium dahliae (Vd) and Thielaviopsis basicola (Tb), and the saprobe Aspergillus nidulans. NaD1 was a more potent antifungal molecule than NaD2 against both the biotrophic and necrotrophic fungal pathogens tested. NaD2 was 5-10 times less effective at killing necrotrophs, but only two-fold less effective on Puccinia species. A new procedure for testing antifungal proteins is described in this study which is applicable to pathogens with spores that are not amenable to liquid culture, such as rust pathogens. Rusts are the most damaging fungal pathogens of many agronomically important crop species (wheat, barley, oats and soybean). NaD1 and NaD2 inhibited urediniospore germination, germ tube growth and germ tube differentiation (appressoria induction) of both Puccinia species tested. NaD1 and NaD2 were fungicidal on Puccinia species and produced stunted germ tubes with a granular cytoplasm. When NaD1 and NaD2 were sprayed onto susceptible oat plants prior to the plants being inoculated with crown rust, they reduced the number of pustules per leaf area, as well as the amount of chlorosis induced by infection. Similar to observations in vitro, NaD1 was more effective as an antifungal control agent than NaD2. Further investigation revealed that both NaD1 and NaD2 permeabilized the plasma membranes of Puccinia spp. This study provides evidence that both secreted (NaD2) and nonsecreted (NaD1) defensins may be useful for broad-spectrum resistance to pathogens. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Nicotinamide riboside, a trace nutrient in foods, is a vitamin B3 with effects on energy metabolism and neuroprotection.

PubMed

Chi, Yuling; Sauve, Anthony A

2013-11-01

This review focuses upon the biology and metabolism of a trace component in foods called nicotinamide riboside. Nicotinamide riboside is a precursor of nicotinamide adenine dinucleotide (NAD), and is a source of Vitamin B3. Evidence indicates that nicotinamide riboside has unique properties as a Vitamin B3. We review knowledge of the metabolism of this substance, as well as recent work suggesting novel health benefits that might be associated with nicotinamide riboside taken in larger quantities than is found naturally in foods. Recent work investigating the effects of nicotinamide riboside in yeast and mammals established that it is metabolized by at least two types of metabolic pathways. The first of these is degradative and produces nicotinamide. The second pathway involves kinases called nicotinamide riboside kinases (Nrk1 and Nrk2, in humans). The likely involvement of the kinase pathway is implicated in the unique effects of nicotinamide riboside in raising tissue NAD concentrations in rodents and for potent effects in eliciting insulin sensitivity, mitochondrial biogenesis, and enhancement of sirtuin functions. Additional studies with nicotinamide riboside in models of Alzheimer's disease indicate bioavailability to brain and protective effects, likely by stimulation of brain NAD synthesis. Initial studies have clarified the potential for a lesser-known Vitamin B3 called nicotinamide riboside that is available in selected foods, and possibly available to humans by supplements. It has properties that are insulin sensitizing, enhancing to exercise, resisting to negative effects of high-fat diet, and neuroprotecting.

Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA) production.

PubMed

Zarei, Adel; Trobacher, Christopher P; Shelp, Barry J

2016-10-11

Polyamines represent a potential source of 4-aminobutyrate (GABA) in plants exposed to abiotic stress. Terminal catabolism of putrescine in Arabidopsis thaliana involves amine oxidase and the production of 4-aminobutanal, which is a substrate for NAD + -dependent aminoaldehyde dehydrogenase (AMADH). Here, two AMADH homologs were chosen (AtALDH10A8 and AtALDH10A9) as candidates for encoding 4-aminobutanal dehydrogenase activity for GABA synthesis. The two genes were cloned and soluble recombinant proteins were produced in Escherichia coli. The pH optima for activity and catalytic efficiency of recombinant AtALDH10A8 with 3-aminopropanal as substrate was 10.5 and 8.5, respectively, whereas the optima for AtALDH10A9 were approximately 9.5. Maximal activity and catalytic efficiency were obtained with NAD + and 3-aminopropanal, followed by 4-aminobutanal; negligible activity was obtained with betaine aldehyde. NAD + reduction was accompanied by the production of GABA and β-alanine, respectively, with 4-aminobutanal and 3-aminopropanal as substrates. Transient co-expression systems using Arabidopsis cell suspension protoplasts or onion epidermal cells and several organelle markers revealed that AtALDH10A9 was peroxisomal, but AtALDH10A8 was cytosolic, although the N-terminal 140 amino acid sequence of AtALDH10A8 localized to the plastid. Root growth of single loss-of-function mutants was more sensitive to salinity than wild-type plants, and this was accompanied by reduced GABA accumulation.

Microwave-assisted synthesis of 3-aminobenzo[b]thiophene scaffolds for the preparation of kinase inhibitors.

PubMed

Bagley, Mark C; Dwyer, Jessica E; Molina, Maria D Beltran; Rand, Alexander W; Rand, Hayley L; Tomkinson, Nicholas C O

2015-06-28

Microwave irradiation of 2-halobenzonitriles and methyl thioglycolate in the presence of triethylamine in DMSO at 130 °C provides rapid access to 3-aminobenzo[b]thiophenes in 58-96% yield. This transformation has been applied in the synthesis of the thieno[2,3-b]pyridine core motif of LIMK1 inhibitors, the benzo[4,5]thieno[3,2-e][1,4]diazepin-5(2H)-one scaffold of MK2 inhibitors and a benzo[4,5]thieno[3,2-d]pyrimidin-4-one inhibitor of the PIM kinases.

Design and synthesis of a series of serine derivatives as small molecule inhibitors of the SARS coronavirus 3CL protease.

PubMed

Konno, Hiroyuki; Wakabayashi, Masaki; Takanuma, Daiki; Saito, Yota; Akaji, Kenichi

2016-03-15

Synthesis of serine derivatives having the essential functional groups for the inhibitor of SARS 3CL protease and evaluation of their inhibitory activities using SARS 3CL R188I mutant protease are described. The lead compounds, functionalized serine derivatives, were designed based on the tetrapeptide aldehyde and Bai's cinnamoly inhibitor, and additionally performed with simulation on GOLD softwear. Structure activity relationship studies of the candidate compounds were given reasonable inhibitors ent-3 and ent-7k against SARS 3CL R188I mutant protease. These inhibitors showed protease selectivity and no cytotoxicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis of the proteinase inhibitor LEKTI domain 6 by the fragment condensation method and regioselective disulfide bond formation.

PubMed

Vasileiou, Zoe; Barlos, Kostas K; Gatos, Dimitrios; Adermann, Knut; Deraison, Celine; Barlos, Kleomenis

2010-01-01

Proteinase inhibitors are of high pharmaceutical interest and are drug candidates for a variety of indications. Specific kallikrein inhibitors are important for their antitumor activity and their potential application to the treatment of skin diseases. In this study we describe the synthesis of domain 6 of the kallikrein inhibitor Lympho-Epithilial Kazal-Type Inhibitor (LEKTI) by the fragment condensation method and site-directed cystine bridge formation. To obtain the linear LEKTI precursor, the condensation was best performed in solution, coupling the protected fragment 1-22 to 23-68. This method yielded LEKTI domain 6 of high purity and equipotent to the recombinantly produced peptide. (c) 2010 Wiley Periodicals, Inc.

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.

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.

An NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme responsive nanocarrier based on mesoporous silica nanoparticles for tumor targeted drug delivery in vitro and in vivo

NASA Astrophysics Data System (ADS)

Gayam, Srivardhan Reddy; Venkatesan, Parthiban; Sung, Yi-Ming; Sung, Shuo-Yuan; Hu, Shang-Hsiu; Hsu, Hsin-Yun; Wu, Shu-Pao

2016-06-01

The synthesis and characterization of an NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme responsive nanocarrier based on mesoporous silica nanoparticles (MSNPs) for on-command delivery applications has been described in this paper. Gatekeeping of MSNPs is achieved by the integration of mechanically interlocked rotaxane nanovalves on the surface of MSNPs. The rotaxane nanovalve system is composed of a linear stalk anchoring on the surface of MSNPs, an α-cyclodextrin ring that encircles it and locks the payload ``cargo'' molecules in the mesopores, and a benzoquinone stopper incorporated at the end of the stalk. The gate opening and controlled release of the cargo are triggered by cleavage of the benzoquinone stopper using an endogenous NQO1 enzyme. In addition to having efficient drug loading and controlled release mechanisms, this smart biocompatible carrier system showed obvious uptake and consequent release of the drug in tumor cells, could selectively induce the tumor cell death and enhance the capability of inhibition of tumor growth in vivo. The controlled drug delivery system demonstrated its use as a potential theranostic material.The synthesis and characterization of an NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme responsive nanocarrier based on mesoporous silica nanoparticles (MSNPs) for on-command delivery applications has been described in this paper. Gatekeeping of MSNPs is achieved by the integration of mechanically interlocked rotaxane nanovalves on the surface of MSNPs. The rotaxane nanovalve system is composed of a linear stalk anchoring on the surface of MSNPs, an α-cyclodextrin ring that encircles it and locks the payload ``cargo'' molecules in the mesopores, and a benzoquinone stopper incorporated at the end of the stalk. The gate opening and controlled release of the cargo are triggered by cleavage of the benzoquinone stopper using an endogenous NQO1 enzyme. In addition to having efficient drug loading and controlled release mechanisms, this smart biocompatible carrier system showed obvious uptake and consequent release of the drug in tumor cells, could selectively induce the tumor cell death and enhance the capability of inhibition of tumor growth in vivo. The controlled drug delivery system demonstrated its use as a potential theranostic material. Electronic supplementary information (ESI) available: Synthesis and characterization of the functional molecules and MSNPs is available in the ESI. See DOI: 10.1039/c6nr03525f

Benzoylurea Chitin Synthesis Inhibitors.

PubMed

Sun, Ranfeng; Liu, Chunjuan; Zhang, Hao; Wang, Qingmin

2015-08-12

Benzoylurea chitin synthesis inhibitors are widely used in integrated pest management (IPM) and insecticide resistance management (IRM) programs due to their low toxicity to mammals and predatory insects. In the past decades, a large number of benzoylurea derivatives have been synthesized, and 15 benzoylurea chitin synthesis inhibitors have been commercialized. This review focuses on the history of commercial benzolyphenylureas (BPUs), synthetic methods, structure-activity relationships (SAR), action mechanism research, environmental behaviors, and ecotoxicology. Furthermore, their disadvantages of high risk to aquatic invertebrates and crustaceans are pointed out. Finally, we propose that the para-substituents at anilide of benzoylphenylureas should be the functional groups, and bipartite model BPU analogues are discussed in an attempt to provide new insight for future development of BPUs.

Design and synthesis of functionalized piperazin-1yl-(E)-stilbenes as inhibitors of 17α-hydroxylase-C17,20-lyase (Cyp17).

PubMed

Blass, Benjamin E; Iyer, Pravin; Abou-Gharbia, Magid; Childers, Wayne E; Gordon, John C; Ramanjulu, Mercy; Morton, George; Arumugam, Premkumar; Boruwa, Joshodeep; Ellingboe, John; Mitra, Sayan; Reddy Nimmareddy, Rajashekar; Paliwal, Shalini; Rajasekhar, Jamallamudi; Shivakumar, Savithiri; Srivastava, Pratima; Tangirala, Raghuram S; Venkataramanaiah, Konda; Bobbala, Ramreddy; Yanamandra, Mahesh; Krishnakanth Reddy, L

2018-07-15

The synthesis of steroid hormones is critical to human physiology and improper regulation of either the synthesis of these key molecules or activation of the associated receptors can lead to disease states. This has led to intense interest in developing compounds capable of modulating the synthesis of steroid hormones. Compounds capable of inhibiting Cyp19 (Aromatase), a key enzyme in the synthesis of estrogens, have been successfully employed as breast cancer therapies, while inhibitors of Cyp17 (17α-hydroxylase-17,20-lyase), a key enzyme in the synthesis of glucocorticoids, mineralocorticoids and steroidal sex hormones, are a key component of prostate cancer therapy. Inhibition of CYP17 has also been suggested as a possible target for the treatment of Cushing Syndrome and Metabolic Syndrome. We have identified two novel series of stilbene based CYP17 inhibitors and demonstrated that exemplary compounds in these series have pharmacokinetic properties consistent with orally delivered drugs. These findings suggest that compounds in these classes may be useful for the treatment of diseases and conditions associated with improper regulation of glucocorticoids synthesis and glucocorticoids receptor activation. Copyright © 2018. Published by Elsevier Ltd.

Functional optical imaging of tracheal health (Conference Presentation)

NASA Astrophysics Data System (ADS)

Gil, Daniel A.; Sharick, Joe T.; Gamm, Ute A.; Choma, Michael A.; Skala, Melissa C.

2017-04-01

The health of the tracheal mucosa is an important, but poorly understood, aspect of critical care medicine. Many critical care patients are mechanically ventilated through an endotracheal tube that can cause local inflammation and blunt damage to the ciliated epithelial cells lining the trachea. These cilia clear mucus and infectious agents from the respiratory tract, so impaired ciliary function may lead to increased susceptibility to respiratory infection. Therefore, a minimally-invasive method to monitor mucosal health and ciliary function in intubated patients would be valuable to critical care medicine. Optical metabolic imaging (OMI) can quantitatively assess the metabolic state of cells by measuring the fluorescence intensities of endogenous metabolic co-enzymes NAD(P)H and FAD. OMI is especially attractive for assessing tracheal health because OMI is label-free, and ciliary function is tightly linked to the levels of NAD(P)H and FAD. In this study, we apply widefield OMI to ex vivo mouse tracheae (n=6), and demonstrate that the optical redox ratio (fluorescence intensity of NAD(P)H divided by the intensity of FAD) is sensitive to changes in the cellular metabolism of the tracheal mucosa. We observed a 46% increase in the redox ratio 20 minutes after treatment with 10mM of sodium cyanide (p<0.001, 95% CI [40%, 52%]), an inhibitor of oxidative cellular respiration. In addition to being a proof-of-concept demonstration, Pseudomonas aeruginosa, an important cause of morbidity and mortality in CF patients and in the ICU, produces hydrogen cyanide. Our results support the development of minimally-invasive fiber-optic probes for in vivo monitoring of tracheal health.

A PHGDH inhibitor reveals coordination of serine synthesis and 1-carbon unit fate

PubMed Central

Pacold, Michael E.; Brimacombe, Kyle R.; Chan, Sze Ham; Rohde, Jason M.; Lewis, Caroline A.; Swier, Lotteke J.Y.M.; Possemato, Richard; Chen, Walter W.; Sullivan, Lucas B.; Fiske, Brian P.; Cho, Sung Won; Freinkman, Elizaveta; Birsoy, Kıvanç; Abu-Remaileh, Monther; Shaul, Yoav D.; Liu, Chieh Min; Zhou, Minerva; Koh, Min Jung; Chung, Haeyoon; Davidson, Shawn M.; Luengo, Alba; Wang, Amy Q.; Xu, Xin; Yasgar, Adam; Liu, Li; Rai, Ganesha; Westover, Kenneth D.; Vander Heiden, Matthew G.; Shen, Min; Gray, Nathanael S.; Boxer, Matthew B.; Sabatini, David M.

2016-01-01

Serine is a both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical glucose-derived serine synthesis pathway, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic towards PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we use a quantitative high-throughput screen to identify small molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and suggest that one-carbon unit wasting may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo. PMID:27110680

Regulation of NAD+ metabolism, signaling and compartmentalization in the yeast Saccharomyces cerevisiae.

PubMed

Kato, Michiko; Lin, Su-Ju

2014-11-01

Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD(+) is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD(+) homeostasis is essential for proper cellular function and aberrant NAD(+) metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD(+) metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD(+) metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD(+) metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD(+) metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD(+) metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD(+)-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD(+) intermediates, and their potential roles in NAD(+) homeostasis. To date, it remains unclear how NAD(+) and NAD(+) intermediates shuttle between different cellular compartments. Together, these studies provide a molecular basis for how NAD(+) homeostasis factors and the interacting signaling pathways confer metabolic flexibility and contribute to maintaining cell fitness and genome stability. Copyright © 2014 Elsevier B.V. All rights reserved.

Regulation of NAD+ metabolism, signaling and compartmentalization in the yeast Saccharomyces cerevisiae

PubMed Central

Kato, Michiko; Lin, Su-Ju

2014-01-01

Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD+ is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD+ homeostasis is essential for proper cellular function and aberrant NAD+ metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD+ metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD+ metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD+ metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD+ metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD+ metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD+-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD+ intermediates, and their potential roles in NAD+ homeostasis. To date, it remains unclear how NAD+ and NAD+ intermediates shuttle between different cellular compartments. Together, these studies provide a molecular basis for how NAD+ homeostasis factors and the interacting signaling pathways confer metabolic flexibility and contribute to maintaining cell fitness and genome stability. PMID:25096760

High-resolution crystal structures of the photoreceptor glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with three and four-bound NAD molecules

PubMed Central

Baker, Bo Y; Shi, Wuxian; Wang, Benlian; Palczewski, Krzysztof

2014-01-01

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of d-glyceraldehyde 3-phosphate (G3P) into 1,3-diphosphoglycerate (BGP) in the presence of the NAD cofactor. GAPDH is an important drug target because of its central role in glycolysis, and nonglycolytic processes such as nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis. Recent studies found that GAPDH participates in the development of diabetic retinopathy and its progression after the cessation of hyperglycemia. Here, we report two structures for native bovine photoreceptor GAPDH as a homotetramer with differing occupancy by NAD, bGAPDH(NAD)4, and bGAPDH(NAD)3. The bGAPDH(NAD)4 was solved at 1.52 Å, the highest resolution for GAPDH. Structural comparison of the bGAPDH(NAD)4 and bGAPDH(NAD)3 models revealed novel details of conformational changes induced by cofactor binding, including a loop region (residues 54–56). Structure analysis of bGAPDH confirmed the importance of Phe34 in NAD binding, and demonstrated that Phe34 was stabilized in the presence of NAD but displayed greater mobility in its absence. The oxidative state of the active site Cys149 residue is regulated by NAD binding, because this residue was found oxidized in the absence of dinucleotide. The distance between Cys149 and His176 decreased upon NAD binding and Cys149 remained in a reduced state when NAD was bound. These findings provide an important structural step for understanding the mechanism of GAPDH activity in vision and its pathological role in retinopathies. PMID:25176140

Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition.

PubMed

Lawlis, V B; Roche, T E

1981-04-28

Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 for alpha-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.

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.

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.

Synthesis, biological evaluation, and molecular docking of Ugi products containing a zinc-chelating moiety as novel inhibitors of histone deacetylases.

PubMed

Grolla, Ambra A; Podestà, Valeria; Chini, Maria Giovanna; Di Micco, Simone; Vallario, Antonella; Genazzani, Armando A; Canonico, Pier Luigi; Bifulco, Giuseppe; Tron, Gian Cesare; Sorba, Giovanni; Pirali, Tracey

2009-05-14

HDAC inhibitors show great promise for the treatment of cancer. As part of a broader effort to explore the SAR of HDAC inhibitors, synthesis, biological evaluation, and molecular docking of novel Ugi products containing a zinc-chelating moiety are presented. One compound shows improved inhibitory potencies compared to SAHA, demonstrating that hindered lipophilic residues grafted on the peptide scaffold of the alpha-aminoacylamides can be favorable in the interaction with the enzyme.

33 CFR 110.1 - General.

Code of Federal Regulations, 2010 CFR

2010-07-01

... North American Datum of 1983 (NAD 83), unless such geographic coordinates are expressly labeled NAD 83. Geographic coordinates without the NAD 83 reference may be plotted on maps or charts referenced to NAD 83...

7 CFR 11.21 - Organization.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Organization. NAD was established on October 13, 1994. Delegation of authority to the Director, NAD, appears at..., Colorado; and the headquarters staff located in Alexandria, Virginia. NAD is headed by a Director. NAD is...

33 CFR 165.8 - Geographic coordinates.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 1983 (NAD 83), unless such geographic coordinates are expressly labeled NAD 83. Geographic coordinates without the NAD 83 reference may be plotted on maps or charts referenced to NAD 83 only after application...

The Commercial Vapor Diffusion Apparatus (CVDA) STS-95

NASA Technical Reports Server (NTRS)

2004-01-01

The Commercial Vapor Diffusion Apparatus will be used to perform 128 individual crystal growth investigations for commercial and science research. These experiments will grow crystals of several different proteins, including HIV-1 Protease Inhibitor, Glycogen Phosphorylase A, and NAD Synthetase. The Commercial Vapor Diffusion Apparatus supports multiple commercial investigations within a controlled environment. The goal of the Commercial Protein Crystal Growth payload on STS-95 is to grow large, high-quality crystals of several different proteins of interest to industry, and to continue to refine the technology and procedures used in microgravity for this important commercial research.

Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.

PubMed

Beauchamp, Justin; Vieille, Claire

2015-01-01

N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N(6)-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N(6)-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N(6)-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N(6)-amine group on NAD.

A spectrophotometric assay for fatty acid amide hydrolase suitable for high-throughput screening.

PubMed

De Bank, Paul A; Kendall, David A; Alexander, Stephen P H

2005-04-15

Signalling via the endocannabinoids anandamide and 2-arachidonylglycerol appears to be terminated largely through the action of the enzyme fatty acid amide hydrolase (FAAH). In this report, we describe a simple spectrophotometric assay to detect FAAH activity in vitro using the ability of the enzyme to hydrolyze oleamide and measuring the resultant production of ammonia with a NADH/NAD+-coupled enzyme reaction. This dual-enzyme assay was used to determine Km and Vmax values of 104 microM and 5.7 nmol/min/mgprotein, respectively, for rat liver FAAH-catalyzed oleamide hydrolysis. Inhibitor potency was determined with the resultant rank order of methyl arachidonyl fluorophosphonate>phenylmethylsulphonyl fluoride>anandamide. This assay system was also adapted for use in microtiter plates and its ability to detect a known inhibitor of FAAH demonstrated, highlighting its potential for use in high-throughput screening.

Human hydroxysteroid dehydrogenases and pre-receptor regulation: Insights into inhibitor design and evaluation

PubMed Central

Penning, Trevor M.

2011-01-01

Hydroxysteroid dehydrogenases (HSDs) represent a major class of NAD(P)(H) dependent steroid hormone oxidoreductases involved in the pre-receptor regulation of hormone action. This is achieved by HSDs working in pairs so that they can interconvert ketosteroids with hydroxysteroids resulting in a change in ligand potency for nuclear receptors. HSDs belong to two protein superfamilies the aldo-keto reductases and the short-chain dehydrogenase/reductases. In humans, many of the important enzymes have been thoroughly characterized including the elucidation of their three-dimensional structures. Because these enzymes play fundamental roles in steroid hormone action they can be considered to be drug targets for a variety of steroid driven diseases: e.g. metabolic syndrome and obesity, inflammation, and hormone dependent malignancies of the endometrium, prostate and breast. This article will review how fundamental knowledge of these enzymes can be exploited in the development of isoform specific HSD inhibitors from both protein superfamilies. PMID:21272640

33 CFR 67.50-1 - Scope.

Code of Federal Regulations, 2010 CFR

2010-07-01

... North American Datum of 1983 (NAD 83), unless such geographic coordinates are expressly labeled NAD 83. Geographic coordinates without the NAD 83 reference may be plotted on maps or charts referenced to NAD 83...

Efficacy ranking of triterpenoids as inducers of a cytoprotective enzyme and as inhibitors of a cellular inflammatory response via their electron affinity and their electrophilicity index

PubMed Central

Bensasson, René V.; Zoete, Vincent; Berthier, Gaston; Talalay, Paul; Dinkova-Kostova, Albena T.

2010-01-01

Electron affinity (EA) and electrophilicity index (ω) of 16 synthetic triterpenoids (TP), previously identified as inducers of cytoprotective enzymes and as inhibitors of cellular inflammatory responses, have been calculated by the molecular orbital method. Linear correlations were obtained by plotting the values of EA, as well as those of ω versus (i) the potencies of induction of NAD(P)H quinone reductase (NQO1, EC 1.6.99.2), a cytoprotective enzyme, expressed via the concentration of TP required to double the specific activity of NQO1 (CD value) and (ii) the values of their anti-inflammatory activity expressed via the IC-50 of TP for suppression of upregulation of inducible nitric oxide synthase (iNOS, EC 1.14.13.39), both previously experimentally determined. The observed correlations demonstrate quantitatively for a series of triterpenoids that their electrophilicity is a major factor determining their potency as inducers of the cytoprotective phase 2 response and as inhibitors of inflammatory processes. PMID:20433811

Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens.

PubMed

Tran, Mai L; McCarthy, Thomas W; Sun, Hao; Wu, Shu-Zon; Norris, Joanna H; Bezanilla, Magdalena; Vidali, Luis; Anderson, Charles T; Roberts, Alison W

2018-01-15

Results from live cell imaging of fluorescently tagged Cellulose Synthase (CESA) proteins in Cellulose Synthesis Complexes (CSCs) have enhanced our understanding of cellulose biosynthesis, including the mechanisms of action of cellulose synthesis inhibitors. However, this method has been applied only in Arabidopsis thaliana and Brachypodium distachyon thus far. Results from freeze fracture electron microscopy of protonemal filaments of the moss Funaria hygrometrica indicate that a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), fragments CSCs and clears them from the plasma membrane. This differs from Arabidopsis, in which DCB causes CSC accumulation in the plasma membrane and a different cellulose synthesis inhibitor, isoxaben, clears CSCs from the plasma membrane. In this study, live cell imaging of the moss Physcomitrella patens indicated that DCB and isoxaben have little effect on protonemal growth rates, and that only DCB causes tip rupture. Live cell imaging of mEGFP-PpCESA5 and mEGFP-PpCESA8 showed that DCB and isoxaben substantially reduced CSC movement, but had no measureable effect on CSC density in the plasma membrane. These results suggest that DCB and isoxaben have similar effects on CSC movement in P. patens and Arabidopsis, but have different effects on CSC intracellular trafficking, cell growth and cell integrity in these divergent plant lineages.

Discovery and gram-scale synthesis of BMS-593214, a potent, selective FVIIa inhibitor

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

Priestley, E. Scott; De Lucca, Indawati; Zhou, Jinglan

A 6-amidinotetrahydroquinoline screening hit was driven to a structurally novel, potent, and selective FVIIa inhibitor through a combination of library synthesis and rational design. An efficient gram-scale synthesis of the active enantiomer BMS-593214 was developed, which required significant optimization of the key Povarov annulation. Importantly, BMS-593214 showed antithrombotic efficacy in a rabbit arterial thrombosis model. A crystal structure of BMS-593214 bound to FVIIa highlights key contacts with Asp 189, Lys 192, and the S2 pocket.

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 Biology, Inc.

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

33 CFR 169.10 - What geographic coordinates are used?

Code of Federal Regulations, 2010 CFR

2010-07-01

... of 1983 (NAD 83), unless such geographic coordinates are expressly labeled NAD 83. Geographic coordinates without the NAD 83 reference may be plotted on maps or charts referenced to NAD 83 only after...

Design, synthesis, X-ray studies, and biological evaluation of novel macrocyclic HIV-1 protease inhibitors involving the P1'-P2' ligands

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

Ghosh, Arun K.; Sean Fyvie, W.; Brindisi, Margherita

Design, synthesis, and evaluation of a new class of HIV-1 protease inhibitors containing diverse flexible macrocyclic P1'-P2' tethers are reported. Inhibitor 5a with a pyrrolidinone-derived macrocycle exhibited favorable enzyme inhibitory and antiviral activity (Ki = 13.2 nM, IC50 = 22 nM). Further incorporation of heteroatoms in the macrocyclic skeleton provided macrocyclic inhibitors 5m and 5o. These compounds showed excellent HIV-1 protease inhibitory (Ki = 62 pM and 14 pM, respectively) and antiviral activity (IC50 = 5.3 nM and 2.0 nM, respectively). Inhibitor 5o also remained highly potent against a DRV-resistant HIV-1 variant.

Haemocompatibility assessment of synthesised platinum nanoparticles and its implication in biology.

PubMed

Shiny, P J; Mukherjee, Amitava; Chandrasekaran, N

2014-06-01

The growing need for advanced treatment of evolving diseases has become a motivation for this study. Among the noble metals, platinum nanoparticles are of importance because of their catalytic property, antioxidant potential, minimal toxicity and diverse applications. Biological process of synthesis has retained its significance, because it is a simple one-step process yielding stable nanoparticles. Herein, we have synthesised platinum nanoparticles through a green process using the unexplored seaweed Padina gymnospora, a brown alga. The course of synthesis was monitored and the nanoparticles were characterised using UV-visible spectroscopy. The synthesised nanoparticles were studied using TEM, XRD and FTIR. The TEM and XRD studies reveal the size of the nanoparticle to be <35 nm. The catalytic nanoparticles were capable of oxidising NADH to NAD(+). The biocompatibility was tested by haemolytic assay for the furtherance of the application of platinum nanoparticles in medicine. This is the first report on the biogenic synthesis of platinum nanoparticles using seaweed.

33 CFR 80.01 - General basis and purpose of demarcation lines.

Code of Federal Regulations, 2010 CFR

2010-07-01

... American Datum of 1983 (NAD 83), unless such geographic coordinates are expressly labeled NAD 83. Geographic coordinates without the NAD 83 reference may be plotted on maps or charts referenced to NAD 83...

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

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

Cyclooxygenase inhibitors are potent sensitizers of prostate tumours to hyperthermia and radiation.

PubMed

Asea, A; Mallick, R; Lechpammer, S; Ara, G; Teicher, B A; Fiorentino, S; Stevenson, M A; Calderwood, S K

2001-01-01

It has previously been demonstrated that hyperthermia can activate prostaglandin synthesis and that prostaglandins are protective against hyperthermia. This study examined the use of inhibitors of prostaglandin synthesis on the response of prostate tumours to hyperthermia. The non-steroidal anti-inflammatory drugs (NSAID) ibuprofen and sulindac, known cyclooxygenase inhibitors that inhibit prostaglandin production, were effective hyperthermia sensitizers and augmented growth delay of DU-145 and PC-3 prostate tumours to combined radiation and hyperthermia treatment protocols. Pre-treatment of mice with ibuprofen and sulindac at hyperthermia sensitizing doses resulted in significant (p < 0.01) inhibition of hyperthemia-induced serum prostaglandin E2. These findings indicate that NSAID may have both sensitizing effects on prostate tumour growth and may function by inhibiting prostaglandin synthesis.

Age-dependent redox status in the brain stem of NO-deficient hypertensive rats.

PubMed

Majzúnová, Miroslava; Pakanová, Zuzana; Kvasnička, Peter; Bališ, Peter; Čačányiová, Soňa; Dovinová, Ima

2017-09-11

The brain stem contains important nuclei that control cardiovascular function via the sympathetic nervous system (SNS), which is strongly influenced by nitric oxide. Its biological activity is also largely determined by oxygen free radicals. Despite many experimental studies, the role of AT1R-NAD(P)H oxidase-superoxide pathway in NO-deficiency is not yet sufficiently clarified. We determined changes in free radical signaling and antioxidant and detoxification response in the brain stem of young and adult Wistar rats during chronic administration of exogenous NO inhibitors. Young (4 weeks) and adult (10 weeks) Wistar rats were treated with 7-nitroindazole (7-NI group, 10 mg/kg/day), a specific nNOS inhibitor, with N G -nitro-L-arginine-methyl ester (L-NAME group, 50 mg/kg/day), a nonspecific NOS inhibitor, and with drinking water (Control group) during 6 weeks. Systolic blood pressure was measured by non-invasive plethysmography. Expression of genes (AT1R, AT2R, p22phox, SOD and NOS isoforms, HO-1, MDR1a, housekeeper GAPDH) was identified by real-time PCR. NOS activity was detected by conversion of [3H]-L-arginine to [3H]-L-citrulline and SOD activity was measured using UV VIS spectroscopy. We observed a blood pressure elevation and decrease in NOS activity only after L-NAME application in both age groups. Gene expression of nNOS (youngs) and eNOS (adults) in the brain stem decreased after both inhibitors. The radical signaling pathway triggered by AT1R and p22phox was elevated in L-NAME adults, but not in young rats. Moreover, L-NAME-induced NOS inhibition increased antioxidant response, as indicated by the observed elevation of mRNA SOD3, HO-1, AT2R and MDR1a in adult rats. 7-NI did not have a significant effect on AT1R-NADPH oxidase-superoxide pathway, yet it affected antioxidant response of mRNA expression of SOD1 and stimulated total activity of SOD in young rats and mRNA expression of AT2R in adult rats. Our results show that chronic NOS inhibition by two different NOS inhibitors has age-dependent effect on radical signaling and antioxidant/detoxificant response in Wistar rats. While 7-NI had neuroprotective effect in the brain stem of young Wistar rats, L-NAME- induced NOS inhibition evoked activation of AT1R-NAD(P)H oxidase pathway in adult Wistar rats. Triggering of the radical pathway was followed by activation of protective compensation mechanism at the gene expression level.

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.

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.

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.

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

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.

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.

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.

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.

Does structured patient education improve the recovery and clinical outcomes of patients with neck pain? A systematic review from the Ontario Protocol for Traffic Injury Management (OPTIMa) Collaboration.

PubMed

Yu, Hainan; Côté, Pierre; Southerst, Danielle; Wong, Jessica J; Varatharajan, Sharanya; Shearer, Heather M; Gross, Douglas P; van der Velde, Gabrielle M; Carroll, Linda J; Mior, Silvano A; Ameis, Arthur; Jacobs, Craig L; Taylor-Vaisey, Anne L

2016-12-01

In 2008, the Bone and Joint Decade 2000 to 2010 Task Force on Neck Pain and Its Associated Disorders recommended patient education for the management of neck pain. However, the effectiveness of education interventions has recently been challenged. To update the findings of the Bone and Joint Decade 2000 to 2010 Task Force on Neck Pain and Its Associated Disorders and evaluate the effectiveness of structured patient education for the management of patients with whiplash-associated disorders (WAD) or neck pain and associated disorders (NAD). Systematic review of the literature and best-evidence synthesis. Randomized controlled trials that compared structured patient education with other conservative interventions. Self-rated recovery, functional recovery (eg, disability, return to activities, work, or school), pain intensity, health-related quality of life, psychological outcomes such as depression or fear, or adverse effects. We systematically searched eight electronic databases (MEDLINE, EMBASE, CINAHL, PsycINFO, the Cochrane Central Register of Controlled Trials, DARE, PubMed, and ICL) from 2000 to 2012. Randomized controlled trials, cohort studies, and case-control studies meeting our selection criteria were eligible for critical appraisal. Random pairs of independent reviewers critically appraised eligible studies using the Scottish Intercollegiate Guidelines Network criteria. Scientifically admissible studies were summarized in evidence tables and synthesized following best-evidence synthesis principles. We retrieved 4,477 articles. Of those, nine were eligible for critical appraisal and six were scientifically admissible. Four admissible articles investigated patients with WAD and two targeted patients with NAD. All structured patient education interventions included advice on activation or exercises delivered orally combined with written information or as written information alone. Overall, as a therapeutic intervention, structured patient education was equal or less effective than other conservative treatments including massage, supervised exercise, and physiotherapy. However, structured patient education may provide small benefits when combined with physiotherapy. Either mode of delivery (ie, oral or written education) provides similar results in patients with recent WAD. This review adds to the Bone and Joint Decade 2000 to 2010 Task Force on Neck Pain and Its Associated Disorders by defining more specifically the role of structured patient education in the management of WAD and NAD. Results suggest that structured patient education alone cannot be expected to yield large benefits in clinical effectiveness compared with other conservative interventions for patients with WAD or NAD. Moreover, structured patient education may be of benefit during the recovery of patients with WAD when used as an adjunct therapy to physiotherapy or emergency room care. These benefits are small and short lived. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

Nicotinamide riboside, a form of vitamin B3, protects against excitotoxicity-induced axonal degeneration.

PubMed

Vaur, Pauline; Brugg, Bernard; Mericskay, Mathias; Li, Zhenlin; Schmidt, Mark S; Vivien, Denis; Orset, Cyrille; Jacotot, Etienne; Brenner, Charles; Duplus, Eric

2017-12-01

NAD + depletion is a common phenomenon in neurodegenerative pathologies. Excitotoxicity occurs in multiple neurologic disorders and NAD + was shown to prevent neuronal degeneration in this process through mechanisms that remained to be determined. The activity of nicotinamide riboside (NR) in neuroprotective models and the recent description of extracellular conversion of NAD + to NR prompted us to probe the effects of NAD + and NR in protection against excitotoxicity. Here, we show that intracortical administration of NR but not NAD + reduces brain damage induced by NMDA injection. Using cortical neurons, we found that provision of extracellular NR delays NMDA-induced axonal degeneration (AxD) much more strongly than extracellular NAD + Moreover, the stronger effect of NR compared to NAD + depends of axonal stress since in AxD induced by pharmacological inhibition of nicotinamide salvage, both NAD + and NR prevent neuronal death and AxD in a manner that depends on internalization of NR. Taken together, our findings demonstrate that NR is a better neuroprotective agent than NAD + in excitotoxicity-induced AxD and that axonal protection involves defending intracellular NAD + homeostasis.-Vaur, P., Brugg, B., Mericskay, M., Li, Z., Schmidt, M. S., Vivien, D., Orset, C., Jacotot, E., Brenner, C., Duplus, E. Nicotinamide riboside, a form of vitamin B 3 , protects against excitotoxicity-induced axonal degeneration. © FASEB.

Structure and Mechanism of ArnA: Conformational Change Implies Ordered Dehydrogenase Mechanism in Key Enzyme for Polymyxin Resistance

PubMed Central

Gatzeva-Topalova, Petia Z.; May, Andrew P.; Sousa, Marcelo C.

2010-01-01

Summary The modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) allows gram-negative bacteria to resist the antimicrobial activity of cationic antimicrobial peptides and antibiotics such as polymyxin. ArnA is the first enzyme specific to the lipid A-Ara4N pathway. It contains two functionally and physically separable domains: a dehydrogenase domain (ArnA_DH) catalyzing the NAD+-dependent oxidative decarboxylation of UDP-Glucuronic acid (UDP-GlcA), and a transformylase domain that formylates UDP-Ara4N. Here, we describe the crystal structure of the full-length bifunctional ArnA with UDP-GlcA and ATP bound to the dehydrogenase domain. Binding of UDP-GlcA triggers a 17 Å conformational change in ArnA_DH that opens the NAD+ binding site while trapping UDP-GlcA. We propose an ordered mechanism of substrate binding and product release. Mutation of residues R619 and S433 demonstrates their importance in catalysis and suggests that R619 functions as a general acid in catalysis. The proposed mechanism for ArnA_DH has important implications for the design of selective inhibitors. PMID:15939024

TNF-{alpha} upregulates the A{sub 2B} adenosine receptor gene: The role of NAD(P)H oxidase 4

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

St Hilaire, Cynthia; Koupenova, Milka; Carroll, Shannon H.

2008-10-24

Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-{alpha}) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A{sub 2B} adenosine receptor (A{sub 2B}AR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-{alpha}. Here, we show a regulatory loop by which TNF-{alpha} upregulates the A{sub 2B}AR in VSMC in vitromore » and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A{sub 2B}AR, and Nox inhibitors dampen the effect of TNF-{alpha}. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A{sub 2B}AR.« less

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase*

PubMed Central

Glasser, Nathaniel R.; Wang, Benjamin X.; Hoy, Julie A.; Newman, Dianne K.

2017-01-01

Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa. Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro, suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG. Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD+ (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD+ in LpdG and other enzymes, achieving the same end by a different mechanism. PMID:28174304

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase.

PubMed

Glasser, Nathaniel R; Wang, Benjamin X; Hoy, Julie A; Newman, Dianne K

2017-03-31

Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro , suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD + (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD + in LpdG and other enzymes, achieving the same end by a different mechanism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Temporal and pharmacological division of fibroblast cyclooxygenase expression into transcriptional and translational phases

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

Raz, A.; Wyche, A.; Needleman, P.

1989-03-01

The authors have recently shown that the synthesis of cyclooxygenase in human dermal fibroblasts is markedly stimulated by the cytokine interleukin 1 (IL-1). They now show that the temporal sequence of the induced synthesis of PG synthase can be separated into an early transcriptional (i.e., actinomycin D inhibitable) phase and a subsequent translational (cycloheximide but not actinomycin D inhibitable) phase and that IL-1 exerts its effect during the transcriptional phase. Phorbol 12-myristate 13-acetate also stimulates synthesis of PG synthase and, together with IL-1, produces a synergistic stimulatory effect. Inhibitors of protein kinase C activation abolished the stimulatory effect of IL-1,more » suggesting that protein kinase C activation is a critical event in the signal-transduction sequence of the IL-1-induced increase of PG synthase synthesis. The antiinflammatory glucocorticosteroids dexamethasone and triamcinolone, but not progesterone or testosterone, were potent inhibitors of PG synthase synthesis when added during the translational phase of the synthesis sequence. The glucocorticosteroid effect was blocked by RNA and protein synthesis inhibitors. This report suggests that glucocorticosteroids exert their effect via a newly synthesized protein, causing a profound translational control of PG synthase synthesis. This novel mechanism of suppression of arachidonate metabolism is distinct from any influence of steroids on phospholipase A{sub 2} activity.« less

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+ : 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.

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

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.

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

Arabinosylthymine: inhibitor of splenic lymphocyte macromolecular synthesis in vitro.

PubMed Central

Barnett, J M; McGowan, J J; Gentry, G A

1979-01-01

The effect of arabinosylthymine on lymphocyte transformation was investigated. Arabinosylthymine was demonstrated not to be cytotoxic for hamster spleen lymphocytes but was found to inhibit the increase in deoxyribonucleic acid and protein synthesis usually observed as a result of mitogen stimulation. These findings suggest that, in addition to being a potent anti-herpesvirus inhibitor, arabinosylthymine is also an immunosuppressive agent. PMID:500206

Design, synthesis and molecular simulation studies of dihydrostilbene derivatives as potent tyrosinase inhibitors.

PubMed

Vontzalidou, Argyro; Zoidis, Grigoris; Chaita, Eliza; Makropoulou, Maria; Aligiannis, Nektarios; Lambrinidis, George; Mikros, Emmanuel; Skaltsounis, Alexios-Leandros

2012-09-01

The synthesis, molecular modeling and biological evaluation of substituted deoxybenzoins and optimized dihydrostilbenes are reported. Preliminary structure-activity relationship data were elucidated and lead compounds suitable for further optimization were discovered. Dihydrostilbene 7 is a particularly potent inhibitor (IC(50)=8.44 μM, more potent than kojic acid). Copyright © 2012 Elsevier Ltd. All rights reserved.

Inhibition of Dengue Virus RNA Synthesis by an Adenosine Nucleoside ▿ †

PubMed Central

Chen, Yen-Liang; Yin, Zheng; Duraiswamy, Jeyaraj; Schul, Wouter; Lim, Chin Chin; Liu, Boping; Xu, Hao Ying; Qing, Min; Yip, Andy; Wang, Gang; Chan, Wai Ling; Tan, Hui Pen; Lo, Melissa; Liung, Sarah; Kondreddi, Ravinder Reddy; Rao, Ranga; Gu, Helen; He, Handan; Keller, Thomas H.; Shi, Pei-Yong

2010-01-01

We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-hydroxy-methyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC50) and cytotoxic concentration (CC50) values of 0.7 μM and >100 μM, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering RNA (siRNA), abolished or reduced the compound's antiviral activity in cell culture. Studies of rats showed that 14C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo. Collectively, the results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which functions as a chain terminator for viral RNA synthesis. PMID:20457821

Glutathione cycle activity and pyridine nucleotide levels in oxidant-induced injury of cells.

PubMed Central

Schraufstätter, I U; Hinshaw, D B; Hyslop, P A; Spragg, R G; Cochrane, C G

1985-01-01

Exposure of target cells to a bolus of H2O2 induced cell lysis after a latent period of several hours, which was prevented only when the H2O2 was removed within the first 30 min of injury by addition of catalase. This indicated that early metabolic events take place that are important in the fate of the cell exposed to oxidants. In this study, we described two early and independent events of H2O2-induced injury in P388D1 macrophagelike tumor cells: activation of the glutathione cycle and depletion of cellular NAD. Glutathione cycle and hexose monophosphate shunt (HMPS) were activated within seconds after the addition of H2O2. High HMPS activity maintained glutathione that was largely reduced. However, when HMPS activity was inhibited--by glucose depletion or by incubation at 4 degrees C--glutathione remained in the oxidized state. Total pyridine nucleotide levels were diminished when cells were exposed to H2O2, and the breakdown product, nicotinamide, was recovered in the extracellular medium. Intracellular NAD levels fell by 80% within 20 min of exposure of cells to H2O2. The loss of NADP(H) and stimulation of the HMPS could be prevented when the glutathione cycle was inhibited by either blocking glutathione synthesis with buthionine sulfoximine (BSO) or by inhibiting glutathione reductase with (1,3-bis) 2 chlorethyl-1-nitrosourea. The loss of NAD developed independently of glutathione cycle and HMPS activity, as it also occurred in BSO-treated cells. PMID:3840176

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.

Cellular antioxidant effects of atorvastatin in vitro and in vivo.

PubMed

Wassmann, Sven; Laufs, Ulrich; Müller, Kirsten; Konkol, Christian; Ahlbory, Katja; Bäumer, Anselm T; Linz, Wolfgang; Böhm, Michael; Nickenig, Georg

2002-02-01

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may exert direct effects on vascular cells and beneficially influence endothelial dysfunction. Because reactive oxygen species (ROS) may lead to vascular damage and dysfunction, we investigated the effect of atorvastatin on ROS production and the underlying mechanisms in vitro and in vivo. Cultured rat aortic vascular smooth muscle cells were incubated with 10 micromol/L atorvastatin. Angiotensin II-induced and epidermal growth factor-induced ROS production were significantly reduced by atorvastatin (dichlorofluorescein fluorescence laser microscopy). Atorvastatin downregulated mRNA expression of the NAD(P)H oxidase subunit nox1, whereas p22phox mRNA expression was not significantly altered (reverse transcription-polymerase chain reaction, Northern analysis). Membrane translocation of rac1 GTPase, which is required for the activation of NAD(P)H oxidase, was inhibited by atorvastatin (Western blot). mRNA expression of superoxide dismutase isoforms and glutathione peroxidase was not modified by atorvastatin, whereas catalase expression was upregulated at mRNA and protein levels, resulting in an increased enzymatic activity. Effects of atorvastatin on ROS production and nox1, rac1, and catalase expression were inhibited by L-mevalonate but not by 25-hydroxycholesterol. In addition, spontaneously hypertensive rats were treated with atorvastatin for 30 days. ROS production in aortic segments was significantly reduced in statin-treated rats (lucigenin chemiluminescence). Treatment with atorvastatin reduced vascular mRNA expression of p22phox and nox1 and increased aortic catalase expression. mRNA expression of superoxide dismutases, glutathione peroxidase, and NAD(P)H oxidase subunits gp91phox, p40phox, p47phox, and p67phox remained unchanged. Translocation of rac1 from the cytosol to the cell membrane was also reduced in vivo. Thus, atorvastatin exerts cellular antioxidant effects in cultured rat vascular smooth muscle cells and in the vasculature of spontaneously hypertensive rats mediated by decreased expression of essential NAD(P)H oxidase subunits and by upregulation of catalase expression. These effects of atorvastatin may contribute to the vasoprotective effects of statins.

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

Schuster, Susanne, E-mail: Susanne.Schuster@medizin.uni-leipzig.de; Penke, Melanie; Gorski, Theresa

Background: Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently, NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here, we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1more » (mTORC1) signaling. Results: FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells, Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN), the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hepatocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPKα activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 significantly activated AMPKα and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment. Conclusion: Taken together, these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a potential treatment option for HCC. - Highlights: • FK866 increases cell death in p53-deficient hepatocarcinoma cells. • AMPK/mTOR signaling is dysregulated in hepatocarcinoma cells. • FK866-induced NAMPT inhibition activates AMPKα and downregulates mTOR signaling. • NMN abrogates the effects of FK866-induced NAMPT inhibition. • Non-cancerous human hepatocytes are less sensitive to FK866.« less

Improvement of the Redox Balance Increases l-Valine Production by Corynebacterium glutamicum under Oxygen Deprivation Conditions

PubMed Central

Hasegawa, Satoshi; Uematsu, Kimio; Natsuma, Yumi; Suda, Masako; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki

2012-01-01

Production of l-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the l-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall l-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of l-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for l-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD+ ratio significantly decreased, and glucose consumption and l-valine production drastically improved. Moreover, l-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD+ ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for l-valine production under oxygen deprivation conditions. The l-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM l-valine after 24 h with a yield of 0.63 mol mol of glucose−1, and the l-valine productivity reached 1,940 mM after 48 h. PMID:22138982

Improvement of the redox balance increases L-valine production by Corynebacterium glutamicum under oxygen deprivation conditions.

PubMed

Hasegawa, Satoshi; Uematsu, Kimio; Natsuma, Yumi; Suda, Masako; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

2012-02-01

Production of L-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the L-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall L-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of L-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for L-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD(+) ratio significantly decreased, and glucose consumption and L-valine production drastically improved. Moreover, L-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD(+) ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for L-valine production under oxygen deprivation conditions. The L-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM L-valine after 24 h with a yield of 0.63 mol mol of glucose(-1), and the L-valine productivity reached 1,940 mM after 48 h.

Oxidoreductases Involved in Cell Carbon Synthesis of Methanobacterium thermoautotrophicum

PubMed Central

Zeikus, J. G.; Fuchs, G.; Kenealy, W.; Thauer, R. K.

1977-01-01

Cell-free extracts of Methanobacterium thermoautotrophicum were found to contain high activities of the following oxidoreductases (at 60°C): pyruvate dehydrogenase (coenzyme A acetylating), 275 nmol/min per mg of protein; α-ketoglutarate dehydrogenase (coenzyme A acylating), 100 nmol/min per mg; fumarate reductase, 360 nmol/min per mg; malate dehydrogenase, 240 nmol/min per mg; and glyceraldehyde-3-phosphate dehydrogenase, 100 nmol/min per mg. The kinetic properties (apparent Vmax and KM values), pH optimum, temperature dependence of the rate, and specificity for electron acceptors/donors of the different oxidoreductases were examined. Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase were shown to be two separate enzymes specific for factor 420 rather than for nicotinamide adenine dinucleotide (NAD), NADP, or ferredoxin as the electron acceptor. Both activities catalyzed the reduction of methyl viologen with the respective α-ketoacid and a coenzyme A-dependent exchange between the carboxyl group of the α-ketoacid and CO2. The data indicate that the two enzymes are similar to pyruvate synthase and α-ketoglutarate synthase, respectively. Fumarate reductase was found in the soluble cell fraction. This enzyme activity coupled with reduced benzyl viologen as the electron donor, but reduced factor 420, NADH, or NADPH was not effective. The cells did not contain menaquinone, thus excluding this compound as the physiological electron donor for fumarate reduction. NAD was the preferred coenzyme for malate dehydrogenase, whereas NADP was preferred for glyceraldehyde-3-phosphate dehydrogenase. The organism also possessed a factor 420-dependent hydrogenase and a factor 420-linked NADP reductase. The involvement of the described oxidoreductases in cell carbon synthesis is discussed. PMID:914779

33 CFR 166.103 - Geographic coordinates.

Code of Federal Regulations, 2010 CFR

2010-07-01

... charts whose referenced horizontal datum is the North American Datum of 1983 (NAD 83), unless such geographic coordinates are expressly labeled NAD 83. Geographic coordinates without the NAD 83 reference may be plotted on maps or charts referenced to NAD 83 only after application of the appropriate...

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 NAD{sup +} or NADH for lifespan extension under CR.« less

Synthesis of HIV-Maturation Inhibitor BMS-955176 from Betulin by an Enabling Oxidation Strategy.

PubMed

Ortiz, Adrian; Soumeillant, Maxime; Savage, Scott A; Strotman, Neil A; Haley, Matthew; Benkovics, Tamas; Nye, Jeffrey; Xu, Zhongmin; Tan, Yichen; Ayers, Sloan; Gao, Qi; Kiau, Susanne

2017-05-05

A concise and scalable second generation synthesis of HIV maturation inhibitor BMS-955176 is described. The synthesis is framed by an oxidation strategy highlighted by a Cu I mediated aerobic oxidation of betulin, a highly selective PIFA mediated dehydrogenation of an oxime, and a subsequent Lossen rearrangement which occurs through a unique reaction mechanism for the installation of the C17 amino functionality. The synthetic route proceeds in 7 steps with 47% overall yield and begins from the abundant and inexpensive natural product betulin.

Improved synthesis of ADAM10 inhibitor GI254023X.

PubMed

Hoettecke, Nicole; Ludwig, Andreas; Foro, Sabine; Schmidt, Boris

2010-01-01

The metalloproteinases ADAM10 and ADAM17 are involved in various diseases: neurodegeneration, cancer and inflammation. The inhibition of these proteases is a promising target in the treatment of inflammation and cancer. In this study, we present an improved synthesis of the ADAM10 reference inhibitor GI254023X with a higher overall yield, enhanced detection ability and increased acid stability, providing easier handling. This upscaled synthesis, free of diastereomeric intermediates, ensures single-batch identity, thus warranting its reproducibility in further biological investigations. 2010 S. Karger AG, Basel.

[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.

Design and Synthesis of New Peptidomimetics as Potential Inhibitors of MurE.

PubMed

Zivec, Matej; Turk, Samo; Blanot, Didier; Gobec, Stanislav

2011-03-01

With the continuing emergence and spread of multidrug-resistant bacteria, there is an urgent need for the development of new antimicrobial agents. One possible source of new antibacterial targets is the biosynthesis of the bacterial cell-wall peptidoglycan. The assembly of the peptide stem is carried out by four essential enzymes, known as the Mur ligases (MurC, D, E and F). We have designed and synthesised a focused library of compounds as potential inhibitors of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase (MurE) from Staphylococcus aureus. This was achieved using two approaches: (i) synthesis of transition-state analogues based on the methyleneamino core; and (ii) synthesis of MurE reaction product analogues. Two methyleneamino-based compounds are identified as initial hits for inhibitors of MurE.

33 CFR 147.10 - Establishment of safety zones.

Code of Federal Regulations, 2010 CFR

2010-07-01

... charts whose referenced horizontal datum is the North American Datum of 1983 (NAD 83), unless such geographic coordinates are expressly labeled NAD 83. Geographic coordinates without the NAD 83 reference may be plotted on maps or charts reference to NAD 83 only after application of the appropriate...

33 CFR 334.6 - Datum.

Code of Federal Regulations, 2010 CFR

2010-07-01

... datum is the North American Datum of 1983 (NAD 83), unless such geographic coordinates are expressly labeled NAD 83. Geographic coordinates without the NAD 83 reference may be plotted on maps or charts referenced to NAD 83 only after application of the appropriate corrections that are published on the...

33 CFR 162.1 - General.

Code of Federal Regulations, 2010 CFR

2010-07-01

... horizontal datum is the North American Datum of 1983 (NAD 83), unless such geographic coordinates are expressly labeled NAD 83. Geographic coordinates without the NAD 83 reference may be plotted on maps or charts referenced to NAD 83 only after application of the appropriate corrections that are published on...

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 EPCs number in healthy individuals. At last, we found that the EPC intracellular NAMPT and NAD(+) levels were reduced in T2DM patients and enhancing NAD pool elevated the circulating blood EPCs number in T2DM patients. Our results indicate that the depletion of NAD pool may contribute to the impairment of EPCs mobilization in diabetic condition, and imply the potential therapeutic value of nicotinamide in the prevention and treatment for cardiovascular complications of diabetes. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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.

Quinolinic acid induces neuritogenesis in SH-SY5Y neuroblastoma cells independently of NMDA receptor activation.

PubMed

Hernandez-Martinez, Juan-Manuel; Forrest, Caroline M; Darlington, L Gail; Smith, Robert A; Stone, Trevor W

2017-03-01

Glutamate and nicotinamide adenine dinucleotide (NAD + ) have been implicated in neuronal development and several types of cancer. The kynurenine pathway of tryptophan metabolism includes quinolinic acid (QA) which is both a selective agonist at N-methyl-D-aspartate (NMDA) receptors and also a precursor for the formation of NAD + . The effect of QA on cell survival and differentiation has therefore been examined on SH-SY5Y human neuroblastoma cells. Retinoic acid (RA, 10 μm) induced differentiation of SH-SY5Y cells into a neuronal phenotype showing neurite growth. QA (50-150 nm) also caused a concentration-dependent increase in the neurite/soma ratio, indicating differentiation. Both RA and QA increased expression of the neuronal marker β3-tubulin in whole-cell homogenates and in the neuritic fraction assessed using a neurite outgrowth assay. Expression of the neuronal proliferation marker doublecortin revealed that, unlike RA, QA did not decrease the number of mitotic cells. QA-induced neuritogenesis coincided with an increase in the generation of reactive oxygen species. Neuritogenesis was prevented by diphenylene-iodonium (an inhibitor of NADPH oxidase) and superoxide dismutase, supporting the involvement of reactive oxygen species. NMDA itself did not promote neuritogenesis and the NMDA antagonist dizocilpine (MK-801) did not prevent quinolinate-induced neuritogenesis, indicating that the effects of QA were independent of NMDA receptors. Nicotinamide caused a significant increase in the neurite/soma ratio and the expression of β3-tubulin in the neuritic fraction. Taken together, these results suggest that QA induces neuritogenesis by promoting oxidizing conditions and affecting the availability of NAD + , independently of NMDA receptors. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Reaction of uridine diphosphate galactose 4-epimerase with a suicide inactivator

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

Flentke, G.R.; Frey, P.A.

UDPgalactose 4-epimerase from Escherichia coli is rapidly inactivated by the compounds uridine 5{prime}-diphosphate chloroacetol (UDC) and uridine 5{prime}-diphosphate bromoacetol (UCB). Both UDC and UDB inactivate the enzyme in neutral solution concomitant with the appearance of chromophores absorbing maximally at 325 and 328 nm, respectively. The reaction of UDC with the enzyme follows saturation kinetics characterized by a K{sub D} of 0.110 mM and k{sub inact} of 0.84 min{sup {minus}1} at pH 8.5 and ionic strength 0.2 M. The inactivation by UDC is competitively inhibited by competitive inhibitors of UDPgalactose 4-epimerase, and it is accompanied by the tight but noncovalent bindingmore » of UDC to the enzyme in a stoichiometry of 1 mol of UDC/mol of enzyme dimer, corresponding to 1 mol of UDC/mol of enzyme-bound NAD{sup +}. The inactivation of epimerase by uridine 5{prime}-diphosphate ({sup 2}H{sub 2})chloroacetol proceeds with a primary kinetic isotope effect (k{sub H}/k{sub D}) of 1.4. The inactivation mechanism is proposed to involve a minimum of three steps: (a) reversible binding of UDC to the active site of UDPgalactose 4-epimerase; (b) enolization of the chloroacetol moiety of enzyme-bound UDC, catalyzed by an enzymic general base at the active site; (c) alkylation of the nicotinamide ring of NAD{sup +} at the active site by the chloroacetol enolate. The resulting adduct between UDC and NAD{sup +} is proposed to be the chromophore with {lambda}{sub max} at 325 nm. The enzymic general base required to facilitate proton transfer in redox catalysis by this enzyme may be the general base that facilitates enolization of the chloroacetol moiety of UDC in the inactivation reaction.« less

The sodium phosphate cotransporter family and nicotinamide phosphoribosyltransferase contribute to the daily oscillation of plasma inorganic phosphate concentration.

PubMed

Miyagawa, Atsumi; Tatsumi, Sawako; Takahama, Wako; Fujii, Osamu; Nagamoto, Kenta; Kinoshita, Emi; Nomura, Kengo; Ikuta, Kayo; Fujii, Toru; Hanazaki, Ai; Kaneko, Ichiro; Segawa, Hiroko; Miyamoto, Ken-Ichi

2018-05-01

Circulating inorganic phosphate exhibits a remarkable daily oscillation based on food intake. In humans and rodents, the daily oscillation in response to food intake may be coordinated to control the intestinal absorption, renal excretion, cellular shifts, and extracellular concentration of inorganic phosphate. However, mechanisms regulating the resulting oscillation are unknown. Here we investigated the roles of the sodium phosphate cotransporter SLC34 (Npt2) family and nicotinamide phosphoribosyltransferase (Nampt) in the daily oscillation of plasma inorganic phosphate levels. First, it is roughly linked to urinary inorganic phosphate excretion. Second, expression of renal Npt2a and Npt2c, and intestinal Npt2b proteins also exhibit a dynamic daily oscillation. Analyses of Npt2a, Npt2b, and Npt2c knockout mice revealed the importance of renal inorganic phosphate reabsorption and cellular inorganic phosphate shifts in the daily oscillation. Third, experiments in which nicotinamide and a specific Nampt inhibitor (FK866) were administered in the active and rest phases revealed that the Nampt/NAD + system is involved in renal inorganic phosphate excretion. Additionally, for cellular shifts, liver-specific Nampt deletion disturbed the daily oscillation of plasma phosphate during the rest but not the active phase. In systemic Nampt +/- mice, NAD levels were significantly reduced in the liver, kidney, and intestine, and the daily oscillation (active and rest phases) of the plasma phosphate concentration was attenuated. Thus, the Nampt/NAD + system for Npt2 regulation and cellular shifts to tissues such as the liver play an important role in generating daily oscillation of plasma inorganic phosphate levels. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Neuroglobin overexpression inhibits oxygen-glucose deprivation-induced mitochondrial permeability transition pore opening in primary cultured mouse cortical neurons.

PubMed

Yu, Zhanyang; Liu, Ning; Li, Yadan; Xu, Jianfeng; Wang, Xiaoying

2013-08-01

Neuroglobin (Ngb) is an endogenous neuroprotective molecule against hypoxic/ischemic brain injury, but the underlying mechanisms remain largely undefined. Our recent study revealed that Ngb can bind to voltage-dependent anion channel (VDAC), a regulator of mitochondria permeability transition (MPT). In this study we examined the role of Ngb in MPT pore (mPTP) opening following oxygen-glucose deprivation (OGD) in primary cultured mouse cortical neurons. Co-immunoprecipitation (Co-IP) and immunocytochemistry showed that the binding between Ngb and VDAC was increased after OGD compared to normoxia, indicating the OGD-enhanced Ngb-VDAC interaction. Ngb overexpression protected primary mouse cortical neurons from OGD-induced neuronal death, to an extent comparable to mPTP opening inhibitor, cyclosporine A (CsA) pretreatment. We further measured the role of Ngb in OGD-induced mPTP opening using Ngb overexpression and knockdown approaches in primary cultured neurons, and recombinant Ngb exposure to isolated mitochondria. Same as CsA pretreatment, Ngb overexpression significantly reduced OGD-induced mPTP opening markers including mitochondria swelling, mitochondrial NAD(+) release, and cytochrome c (Cyt c) release in primary cultured neurons. Recombinant Ngb incubation significantly reduced OGD-induced NAD(+) release and Cyt c release from isolated mitochondria. In contrast, Ngb knockdown significantly increased OGD-induced neuron death, and increased OGD-induced mitochondrial NAD(+) release and Cyt c release as well, and these outcomes could be rescued by CsA pretreatment. In summary, our results demonstrated that Ngb overexpression can inhibit OGD-induced mPTP opening in primary cultured mouse cortical neurons, which may be one of the molecular mechanisms of Ngb's neuroprotection. Copyright © 2013 Elsevier Inc. All rights reserved.

Elimination of glycerol production in anaerobic cultures of a Saccharomyces cerevisiae strain engineered to use acetic acid as an electron acceptor.

PubMed

Guadalupe Medina, Víctor; Almering, Marinka J H; van Maris, Antonius J A; Pronk, Jack T

2010-01-01

In anaerobic cultures of wild-type Saccharomyces cerevisiae, glycerol production is essential to reoxidize NADH produced in biosynthetic processes. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, the single largest fermentation process in industrial biotechnology. The present study investigates the possibility of completely eliminating glycerol production by engineering S. cerevisiae such that it can reoxidize NADH by the reduction of acetic acid to ethanol via NADH-dependent reactions. Acetic acid is available at significant amounts in lignocellulosic hydrolysates of agricultural residues. Consistent with earlier studies, deletion of the two genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase (GPD1 and GPD2) led to elimination of glycerol production and an inability to grow anaerobically. However, when the E. coli mhpF gene, encoding the acetylating NAD-dependent acetaldehyde dehydrogenase (EC 1.2.1.10; acetaldehyde+NAD++coenzyme A<-->acetyl coenzyme A+NADH+H+), was expressed in the gpd1Delta gpd2Delta strain, anaerobic growth was restored by supplementation with 2.0 g liter(-1) acetic acid. The stoichiometry of acetate consumption and growth was consistent with the complete replacement of glycerol formation by acetate reduction to ethanol as the mechanism for NADH reoxidation. This study provides a proof of principle for the potential of this metabolic engineering strategy to improve ethanol yields, eliminate glycerol production, and partially convert acetate, which is a well-known inhibitor of yeast performance in lignocellulosic hydrolysates, to ethanol. Further research should address the kinetic aspects of acetate reduction and the effect of the elimination of glycerol production on cellular robustness (e.g., osmotolerance).

NCLX protein, but not LETM1, mediates mitochondrial Ca2+ extrusion, thereby limiting Ca2+-induced NAD(P)H production and modulating matrix redox state.

PubMed

De Marchi, Umberto; Santo-Domingo, Jaime; Castelbou, Cyril; Sekler, Israel; Wiederkehr, Andreas; Demaurex, Nicolas

2014-07-18

Mitochondria capture and subsequently release Ca(2+) ions, thereby sensing and shaping cellular Ca(2+) signals. The Ca(2+) uniporter MCU mediates Ca(2+) uptake, whereas NCLX (mitochondrial Na/Ca exchanger) and LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) were proposed to exchange Ca(2+) against Na(+) or H(+), respectively. Here we study the role of these ion exchangers in mitochondrial Ca(2+) extrusion and in Ca(2+)-metabolic coupling. Both NCLX and LETM1 proteins were expressed in HeLa cells mitochondria. The rate of mitochondrial Ca(2+) efflux, measured with a genetically encoded indicator during agonist stimulations, increased with the amplitude of mitochondrial Ca(2+) ([Ca(2+)]mt) elevations. NCLX overexpression enhanced the rates of Ca(2+) efflux, whereas increasing LETM1 levels had no impact on Ca(2+) extrusion. The fluorescence of the redox-sensitive probe roGFP increased during [Ca(2+)]mt elevations, indicating a net reduction of the matrix. This redox response was abolished by NCLX overexpression and restored by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The [Ca(2+)]mt elevations were associated with increases in the autofluorescence of NAD(P)H, whose amplitude was strongly reduced by NCLX overexpression, an effect reverted by Na(+)/Ca(2+) exchange inhibition. We conclude that NCLX, but not LETM1, mediates Ca(2+) extrusion from mitochondria. By controlling the duration of matrix Ca(2+) elevations, NCLX contributes to the regulation of NAD(P)H production and to the conversion of Ca(2+) signals into redox changes. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

NCLX Protein, but Not LETM1, Mediates Mitochondrial Ca2+ Extrusion, Thereby Limiting Ca2+-induced NAD(P)H Production and Modulating Matrix Redox State*

PubMed Central

De Marchi, Umberto; Santo-Domingo, Jaime; Castelbou, Cyril; Sekler, Israel; Wiederkehr, Andreas; Demaurex, Nicolas

2014-01-01

Mitochondria capture and subsequently release Ca2+ ions, thereby sensing and shaping cellular Ca2+ signals. The Ca2+ uniporter MCU mediates Ca2+ uptake, whereas NCLX (mitochondrial Na/Ca exchanger) and LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) were proposed to exchange Ca2+ against Na+ or H+, respectively. Here we study the role of these ion exchangers in mitochondrial Ca2+ extrusion and in Ca2+-metabolic coupling. Both NCLX and LETM1 proteins were expressed in HeLa cells mitochondria. The rate of mitochondrial Ca2+ efflux, measured with a genetically encoded indicator during agonist stimulations, increased with the amplitude of mitochondrial Ca2+ ([Ca2+]mt) elevations. NCLX overexpression enhanced the rates of Ca2+ efflux, whereas increasing LETM1 levels had no impact on Ca2+ extrusion. The fluorescence of the redox-sensitive probe roGFP increased during [Ca2+]mt elevations, indicating a net reduction of the matrix. This redox response was abolished by NCLX overexpression and restored by the Na+/Ca2+ exchanger inhibitor CGP37157. The [Ca2+]mt elevations were associated with increases in the autofluorescence of NAD(P)H, whose amplitude was strongly reduced by NCLX overexpression, an effect reverted by Na+/Ca2+ exchange inhibition. We conclude that NCLX, but not LETM1, mediates Ca2+ extrusion from mitochondria. By controlling the duration of matrix Ca2+ elevations, NCLX contributes to the regulation of NAD(P)H production and to the conversion of Ca2+ signals into redox changes. PMID:24898248

AMPK and SIRT1 activation contribute to inhibition of neuroinflammation by thymoquinone in BV2 microglia.

PubMed

Velagapudi, Ravikanth; El-Bakoush, Abdelmeneim; Lepiarz, Izabela; Ogunrinade, Folashade; Olajide, Olumayokun A

2017-11-01

Thymoquinone is a known inhibitor of neuroinflammation. However, the mechanism(s) involved in its action remain largely unknown. In this study, we investigated the roles of cellular reactive oxygen species (ROS), 5' AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) in the anti-neuroinflammatory activity of thymoquinone. We investigated effects of the compound on ROS generation in LPS-activated microglia using the fluorescent 2',7'-dichlorofluorescin diacetate (DCFDA)-cellular ROS detection. Immunoblotting was used to detect protein levels of p40 phox , gp91 phox , AMPK, LKB1 and SIRT1. Additionally, ELISA and immunofluorescence were used to detect nuclear accumulation of SIRT1. NAD + /NADH assay was also performed. The roles of AMPK and SIRT1 in anti-inflammatory activity of thymoquinone were investigated using RNAi and pharmacological inhibition. Our results show that thymoquinone reduced cellular ROS generation, possibly through inhibition of p40 phox and gp91 phox protein. Treatment of BV2 microglia with thymoquinone also resulted in elevation in the levels of LKB1 and phospho-AMPK proteins. We further observed that thymoquinone reduced cytoplasmic levels and increased nuclear accumulation of SIRT1 protein and increased levels of NAD + . Results also show that the anti-inflammatory activity of thymoquinone was abolished when the expressions of AMPK and SIRT1 were suppressed by RNAi or pharmacological antagonists. Pharmacological antagonism of AMPK reversed thymoquinone-induced increase in SIRT1. Taken together, we propose that thymoquinone inhibits cellular ROS generation in LPS-activated BV2 microglia. It is also suggested that activation of both AMPK and NAD + /SIRT1 may contribute to the anti-inflammatory, but not antioxidant activity of the compound in BV2 microglia.

Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis.

PubMed

Dulermo, Thierry; Lazar, Zbigniew; Dulermo, Rémi; Rakicka, Magdalena; Haddouche, Ramedane; Nicaud, Jean-Marc

2015-09-01

The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica. Copyright © 2015. Published by Elsevier B.V.

33 CFR 100.01 - Purpose and intent.

Code of Federal Regulations, 2010 CFR

2010-07-01

... plotting on maps or charts whose referenced horizontal datum is the North American Datum of 1983 (NAD 83), unless such geographic coordinates are expressly labeled NAD 83. Geographic coordinates without the NAD 83 reference may be plotted on maps or charts referenced to NAD 83 only after application of the...

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

[Effect of K-ATP channel opener-pinacidil on the liver mitochondria function in rats with different resistance to hypoxia during stress].

PubMed

Tkachenko, H M; Kurhaliuk, N M; Vovkanych, L S

2004-01-01

We have examined the influence of ATP-sensitive potassium (KATP) channel opener pinacidil (0.06 mg/kg) and inhibitor glibenclamide (1 mg/kg) on the changes of energy metabolism in the liver of rats under the stress conditions. The rats were divided in two groups with high and low resistance to hypoxia. The stress was modeled by placing the rats in a cage filled with water and closed with a net. The distance from water to the net was only 5 cm. The effects of KATP opener pinacidil (0.06 mg/kg) and inhibitor glibenclamide (1 mg/kg) on ADP-stimulating mitochondrial respiration by Chance, calcium capacity of organellas and processes of lipid peroxidation in the liver of rats with different resistance to hypoxia under the stress condition have been investigated. We have used the next substrates of oxidation: 0.35 mM succinate and 1 mM alpha-ketoglutarate. The additional analyses were conducted with the use of inhibitors: mitochondrial enzyme complex I 10 mM rotenone and succinate dehydrohenase 2 mM malonic acid. It was shown that the stress condition evoked the succinate oxidation and the decrease of alpha-ketoglutarate efficacy, the increase of calcium mitochondrial capacity and the intensification of lipid peroxidation processes. Under the presence of succinate, the increase of O2 uptake with simultaneous decrease of ADP/O ratio in rats with high resistance under stress was observed. Simultaneously, oxidation of alpha-ketoglutarate, a NAD-dependent substrate, was inhibited. Pinacidil caused the reorganization of mitochondrial energy metabolism in favour of NAD-dependent oxidation and the improvment of the protection against stress. The decrease of the efficacy of mitochondrial energy processes functioning was shown in animals with low resistance to hypoxia. KATP channel opener pinacidil has a protective effect on the processes of mitochondrial liver energy support under stress. These changes deal with the increase of alpha-ketoglutarate oxidation (respiratory rate and ADP/O) and the decrease of lipid peroxidation processes. We concluded about protective effect ofpinacidil on mitochondrial functioning under stress.

Novel contraceptive targets to inhibit ovulation: the prostaglandin E2 pathway

PubMed Central

Duffy, Diane M.

2015-01-01

BACKGROUND Prostaglandin E2 (PGE2) is an essential intrafollicular regulator of ovulation. In contrast with the one-gene, one-protein concept for synthesis of peptide signaling molecules, production and metabolism of bioactive PGE2 requires controlled expression of many proteins, correct subcellular localization of enzymes, coordinated PGE2 synthesis and metabolism, and prostaglandin transport in and out of cells to facilitate PGE2 action and degradation. Elevated intrafollicular PGE2 is required for successful ovulation, so disruption of PGE2 synthesis, metabolism or transport may yield effective contraceptive strategies. METHODS This review summarizes case reports and studies on ovulation inhibition in women and macaques treated with cyclooxygenase inhibitors published from 1987 to 2014. These findings are discussed in the context of studies describing levels of mRNA, protein, and activity of prostaglandin synthesis and metabolic enzymes as well as prostaglandin transporters in ovarian cells. RESULTS The ovulatory surge of LH regulates the expression of each component of the PGE2 synthesis-metabolism-transport pathway within the ovulatory follicle. Data from primary ovarian cells and cancer cell lines suggest that enzymes and transporters can cooperate to optimize bioactive PGE2 levels. Elevated intrafollicular PGE2 mediates key ovulatory events including cumulus expansion, follicle rupture and oocyte release. Inhibitors of the prostaglandin-endoperoxide synthase 2 (PTGS2) enzyme (also known as cyclooxygenase-2 or COX2) reduce ovulation rates in women. Studies in macaques show that PTGS2 inhibitors can reduce the rates of cumulus expansion, oocyte release, follicle rupture, oocyte nuclear maturation and fertilization. A PTGS2 inhibitor reduced pregnancy rates in breeding macaques when administered to simulate emergency contraception. However, PTGS2 inhibition did not prevent pregnancy in monkeys when administered to simulate monthly contraceptive use. CONCLUSION PTGS2 inhibitors alone may be suitable for use as emergency contraceptives. However, drugs of this class are unlikely to be effective as monthly contraceptives. Inhibitors of additional PGE2 synthesis enzymes or modulation of PGE2 metabolism or transport also hold potential for reducing follicular PGE2 and preventing ovulation. Approaches which target multiple components of the PGE2 synthesis-metabolism-transport pathway may be required to effectively block ovulation and lead to the development of novel contraceptive options for women. Therapies which target PGE2 may also impact disorders of the uterus and could also have benefits for women's health in addition to contraception. PMID:26025453

Enhanced poly(γ-glutamic acid) production by H2 O2 -induced reactive oxygen species in the fermentation of Bacillus subtilis NX-2.

PubMed

Tang, Bao; Zhang, Dan; Li, Sha; Xu, Zongqi; Feng, Xiaohai; Xu, Hong

2016-09-01

Effects of reactive oxygen species (ROS) on cell growth and poly(γ-glutamic acid) (γ-PGA) synthesis were studied by adding hydrogen peroxide to a medium of Bacillus subtilis NX-2. After optimizing the addition concentration and time of H 2 O 2 , a maximum concentration of 33.9 g/L γ-PGA was obtained by adding 100 µM H 2 O 2 to the medium after 24 H. This concentration was 20.6% higher than that of the control. The addition of diphenyleneiodonium chloride (ROS inhibitor) can interdict the effect of H 2 O 2 -induced ROS. Transcriptional levels of the cofactors and relevant genes were also determined under ROS stress to illustrate the possible metabolic mechanism contributing to the improve γ-PGA production. The transcriptional levels of genes belonging to the tricarboxylic acid cycle and electron transfer chain system were significantly increased by ROS, which decreased the NADH/NAD + ratio and increased the ATP levels, thereby providing more reducing power and energy for γ-PGA biosynthesis. The enhanced γ-PGA synthetic genes also directly promoted the formation of γ-PGA. This study was the first to use the ROS control strategy for γ-PGA fermentation and provided valuable information on the possible mechanism by which ROS regulated γ-PGA biosynthesis in B. subtilis NX-2. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Quantification of diphtheria toxin mediated ADP-ribosylation in a solid-phase assay.

PubMed

Bachran, Christopher; Sutherland, Mark; Bachran, Diana; Fuchs, Hendrik

2007-09-01

Because of reduced vaccination programs, the number of diphtheria infections has increased in the last decade. Diphtheria toxin (DT) is expressed by Corynebacterium diphtheriae and is responsible for the lethality of diphtheria. DT inhibits cellular protein synthesis by ADP-ribosylation of the eukaryotic elongation factor 2 (eEF2). No in vitro system for the quantification of DT enzymatic activity exists. We developed a solid-phase assay for the specific detection of ADP-ribosylation by DT. Solid phase-bound his-tag eEF2 is ADP-ribosylated by toxins using biotinylated NAD(+) as substrate, and the transferred biotinylated ADP-ribose is detected by streptavidin-peroxidase. DT enzymatic activity correlated with absorbance. We measured the amount of ADP-ribosylated eEF2 after precipitation with streptavidin-Sepharose. Quantification was done after Western blotting and detection with anti-his-tag antibody using an LAS-1000 System. The assay detected enzymatically active DT at 30 ng/L, equivalent to 5 mU/L ADP-ribosylating activity. Pseudomonas exotoxin A (PE) activity was also detected at 100 ng/L. We verified the assay with chimeric toxins composed of the catalytic domain of DT or PE and a tumor-specific ligand. These chimeric toxins revealed increased signals at 1000 ng/L. Heat-inactivated DT and cholera toxin that ADP-ribosylates G-proteins did not show any signal increase. The assay may be the basis for the development of a routine diagnostic assay for the detection of DT activity and highly specific inhibitors of DT.

New PARP targets for cancer therapy

PubMed Central

Vyas, Sejal; Chang, Paul

2015-01-01

Poly(ADP-ribose) polymerases (PARPs) modify target proteins post-translationally with poly(ADP-ribose) (PAR) or mono(ADP-ribose) (MAR) using NAD+ as substrate. The best-studied PARPs generate PAR modifications and include PARP1 and the tankyrase PARP5a, both of which are targets for cancer therapy with inhibitors in either clinical trials or preclinical development. There are 15 additional PARPs, the majority of which modify proteins with MAR, and their biology is less well understood. Recent data identify potentially cancer relevant functions for these PARPs, indicating that we need to understand more about these PARPs in order to target them effectively. PMID:24898058

Neo-synthesis of estrogenic or androgenic neurosteroids determine whether long-term potentiation or depression is induced in hippocampus of male rat.

PubMed

Di Mauro, Michela; Tozzi, Alessandro; Calabresi, Paolo; Pettorossi, Vito Enrico; Grassi, Silvarosa

2015-01-01

Estrogenic and androgenic steroids synthesized in the brain may rapidly modulate synaptic plasticity interacting with specific membrane receptors. We explored by electrophysiological recordings in hippocampal slices of male rat the influence of 17β-estradiol (E2) and 5α-dihydrotestosterone (DHT) neo-synthesis on the synaptic changes induced in the CA1 region. Induction of long-term depression (LTD) and depotentiation (DP) by low frequency stimulation (LFS, 15 min-1 Hz) and of long-term potentiation (LTP) by high frequency stimulation (HFS, 1 s-100 Hz), medium (MFS, 1 s-50 Hz), or weak (WFS, 1 s-25 Hz) frequency stimulation was assayed under inhibitors of enzymes converting testosterone (T) into DHT (5α-reductase) and T into E2 (P450-aromatase). We found that LFS-LTD depends on DHT synthesis, since it was fully prevented under finasteride, an inhibitor of DHT synthesis, and rescued by exogenous DHT, while the E2 synthesis was not involved. Conversely, the full development of HFS-LTP requires the synthesis of E2, as demonstrated by the LTP reduction observed under letrozole, an inhibitor of E2 synthesis, and its full rescue by exogenous E2. For intermediate stimulation protocols DHT, but not E2 synthesis, was involved in the production of a small LTP induced by WFS, while the E2 synthesis was required for the MFS-dependent LTP. Under the combined block of DHT and E2 synthesis all stimulation frequencies induced partial LTP. Overall, these results indicate that DHT is required for converting the partial LTP into LTD whereas E2 is needed for the full expression of LTP, evidencing a key role of the neo-synthesis of sex neurosteroids in determining the direction of synaptic long-term effects.

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.

Nicotinamide riboside is uniquely and orally bioavailable in mice and humans.

PubMed

Trammell, Samuel A J; Schmidt, Mark S; Weidemann, Benjamin J; Redpath, Philip; Jaksch, Frank; Dellinger, Ryan W; Li, Zhonggang; Abel, E Dale; Migaud, Marie E; Brenner, Charles

2016-10-10

Nicotinamide riboside (NR) is in wide use as an NAD + precursor vitamin. Here we determine the time and dose-dependent effects of NR on blood NAD + metabolism in humans. We report that human blood NAD + can rise as much as 2.7-fold with a single oral dose of NR in a pilot study of one individual, and that oral NR elevates mouse hepatic NAD + with distinct and superior pharmacokinetics to those of nicotinic acid and nicotinamide. We further show that single doses of 100, 300 and 1,000 mg of NR produce dose-dependent increases in the blood NAD + metabolome in the first clinical trial of NR pharmacokinetics in humans. We also report that nicotinic acid adenine dinucleotide (NAAD), which was not thought to be en route for the conversion of NR to NAD + , is formed from NR and discover that the rise in NAAD is a highly sensitive biomarker of effective NAD + repletion.

Synthesis and Biochemical Evaluation of Thiochromanone Thiosemicarbazone Analogues as Inhibitors of Cathepsin L

PubMed Central

2012-01-01

A series of 36 thiosemicarbazone analogues containing the thiochromanone molecular scaffold functionalized primarily at the C-6 position were prepared by chemical synthesis and evaluated as inhibitors of cathepsins L and B. The most promising inhibitors from this group are selective for cathepsin L and demonstrate IC50 values in the low nanomolar range. In nearly all cases, the thiochromanone sulfide analogues show superior inhibition of cathepsin L as compared to their corresponding thiochromanone sulfone derivatives. Without exception, the compounds evaluated were inactive (IC50 > 10000 nM) against cathepsin B. The most potent inhibitor (IC50 = 46 nM) of cathepsin L proved to be the 6,7-difluoro analogue 4. This small library of compounds significantly expands the structure–activity relationship known for small molecule, nonpeptidic inhibitors of cathepsin L. PMID:24900494

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

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

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.

Hexavalent chromium emissions from aerospace operations: A case study

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

Chaurushia, A.; Bajza, C.

1994-12-31

Northrop Aircraft Division (NAD) is subject to several air toxic regulations such as EPA SARA Title 3, California Assembly Bill 2588 (AB2588), and Proposition 65 and is a voluntary participant in air toxic emissions reduction programs such as the EPA 33/50 and MERIT Program. To quantify emissions, NAD initially followed regulatory guidelines which recommend that emission inventories of air toxics be based on engineering assumptions and conservative emission factors in absence of specific source test data. NAD was concerned that Chromium VI emissions from NAD`s spray coating and chemical tank line operations were not representative due to these techniques. Moremore » recently, NAD has relied upon information from its ongoing source testing program to determine emission rates of Chromium VI. Based on these source test results, NAD revised emission calculations for use in Chromium VI inventories, impact assessments and control strategies. NAD has been successful in demonstrating a significant difference between emissions calculated utilizing the source test results and emissions based on the traditional mass balance using agency suggested methods.« less

Synthesis of Barbiturate-Based Methionine Aminopeptidase-1 Inhibitors

PubMed Central

Haldar, Manas K.; Scott, Michael D.; Sule, Nitesh; Srivastava, D. K.; Mallik, Sanku

2008-01-01

The syntheses of a new class of barbiturate-based inhibitors for human and E. Coli Methionine Aminopeptidase -1 (MetAP-1) are described. Some of the synthesized inhibitors show selective inhibition of the human enzyme with high potency. PMID:18343108

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

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.

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.

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

Design of HIV-1 Protease Inhibitors with Amino-bis-tetrahydrofuran Derivatives as P2-Ligands to Enhance Backbone-Binding Interactions. Synthesis, Biological Evaluation, and Protein-Ligand X-ray Studies

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

Ghosh, Arun K.; Martyr, Cuthbert D.; Osswald, Heather L.

Structure-based design, synthesis, and biological evaluation of a series of very potent HIV-1 protease inhibitors are described. In an effort to improve backbone ligand–binding site interactions, we have incorporated basic-amines at the C4 position of the bis-tetrahydrofuran (bis-THF) ring. We speculated that these substituents would make hydrogen bonding interactions in the flap region of HIV-1 protease. Synthesis of these inhibitors was performed diastereoselectively. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 25f, 25i, and 25j were evaluated against a number of highly-PI-resistant HIV-1 strains, and they exhibited improved antiviral activity over darunavir. Two high resolutionmore » X-ray structures of 25f- and 25g-bound HIV-1 protease revealed unique hydrogen bonding interactions with the backbone carbonyl group of Gly48 as well as with the backbone NH of Gly48 in the flap region of the enzyme active site. These ligand–binding site interactions are possibly responsible for their potent activity.« 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. These findings have a high translational potential as the pharmacologic strategy of increasing NAD(+) precursors are feasible in humans. © 2016 American Heart Association, Inc.

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.

Synthesis of (S)-(+)-decursin and its analogues as potent inhibitors of melanin formation in B16 murine melanoma cells.

PubMed

Lee, Kyeong; Lee, Jee-Hyun; Boovanahalli, Shanthaveerappa K; Choi, Yongseok; Choo, Soo-Jin; Yoo, Ick-dong; Kim, Dong Hee; Yun, Mi Young; Lee, Gye Won; Song, Gyu-Yong

2010-12-01

We report the synthesis of a novel series of highly potent melanin inhibitors which were obtained through structural modification of an anticancer compound S-(+)-decursinol. The in vitro inhibitory potencies of the newly synthesized compounds were evaluated against α-MSH induced melanin production in B16 murine melanoma cells. Among the compounds evaluated, compounds 2, 3, 6b, 7a, 7b, 8a and 8b emerged as highly potent inhibitors of melanin production. Besides, these compounds demonstrated significantly low cytotoxicity. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

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.

Cell cycle progression is regulated by intertwined redox oscillators.

PubMed

da Veiga Moreira, Jorgelindo; Peres, Sabine; Steyaert, Jean-Marc; Bigan, Erwan; Paulevé, Loïc; Nogueira, Marcel Levy; Schwartz, Laurent

2015-05-29

The different phases of the eukaryotic cell cycle are exceptionally well-preserved phenomena. DNA decompaction, RNA and protein synthesis (in late G1 phase) followed by DNA replication (in S phase) and lipid synthesis (in G2 phase) occur after resting cells (in G0) are committed to proliferate. The G1 phase of the cell cycle is characterized by an increase in the glycolytic metabolism, sustained by high NAD+/NADH ratio. A transient cytosolic acidification occurs, probably due to lactic acid synthesis or ATP hydrolysis, followed by cytosolic alkalinization. A hyperpolarized transmembrane potential is also observed, as result of sodium/potassium pump (NaK-ATPase) activity. During progression of the cell cycle, the Pentose Phosphate Pathway (PPP) is activated by increased NADP+/NADPH ratio, converting glucose 6-phosphate to nucleotide precursors. Then, nucleic acid synthesis and DNA replication occur in S phase. Along with S phase, unpublished results show a cytosolic acidification, probably the result of glutaminolysis occurring during this phase. In G2 phase there is a decrease in NADPH concentration (used for membrane lipid synthesis) and a cytoplasmic alkalinization occurs. Mitochondria hyperfusion matches the cytosolic acidification at late G1/S transition and then triggers ATP synthesis by oxidative phosphorylation. We hypothesize here that the cytosolic pH may coordinate mitochondrial activity and thus the different redox cycles, which in turn control the cell metabolism.

Role of quinate dehydrogenase in quinic acid metabolism in conifers

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

Osipov, V.I.; Shein, I.V.

1986-08-10

Quinate dehydrogenase was isolated from young needles of the Siberian larch and partially purified by ammonium sulfate fractionation. It was found that in conifers, in contrast to other plants, quinate dehydrogenase is active both with NAD and with NADP. The values of K/sub m/ for quinate and NADP were 1.8 and 0.18 mM. The enzyme exhibits maximum activity at pH 9.0. It was assumed that NADP-dependent quinate dehydrogenase is responsible for quinic acid synthesis. The special features of the organization and regulation of the initial stages of the shikimate pathway in conifers are discussed.

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…

FR258900, a novel glycogen phosphorylase inhibitor isolated from Fungus No. 138354. I. Taxonomy, fermentation, isolation and biological activities.

PubMed

Furukawa, Shigetada; Tsurumi, Yasuhisa; Murakami, Kana; Nakanishi, Tomoko; Ohsumi, Keisuke; Hashimoto, Michizane; Nishikawa, Motoaki; Takase, Shigehiro; Nakayama, Osamu; Hino, Motohiro

2005-08-01

FR258900 is a novel glycogen synthesis activator produced by Fungus No. 138354. This compound was isolated from the culture broth by solvent extraction and reverse-phase column chromatography. FR258900 stimulated glycogen synthesis and glycogen synthase activity in primary rat hepatocytes. FR258900 exhibited a potent inhibitory effect on the activity of liver glycogen phosphorylase, suggesting that this compound may activate hepatic glycogen synthesis via glycogen phosphorylase inhibition. Thus, this glycogen phosphorylase inhibitor may be useful in the treatment of postprandial hyperglycemia in type 2 diabetes.

Chemoprevention of Prostate Cancer by Phenethyl Isothiocyanate

DTIC Science & Technology

2007-03-01

AD cells were exposed to PEITC, protein synthesis inhibitor cycloheximide (CHX), or proteasome inhibitor MG-132 alone, or to the combination of...The results indicated that down-regulation of Sp1 may require protein synthesis for a proteasome activity, thereby enhancing Sp1 protein degradation...carcinogen N- methyl-N- nitrosourea (MNU) for the formation of prostate tumors. The experimental group of rats was provided with PEITC, to evaluate the

Inhibition of Fatty Acid Synthesis Induces Apoptosis of Human Pancreatic Cancer Cells.

PubMed

Nishi, Koji; Suzuki, Kenta; Sawamoto, Junpei; Tokizawa, Yuma; Iwase, Yumiko; Yumita, Nagahiko; Ikeda, Toshihiko

2016-09-01

Cancer cells tend to have a high requirement for lipids, including fatty acids, cholesterol and triglyceride, because of their rapid proliferative rate compared to normal cells. In this study, we investigated the effects of inhibition of lipid synthesis on the proliferation and viability of human pancreatic cancer cells. Of the inhibitors of lipid synthesis that were tested, 5-(tetradecyloxy)-2-furoic acid (TOFA), which is an inhibitor of acetyl-CoA carboxylase, and the fatty acid synthase (FAS) inhibitors cerulenin and irgasan, significantly suppressed the proliferation of MiaPaCa-2 and AsPC-1 cells. Treatment of MiaPaCa-2 cells with these inhibitors significantly increased the number of apoptotic cells. In addition, TOFA increased caspase-3 activity and induced cleavage of poly (ADP-ribose) polymerase in MiaPaCa-2 cells. Moreover, addition of palmitate to MiaPaCa-2 cells treated with TOFA rescued cells from apoptotic cell death. These results suggest that TOFA induces apoptosis via depletion of fatty acids and that, among the various aspects of lipid metabolism, inhibition of fatty acid synthesis may be a notable target for the treatment of human pancreatic cancer cells. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Homogeneous screening assay for human tankyrase.

PubMed

Narwal, Mohit; Fallarero, Adyary; Vuorela, Pia; Lehtiö, Lari

2012-06-01

Tankyrase, a member of human PARP protein superfamily, catalyzes a covalent post-translational modification of substrate proteins. This modification, poly(ADP-ribos)ylation, leads to changes in protein interactions and modifies downstream signaling events. Tankyrase 1 is a potential drug target due to its functions in telomere homeostasis and in Wnt signaling. We describe here optimization and application of an activity-based homogenous assay for tankyrase inhibitors in a high-throughput screening format. The method measures the consumption of substrate by the chemical conversion of the remaining NAD(+) into a stable fluorescent condensation product. Conditions were optimized to measure the enzymatic auto-modification of a recombinant catalytic fragment of tankyrase 1. The fluorescence assay is inexpensive, operationally easy and performs well according to the statistical analysis (Z'= 0.7). A validatory screen with a natural product library confirmed suitability of the assay for finding new tankyrase inhibitors. Flavone was the most potent (IC(50)=325 nM) hit from the natural compounds. A flavone derivative, apigenin, and isopropyl gallate showed potency on the micromolar range, but displayed over 30-fold selectivity for tankyrase over the studied isoenzymes PARP1 and PARP2. The assay is robust and will be useful for screening new tankyrase inhibitors.

Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

PubMed

Clemens, Michael J; Elia, Androulla; Morley, Simon J

2013-01-01

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

Regulation of aflatoxin biosynthesis: effect of glucose on activities of various glycolytic enzymes.

PubMed Central

Buchanan, R L; Lewis, D F

1984-01-01

Catabolism of carbohydrates has been implicated in the regulation of aflatoxin synthesis. To characterize this effect further, the activities of various enzymes associated with glucose catabolism were determined in Aspergillus parasiticus organisms that were initially cultured in peptone-mineral salts medium and then transferred to glucose-mineral salts and peptone-mineral salts media. After an initial increase in activity, the levels of glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and malate dehydrogenase were lowered in the presence of glucose. Phosphofructokinase activity was greater in the peptone-grown mycelium, but fructose diphosphatase was largely unaffected by carbon source. Likewise, carbon source had relatively little effect on the activities of pyruvate kinase, malic enzyme, isocitrate-NADP dehydrogenase, and isocitrate-NAD dehydrogenase. The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes. PMID:6091545

Biological-inorganic hybrid systems as a generalized platform for chemical production.

PubMed

Nangle, Shannon N; Sakimoto, Kelsey K; Silver, Pamela A; Nocera, Daniel G

2017-12-01

An expanding renewable energy market to supplant petrochemicals has motivated synthesis technologies that use renewable feedstocks, such as CO 2 . Hybrid biological-inorganic systems provide a sustainable, efficient, versatile, and inexpensive chemical synthesis platform. These systems comprise biocompatible electrodes that transduce electrical energy either directly or indirectly into bioavailable energy, such as H 2 and NAD(P)H. In combination, specific bacteria use these energetic reducing equivalents to fix CO 2 into multi-carbon organic compounds. As hybrid biological-inorganic technologies have developed, the focus has shifted from phenomenological and proof-of-concept discovery towards enhanced energy efficiency, production rate, product scope, and industrial robustness. In this review, we highlight the progress and the state-of-the-art of this field and describe the advantages and challenges involved in designing bio- and chemo- compatible systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

Light- induced electron transfer and ATP synthesis in a carotene synthesizing insect

NASA Astrophysics Data System (ADS)

Valmalette, Jean Christophe; Dombrovsky, Aviv; Brat, Pierre; Mertz, Christian; Capovilla, Maria; Robichon, Alain

2012-08-01

A singular adaptive phenotype of a parthenogenetic insect species (Acyrthosiphon pisum) was selected in cold conditions and is characterized by a remarkable apparition of a greenish colour. The aphid pigments involve carotenoid genes well defined in chloroplasts and cyanobacteria and amazingly present in the aphid genome, likely by lateral transfer during evolution. The abundant carotenoid synthesis in aphids suggests strongly that a major and unknown physiological role is related to these compounds beyond their canonical anti-oxidant properties. We report here that the capture of light energy in living aphids results in the photo induced electron transfer from excited chromophores to acceptor molecules. The redox potentials of molecules involved in this process would be compatible with the reduction of the NAD+ coenzyme. This appears as an archaic photosynthetic system consisting of photo-emitted electrons that are in fine funnelled into the mitochondrial reducing power in order to synthesize ATP molecules.

Stimulatory effects of calcium on respiration and NAD(P)H synthesis in intact rat heart mitochondria utilizing physiological substrates cannot explain respiratory control in vivo.

PubMed

Vinnakota, Kalyan C; Dash, Ranjan K; Beard, Daniel A

2011-09-02

Mitochondrial TCA cycle dehydrogenase enzymes have been shown to be stimulated by Ca(2+) under various substrate and ADP incubation conditions in an attempt to determine and understand the role of Ca(2+) in maintaining energy homeostasis in working hearts. In this study, we tested the hypothesis that, at physiological temperature and 1 mM extramitochondrial free magnesium, Ca(2+) can stimulate the overall mitochondrial NAD(P)H generation flux in rat heart mitochondria utilizing pyruvate and malate as substrates at both subsaturating and saturating concentrations. In both cases, we found that, in the physiological regime of mitochondrial oxygen consumption observed in the intact animal and in the physiological range of cytosolic Ca(2+) concentration averaged per beat, Ca(2+) had no observable stimulatory effect. A modest apparent stimulatory effect (22-27%) was observable at supraphysiological maximal ADP-stimulated respiration at 2.5 mM initial phosphate. The stimulatory effects observed over the physiological Ca(2+) range are not sufficient to make a significant contribution to the control of oxidative phosphorylation in the heart in vivo.

Stimulatory Effects of Calcium on Respiration and NAD(P)H Synthesis in Intact Rat Heart Mitochondria Utilizing Physiological Substrates Cannot Explain Respiratory Control in Vivo*

PubMed Central

Vinnakota, Kalyan C.; Dash, Ranjan K.; Beard, Daniel A.

2011-01-01

Mitochondrial TCA cycle dehydrogenase enzymes have been shown to be stimulated by Ca2+ under various substrate and ADP incubation conditions in an attempt to determine and understand the role of Ca2+ in maintaining energy homeostasis in working hearts. In this study, we tested the hypothesis that, at physiological temperature and 1 mm extramitochondrial free magnesium, Ca2+ can stimulate the overall mitochondrial NAD(P)H generation flux in rat heart mitochondria utilizing pyruvate and malate as substrates at both subsaturating and saturating concentrations. In both cases, we found that, in the physiological regime of mitochondrial oxygen consumption observed in the intact animal and in the physiological range of cytosolic Ca2+ concentration averaged per beat, Ca2+ had no observable stimulatory effect. A modest apparent stimulatory effect (22–27%) was observable at supraphysiological maximal ADP-stimulated respiration at 2.5 mm initial phosphate. The stimulatory effects observed over the physiological Ca2+ range are not sufficient to make a significant contribution to the control of oxidative phosphorylation in the heart in vivo. PMID:21757763

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.

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.

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

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.

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.

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.

Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed.

PubMed

Wang, Xiu-Ling; Hu, Zi-Ying; You, Chun-Xiang; Kong, Xiu-Zhen; Shi, Xiao-Pu

2013-09-01

Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Structural Implications for Selective Targeting of PARPs.

PubMed

Steffen, Jamin D; Brody, Jonathan R; Armen, Roger S; Pascal, John M

2013-12-20

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that use NAD(+) as a substrate to synthesize polymers of ADP-ribose (PAR) as post-translational modifications of proteins. PARPs have important cellular roles that include preserving genomic integrity, telomere maintenance, transcriptional regulation, and cell fate determination. The diverse biological roles of PARPs have made them attractive therapeutic targets, which have fueled the pursuit of small molecule PARP inhibitors. The design of PARP inhibitors has matured over the past several years resulting in several lead candidates in clinical trials. PARP inhibitors are mainly used in clinical trials to treat cancer, particularly as sensitizing agents in combination with traditional chemotherapy to reduce side effects. An exciting aspect of PARP inhibitors is that they are also used to selectivity kill tumors with deficiencies in DNA repair proteins (e.g., BRCA1/2) through an approach termed "synthetic lethality." In the midst of the tremendous efforts that have brought PARP inhibitors to the forefront of modern chemotherapy, most clinically used PARP inhibitors bind to conserved regions that permits cross-selectivity with other PARPs containing homologous catalytic domains. Thus, the differences between therapeutic effects and adverse effects stemming from pan-PARP inhibition compared to selective inhibition are not well understood. In this review, we discuss current literature that has found ways to gain selectivity for one PARP over another. We furthermore provide insights into targeting other domains that make up PARPs, and how new classes of drugs that target these domains could provide a high degree of selectivity by affecting specific cellular functions. A clear understanding of the inhibition profiles of PARP inhibitors will not only enhance our understanding of the biology of individual PARPs, but may provide improved therapeutic options for patients.

Structural Implications for Selective Targeting of PARPs

PubMed Central

Steffen, Jamin D.; Brody, Jonathan R.; Armen, Roger S.; Pascal, John M.

2013-01-01

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that use NAD+ as a substrate to synthesize polymers of ADP-ribose (PAR) as post-translational modifications of proteins. PARPs have important cellular roles that include preserving genomic integrity, telomere maintenance, transcriptional regulation, and cell fate determination. The diverse biological roles of PARPs have made them attractive therapeutic targets, which have fueled the pursuit of small molecule PARP inhibitors. The design of PARP inhibitors has matured over the past several years resulting in several lead candidates in clinical trials. PARP inhibitors are mainly used in clinical trials to treat cancer, particularly as sensitizing agents in combination with traditional chemotherapy to reduce side effects. An exciting aspect of PARP inhibitors is that they are also used to selectivity kill tumors with deficiencies in DNA repair proteins (e.g., BRCA1/2) through an approach termed “synthetic lethality.” In the midst of the tremendous efforts that have brought PARP inhibitors to the forefront of modern chemotherapy, most clinically used PARP inhibitors bind to conserved regions that permits cross-selectivity with other PARPs containing homologous catalytic domains. Thus, the differences between therapeutic effects and adverse effects stemming from pan-PARP inhibition compared to selective inhibition are not well understood. In this review, we discuss current literature that has found ways to gain selectivity for one PARP over another. We furthermore provide insights into targeting other domains that make up PARPs, and how new classes of drugs that target these domains could provide a high degree of selectivity by affecting specific cellular functions. A clear understanding of the inhibition profiles of PARP inhibitors will not only enhance our understanding of the biology of individual PARPs, but may provide improved therapeutic options for patients. PMID:24392349

Photolabeling of Glu-129 of the S-1 subunit of pertussis toxin with NAD

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

Barbieri, J.T.; Mende-Mueller, L.M.; Rappuoli, R.

1989-11-01

UV irradiation was shown to induce efficient transfer of radiolabel from nicotinamide-labeled NAD to a recombinant protein (C180 peptide) containing the catalytic region of the S-1 subunit of pertussis toxin. Incorporation of label from (3H-nicotinamide)NAD was efficient (0.5 to 0.6 mol/mol of protein) relative to incorporation from (32P-adenylate)NAD (0.2 mol/mol of protein). Label from (3H-nicotinamide)NAD was specifically associated with Glu-129. Replacement of Glu-129 with glycine or aspartic acid made the protein refractory to photolabeling with (3H-nicotinamide)NAD, whereas replacement of a nearby glutamic acid, Glu-139, with serine did not. Photolabeling of the C180 peptide with NAD is similar to that observedmore » with diphtheria toxin and exotoxin A of Pseudomonas aeruginosa, in which the nicotinamide portion of NAD is transferred to Glu-148 and Glu-553, respectively, in the two toxins. These results implicate Glu-129 of the S-1 subunit as an active-site residue and a potentially important site for genetic modification of pertussis toxin for development of an acellular vaccine against Bordetella pertussis.« less

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.

Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829.

PubMed

Ali, Thanaa Hamed; El-Ghonemy, Dina Helmy

2016-06-01

The present study was conducted to investigate a new pathway for the degradation of nicotinamide adenine dinucleotide (NAD) by Penicillium brevicompactum NRC 829 extracts. Enzymes involved in the hydrolysis of NAD, i.e. alkaline phosphatase, aminohydrolase and glycohydrolase were determined. Alkaline phosphatase was found to catalyse the sequential hydrolysis of two phosphate moieties of NAD molecule to nicotinamide riboside plus adenosine. Adenosine was then deaminated by aminohydrolase to inosine and ammonia. While glycohydrolase catalyzed the hydrolysis of the nicotinamide-ribosidic bond of NAD+ to produce nicotinamide and ADP-ribose in equimolar amounts, enzyme purification through a 3-step purification procedure revealed the existence of two peaks of alkaline phosphatases, and one peak contained deaminase and glycohydrolase activities. NAD deaminase was purified to homogeneity as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with an apparent molecular mass of 91 kDa. Characterization and determination of some of NAD aminohydrolase kinetic properties were conducted due to its biological role in the regulation of cellular NAD level. The results also revealed that NAD did not exert its feedback control on nicotinamide amidase produced by P. brevicompactum.

Rational Design Synthesis and Evaluation of New Selective Inhibitors of Microbial Class II (Zinc Dependent) Fructose Bis-phosphate Aldolases

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

R Daher; M Coincon; M Fonvielle

2011-12-31

We report the synthesis and biochemical evaluation of several selective inhibitors of class II (zinc dependent) fructose bis-phosphate aldolases (Fba). The products were designed as transition-state analogues of the catalyzed reaction, structurally related to the substrate fructose bis-phosphate (or sedoheptulose bis-phosphate) and based on an N-substituted hydroxamic acid, as a chelator of the zinc ion present in active site. The compounds synthesized were tested on class II Fbas from various pathogenic microorganisms and, by comparison, on a mammalian class I Fba. The best inhibitor shows Ki against class II Fbas from various pathogens in the nM range, with very highmore » selectivity (up to 105). Structural analyses of inhibitors in complex with aldolases rationalize and corroborate the enzymatic kinetics results. These inhibitors represent lead compounds for the preparation of new synthetic antibiotics, notably for tuberculosis prophylaxis.« less

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.

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.

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

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.

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

Discovery of 8-Membered Ring Sulfonamides as Inhibitors of Oncogenic Mutant Isocitrate Dehydrogenase 1.

PubMed

Law, Jason M; Stark, Sebastian C; Liu, Ke; Liang, Norah E; Hussain, Mahmud M; Leiendecker, Matthias; Ito, Daisuke; Verho, Oscar; Stern, Andrew M; Johnston, Stephen E; Zhang, Yan-Ling; Dunn, Gavin P; Shamji, Alykhan F; Schreiber, Stuart L

2016-10-13

Evidence suggests that specific mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) are critical for the initiation and maintenance of certain tumor types and that inhibiting these mutant enzymes with small molecules may be therapeutically beneficial. In order to discover mutant allele-selective IDH1 inhibitors with chemical features distinct from existing probes, we screened a collection of small molecules derived from diversity-oriented synthesis. The assay identified compounds that inhibit the IDH1-R132H mutant allele commonly found in glioma. Here, we report the discovery of a potent (IC 50 = 50 nM) series of IDH1-R132H inhibitors having 8-membered ring sulfonamides as exemplified by the compound BRD2879. The inhibitors suppress ( R )-2-hydroxyglutarate production in cells without apparent toxicity. Although the solubility and pharmacokinetic properties of the specific inhibitor BRD2879 prevent its use in vivo , the scaffold presents a validated starting point for the synthesis of future IDH1-R132H inhibitors having improved pharmacological properties.

A conserved NAD+ binding pocket that regulates protein-protein interactions during aging

PubMed Central

Li, Jun; Bonkowski, Michael S.; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P.; Ling, Alvin J. Y.; Rajman, Luis A.; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L.; Steegborn, Clemens; Sinclair, David A.

2017-01-01

DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD+ (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD+ to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate–ribose) polymerase], a critical DNA repair protein. As mice age and NAD+ concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD+. Thus, NAD+ directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. PMID:28336669

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.

Inhibitory effects of Aphanizomenon flos-aquae constituents on human UDP-glucose dehydrogenase activity.

PubMed

Scoglio, Stefano; Lo Curcio, Valeria; Catalani, Simona; Palma, Francesco; Battistelli, Serafina; Benedetti, Serena

2016-12-01

The purpose of this study was to investigate the in vitro inhibitory effects of the edible microalga Aphanizomenon flos-aquae (AFA) on human UDP-α-d-glucose 6-dehydrogenase (UGDH) activity, a cytosolic enzyme involved both in tumor progression and in phytochemical bioavailability. Both the hydrophilic and ethanolic AFA extracts as well as the constitutive active principles phycocyanin (PC), phycocyanobilin (PCB) and mycosporine-like amino acids (MAAs) were tested. Among AFA components, PCB presented the strongest inhibitory effect on UGDH activity, acting as a competitive inhibitor with respect to UDP-glucose and a non-competitive inhibitor with respect to NAD(+). In preliminary experiments, AFA PCB was also effective in reducing the colony formation capacity of PC-3 prostate cancer cells and FTC-133 thyroid cancer cells. Overall, these findings confirmed that AFA and its active principles are natural compounds with high biological activity. Further studies evaluating the effects of AFA PCB in reducing tumor cell growth and phytochemical glucuronidation are encouraged.

Phenazine redox cycling enhances anaerobic survival in Pseudomonas aeruginosa by facilitating generation of ATP and a proton-motive force

PubMed Central

Glasser, Nathaniel R.; Kern, Suzanne E.

2014-01-01

Summary While many studies have explored the growth of Pseudomonas aeruginosa, comparatively few have focused on its survival. Previously, we reported that endogenous phenazines support the anaerobic survival of P. aeruginosa, yet the physiological mechanism underpinning survival was unknown. Here, we demonstrate that phenazine redox cycling enables P. aeruginosa to oxidize glucose and pyruvate into acetate, which promotes survival by coupling acetate and ATP synthesis through the activity of acetate kinase. By measuring intracellular NAD(H) and ATP concentrations, we show that survival is correlated with ATP synthesis, which is tightly coupled to redox homeostasis during pyruvate fermentation but not during arginine fermentation. We also show that ATP hydrolysis is required to generate a proton-motive force using the ATP synthase complex during fermentation. Together, our results suggest that phenazines enable maintenance of the proton-motive force by promoting redox homeostasis and ATP synthesis. This work demonstrates the more general principle that extracellular redox-active molecules, such as phenazines, can broaden the metabolic versatility of microorganisms by facilitating energy generation. PMID:24612454

40 CFR 725.421 - Introduced genetic material.

Code of Federal Regulations, 2010 CFR

2010-07-01

... elongation factor 2, leading to inhibition of protein synthesis in target respiratory, heart, kidney, and... protein synthesis inhibitor. Sequence Source Toxin Name Corynebacterium diphtheriae & C. ulcerans...

Synthesis and sensing integration: A novel enzymatic reaction modulated Nanoclusters Beacon (NCB) "Illumination" strategy for label-free biosensing and logic gate operation.

PubMed

Hong, Lu; Zhou, Fu; Wang, Guangfeng; Zhang, Xiaojun

2016-12-15

A novel fluorescent label-free "turn-on" NAD(+) and adenosine triphosphate (ATP) biosensing strategy is proposed by fully exploiting ligation triggered Nanocluster Beacon (NCB). In the presence of the target, the split NCB was brought to intact, which brought the C-rich sequence and enhancer sequence in close proximity resulting in the lightening of dark DNA/AgNCs ("On" mode). Further application was presented for logic gate operation and aptasensor construction. The feasibility was investigated by Ultraviolet-visible spectroscopy (UV-vis), Fluorescence, lifetime and High Resolution Transmission Electron Microscopy (HRTEM) etc. The strategy displayed good performance in the detection of NAD(+) and ATP, with the detection limit of 0.002nM and 0.001mM, the linear range of 10-1000nM and 0.003-0.01mM, respectively. Due to the DNA/AgNCs as fluorescence reporter, the completely label-free fluorescent strategy boasts the features of simplicity and low cost, and showing little reliance on the sensing environment. Meanwhile, the regulation by overhang G-rich sequence not relying on Förster energy transfer quenching manifests the high signal-to-background ratios (S/B ratios). This method not only provided a simple, economical and reliable fluorescent NAD(+) assay but also explored a flexible G-rich sequence regulated NCB probe for the fluorescent biosensors. Furthermore, this sensing mode was expanded to the application of a logic gate design, which exhibited a high performance for not only versatile biosensors construction but also for molecular computing application. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of a nicotinamide/nicotinate mononucleotide adenylyltransferase in Giardia lamblia (GlNMNAT).

PubMed

Forero-Baena, Nicolás; Sánchez-Lancheros, Diana; Buitrago, July Constanza; Bustos, Victor; Ramírez-Hernández, María Helena

2015-01-01

Giardia lamblia is an intestinal protozoan parasite that causes giardiasis, a disease of high prevalence in Latin America, Asia and Africa. Giardiasis leads to poor absorption of nutrients, severe electrolyte loss and growth retardation. In addition to its clinical importance, this parasite is of special biological interest due to its basal evolutionary position and simplified metabolism, which has not been studied thoroughly. One of the most important and conserved metabolic pathways is the biosynthesis of nicotinamide adenine dinucleotide (NAD). This molecule is widely known as a coenzyme in multiple redox reactions and as a substrate in cellular processes such as synthesis of Ca 2+ mobilizing agents, DNA repair and gene expression regulation. There are two pathways for NAD biosynthesis, which converge at the step catalyzed by nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18). Using bioinformatics tools, we found two NMNAT sequences in Giardia lamblia ( glnmnat-a and glnmnat-b ). We first verified the identity of the sequences in silico . Subsequently, glnmnat-a was cloned into an expression vector. The recombinant protein (His-GlNMNAT) was purified by nickel-affinity binding and was used in direct in vitro enzyme assays assessed by C18-HPLC, verifying adenylyltransferase activity with both nicotinamide (NMN) and nicotinic acid (NAMN) mononucleotides. Optimal reaction pH and temperature were 7.3 and 26 °C. Michaelis-Menten kinetics were observed for NMN and ATP, but saturation was not accomplished with NAMN, implying low affinity yet detectable activity with this substrate. Double-reciprocal plots showed no cooperativity for this enzyme. This represents an advance in the study of NAD metabolism in Giardia spp.

78 FR 48421 - Publication of North American Datum of 1983 (2011) Epoch 2010.00, North American Datum of 1983...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-08-08

... of 1983'' (or ``NAD 83''). The new realizations are NAD 83 (2011) epoch 2010.00 [for the North America and Caribbean tectonic plates], NAD 83 (MA11) epoch 2010.00 [for the Mariana tectonic plate] and NAD 83 (PA11) epoch 2010.00 [for the Pacific tectonic plate]. These three realizations supersede all...

Synthesis and Biological Evaluation of Benzochromenopyrimidinones as Cholinesterase Inhibitors and Potent Antioxidant, Non-Hepatotoxic Agents for Alzheimer's Disease.

PubMed

Dgachi, Youssef; Bautista-Aguilera, Oscar M; Benchekroun, Mohamed; Martin, Hélène; Bonet, Alexandre; Knez, Damijan; Godyń, Justyna; Malawska, Barbara; Gobec, Stanislav; Chioua, Mourad; Janockova, Jana; Soukup, Ondrej; Chabchoub, Fakher; Marco-Contelles, José; Ismaili, Lhassane

2016-05-14

We report herein the straightforward two-step synthesis and biological assessment of novel racemic benzochromenopyrimidinones as non-hepatotoxic, acetylcholinesterase inhibitors with antioxidative properties. Among them, compound 3Bb displayed a mixed-type inhibition of human acetylcholinesterase (IC50 = 1.28 ± 0.03 μM), good antioxidant activity, and also proved to be non-hepatotoxic on human HepG2 cell line.

Direct reductive amination of aldehyde bisulfite adducts induced by 2-picoline borane: application to the synthesis of a DPP-IV inhibitor.

PubMed

Faul, Margaret; Larsen, Rob; Levinson, Adam; Tedrow, Jason; Vounatsos, Filisaty

2013-02-15

Aldehyde-bisulfite adducts dervied from unstable parent aldehydes were reductively alkylated in a direct fashion with a variety of amines. This approach features the use of 2-picoline borane as the reducing agent and a protic solvent for the reaction media and has been successfully applied to the synthesis of a DPP-IV inhibitor and a variety of other amines.

Novel diamide-based inhibitors of IMPDH.

PubMed

Gu, Henry H; Iwanowicz, Edwin J; Guo, Junqing; Watterson, Scott H; Shen, Zhongqi; Pitts, William J; Dhar, T G Murali; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Witmer, Mark; Tredup, Jeffrey; Hollenbaugh, Diane

2002-05-06

A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase is described. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are presented.

Treatment with protein synthesis inhibitors improves outcomes from secondary bacterial pneumonia following influenza

PubMed Central

Karlström, Åsa; Boyd, Kelli L.; English, B. Keith; McCullers, Jonathan A.

2008-01-01

Pneumonia as a secondary infection after influenza is a major cause of excess morbidity and mortality despite the availability and use of antibiotics active against Streptococcus pneumoniae. We hypothesized that use of a bacteriostatic protein synthesis inhibitor would improve outcomes by reducing the inflammatory response. BALB/c mice infected with influenza virus and super-infected with Streptococcus pneumoniae were treated with either the cell wall active antibiotic ampicillin or protein synthesis inhibitors clindamycin or azithromycin. Ampicillin therapy performed significantly worse (56% survival) in the model compared to clindamycin therapy either alone (82%) or in combination with ampicillin (80%) and to azithromycin (92%). Improved survival appeared to be mediated by decreased inflammation manifested as lower levels of inflammatory cells and pro-inflammatory cytokines in the lungs, and less severe histopathology. These data suggest that beta-lactam therapy may not be optimal as first line treatment of community acquired pneumonia when it follows influenza. PMID:19113989

Microwave-Assisted Synthesis of a MK2 Inhibitor by Suzuki-Miyaura Coupling for Study in Werner Syndrome Cells.

PubMed

Bagley, Mark C; Baashen, Mohammed; Chuckowree, Irina; Dwyer, Jessica E; Kipling, David; Davis, Terence

2015-06-03

Microwave-assisted Suzuki-Miyaura cross-coupling reactions have been employed towards the synthesis of three different MAPKAPK2 (MK2) inhibitors to study accelerated aging in Werner syndrome (WS) cells, including the cross-coupling of a 2-chloroquinoline with a 3-pyridinylboronic acid, the coupling of an aryl bromide with an indolylboronic acid and the reaction of a 3-amino-4-bromopyrazole with 4-carbamoylphenylboronic acid. In all of these processes, the Suzuki-Miyaura reaction was fast and relatively efficient using a palladium catalyst under microwave irradiation. The process was incorporated into a rapid 3-step microwave-assisted method for the synthesis of a MK2 inhibitor involving 3-aminopyrazole formation, pyrazole C-4 bromination using N-bromosuccinimide (NBS), and Suzuki-Miyaura cross-coupling of the pyrazolyl bromide with 4-carbamoylphenylboronic acid to give the target 4-arylpyrazole in 35% overall yield, suitable for study in WS cells.

Podoscyphic acid, a new inhibitor of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptase from a Podoscypha species.

PubMed

Erkel, G; Anke, T; Velten, R; Steglich, W

1991-01-01

A novel enzyme inhibitor of RNA-directed DNA-polymerases of avian myeloblastosis and murine leukemia virus was isolated from fermentations of an tasmanian Podoscypha species. Its structure was elucidated by spectroscopic methods and oxidative degradation as (E)-4,5-dioxo-2-hexadecenoic acid (1). The enzyme inhibitor, which was named podoscyphic acid, did not inhibit DNA and RNA synthesis in permeabilized L 1210 cells nor did it affect RNA synthesis in isolated nuclei of L 1210 cells. 1 inhibits protein synthesis in whole L 1210 cells and rabbit reticulocyte lysate and shows very weak antimicrobial and cytotoxic properties. The testing of ethyl (E)-4,5-dioxo-2-hexadecenoate (2) and (E)-4-oxo-2-tetradecenoic acid (11) revealed the importance of the free gamma-oxoacrylic acid unit for the biological activities of 1.

Design and Synthesis of 4-Heteroaryl 1,2,3,4-Tetrahydroisoquinolines as Triple Reuptake Inhibitors

PubMed Central

2014-01-01

A series of 4-bicyclic heteroaryl 1,2,3,4-tetrahydroisoquinoline inhibitors of the serotonin transporter (SERT), norepinephrine transporter (NET), and dopamine transporter (DAT) was discovered. The synthesis and structure–activity relationship (SAR) of these triple reuptake inhibitors (TRIs) will be discussed. Compound 10i (AMR-2), a very potent inhibitor of SERT, NET, and DAT, showed efficacy in the rat forced-swim and mouse tail suspension models with minimum effective doses of 0.3 and 1 mg/kg (po), respectively. At efficacious doses in these assays, 10i exhibited substantial occupancy levels at the three transporters in both rat and mouse brain. The study of the metabolism of 10i revealed the formation of a significant active metabolite, compound 13. PMID:25050161

Design and synthesis of 4-heteroaryl 1,2,3,4-tetrahydroisoquinolines as triple reuptake inhibitors.

PubMed

Liu, Shuang; Zha, Congxiang; Nacro, Kassoum; Hu, Min; Cui, Wenge; Yang, Yuh-Lin; Bhatt, Ulhas; Sambandam, Aruna; Isherwood, Matthew; Yet, Larry; Herr, Michael T; Ebeltoft, Sarah; Hassler, Carla; Fleming, Linda; Pechulis, Anthony D; Payen-Fornicola, Anne; Holman, Nicholas; Milanowski, Dennis; Cotterill, Ian; Mozhaev, Vadim; Khmelnitsky, Yuri; Guzzo, Peter R; Sargent, Bruce J; Molino, Bruce F; Olson, Richard; King, Dalton; Lelas, Snjezana; Li, Yu-Wen; Johnson, Kim; Molski, Thaddeus; Orie, Anitra; Ng, Alicia; Haskell, Roy; Clarke, Wendy; Bertekap, Robert; O'Connell, Jonathan; Lodge, Nicholas; Sinz, Michael; Adams, Stephen; Zaczek, Robert; Macor, John E

2014-07-10

A series of 4-bicyclic heteroaryl 1,2,3,4-tetrahydroisoquinoline inhibitors of the serotonin transporter (SERT), norepinephrine transporter (NET), and dopamine transporter (DAT) was discovered. The synthesis and structure-activity relationship (SAR) of these triple reuptake inhibitors (TRIs) will be discussed. Compound 10i (AMR-2), a very potent inhibitor of SERT, NET, and DAT, showed efficacy in the rat forced-swim and mouse tail suspension models with minimum effective doses of 0.3 and 1 mg/kg (po), respectively. At efficacious doses in these assays, 10i exhibited substantial occupancy levels at the three transporters in both rat and mouse brain. The study of the metabolism of 10i revealed the formation of a significant active metabolite, compound 13.

Metabolic Interplay between Peroxisomes and Other Subcellular Organelles Including Mitochondria and the Endoplasmic Reticulum

PubMed Central

Wanders, Ronald J. A.; Waterham, Hans R.; Ferdinandusse, Sacha

2016-01-01

Peroxisomes are unique subcellular organelles which play an indispensable role in several key metabolic pathways which include: (1.) etherphospholipid biosynthesis; (2.) fatty acid beta-oxidation; (3.) bile acid synthesis; (4.) docosahexaenoic acid (DHA) synthesis; (5.) fatty acid alpha-oxidation; (6.) glyoxylate metabolism; (7.) amino acid degradation, and (8.) ROS/RNS metabolism. The importance of peroxisomes for human health and development is exemplified by the existence of a large number of inborn errors of peroxisome metabolism in which there is an impairment in one or more of the metabolic functions of peroxisomes. Although the clinical signs and symptoms of affected patients differ depending upon the enzyme which is deficient and the extent of the deficiency, the disorders involved are usually (very) severe diseases with neurological dysfunction and early death in many of them. With respect to the role of peroxisomes in metabolism it is clear that peroxisomes are dependent on the functional interplay with other subcellular organelles to sustain their role in metabolism. Indeed, whereas mitochondria can oxidize fatty acids all the way to CO2 and H2O, peroxisomes are only able to chain-shorten fatty acids and the end products of peroxisomal beta-oxidation need to be shuttled to mitochondria for full oxidation to CO2 and H2O. Furthermore, NADH is generated during beta-oxidation in peroxisomes and beta-oxidation can only continue if peroxisomes are equipped with a mechanism to reoxidize NADH back to NAD+, which is now known to be mediated by specific NAD(H)-redox shuttles. In this paper we describe the current state of knowledge about the functional interplay between peroxisomes and other subcellular compartments notably the mitochondria and endoplasmic reticulum for each of the metabolic pathways in which peroxisomes are involved. PMID:26858947

Design, Synthesis, and Biological Activity of 1,2,3-Triazolobenzodiazepine BET Bromodomain Inhibitors.

PubMed

Sharp, Phillip P; Garnier, Jean-Marc; Hatfaludi, Tamas; Xu, Zhen; Segal, David; Jarman, Kate E; Jousset, Hélène; Garnham, Alexandra; Feutrill, John T; Cuzzupe, Anthony; Hall, Peter; Taylor, Scott; Walkley, Carl R; Tyler, Dean; Dawson, Mark A; Czabotar, Peter; Wilks, Andrew F; Glaser, Stefan; Huang, David C S; Burns, Christopher J

2017-12-14

A number of diazepines are known to inhibit bromo- and extra-terminal domain (BET) proteins. Their BET inhibitory activity derives from the fusion of an acetyl-lysine mimetic heterocycle onto the diazepine framework. Herein we describe a straightforward, modular synthesis of novel 1,2,3-triazolobenzodiazepines and show that the 1,2,3-triazole acts as an effective acetyl-lysine mimetic heterocycle. Structure-based optimization of this series of compounds led to the development of potent BET bromodomain inhibitors with excellent activity against leukemic cells, concomitant with a reduction in c- MYC expression. These novel benzodiazepines therefore represent a promising class of therapeutic BET inhibitors.

ROLE OF NEUROTRANSMITTERS AND PROTEIN SYNTHESIS IN SHORT- AND LONG-TERM MEMORY

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

Bennett, E.L.; Rosenzweig, M.R.; Flood, J.F.

1978-10-01

Anisomycin is an effective inhibitor of cerebral protein synthesis in mice and is also an effective amnestic agent for both passive and active behavioral tasks. From use of anisomycin in combination with a variety of stimulant and depressant drugs, we conclude that the level of arousal following acquisition plays an important role in determining the duration and the rate of the biosynthetic phase of memory formation. While we have interpreted the experiments with anisomycin as evidence for an essential role of protein in memory storage, others have suggested that side effects of inhibitors of protein synthesis on catecholamine metabolism aremore » the main cause of amnesia. Several experiments were therefore done to compare the effects of anisemycin and catecholamine inhibitors on memory. We conclude that anisomycin's principal amnestic mechanism does not involve inhibition of the catecholamine system. The results strengthen our conclusion that protein synthesis is an essential component for longterm memory trace formation. Also, it is suggested that proteins synthesized in the neuronal cell body are used, in conjunction with other molecules, to produce permanent and semi-permanent anatomical changes.« less

Comparison of specificity of inhibition of polyamine synthesis in bovine lymphocytes by ethylglyoxal bis(guanylhydrazone) and methylglyoxal bis(guanylhydrazone).

PubMed

Igarashi, K; Porter, C W; Morris, D R

1984-11-01

Ethylglyoxal bis(guanylhydrazone) (EGBG) was compared as an inhibitor of polyamine biosynthesis with methylglyoxal bis(guanylhydrazone) (MGBG) in bovine small lymphocytes stimulated by concanavalin A. EGBG brought about a decrease in spermidine and spermine levels equal to that found with MGBG, but at a 5-fold lower intracellular drug concentration. Despite identical polyamine levels, the degree of inhibition of DNA and protein synthesis by EGBG was smaller than that observed with MGBG, in either the presence or absence of the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. It was found that in vitro protein synthesis and in vivo mitochondrial function were inhibited by concentrations of MGBG necessary to inhibit polyamine synthesis in cells (1 to 3 mM), but not by efficacious levels of EGBG (0.2 to 0.6 mM). These results suggest that EGBG is more suitable as a specific inhibitor of polyamine biosynthesis and that use of this drug, rather than MGBG, in combination with alpha-difluoromethylornithine may be useful for studying the physiological functions of polyamines in animal cells.

Genetic variability among Schistosoma japonicum isolates from the Philippines, Japan and China revealed by sequence analysis of three mitochondrial genes.

PubMed

Chen, Fen; Li, Juan; Sugiyama, Hiromu; Zhou, Dong-Hui; Song, Hui-Qun; Zhao, Guang-Hui; Zhu, Xing-Quan

2015-02-01

The present study examined sequence variability in the mitochondrial (mt) protein-coding genes cytochrome b (cytb), NADH dehydrogenase subunits 2 and 6 (nad2 and nad6) among 24 isolates of Schistosoma japonicum from different endemic regions in the Philippines, Japan and China. The complete cytb, nad2 and nad6 genes were amplified and sequenced separately from individual schistosome. Sequence variations for isolates from the Philippines were 0-0.5% for cytb, 0-0.6% for nad2, and 0-0.9% for nad6. Variation was 0-0.5%, 0.1-0.8%, 0-0.7% for corresponding genes for schistosome samples from mainland China. For worms in Japan, genetic variations were 0-0.2%, 0.1-0.2% and 0 for the three genes, respectively. Sequence variations were 0-1.0%, 0-1.8% and 0-1.1% for cytb, nad2 and nad6, respectively, among schistosome isolates from different geographical strains in the Philippines, Japan and China. Of the three countries, lowest sequence variations were found between isolates from mainland China and the Philippines and highest were detected between Japan and the Philippines in three mtDNA genes. Phylogenetic analyses based on the combined sequences of cytb, nad2 and nad6 revealed that all isolates in the Philippines clustered together sistered to samples from Yunnan and Zhejiang provinces in China, while isolates from Yamanashi in Japan were in a solitary clade. These results demonstrated the usefulness of the combined three mtDNA sequences for studying genetic diversity and population structure among S. japonicum isolates from the Philippines, China and Japan.

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.

Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function.

PubMed

Niehaus, Thomas D; Elbadawi-Sidhu, Mona; Huang, Lili; Prunetti, Laurence; Gregory, Jesse F; de Crécy-Lagard, Valérie; Fiehn, Oliver; Hanson, Andrew D

2018-06-29

NAD(P)H-hydrate epimerase (EC 5.1.99.6) is known to help repair NAD(P)H hydrates (NAD(P)HX), which are damage products existing as R and S epimers. The S epimer is reconverted to NAD(P)H by a dehydratase; the epimerase facilitates epimer interconversion. Epimerase deficiency in humans causes a lethal disorder attributed to NADHX accumulation. However, bioinformatic evidence suggest caution about this attribution by predicting that the epimerase has a second function connected to vitamin B 6 (pyridoxal 5'-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B 6 salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B 6 -related genes in bacteria, and (iii) epimerase and B 6 -related genes are coexpressed in yeast and Arabidopsis The predicted second function was explored in Escherichia coli , whose epimerase and dehydratase are fused and encoded by yjeF The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in E. coli YjeF). Changing this residue to alanine cut in vitro epimerase activity by ≥95% but did not affect dehydratase activity. Mutant cells carrying the K192A mutation had essentially normal NAD(P)HX dehydratase activity and NAD(P)HX levels, showing that the mutation had little impact on NAD(P)HX repair in vivo However, these cells showed metabolome changes, particularly in amino acids, which exceeded those in cells lacking the entire yjeF gene. The K192A mutant cells also had reduced levels of 'free' (i.e. weakly bound or unbound) pyridoxal 5'-phosphate. These results provide circumstantial evidence that the epimerase has a metabolic function beyond NAD(P)HX repair and that this function involves vitamin B 6 . © 2018 The Author(s).

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

Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function

PubMed Central

Niehaus, Thomas D.; Elbadawi-Sidhu, Mona; Huang, Lili; Prunetti, Laurence; Gregory, Jesse F.; de Crécy-Lagard, Valérie; Fiehn, Oliver; Hanson, Andrew D.

2018-01-01

NAD(P)H-hydrate epimerase (EC 5.1.99.6) is known to help repair NAD(P)H hydrates (NAD(P)HX), which are damage products existing as R and S epimers. The S epimer is reconverted to NAD(P)H by a dehydratase; the epimerase facilitates epimer interconversion. Epimerase deficiency in humans causes a lethal disorder attributed to NADHX accumulation. However, bioinformatic evidence suggest caution about this attribution by predicting that the epimerase has a second function connected to vitamin B6 (pyridoxal 5′-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B6 salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B6-related genes in bacteria, and (iii) epimerase and B6-related genes are coexpressed in yeast and Arabidopsis. The predicted second function was explored in Escherichia coli, whose epimerase and dehydratase are fused and encoded by yjeF. The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in E. coli YjeF). Changing this residue to alanine cut in vitro epimerase activity by ≥95% but did not affect dehydratase activity. Mutant cells carrying the K192A mutation had essentially normal NAD(P)HX dehydratase activity and NAD(P)HX levels, showing that the mutation had little impact on NAD(P)HX repair in vivo. However, these cells showed metabolome changes, particularly in amino acids, which exceeded those in cells lacking the entire yjeF gene. The K192A mutant cells also had reduced levels of ‘free’ (i.e. weakly bound or unbound) pyridoxal 5'-phosphate. These results provide circumstantial evidence that the epimerase has a metabolic function beyond NAD(P)HX repair and that this function involves vitamin B6. PMID:29654173