Photocatalyzed Anaerobic Oxidation of Nicotinamide Coenzyme Dimers to NAD+ by Adriamycin

The nicotinamide adenine dinucleotide dimers (NAD)₂ obtained by electrochemical reduction of NAD⁺ are oxidized by adriamycin in anaerobic photocatalyzed reaction yielding NAD⁺ and 7-deoxyadriamyci-none. Under the same conditions NADH is not oxidized.     

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

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.     

Metabolism of NAD+ in Nuclei of Saccharomyces cerevisiae during Stimulation of Its Biosynthesis by Nicotinamide

The activities of nuclear enzymes involved in NAD⁺ metabolism in Saccharomyces cerevisiae strain 913a-1 and its mutant 110 previously selected as an NAD⁺ producer were investigated. The presence of extracellular nicotinamide increased the total NAD⁺ pool in the cells and increased [³H]nicotinic acid incorporation; however, NAD⁺ concentration in isolated nuclei decreased slightly. The stimulating effect of nicotinamide on intracellular synthesis of NAD⁺ correlated with increases in ADP-ribosyl transferase, NAD⁺-pyrophosphorylase, and NAD⁺ ase activities.     

PARP-1 inhibition does not restore oxidant-mediated reduction in SIRT1 activity

Sirtuin1 (SIRT1) deacetylase and poly(ADP-ribose)-polymerase-1 (PARP-1) respond to environmental cues, and both require NAD⁺ cofactor for their enzymatic activities. However, the functional link between environmental/oxidative stress-mediated activation of PARP-1 and SIRT1 through NAD⁺ cofactor availability is not known. We investigated whether NAD⁺ depletion by PARP-1 activation plays a role in environmental stimuli/oxidant-induced reduction in SIRT1 activity. Both H₂O₂ and cigarette smoke (CS) decreased intracellular NAD⁺ levels in vitro in lung epithelial cells and in vivo in lungs of mice exposed to CS. Pharmacological PARP-1 inhibition prevented oxidant-induced NAD⁺ loss and attenuated loss of SIRT1 activity. Oxidants decreased SIRT1 activity in lung epithelial cells; however increasing cellular NAD⁺ cofactor levels by PARP-1 inhibition or NAD⁺ precursors was unable to restore SIRT1 activity. SIRT1 was found to be carbonylated by CS, which was not reversed by PARP-1 inhibition or selective SIRT1 activator. Overall, these data suggest that environmental/oxidant stress-induced SIRT1 down-regulation and PARP-1 activation are independent events despite both enzymes sharing the same cofactor.     

Extracellular NAD induces a rise in [Ca]i in activated human monocytes via engagement of P2Y1 and P2Y11 receptors

Extracellular nicotinamide adenine dinucleotide (NAD⁺) is known to increase the intracellular calcium concentration [Ca²⁺]_i in different cell types and by various mechanisms. Here we show that NAD⁺ triggers a transient rise in [Ca²⁺]_i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca²⁺ from IP₃-responsive intracellular stores and an influx of extracellular Ca²⁺. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD⁺-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD⁺. The identification of P2Y₁ and P2Y₁₁ as receptor subtypes responsible for the NAD⁺-triggered increase in [Ca²⁺]_i was supported by several lines of evidence. First, specific P2Y₁ and P2Y₁₁ receptor antagonists inhibited the NAD⁺-induced increase in [Ca²⁺]_i. Second, NAD⁺ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD⁺ caused an increase in [cAMP]_i, prevented by the P2Y₁₁ receptor-specific antagonist NF157.     

Pocket analysis of the full-length cholix toxin. An assessment of the structure–dynamics of the apo catalytic domain

Cholix toxin from Vibrio cholerae is the third member of the diphtheria toxin (DT) group of mono-ADP-ribosyltransferase (mART) bacterial toxins. It shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae DT. Cholix toxin is an important model for the development of antivirulence approaches and therapeutics against these toxins from pathogenic bacteria. Herein, we have used the high-resolution X-ray structure of full-length cholix complexed with NAD⁺ to describe the properties of the NAD⁺-binding pocket at the residue level, including the role of crystallographic water molecules in the NAD⁺ substrate interaction. The full-length apo cholix structure is used to describe the putative NAD⁺-binding site(s) and to correlate biochemical with crystallographic data to study the stoichiometry and orientation of bound NAD⁺ molecules. We quantitatively describe the NAD⁺ substrate interactions on a residue basis for the main 22 pocket residues in cholix_f, a glycerol and 5 contact water molecules as part of the recognition surface by the substrate according to the conditions of crystallization. In addition, the dynamic properties of an in silico version of the catalytic domain were investigated in order to understand the lack of electronic density for one of the main flexible loops (R-loop) in the pocket of X-ray complexes. Implications for a rational drug design approach for mART toxins are derived.     

Tankyrase-1 overexpression reduces genotoxin-induced cell death by inhibiting PARP1

Poly(ADP-ribose) polymerases or PARPs are a family of NAD⁺-dependent enzymes that modify themselves and other substrate proteins with ADP-ribose polymers. The founding member PARP1 is localized predominantly in the nucleus and is activated by binding to DNA lesions. Excessive PARP1 activation following genotoxin treatment causes NAD⁺ depletion and cell death, whereas pharmacological PARP1 inhibition protects cells from genotoxicity. This study investigates whether cellular viability and NAD⁺ metabolism are regulated by tankyrase-1, a PARP member localized predominantly in the cytosol. Using a tetracycline-sensitive promoter to regulate tankyrase-1 expression in Madin–Darby canine kidney (MDCK) cells, we found that a 40-fold induction of tankyrase-1 (from 1500 to 60,000 copies per cell) lowers steady-state NAD⁺ levels but does not affect basal cellular viability. Moreover, the induction confers protection against the oxidative agent H₂O₂ and the alkylating agent MNNG, genotoxins that kill cells by activating PARP1. The cytoprotective effect of tankyrase-1 is not due to enhanced scavenging of oxidants or altered expression of Mcl-1, an anti-apoptotic molecule previously shown to be down-regulated by tankyrase-1 in CHO cells. Instead, tankyrase-1 appears to protect cells by preventing genotoxins from activating PARP1-mediated reactions such as PARP1 automodification and NAD⁺ consumption. Our findings therefore indicate a cytoprotective function of tankyrase-1 mediated through altered NAD⁺ homeostasis and inhibition of PARP1 function.     

Isolation and characterization of an NAD+-degrading bacterium PTX1 and its role in chromium biogeochemical cycle

Microorganisms can reduce toxic chromate to less toxic trivalent chromium [Cr(III)]. Besides Cr(OH)₃ precipitates, some soluble organo-Cr(III) complexes are readily formed upon microbial, enzymatic, and chemical reduction of chromate. However, the biotransformation of the organo-Cr(III) complexes has not been characterized. We have previously reported the formation of a nicotinamide adenine dinucleotide (NAD⁺)-Cr(III) complex after enzymatic reduction of chromate. Although the NAD⁺-Cr(III) complex was stable under sterile conditions, microbial cells were identified as precipitates in a non-sterile NAD⁺-Cr(III) solution after extended incubation. The most dominant bacterium PTX1 was isolated and assigned to Leifsonia genus by phylogenetic analysis of 16S rRNA gene sequence. PTX1 grew slowly on NAD⁺ with a doubling time of 17 h, and even more slowly on the NAD⁺-Cr(III) complex with an estimated doubling time of 35 days. The slow growth suggests that PTX1 passively grew on trace NAD⁺ dissociated from the NAD⁺-Cr(III) complex, facilitating further dissociation of the complex and formation of Cr(III) precipitates. Thus, organo-Cr(III) complexes might be an intrinsic link of the chromium biogeochemical cycle; they can be produced during chromate reduction and then further mineralized by microorganisms.     

Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes

In the present study, we show that the extracellular addition of nicotinamide adenine dinucleotide (NAD⁺) induces a transient rise in [Ca²⁺]_i in human monocytes caused by an influx of extracellular calcium. The NAD⁺-induced Ca²⁺ response was prevented by adenosine triphosphate (ATP), suggesting the involvement of ATP receptors. Of the two subtypes of ATP receptors (P2X and P2Y), the P2X receptors were considered the most likely candidates. By the use of subtype preferential agonists and antagonists, we identified P2X₁, P2X₄, and P2X₇ receptors being engaged in the NAD⁺-induced rise in [Ca²⁺]_i. Among the P2X receptor subtypes, the P2X₇ receptor is unique in facilitating the induction of nonselective pores that allow entry of ethidium upon stimulation with ATP. In monocytes, opening of P2X₇ receptor-dependent pores strongly depends on specific ionic conditions. Measuring pore formation in response to NAD⁺, we found that NAD⁺ unlike ATP lacks the ability to induce this pore-forming response. Whereas as little as 100 μM ATP was sufficient to activate the nonselective pore, NAD⁺ at concentrations up to 2 mM had no effect. Taken together, these data indicate that despite similarities in the action of extracellular NAD⁺ and ATP there are nucleotide-specific variations. So far, common and distinct features of the two nucleotides are only beginning to be understood.     

NAD+ metabolism and oxidative stress: the golden nucleotide on a crown of thorns

Abstract

In the twentieth century, NAD⁺ research generated multiple discoveries. Identification of the important role of NAD⁺ as a cofactor in cellular respiration and energy production was followed by discoveries of numerous NAD⁺ biosynthesis pathways. In recent years, NAD⁺ has been shown to play a unique role in DNA repair and protein deacetylation. As discussed in this review, there are close interactions between oxidative stress and immune activation, energy metabolism, and cell viability in neurodegenerative disorders and ageing. Profound interactions with regard to oxidative stress and NAD⁺ have been highlighted in the present work. This review emphasizes the pivotal role of NAD⁺ in the regulation of DNA repair, stress resistance, and cell death, suggesting that NAD⁺ synthesis through the kynurenine pathway and/or salvage pathway is an attractive target for therapeutic intervention in age-associated degenerative disorders. NAD⁺ precursors have been shown to slow down ageing and extend lifespan in yeasts, and protect severed axons from degeneration in animal models neurodegenerative diseases.     

172 Role of conserved water molecular triad in the recognition of IMP,NAD+ with Asp 274, Asn 303, Arg 322, and Asp 364 in both the isoform of hIMPDH

Inosine monophosphate dehydrogenase (IMPDH) plays an important role in the Guanosine monophosphate (GMP) biosynthesis pathway. As hIMPDH-II is involved in CML-Cancer, it is thought to be an active target for leukemic drug design. The importance of conserved water molecules in the salt-bridge-mediated interdomain recognition and loop-flap recognition of hIMPDH has already been indicated in some simulation studies (Bairagya et al., 2009, 2011a, 2011b, 2012; Mishra et al., 2012). In this work, the role of conserved water molecules in the recognition of Inosine monophosphate (IMP) and NAD⁺ (co-factor) to active site residues of both the isoforms has been investigated by all atoms MD-Simulation studies. During 25-ns dynamics of the solvated hIMPDH-II and I (1B3O and 1JCN PDB structures), the involvement of conserved water molecular triad (W _M, W _L and W _C) in the recognition of active site residues (Asp 274, Asn 303, Arg 322, and Asp 364), IMP and NAD⁺ has been observed (Figure 1). The H-bonding co-ordination of all three conserved water molecular centers is within 4–7 and their occupation frequency is 1.0. The H-bonding geometry and the electronic consequences of the water molecular interaction at the different residues (and also IMP and NAD⁺) may put forward some rational clues on antileukemic agent design.     

Probing the catalytic mechanism of bovine CD38/NADglycohydrolase by site directed mutagenesis of key active site residues

Bovine CD38/NAD⁺ glycohydrolase catalyzes the hydrolysis of NAD⁺ to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism. Our recent crystallographic study of its Michaelis complex and covalently-trapped intermediates provided insights into the modalities of substrate binding and the molecular mechanism of bCD38. The aim of the present work was to determine the precise role of key conserved active site residues (Trp118, Glu138, Asp147, Trp181 and Glu218) by focusing mainly on the cleavage of the nicotinamide–ribosyl bond. We analyzed the kinetic parameters of mutants of these residues which reside within the bCD38 subdomain in the vicinity of the scissile bond of bound NAD⁺. To address the reaction mechanism we also performed chemical rescue experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. The crucial role of Glu218, which orients the substrate for cleavage by interacting with the N-ribosyl 2′-OH group of NAD⁺, was highlighted. This contribution to catalysis accounts for almost half of the reaction energy barrier. Other contributions can be ascribed notably to Glu138 and Asp147 via ground-state destabilization and desolvation in the vicinity of the scissile bond. Key interactions with Trp118 and Trp181 were also proven to stabilize the ribooxocarbenium ion-like transition state. Altogether we propose that, as an alternative to a covalent acylal reaction intermediate with Glu218, catalysis by bCD38 proceeds through the formation of a discrete and transient ribooxocarbenium intermediate which is stabilized within the active site mostly by electrostatic interactions.     

Structures of iron-dependent alcohol dehydrogenase 2 from Zymomonas mobilis ZM4 with and without NAD+ cofactor

The ethanologenic bacterium Zymomonas mobilis ZM4 is of special interest because it has a high ethanol yield. This is made possible by the two alcohol dehydrogenases (ADHs) present in Z. mobilis ZM4 (zmADHs), which shift the equilibrium of the reaction toward the synthesis of ethanol. They are metal-dependent enzymes: zinc for zmADH1 and iron for zmADH2. However, zmADH2 is inactivated by oxygen, thus implicating zmADH2 as the component of the cytosolic respiratory system in Z. mobilis. Here, we show crystal structures of zmADH2 in the form of an apo-enzyme and an NAD⁺-cofactor complex. The overall folding of the monomeric structure is very similar to those of other functionally related ADHs with structural variations around the probable substrate and NAD⁺ cofactor binding region. A dimeric structure is formed by the limited interactions between the two subunits with the bound NAD⁺ at the cleft formed along the domain interface. The catalytic iron ion binds near to the nicotinamide ring of NAD⁺, which is likely to restrict and locate the ethanol to the active site together with the oxidized Cys residue and several nonpolar bulky residues. The structures of the zmADH2 from the proficient ethanologenic bacterium Z. mobilis, with and without NAD⁺ cofactor, and modeling ethanol in the active site imply that there is a typical metal-dependent catalytic mechanism.     

Genetically encoded fluorescent indicator for imaging NAD/NADH ratio changes in different cellular compartments

Background

The ratio of NAD⁺/NADH is a key indicator that reflects the overall redox state of the cells. Until recently, there were no methods for real time NAD⁺/NADH monitoring in living cells. Genetically encoded fluorescent probes for NAD⁺/NADH are fundamentally new approach for studying the NAD⁺/NADH dynamics.

Methods

We developed a genetically encoded probe for the nicotinamide adenine dinucleotide, NAD(H), redox state changes by inserting circularly permuted YFP into redox sensor T-REX from Thermus aquaticus. We characterized the sensor in vitro using spectrofluorometry and in cultured mammalian cells using confocal fluorescent microscopy.

Results

The sensor, named RexYFP, reports changes in the NAD⁺/NADH ratio in different compartments of living cells. Using RexYFP, we were able to track changes in NAD⁺/NADH in cytoplasm and mitochondrial matrix of cells under a variety of conditions. The affinity of the probe enables comparison of NAD⁺/NADH in compartments with low (cytoplasm) and high (mitochondria) NADH concentration. We developed a method of eliminating pH-driven artifacts by normalizing the signal to the signal of the pH sensor with the same chromophore.

Conclusion

RexYFP is suitable for detecting the NAD(H) redox state in different cellular compartments.

General significance

RexYFP has several advantages over existing NAD⁺/NADH sensors such as smallest size and optimal affinity for different compartments. Our results show that normalizing the signal of the sensor to the pH changes is a good strategy for overcoming pH-induced artifacts in imaging.     

Skeletal muscle hexokinase: regulation in mammalian hibernation

The perfused rat liver responds in several ways to NAD⁺ infusion (20–100 μM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption and gluconeogenesis are some of the effects that were observed. Extracellular NAD⁺ is also extensively transformed in the liver. The purpose of the present work was to determine the main products of extracellular NAD⁺ transformation under various conditions and to investigate the possible contribution of these products for the metabolic effects of the parent compound. The experiments were done with the isolated perfused rat liver. The NAD⁺ transformation was monitored by HPLC. Confirming previous findings, the single-pass transformation of 100 μM NAD⁺ ranged between 75% at 1.5 min after starting infusion to 95% at 8 min. The most important products of single-pass NAD⁺ transformation appearing in the outflowing perfusate were nicotinamide, ADP-ribose, uric acid, and inosine. The relative proportions of these products presented some variations with the time after initiation of NAD⁺ infusion and the perfusion conditions, but ADP-ribose was always more abundant than uric acid and inosine. Cyclic ADP-ribose (cADP-ribose) as well as adenosine were not detected in the outflowing perfusate. The metabolic effects of ADP-ribose were essentially those already described for NAD⁺. These effects were sensitive to suramin (P2_XY purinergic receptor antagonist) and insensitive to 3,7-dimethyl-1-(2-propargyl)-xanthine (A2 purinergic receptor antagonist). Inosine, a known purinergic A3 agonist, was also active on metabolism, but uric acid and nicotinamide were inactive. It was concluded that the metabolic and hemodynamic effects of extracellular NAD⁺ are caused mainly by interactions with purinergic receptors with a highly significant participation of its main transformation product ADP-ribose.     

A 3-aminobenzamide-resistant labeled protein in [P]NAD-labeled cells

A series of proteins are covalently labeled when human lymphocytes are incubated with [³²P]NAD⁺. The majority of this labeling is effectively inhibited when the lymphocytes are coincubated with 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase. However, labeling of a 72 000 molecular weight protein was resistant to the inhibitory effect of 3-aminobenzamide. Labeling of this protein from [³²P]NAD⁺ was shown to be Mg²⁺-dependent. The 72 000 molecular weight protein could also be labeled on incubation with [α-³²P]ATP, [γ-³²P]ATP and [³²P]orthophosphate, but not from [³H]NAD⁺ or [¹⁴C]NAD⁺. In the present study, we show that the 72 000 molecular weight protein is not ADP-ribosylated but rather, phosphorylated on incubation with [³²P]NAD⁺. This phosphorylation appears to occur via an Mg²⁺-dependent conversion of NAD⁺ to AMP with the eventual utilization of the α-phosphate for phosphorylation of the 72 000 molecular weight protein.     

NAD+ ameliorates endotoxin‐induced acute kidney injury in a sirtuin1–dependent manner via GSK‐3β/Nrf2 signalling pathway

Acute kidney injury (AKI) is a substantial worldwide public health concern with no specific and effective therapies in clinic. NAD⁺ is a pivotal determinant of cellular energy metabolism involved in the progression of AKI; however, its mechanism in kidney injury remains poorly understood. Sirtuin 1 (SIRT1) is an NAD⁺‐dependent deacetylase associated with renal protection and acute stress resistance. In this study, we have investigated the role of NAD⁺ in AKI and the potential mechanism(s) involved in its renoprotective effect. NAD⁺ was notably decreased and negatively correlated with kidney dysfunction in AKI, restoring NAD⁺ with NMN significantly ameliorates LPS‐induced oxidative stress and apoptosis and attenuates renal damage. We also found that the protection of NAD⁺ is associated with SIRT1 expressions and performs in a SIRT1‐dependent manner. Inhibition of SIRT1 blunted the protective effect of NAD⁺ and up‐regulated the activity of glycogen synthase kinase‐3β (GSK‐3β) that was concomitant with mitigated Nrf2 nuclear accumulation, thereby exacerbates AKI. These findings suggest that NAD⁺/SIRT1/GSK‐3β/Nrf2 axis is an important mechanism that can protect against AKI which might be a potential therapeutic target for the treatment of AKI.