Coenzyme Nicotinamide Adenine Dinucleotide Research Articles

Local redox conditions in cells imaged via non-fluorescent transient states of NAD(P)H

The autofluorescent coenzyme nicotinamide adenine dinucleotide (NADH) and its phosphorylated form (NADPH) are major determinants of cellular redox balance. Both their fluorescence intensities and lifetimes are extensively used as label-free readouts in cellular metabolic imaging studies. Here, we introduce fluorescence blinking of NAD(P)H, as an additional, orthogonal readout in such studies. Blinking of fluorophores and their underlying dark state transitions are specifically sensitive to redox conditions and oxygenation, parameters of particular relevance in cellular metabolic studies. We show that such dark state transitions in NAD(P)H can be quantified via the average fluorescence intensity recorded upon modulated one-photon excitation, so-called transient state (TRAST) monitoring. Thereby, transitions in NAD(P)H, previously only accessible from elaborate spectroscopic cuvette measurements, can be imaged at subcellular resolution in live cells. We then demonstrate that these transitions can be imaged with a standard laser-scanning confocal microscope and two-photon excitation, in parallel with regular fluorescence lifetime imaging (FLIM). TRAST imaging of NAD(P)H was found to provide additional, orthogonal information to FLIM and allows altered oxidative environments in cells treated with a mitochondrial un-coupler or cyanide to be clearly distinguished. We propose TRAST imaging as a straightforward and widely applicable modality, extending the range of information obtainable from cellular metabolic imaging of NAD(P)H fluorescence.

Highly sensitive and selective localized surface plasmon resonance biosensor for detecting glutamate realized on optical fiber substrate using gold nanoparticles

In this study, we fabricated and characterized a localized surface plasmon resonance-based biosensor comprising gold nanoparticles (AuNPs) on an optical fiber substrate to detect glutamate at concentrations ranging from 0 to 10 mM. To fabricate the probe, AuNPs were decorated on an unclad portion of an optical fiber, and the enzyme glutamate dehydrogenase and coenzyme nicotinamide adenine dinucleotide were co-immobilized over the AuNPs. The sensor was designed to operate according to an intensity modulation scheme where the absorbance changed as the glutamate concentration varied in the sample around the probe. The change in the absorbance was due to the chemical reaction between glutamate dehydrogenase, glutamate, water and NAD+ which affected the refractive index of the medium around the AuNPs. The changes in the local refractive index led to significant variations in the absorbance of the AuNPs present in the sensing layer. The change recorded in the absorbance by AuNPs was 0.019 for the glutamate concentration range from 0 to 10 mM. The limit of detection and sensitivity of the sensor were determined as 0.36 mM and 0.0048 AU/mM, respectively. The sensor has advantages in terms of its low cost, immunity to electromagnetic interference, miniaturized probe, and capability of online monitoring and remote sensing because an optical fiber is used as the substrate for probe fabrication.

From the Literature

From the Literature

Does Nitrate Reductase Play a Role in Silver Nanoparticle Synthesis? Evidence for NADPH as the Sole Reducing Agent

A leading hypothesis for silver nanoparticle biosynthesis suggests that the enzyme nitrate reductase plays a key role in the process, assisted by the coenzyme nicotinamide adenine dinucleotide phos...

Synthetic Biomimetic Coenzymes and Alcohol Dehydrogenases for Asymmetric Catalysis

Redox reactions catalyzed by highly selective nicotinamide-dependent oxidoreductases are rising to prominence in industry. The cost of nicotinamide adenine dinucleotide coenzymes has led to the use of well-established elaborate regeneration systems and more recently alternative synthetic biomimetic cofactors. These biomimetics are highly attractive to use with ketoreductases for asymmetric catalysis. In this work, we show that the commonly studied cofactor analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) can be used with alcohol dehydrogenases (ADHs) under certain conditions. First, we carried out the rhodium-catalyzed recycling of BNAH with horse liver ADH (HLADH), observing enantioenriched product only with unpurified enzyme. Then, a series of cell-free extracts and purified ketoreductases were screened with BNAH. The use of unpurified enzyme led to product formation, whereas upon dialysis or further purification no product was observed. Several other biomimetics were screened with various ADHs and showed no or very low activity, but also no inhibition. BNAH as a hydride source was shown to directly reduce nicotinamide adenine dinucleotide (NAD) to NADH. A formate dehydrogenase could also mediate the reduction of NAD from BNAH. BNAH was established to show no or very low activity with ADHs and could be used as a hydride donor to recycle NADH.

Stem cell-like breast cancer cells with acquired resistance to metformin are sensitive to inhibitors of NADH-dependent CtBP dimerization.

Altered flux through major metabolic pathways is a hallmark of cancer cells and provides opportunities for therapy. Stem cell-like cancer (SCLC) cells can cause metastasis and therapy resistance. They possess metabolic plasticity, theoretically enabling resistance to therapies targeting a specific metabolic state. The C-terminal binding protein (CtBP) transcriptional regulators are potential therapeutic targets in highly glycolytic cancer cells, as they are activated by the glycolytic coenzyme nicotinamide adenine dinucleotide (NADH). However, SCLC cells commonly exist in an oxidative state with low rates of glycolysis. Metformin inhibits complex I of the mitochondrial electron transport chain; it can kill oxidative SCLC cells and has anti-cancer activity in patients. SCLC cells can acquire resistance to metformin through increased glycolysis. Given the potential for long-term metformin therapy, we have studied acquired metformin resistance in cells from the claudin-low subtype of breast cancer. Cells cultured for 8 weeks in sub-IC50 metformin concentration proliferated comparably to untreated cells and exhibited higher rates of glucose uptake. SCLC cells were enriched in metformin-adapted cultures. These SCLC cells acquired sensitivity to multiple methods of inhibition of CtBP function, including a cyclic peptide inhibitor of NADH-induced CtBP dimerization. Single-cell mRNA sequencing identified a reprogramming of epithelial-mesenchymal and stem cell gene expression in the metformin-adapted SCLC cells. These SCLC cells demonstrated an acquired dependency on one of these genes, Tenascin C. Thus, in addition to acquisition of sensitivity to glycolysis-targeting therapeutic strategies, the reprograming of gene expression in the metformin-adapted SCLC cells renders them sensitive to potential therapeutic approaches not directly linked to cell metabolism.

Kynurenine 3-Monooxygenase Activity in Human Primary Neurons and Effect on Cellular Bioenergetics Identifies New Neurotoxic Mechanisms.

Upregulation of the kynurenine pathway (KP) of tryptophan metabolism is commonly observed in neurodegenerative disease. When activated, L-kynurenine (KYN) increases in the periphery and central nervous system where it is further metabolised to other neuroactive metabolites including 3-hydroxykynurenine (3-HK), kynurenic acid (KYNA) and quinolinic acid (QUIN). Particularly biologically relevant metabolites are 3-HK and QUIN, formed downstream of the enzyme kynurenine 3-monooxygenase (KMO) which plays a pivotal role in maintaining KP homeostasis. Indeed, excessive production of 3-HK and QUIN has been described in neurodegenerative disease including Alzheimer's disease and Huntington's disease. In this study, we characterise KMO activity in human primary neurons and identified new mechanisms by which KMO activation mediates neurotoxicity. We show that while transient activation of the KP promotes synthesis of the essential co-enzyme nicotinamide adenine dinucleotide (NAD+), allowing cells to meet short-term increased energy demands, chronic KMO activation induces production of reactive oxygen species (ROS), mitochondrial damage and decreases spare-respiratory capacity (SRC). We further found that these events generate a vicious-cycle, as mitochondrial dysfunction further shunts the KP towards the KMO branch of the KP to presumably enhance QUIN production. These mechanisms may be especially relevant in neurodegenerative disease as neurons are highly sensitive to oxidative stress and mitochondrial impairment.

Niacin Regulates Glucose Reactive Protein (GRP78), Protein Carbonyl Content (PCC) and Malondialdehyde (MDA) in the Hyperglycemic Human Lens Epithelial Cells

Introduction: Niacin is part of the chemical structure of coenzymes nicotinamide adenine nucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). Previous studies suggested that a high niacin intake could decrease the prevalence of cataracts, which may delay the onset of diabetic cataract. Aim: The aim of this study was to evaluate the effect of niacin on the hyperglycemia-induced osmotic stress and oxidative stress in human lens epithelial cells. Materials and Methods: Human lens epithelial cells were cultured in a high glucose condition. Oxidative stress markers, including malondialdehyde (MDA), protein carbonyl content (PCC) and glucose reactive protein (GRP), were measured using TBARS analysis (MDA) and ELISA (PCC and GRP) after 72 h incubation. Results: The MDA levels increased after high glucose administration relative to that in the control group (p <0.05). Further, the groups that were co-treated with niacin showed decrease in the MDA levels for all doses of niacin and the lowest mean MDA level was obtained with 100 μM niacin. There was a decrease in the PCC levels for all doses, whereas the lowest mean PCC level was observed at a 100 μM niacin dose. The GRP levels increased after high glucose administration as compared with the control group. Also, the groups that were co-treated with niacin exhibited statistically significant reduction. Conclusion: These results suggest that niacin can inhibit the osmotic stress and oxidative stress which may lead to the progression of a diabetic cataract. Also, it may maintain lens transparency by acting as a precursor for glutathione biosynthesis and an antioxidant.

Why Is Mom Stressed: Homeorhesis as the Potential Problem and Nicotinamide Riboside as the Potential Solution

The remodeling of female mammalian physiology to support the development of a fertilized egg into an externally breathing individual and then to provide all the nutrition to this individual while remodeling back to nearly her pregestational state is without parallel in male mammalian physiological transitions. While it is common parlance to refer to postpartum depression as a not infrequent stress in women, the postpartum physiological changes after every birth constitute profound metabolic stresses that are understudied and have important nutritional, behavioral, and neurodevelopmental implications for the maternal and neonatal health of every mammalian species. We discovered that the postpartum liver of a lactating female mouse has a depressed nicotinamide adenine dinucleotide (NAD) metabolome linked to circulation of higher levels of NAD metabolites in support of a >20-fold increase in NAD coenzymes in the mammary. Furthermore, by supporting a new mother’s apparent higher demand for NAD precursors, we increased circulation of prolactin, superinduced mammary biosynthetic programs, increased her time of arched-back nursing, enhanced mammary production of brain-derived neurotrophic factor, promoted postgestational weight loss, advanced the neurobehavioral development of her offspring, and allowed them to mature as stronger and more resilient adults with advantages in hippocampal neurogenesis and body composition. These results show that a new mother’s capacity for biosynthesis and functionally important nurturing is apparently limited by NAD. Here, we discuss homeorhetic flow of resources from a new mother to her offspring in the context of NAD metabolism and suggest avenues for future investigation.

NAD-modifizierte RNA: Redox - biochemie trifft RNA-Prozessierung

RNA is an important regulator in biology. Its simple architecture is augmented by diverse chemical modifications. Recently we discovered the ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) to be attached to a specific set of regulatory RNAs in bacteria in a cap-like manner, and to modulate the cellular functions of these RNAs. This article highlights the biosynthesis, removal, and biological function of this novel RNA modification.

Identification, Biosynthesis, and Decapping of NAD-Capped RNAs in B. subtilis

The ubiquitous coenzyme nicotinamide adenine dinucleotide (NAD) decorates various RNAs in different organisms. In the proteobacterium Escherichia coli, the NAD-cap confers stability against RNA degradation. To date, NAD-RNAs have not been identified in any other bacterial microorganism. Here, we report the identification of NAD-RNA in the firmicute Bacillus subtilis. In the late exponential growth phase, predominantly mRNAs are NAD modified. NAD is incorporated de novo into RNA by the cellular RNA polymerase using non-canonical transcription initiation. The incorporation efficiency depends on the -1 position of the promoter but is independent of sigma factors or mutations in the rifampicin binding pocket. RNA pyrophosphohydrolase BsRppH is found to decap NAD-RNA. Invitro, the decapping activity is facilitated by manganese ions and single-stranded RNA 5' ends. Depletion of BsRppH influences the gene expression of ∼13% of transcripts in B.subtilis. The NAD-cap stabilizes RNA against 5'-to-3'-exonucleolytic decay by RNase J1.

New Insight into the Mechanism of NADH Model Oxidation by Metal Ions in Nonalkaline Media.

For a long time, it has been controversial that the three-step (e-H+-e) or two-step (e-H•) mechanism was used for the oxidation of nicotinamide adenine dinucleotide coenzyme (NADH) and its models by metal ions in nonalkaline media. The latter mechanism has been accepted by the majority of researchers. In this work, 1-benzyl-1,4-dihydronicotinamide (BNAH) and 1-phenyl-l,4-dihydronicotinamide are used as NADH models and ferrocenium (Fc+) metal ion as an electron acceptor. The kinetics for oxidation of the NADH models by Fc+ in pure acetonitrile was monitored by using UV-vis absorption and a quadratic relationship between kobs and the concentrations of NADH models was found for the first time. The rate expression of the reactions developed according to the three-step mechanism is quite consistent with the quadratic curves. The rate constants, thermodynamic driving forces, and kinetic isotope effects of each elementary step for the reactions were estimated. All results supported the three-step mechanism. The intrinsic kinetic barriers of the proton transfer from BNAH+• to BNAH and the hydrogen-atom transfer from BNAH+• to BNAH+• were estimated by using Zhu equation; the results showed that the former is 11.8 kcal/mol and the latter is larger than 24.3 kcal/mol. It is the large intrinsic kinetic barrier of the hydrogen-atom transfer that makes the reactions choose the three-step rather than two-step mechanism. Further investigation of the factors affecting the intrinsic kinetic barrier of chemical reactions indicated that the large intrinsic kinetic barrier of the hydrogen-atom transfer originated from the repulsion of positive charges between BNAH+• and BNAH+•. The greatest contribution of this work is the discovery of the quadratic dependence of kobs on the concentrations of the NADH models, which is inconsistent with the conventional viewpoint of the "two-step mechanism" on the oxidation of NADH and its models by metal ions in the nonalkaline media.

Integrating glucose and fat

Adipogenic Regulation Consuming too much glucose makes you fat, but it is unclear how this conversion is mediated by the body. Glycolysis links to gene transcription via the essential coenzyme nicotinamide adenine dinucleotide in its oxidized state (NAD+). Ryu et al. found that compartmentalized NAD+ synthesis and consumption integrate glucose metabolism and adipogenic (fat-promoting) transcription during adipocyte differentiation (see the Perspective by Trefely and Wellen). Competition between the NAD+ precursors—nuclear NMNAT-1 and cytosolic NMNAT-2—for their common substrate, nicotinamide mononucleotide, regulates the balance between nuclear NAD+ synthesis for adipogenic gene regulation and cytosolic NAD+ synthesis used in metabolism. Science , this issue p. [eaan5780][1]; see also p. [603][2] [1]: /lookup/doi/10.1126/science.aan5780 [2]: /lookup/doi/10.1126/science.aat6663

Metabolic regulation of transcription through compartmentalized NAD+ biosynthesis.

NAD+ (nicotinamide adenine dinucleotide in its oxidized state) is an essential molecule for a variety of physiological processes. It is synthesized in distinct subcellular compartments by three different synthases (NMNAT-1, -2, and -3). We found that compartmentalized NAD+ synthesis by NMNATs integrates glucose metabolism and adipogenic transcription during adipocyte differentiation. Adipogenic signaling rapidly induces cytoplasmic NMNAT-2, which competes with nuclear NMNAT-1 for the common substrate, nicotinamide mononucleotide, leading to a precipitous reduction in nuclear NAD+ levels. This inhibits the catalytic activity of poly[adenosine diphosphate (ADP)-ribose] polymerase-1 (PARP-1), a NAD+-dependent enzyme that represses adipogenic transcription by ADP-ribosylating the adipogenic transcription factor C/EBPβ. Reversal of PARP-1-mediated repression by NMNAT-2-mediated nuclear NAD+ depletion in response to adipogenic signals drives adipogenesis. Thus, compartmentalized NAD+ synthesis functions as an integrator of cellular metabolism and signal-dependent transcriptional programs.

In vivo metabolic imaging and monitoring of brown and beige fat.

Activation of the thermogenic brown and beige fat is an effective means to increasing whole-body energy expenditure. In this work, a unique label-free method was developed to quantitatively assess the metabolism and thermogenesis of mouse adipose tissues in vivo. Specifically, an optical redox ratio (ORR) based on the endogenous fluorescence of mitochondrial metabolic coenzymes (nicotinamide adenine dinucleotide and flavin adenine dinucleotide) was used to measure the metabolic state of adipocytes. Our findings demonstrate that the ORR provides a label-free and real-time biomarker to determine the thermogenic response of brown, beige and white adipose tissues to a variety of physiological stimulations. In addition, the redox ratio also can be used to evaluate the degree of browning in the white fat of cold-acclimated mice. This technique is important to understand the recruitment and activation of thermogenic adipocytes in mammals and thus can help to develop therapeutic strategies against obesity.

Hierarchically Engineered Mesoporous Metal-Organic Frameworks toward Cell-free Immobilized Enzyme Systems

Summary Highly efficient cell-free enzymatic systems are typically difficult to achieve in traditional immobilized enzyme systems because of the lack of optimal spatial control of enzyme localization, substrate and product diffusion, and enzyme and coenzyme accessibility. Here, we report a strategy for expanding the pore apertures (from 3.3 to 6.7 nm) of a series of Zr-based metallic-organic frameworks (MOFs) (termed NU-100x , x = 3, 4, 5, 6, 7) with interconnected hierarchical pores by maintaining precise control of torsional angles associated with the linkers. As a proof of concept, we use the expanded NU-100x MOF structures to encapsulate lactate dehydrogenase (LDH) and demonstrate the use of the captured protein in a cell-free biosynthetic catalytic system. Remarkably, LDH immobilized in the large pores of the MOF is accessible to nicotinamide adenine dinucleotide coenzymes (NAD and NADH), allowing for in situ coenzyme regeneration leading to higher activity than that of the free enzyme.

Surface enhanced Raman spectroscopy of Chlamydia trachomatis and Neisseria gonorrhoeae for diagnostics, and extra-cellular metabolomics and biochemical monitoring

SERS spectra excited at 785 nm of the bacteria Chlamydia trahomatis (elementary bodies, EB) and Neisseria gonorrheoae, the causative pathogens for the two most common sexually transmitted diseases (STD), chlamydia and gonorrhea, respectively, are reported. Although both are Gram-negative bacteria, the SERS signatures of C. trachomatis and N. gonorrheoae are completely different. N. gonorrheoae SERS spectra are due to the starvation induced nucleotide metabolites adenine and guanine, and the surface associated co-enzyme nicotinamide adenine dinucleotide and are very similar on Au and Ag although the spectrum appears more rapidly on Ag. The C. trachomatis SERS spectrum is dominated by the vibrational features of cell surface proteins. While features attributable to specific residues and the amide backbone characterize the C. trachomatis spectrum on Ag, the corresponding SERS spectrum on Au substrates displays vibrational characteristics of aggregated proteins. The prospects for the development of a SERS based platform for rapid (<one hour), low-cost bacterial STD diagnostics are promising based on these initial studies. Furthermore, this biomedical application demonstrates the potential for SERS to be a sensitive real time probe of the dynamics of biochemical activity in the cell wall and extracellular regions of microorganisms.

Bioinspired Design of Alcohol Dehydrogenase@nano TiO₂ Microreactors for Sustainable Cycling of NAD⁺/NADH Coenzyme.

The bioinspired design and construction of enzyme@capsule microreactors with specific cell-like functionality has generated tremendous interest in recent years. Inspired by their fascinating complexity, scientists have endeavored to understand the essential aspects of a natural cell and create biomimicking microreactors so as to immobilize enzymes within the hierarchical structure of a microcapsule. In this study, simultaneous encapsulation of alcohol dehydrogenase (ADH) was achieved during the preparation of microcapsules by the Pickering emulsion method using amphiphilic modified TiO2 nanoparticles (NPs) as building blocks for assembling the photocatalytic microcapsule membrane. The ADH@TiO2 NP microreactors exhibited dual catalytic functions, i.e., spatially confined enzymatic catalysis and the membrane-associated photocatalytic oxidation under visible light. The sustainable cycling of nicotinamide adenine dinucleotide (NAD) coenzyme between NADH and NAD+ was realized by enzymatic regeneration of NADH from NAD+ reduction, and was provided in a form that enabled further photocatalytic oxidation to NAD+ under visible light. This bioinspired ADH@TiO2 NP microreactor allowed the linking of a semiconductor mineral-based inorganic photosystem to enzymatic reactions. This is a first step toward the realization of sustainable biological cycling of NAD+/NADH coenzyme in synthetic functional microsystems operating under visible light irradiation.

Porphyrin/SiO2/Cp*Rh(bpy)Cl Hybrid Nanoparticles Mimicking Chloroplast with Enhanced Electronic Energy Transfer for Biocatalyzed Artificial Photosynthesis

AbstractA biocatalyzed artificial photosynthesis system (APS) based on porphyrin/SiO2/Cp*Rh(bpy)Cl hybrid nanoparticles (TCPP/SiO2/Rh HNPs) to mimic chloroplasts in green plant is reported. The TCPP/SiO2/Rh HNPs are fabricated via sol–gel reaction of silica precursors functionalized with photosensitizer (porphyrin, TCPP) and electron mediator (Cp*Rh(bpy)Cl, M); while the integration of enzyme and coenzyme nicotinamide adenine dinucleotide (NAD)(H), on the outer surface of the HNPs is achieved through electrostatic‐interaction‐driven assembling under the entanglement of a negatively charged polyelectrolyte. The chloroplast‐mimicking, highly integrated APS exhibits remarkably superior performance over a free system such that the regeneration of NADH is improved from 11% to 75%, and the synthesis of formic acid from CO2 increased from 15 to 100 µmol. Based on the detailed investigations into the photochemical and electrochemical properties, it is speculated that the covalent linking of the photosensitizer and electron mediator via silicon hydride bonds, and the formed SiO2 network through sol–gel reaction, may form intramolecular and intermolecular electron transfer chains to direct more efficient electron transfer from TCPP to M. Such intramolecular and intermolecular electrons and energy transfer, cooperated with the integrated biocatalytic process, lead to the significantly enhanced overall reaction efficiency. Moreover, the integrated APS also allows facile recycling of expensive M, enzymes, and cofactors.

Spectrofluorimeter for estimating the metabolic state of organs and tissues

A spectrofluorimeter has been developed that measures the intrinsic fluorescence intensity in the emission regions of respiratory coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) at wavelengths 465 and 515 nm with excitation at 365 nm and 440 nm. The redox ratio NADH/FAD is used as the main index of the metabolic state of a bio-object. This parameter, because of the different directionality of the NADH and FAD reactions in response to oxidation, possesses higher sensitivity to the change of state of biotissue and depends less on absorption and scattering variations. The diffuse reflectance at 440 nm serves as a supplementary parameter. All the parameters are normalized to normoxia conditions. The NADH-excitation effect is detected at wavelength 440 nm. Using a study of rat heart under ex vivo and in vivo conditions as an example, it is demonstrated that early damage of the myocardium accompanying regional ischemia can be detected.