ABSTRACT
KEY MESSAGE: The CcGRXS12 gene protects plants from cellular oxidative damage that are caused by both biotic and abiotic stresses. The protein possesses GSH-disulphide oxidoreductase property but lacks Fe-S cluster assembly mechanism. Glutaredoxins (Grxs) are small, ubiquitous and multi-functional proteins. They are present in different compartments of plant cells. A chloroplast targeted Class I GRX (CcGRXS12) gene was isolated from Capsicum chinense during the pepper mild mottle virus (PMMoV) infection. Functional characterization of the gene was performed in Nicotiana benthamiana transgenic plants transformed with native C. chinense GRX (Nb:GRX), GRX-fused with GFP (Nb:GRX-GFP) and GRX-truncated for chloroplast sequences fused with GFP (Nb:Δ2MGRX-GFP). Overexpression of CcGRXS12 inhibited the PMMoV-I accumulation at the later stage of infection, accompanied with the activation of salicylic acid (SA) pathway pathogenesis-related (PR) transcripts and suppression of JA/ET pathway transcripts. Further, the reduced accumulation of auxin-induced Glutathione-S-Transferase (pCNT103) in CcGRXS12 overexpressing lines indicated that the protein could protect the plants from the oxidative stress caused by the virus. PMMoV-I infection increased the accumulation of pyridine nucleotides (PNs) mainly due to the reduced form of PNs (NAD(P)H), and it was high in Nb:GRX-GFP lines compared to other transgenic lines. Apart from biotic stress, CcGRXS12 protects the plants from abiotic stress conditions caused by H2O2 and herbicide paraquat. CcGRXS12 exhibited GSH-disulphide oxidoreductase activity in vitro; however, it was devoid of complementary Fe-S cluster assembly mechanism found in yeast. Overall, this study proves that CcGRXS12 plays a crucial role during biotic and abiotic stress in plants.
Subject(s)
Capsicum , Tobamovirus , Capsicum/genetics , Capsicum/metabolism , Glutaredoxins/genetics , Glutaredoxins/metabolism , Hydrogen Peroxide , Oxidation-Reduction , DisulfidesABSTRACT
The reconfiguration of the primary metabolism is essential in plant-pathogen interactions. We compared the local metabolic responses of cucumber leaves inoculated with Pseudomonas syringae pv lachrymans (Psl) with those in non-inoculated systemic leaves, by examining the changes in the nicotinamide adenine dinucleotides pools, the concentration of soluble carbohydrates and activities/gene expression of carbohydrate metabolism-related enzymes, the expression of photosynthesis-related genes, and the tricarboxylic acid cycle-linked metabolite contents and enzyme activities. In the infected leaves, Psl induced a metabolic signature with an altered [NAD(P)H]/[NAD(P)+] ratio; decreased glucose and sucrose contents, along with a changed invertase gene expression; and increased glucose turnover and accumulation of raffinose, trehalose, and myo-inositol. The accumulation of oxaloacetic and malic acids, enhanced activities, and gene expression of fumarase and l-malate dehydrogenase, as well as the increased respiration rate in the infected leaves, indicated that Psl induced the tricarboxylic acid cycle. The changes in gene expression of ribulose-l,5-bis-phosphate carboxylase/oxygenase large unit, phosphoenolpyruvate carboxylase and chloroplast glyceraldehyde-3-phosphate dehydrogenase were compatible with a net photosynthesis decline described earlier. Psl triggered metabolic changes common to the infected and non-infected leaves, the dynamics of which differed quantitatively (e.g., malic acid content and metabolism, glucose-6-phosphate accumulation, and glucose-6-phosphate dehydrogenase activity) and those specifically related to the local or systemic response (e.g., changes in the sugar content and turnover). Therefore, metabolic changes in the systemic leaves may be part of the global effects of local infection on the whole-plant metabolism and also represent a specific acclimation response contributing to balancing growth and defense.
Subject(s)
Carbon-Nitrogen Ligases , Cucumis sativus , Pseudomonas syringae/physiology , Cucumis sativus/genetics , Cucumis sativus/metabolism , Carbon/metabolism , Phosphoenolpyruvate Carboxylase/genetics , beta-Fructofuranosidase/metabolism , Malate Dehydrogenase/metabolism , Raffinose/metabolism , Trehalose/metabolism , NAD/metabolism , Fumarate Hydratase , Glucose-6-Phosphate/metabolism , Glucosephosphate Dehydrogenase/metabolism , Plant Leaves/metabolism , Photosynthesis/physiology , Carbohydrate Metabolism , Sucrose/metabolism , Phosphates/metabolism , Oxygenases/metabolism , Inositol/metabolism , Carbon-Nitrogen Ligases/metabolism , Niacinamide/metabolism , Adenine/metabolism , Glucose/metabolismABSTRACT
Desiccation tolerance is a developmental program enabling seed survival in a dry state and is common in seeds categorized as orthodox. We focused on NAD and its phosphorylated form (NADP) because their continual switching between reduced (NAD(P)H) and oxidized (NAD(P)+) forms is involved in the modulation of redox signaling and the determination of the reducing power and further antioxidant responses. Norway maple and sycamore seeds representing the orthodox and recalcitrant categories, respectively, were used as models in a comparison of responses to water loss. The process of desiccation up to 10% water content (WC) was monitored in Norway maple seeds, while dehydration up to 30% WC was monitored in desiccation-sensitive sycamore seeds. Norway maple and sycamore seeds, particularly their embryonic axes, exhibited a distinct redox status during dehydration and desiccation. High NADPH levels, NAD+ accumulation, low and stable NAD(P)H/NAD(P)+ ratios expressed as reducing power and high NADPH-dependent enzyme activity were reported in Norway maple seeds and were considered attributes of orthodox-type seeds. The contrasting results of sycamore seeds contributed to their low antioxidant capacity and high sensitivity to desiccation. NADPH deficiency, low NADPH-dependent enzyme activity and lack of NAD+ accumulation were primary features of sycamore seeds, with implications for their NAD(P)H/NAD(P)+ ratios and reducing power and with effects on many seed traits. Thus, we propose that the distinct levels of pyridine nucleotides and their redox status contribute to orthodox and recalcitrant phenotype differentiation in seeds by affecting cellular redox signaling, metabolism and the antioxidant system.
Subject(s)
Acer/metabolism , NADP/metabolism , Oxidation-Reduction , Seeds/metabolism , Acer/physiology , Dehydration , NADP/physiology , Seeds/physiologyABSTRACT
6-NADH and 6-NADPH are strong inhibitors of several dehydrogenases that may form spontaneously from NAD(P)H. They are known to be oxidized to NAD(P)+ by mammalian renalase, an FAD-linked enzyme mainly present in heart and kidney, and by related bacterial enzymes. We partially purified an enzyme oxidizing 6-NADPH from rat liver, and, surprisingly, identified it as pyridoxamine-phosphate oxidase (PNPO). This was confirmed by the finding that recombinant mouse PNPO oxidized 6-NADH and 6-NADPH with catalytic efficiencies comparable to those observed with pyridoxine- and pyridoxamine-5'-phosphate. PNPOs from Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana also displayed 6-NAD(P)H oxidase activity, indicating that this 'side-activity' is conserved. Remarkably, 'pyridoxamine-phosphate oxidase-related proteins' (PNPO-RP) from Nostoc punctiforme, A. thaliana and the yeast S. cerevisiae (Ygr017w) were not detectably active on pyridox(am)ine-5'-P, but oxidized 6-NADH, 6-NADPH and 2-NADH suggesting that this may be their main catalytic function. Their specificity profiles were therefore similar to that of renalase. Inactivation of renalase and of PNPO in mammalian cells and of Ygr017w in yeasts led to the accumulation of a reduced form of 6-NADH, tentatively identified as 4,5,6-NADH3, which can also be produced in vitro by reduction of 6-NADH by glyceraldehyde-3-phosphate dehydrogenase or glucose-6-phosphate dehydrogenase. As 4,5,6-NADH3 is not a substrate for renalase, PNPO or PNPO-RP, its accumulation presumably reflects the block in the oxidation of 6-NADH. These findings indicate that two different classes of enzymes using either FAD (renalase) or FMN (PNPOs and PNPO-RPs) as a cofactor play an as yet unsuspected role in removing damaged forms of NAD(P).
Subject(s)
Biocatalysis , NADPH Oxidases/metabolism , NAD/metabolism , Pyridoxaminephosphate Oxidase/metabolism , Animals , Arabidopsis/enzymology , Catalytic Domain , Escherichia coli/enzymology , Gene Knockout Techniques , HCT116 Cells , Humans , Liver/enzymology , Mice , Monoamine Oxidase/chemistry , Monoamine Oxidase/metabolism , NADPH Oxidases/isolation & purification , Nostoc/enzymology , Oxidation-Reduction , Pyridoxaminephosphate Oxidase/chemistry , Rats , Saccharomyces cerevisiae/enzymology , TransfectionABSTRACT
The present study investigates the physiological role of Kvß1 subunit for sensing pyridine nucleotide (NADH/NAD+) changes in the heart. We used Kvß1.1 knockout (KO) or wild-type (WT) mice and established that Kvß1.1 preferentially binds with Kv4.2 and senses the pyridine nucleotide changes in the heart. The cellular action potential duration (APD) obtained from WT cardiomyocytes showed longer APDs with lactate perfusion, which increases intracellular NADH levels, while the APDs remained unaltered in the Kvß1.1 KO. Ex vivo monophasic action potentials showed a similar response, in which the APDs were prolonged in WT mouse hearts with lactate perfusion; however, the Kvß1.1 KO mouse hearts did not show APD changes upon lactate perfusion. COS-7 cells coexpressing Kv4.2 and Kvß1.1 were used for whole cell patch-clamp recordings to evaluate changes caused by NADH (lactate). These data reveal that Kvß1.1 is required in the mediated inactivation of Kv4.2 currents, when NADH (lactate) levels are increased. In vivo, isoproterenol infusion led to increased NADH in the heart along with QTc prolongation in wild-type mice; regardless of the approach, our data show that Kvß1.1 recognizes NADH changes and modulates Kv4.2 currents affecting AP and QTc durations. Overall, this study uses multiple levels of investigation, including the heterologous overexpression system, cardiomyocyte, ex vivo, and ECG, and clearly depicts that Kvß1.1 is an obligatory sensor of NADH/NAD changes in vivo, with a physiological role in the heart.NEW & NOTEWORTHY Cardiac electrical activity is mediated by ion channels, and Kv4.2 plays a significant role, along with its binding partner, the Kvß1.1 subunit. In the present study, we identify Kvß1.1 as a sensor of pyridine nucleotide changes and as a modulator of Kv4.2 gating, action potential duration, and ECG in the mouse heart.
Subject(s)
Heart/drug effects , Kv1.1 Potassium Channel/metabolism , Myocardium/metabolism , Nucleotides/metabolism , Pyridines/metabolism , Action Potentials/drug effects , Adrenergic beta-Agonists/pharmacology , Animals , COS Cells , Chlorocebus aethiops , Electrophysiological Phenomena/drug effects , Isoproterenol/pharmacology , Lactic Acid/metabolism , Male , Mice , Mice, Knockout , NAD/metabolism , Patch-Clamp Techniques , Rats , Shal Potassium ChannelsABSTRACT
Activation of enzymes by low concentrations of denaturants has been reported for a limited number of enzymes including lipocalin-type prostaglandin D synthase (L-PGDS) and adenylate kinase. During unfolding studies on human biliverdin-IXα reductase it was discovered that the enzyme is activated at low concentrations of urea. Under standard assay conditions the native enzyme displays pronounced substrate inhibition with biliverdin as variable substrate; however in the presence of 3M urea, the substrate inhibition is abolished and the enzyme exhibits Michaelian kinetics. When the initial rate kinetics with NADPH as variable substrate are conducted in 3M urea, the Vmax is increased 11-fold to 1.8µmol/min/mg and the apparent Km for biliverdin increases from 1 to 3µM. We report the existence of two kinetically distinct folded intermediates between the native and unfolded forms. When the period of incubation with urea was varied prior to measuring enzyme activity, the apparent Vmax was shown to decay to half that seen at zero time with a half life of 5.8minutes, while the apparent Km for NADPH remains constant at approximately 5µM. With NADH as cofactor the half life of the activated (A) form was 2.9minutes, and this form decays in 3M urea to a less active (LA) form. The apparent Km for NADH increases from 0.33mM to 2mM for the A and LA forms. These kinetically distinct species are reminiscent of the activity-enhanced and inactive forms of L-PGDS observed in the presence of urea and guanidine hydrochloride.
Subject(s)
Biliverdine , Oxidoreductases/chemistry , Protein Unfolding , Urea/chemistry , Enzyme Activation , Humans , Kinetics , Oxidoreductases/metabolismABSTRACT
NAD is an important cofactor involved in multiple metabolic reactions and as a substrate for several NAD-dependent signalling enzymes. One such enzyme is CD38 which, alongside synthesising Ca(2+)-releasing second messengers and acting as a cell surface receptor, has also been suggested to play a key role in NAD(+) homeostasis. CD38 is well known as a negative prognostic marker in B-CLL but the role of its enzymatic activity has not been studied in depth to date. We have exploited the HL-60 cell line as a model of inducible CD38 expression, to investigate CD38-mediated regulation intracellular NAD(+) levels and the consequences of changes in NAD(+) levels on cell physiology. Intracellular NAD(+) levels fell with increasing CD38 expression and this was reversed with the CD38 inhibitor, kuromanin confirming the key role of CD38 in NAD(+) homeostasis. We also measured the consequences of CD38 expression during the differentiation on a number of functions linked to NAD(+) and we show that some but not all NAD(+)-dependent processes are significantly affected by the lowered NAD(+) levels. These data suggest that both functional roles of CD38 might be important in the pathogenesis of B-CLL.
Subject(s)
ADP-ribosyl Cyclase 1/biosynthesis , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , NAD/metabolism , ADP-ribosyl Cyclase 1/genetics , Cell Differentiation , HL-60 Cells , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , NAD/analysis , RNA, Messenger/biosynthesis , RNA, Messenger/geneticsABSTRACT
Besides defence pathways regulated by classical stress hormones, distinct amino acid metabolic pathways constitute integral parts of the plant immune system. Mutations in several genes involved in Asp-derived amino acid biosynthetic pathways can have profound impact on plant resistance to specific pathogen types. For instance, amino acid imbalances associated with homoserine or threonine accumulation elevate plant immunity to oomycete pathogens but not to pathogenic fungi or bacteria. The catabolism of Lys produces the immune signal pipecolic acid (Pip), a cyclic, non-protein amino acid. Pip amplifies plant defence responses and acts as a critical regulator of plant systemic acquired resistance, defence priming and local resistance to bacterial pathogens. Asp-derived pyridine nucleotides influence both pre- and post-invasion immunity, and the catabolism of branched chain amino acids appears to affect plant resistance to distinct pathogen classes by modulating crosstalk of salicylic acid- and jasmonic acid-regulated defence pathways. It also emerges that, besides polyamine oxidation and NADPH oxidase, Pro metabolism is involved in the oxidative burst and the hypersensitive response associated with avirulent pathogen recognition. Moreover, the acylation of amino acids can control plant resistance to pathogens and pests by the formation of protective plant metabolites or by the modulation of plant hormone activity.
Subject(s)
Amino Acids/metabolism , Metabolic Networks and Pathways , Plant Immunity , Homeostasis , Host-Pathogen Interactions , Oxidation-ReductionABSTRACT
In previous studies, we observed that aspirin, a promising cancer-preventive agent, induces apoptosis in mitochondrial manganese superoxide dismutase (MnSOD)-deficient Saccharomyces cerevisiae cells grown aerobically in ethanol medium. In this study, we show that aspirin-induced apoptosis is associated with a significant increase in mitochondrial and cytosolic O2 ·- and oxidation of mitochondrial NAD(P)H. A concomitant rise in the level of cytosolic CuZnSOD activity failed to compensate for mitochondrial MnSOD deficiency. However, an observed increase in activity of Escherichia coli FeSOD targeted to the mitochondrial matrix of the MnSOD-deficient yeast cells, markedly decreased aspirin-induced accumulation of mitochondrial O2 ·-, significantly increased the mitochondrial NAD(P)H level and rescued the apoptotic phenotype. Indeed, recombinant yeast cells expressing E. coli FeSOD behaved in a similar manner to the parent wild-type yeast cells with native mitochondrial MnSOD activity. Wild-type cells consistently showed a decrease in mitochondrial O2 ·- and an increase in mitochondrial NAD(P)H levels in the presence of aspirin in ethanol medium. In fact, in wild-type cells, our studies supported an antioxidant action of aspirin. Taken together, our results indicate that a pro-oxidant effect of aspirin occurring predominantly in cells with compromised mitochondrial redox balance may be enough to overcome antioxidant defences resulting in apoptosis, as observed in MnSOD-deficient yeast cells.
Subject(s)
Apoptosis/drug effects , Aspirin/pharmacology , Mitochondria/drug effects , Mitochondria/metabolism , NADP/metabolism , Superoxides/metabolism , Cytosol/metabolism , Enzyme Activation , Escherichia coli/metabolism , Oxidation-Reduction/drug effects , Phenotype , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Superoxide Dismutase/deficiency , Superoxide Dismutase/metabolismABSTRACT
The pyridine nucleotides nicotinamide adenine dinucleotide [NAD(H)] and nicotinamide adenine dinucleotide phosphate [NADP(H)] simultaneously act as energy transducers, signalling molecules, and redox couples. Recent research into photosynthetic optimisation, photorespiration, immunity, hypoxia/oxygen signalling, development, and post-harvest metabolism have all identified pyridine nucleotides as key metabolites. Further understanding will require accurate description of NAD(P)(H) metabolism, and genetically encoded fluorescent biosensors have recently become available for this purpose. Although these biosensors have begun to provide novel biological insights, their limitations must be considered and the information they provide appropriately interpreted. We provide a framework for understanding NAD(P)(H) metabolism and explore what fluorescent biosensors can, and cannot, tell us about plant biology, looking ahead to the pressing questions that could be answered with further development of these tools.
Subject(s)
Energy Metabolism , NADP , NAD , Plants/metabolism , NAD/metabolism , NADP/metabolism , Oxidation-Reduction , Signal TransductionABSTRACT
European beech is an important component of European lowland forests in terms of ecology, and produces irregular seeds categorized as intermediate due to their limited longevity. Removal of the excess of reactive oxygen species is crucial for redox homeostasis in growing plant tissues. Hydrogen peroxide (H2O2) is detoxified via the plant-specific ascorbate-glutathione cycle, and enzymatically, mainly by catalase (CAT). The reduced and oxidized (redox) forms of ascorbate (AsA, DHA) and glutathione (GSH, GSSG) decreased during maturation as the content of redox forms of nicotinamide adenine dinucleotide (NADH, NAD+) phosphate (NADPH, NADP+), cofactors of ascorbate-glutathione enzymes, declined and limited this cycle. The degree of oxidation of glutathione peaked at approximately 80%, at the exact time when the NADP content was the lowest and the NADPH/NADP+ ratio reached the highest values. The glutathione pool was reflected in changes in the NADP pool, both in embryonic axes (R2 = 0.61) and in cotyledons (R2 = 0.98). A large excess of NADPH was reported in embryonic axes, whereas cotyledons displayed more unified levels of NADP redox forms. As a result, anabolic redox charge and reducing power were higher in embryonic axes. CAT was recognized as two proteins, and the abundance of the 55 kDa protein was correlated with all redox forms of ascorbate, glutathione, NAD, and NADP, whereas the 37 kDa protein was oppositely regulated in embryonic axes and cotyledons. Here, we discuss the role of NAD(P) in the regulation of the ascorbate-glutathione cycle, catalase, and seed longevity concerning a putative role of NAD(P)H as a redox biomarker involved in predefining seed quality, because NAD(P)H-derived redox homeostasis was found to be better controlled in embryonic axes than cotyledons.
ABSTRACT
The localization of NQO1 near acetylated microtubules has led to the hypothesis that NQO1 may work in concert with the NAD+-dependent deacetylase SIRT2 to regulate acetyl α-tubulin (K40) levels on microtubules. NQO1 catalyzes the oxidation of NADH to NAD+ and may supplement levels of NAD+ near microtubules to aid SIRT2 deacetylase activity. While HDAC6 has been shown to regulate the majority of microtubule acetylation at K40, SIRT2 is also known to modulate microtubule acetylation (K40) in the perinuclear region. In this study we examined the potential roles NQO1 may play in modulating acetyl α-tubulin levels. Knock-out or knock-down of NQO1 or SIRT2 did not change the levels of acetyl α-tubulin in 16HBE human bronchial epithelial cells and 3T3-L1 fibroblasts; however, treatment with a mechanism-based inhibitor of NQO1 (MI2321) led to a short-lived temporal increase in acetyl α-tubulin levels in both cell lines without impacting the intracellular pools of NADH or NAD+. Inactivation of NQO1 by MI2321 resulted in lower levels of NQO1 immunostaining on microtubules, consistent with redox-dependent changes in NQO1 conformation as evidenced by the use of redox-specific, anti-NQO1 antibodies in immunoprecipitation studies. Given the highly dynamic nature of acetylation-deacetylation reactions at α-tubulin K40 and the crowded protein environment surrounding this site, disruption in the binding of NQO1 to microtubules may temporally disturb the physical interactions of enzymes responsible for maintaining the microtubule acetylome.
Subject(s)
Microtubules , Tubulin , 3T3-L1 Cells , Acetylation , Animals , Humans , Mice , Microtubules/metabolism , NAD(P)H Dehydrogenase (Quinone)/genetics , NAD(P)H Dehydrogenase (Quinone)/metabolism , Oxidation-Reduction , Sirtuin 2/metabolism , Tubulin/metabolismABSTRACT
Kvß subunits belong to the aldo-keto reductase superfamily, which plays a significant role in ion channel regulation and modulates the physiological responses. However, the role of Kvß2 in cardiac pathophysiology was not studied, and therefore, in the present study, we hypothesized that Kvß2 plays a significant role in cardiovascular pathophysiology by modulating the cardiac excitability and gene responses. We utilized an isoproterenol-infused mouse model to investigate the role of Kvß2 and the cardiac function, biochemical changes, and molecular responses. The deletion of Kvß2 attenuated the QTc (corrected QT interval) prolongation at the electrocardiographic (ECG) level after a 14-day isoproterenol infusion, whereas the QTc was significantly prolonged in the littermate wildtype group. Monophasic action potentials verified the ECG changes, suggesting that cardiac changes and responses due to isoproterenol infusion are mediated similarly at both the in vivo and ex vivo levels. Moreover, the echocardiographic function showed no further decrease in the ejection fraction in the isoproterenol-stimulated Kvß2 knockout (KO) group, whereas the wildtype mice showed significantly decreased function. These experiments revealed that Kvß2 plays a significant role in cardiovascular pathophysiology. Furthermore, the present study revealed SLC41a3, a major solute carrier transporter affected with a significantly decreased expression in KO vs. wildtype hearts. The electrical function showed that the decreased expression of SLC41a3 in Kvß2 KO hearts led to decreased Mg2+ responses, whereas, in the wildtype hearts, Mg2+ caused action potential duration (APD) shortening. Based on the in vivo, ex vivo, and molecular evaluations, we identified that the deletion of Kvß2 altered the cardiac pathophysiology mediated by SLC41a3 and altered the NAD (nicotinamide adenine dinucleotide)-dependent gene responses.
ABSTRACT
The pyridine nucleotides nicotineamide adenine dinucleotide (NAD) and nicotineamide adenine dinucleotide phosphate (NADP) are conserved coenzymes across all domains of life, and are involved in more than 200 different hydride transfer reactions supporting essential catabolic and anabolic functions. The intracellular levels of these metabolites, and the ratio of their oxidized to reduced forms regulate an extensive network of reactions ranging beyond metabolism. Hence, monitoring their intracellular levels provides information about, but not limited to, the metabolic state of a cell or tissue. Interconversion between oxidized and reduced forms, varying pH liability and varying intracellular concentrations of the different species leaves absolute quantification of the pyridine nucleotides analytically challenging. These polar metabolites are poorly retained on conventional reverseed-phase stationary phases without ion-pair reagents that contaminates the LC-system. Herein we demonstrate that zwitterionic HILIC-tandem mass spectroemtry can be applied to successfully resolve the pyridine nucleotides in biological extracts in a fast, robust and highly sensitive way. The presented method applies isotope dilution to compensate potential loss of these labile metabolites and is validated for low, medium and high biomass samples of two popular biological model systems; Escherichia coli and the human cell line JJN-3. High stability and rapid sample preparation without solvent removal allows for long sequence runs, making this method ideal for high-throughput analysis of biological extracts.
Subject(s)
Isotopes/analysis , Nucleotides/analysis , Plant Extracts/analysis , Pyridines/analysis , Adenine Nucleotides/chemistry , Cell Line , Chromatography, High Pressure Liquid , Escherichia coli , Humans , Limit of Detection , NAD/metabolism , Oxidation-Reduction , Reproducibility of Results , Tandem Mass SpectrometryABSTRACT
It is a textbook definition that in the absence of oxygen or inhibition of the mitochondrial respiratory chain by pharmacologic or genetic means, hyper-reduction of the matrix pyridine nucleotide pool ensues due to impairment of complex I oxidizing NADH, leading to reductive stress. However, even under these conditions, the ketoglutarate dehydrogenase complex (KGDHC) is known to provide succinyl-CoA to succinyl-CoA ligase, thus supporting mitochondrial substrate-level phosphorylation (mSLP). Mindful that KGDHC is dependent on provision of NAD+, hereby sources of acute NADH oxidation are reviewed, namely i) mitochondrial diaphorases, ii) reversal of mitochondrial malate dehydrogenase, iii) reversal of the mitochondrial isocitrate dehydrogenase as it occurs under acidic conditions, iv) residual complex I activity and v) reverse operation of the malate-aspartate shuttle. The concept of NAD+ import through the inner mitochondrial membrane as well as artificial means of manipulating matrix NAD+/NADH are also discussed. Understanding the above mechanisms providing NAD+ to KGDHC thus supporting mSLP may assist in dampening mitochondrial dysfunction underlying neurological disorders encompassing impairment of the electron transport chain.
Subject(s)
Electron Transport/physiology , Mitochondria/metabolism , NAD/metabolism , Oxidative Stress/physiology , Oxygen Consumption/physiology , Animals , Cell Respiration , Oxidation-ReductionABSTRACT
Pyridine nucleotides serve an array of intracellular metabolic functions such as, to name a few, shuttling electrons in enzymatic reactions, safeguarding the redox state against reactive oxygen species, cytochrome P450 (CYP) enzyme detoxification pathways and, relevant to this study, the regulation of ion fluxes. In particular, the maintenance of a steep calcium gradient between the cytosol and endoplasmic reticulum (ER), without which apoptosis ensues, is achieved by an elaborate combination of energy-requiring ER membrane pumps and efflux channels. In liver microsomes, net calcium uptake was inhibited by physiological concentrations of NADP. In the presence of 1 mmol/L NADP, calcium uptake was attenuated by nearly 80%, additionally, this inhibitory effect was blunted by concomitant addition of NADPH. No other nicotinamide containing compounds -save a slight inhibition by NAADP-hindered calcium uptake; thus, only oxidized pyridine nucleotides, or related compounds with a phosphate moiety, had an imposing effect. Moreover, the NADP inhibition was evident even after selectively blocking ER calcium efflux channels. Given the fundamental role of endoplasmic calcium homeostasis, it is plausible that changes in cytosolic NADP concentration, for example, during anabolic processes, could regulate net ER calcium uptake.
Subject(s)
Calcium/metabolism , Endoplasmic Reticulum/metabolism , Liver/metabolism , NADP/physiology , NAD/physiology , Animals , Calcium Channel Blockers/pharmacology , Endoplasmic Reticulum/drug effects , Liver/drug effects , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , NAD/pharmacology , NADP/pharmacology , Oxidation-Reduction , Rats, Sprague-DawleyABSTRACT
Pyridine nucleotides which include NAD+, NADH, NADP, and NADPH play vital roles in many different biological processes. These metabolites can be accurately quantified in a wide variety of biological samples using LC-MS/MS. The quality and precision of these measurements was enhanced using heavy isotope-labeled internal standards and carefully crafted protocols for sample processing.
Subject(s)
Metabolomics/methods , NADP/analysis , NAD/analysis , Tandem Mass Spectrometry/methods , Animals , Cells, Cultured , Chromatography, High Pressure Liquid/methods , Metabolomics/standards , NAD/chemistry , NAD/metabolism , NADP/chemistry , NADP/metabolism , Oxidation-Reduction , Oxygen Isotopes/chemistry , Reference StandardsABSTRACT
The mitochondrial, inner-membrane-associated, reversible NADPHâNAD+ transhydrogenase of the energetically anaerobic adult cestode Hymenolepis diminuta connects NADPH generation, via a mitochondrial NADP+-specific "malic" enzyme, with NADH formation needed for electron transport. In reducing the pyridine nucleotide, the enzyme concomitantly catalyzes transmembrane proton translocation, thereby coupling NADH formation to ATP generation or NADPH formation to ATP hydrolysis. Detergent-solubilized transhydrogenase, from isolated mitochondrial membranes, was purified to apparent homogeneity using ion exchange and hydroxylapatite chromatographies. The enzyme displayed a monomeric Mr of â¼110 kDa and required phospholipid, without which activity was rapidly lost. Of the phospholipids examined, phosphatidylcholine was the most effective. Transhydrogenase-catalyzed NADH formation was inhibited by NAD(P)+ and adenylates, suggesting regulatory effects of the pyridine nucleotides and effects of pyridine nucleotide-simulating molecules. In keeping with its proton-translocating function, the enzyme was inhibited by dicyclohexylcarbodiimide. The isolated enzyme catalyzed neither NADHâNADP+ nor NADHâNAD+ transhydrogenations, thereby suggesting a need for a minimal coupling to electron transport for the NADHâNADP+ reaction as well as enzyme specificity. Anti-transhydrogenase monospecific antibodies proved inhibitory to NADPHâNAD+ transhydrogenation catalyzed by both isolated and membrane-associated enzymes. This purification study apparently represents a first for parasitic helminths or multicellular invertebrates generally and establishes a framework for evaluating the transhydrogenase as a potential site for specific chemotherapeutic attack.
Subject(s)
Hymenolepis diminuta/enzymology , Mitochondria/enzymology , NADP Transhydrogenases/isolation & purification , NADP/metabolism , NAD/metabolism , Animals , Electrophoresis, Polyacrylamide Gel , Female , Immunoblotting , Immunoglobulin G/immunology , Male , NADP Transhydrogenases/antagonists & inhibitors , NADP Transhydrogenases/immunology , NADP Transhydrogenases/metabolism , Phospholipids/metabolism , Phospholipids/pharmacology , Rabbits , Rats , Rats, Sprague-DawleyABSTRACT
SIGNIFICANCE: It is increasingly clear that proline metabolism plays an important role in metabolic reprogramming, not only in cancer but also in related fields such as aging, senescence, and development. Although first focused on proline catabolism, recent studies from a number of laboratories have emphasized the regulatory effects of proline synthesis and proline cycling. Recent Advances: Although proline dehydrogenase/proline oxidase (PRODH/POX) has been known as a tumor protein 53 (P53)-activated source of redox signaling for initiating apoptosis and autophagy, senescence has been added to the responses. On the biosynthetic side, two well-recognized oncogenes, c-MYC and phosphoinositide 3-kinase (PI3K), markedly upregulate enzymes of proline synthesis; mechanisms affected include augmented redox cycling and maintenance of pyridine nucleotides. The reprogramming has been shown to shift in clonogenesis and/or metastasis. CRITICAL ISSUES: Although PRODH/POX generates reactive oxygen species (ROS) for signaling, the cellular endpoint is variable and dependent on metabolic context; the switches for these responses remain unknown. On the synthetic side, the enzymes require more complete characterization in various cancers, and demonstration of coupling of proline metabolites to other pathways may require studies of protein-protein interactions, membrane transporters, and shuttles. FUTURE DIRECTIONS: The proline metabolic axis can serve as a scaffold on which a variety of regulatory mechanisms are integrated. Once understood as a central mechanism in cancer metabolism, proline metabolism may be a good target for adjunctive cancer therapy.
Subject(s)
Neoplasms/metabolism , Proline/metabolism , Humans , Neoplasms/pathology , Oxidation-Reduction , Proline/chemistry , Proline Oxidase/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
The predicted future increase in tropospheric carbon dioxide (CO2) levels will have major effects on C3 plants and their interactions with other organisms in the biosphere. In response to attack by chewing arthropod herbivores or nectrotrophic pathogens, many plants mount a rapid and intense increase in jasmonate-related phytohormones that results in a robust defense response; however, previous studies have shown that C3 plants grown at elevated CO2 may have lower induced jasmonate levels, particularly in well nitrate-fertilized plants. Given the relationship between atmospheric CO2, photorespiration, cellular reductant and redox status, nitrogen assimilation and phytohormones, we compared wound-induced responses of the C3 plant Arabidopsis thaliana. These plants were fertilized at two different rates (1 or 10 mM) with nitrate or ammonium and grown at ambient or elevated CO2. In response to artificial wounding, an increase in cellular oxidative status leads to a strong increase in jasmonate phytohormones. At ambient CO2, increased oxidative state of nitrate-fertilized plants leads to a robust 7-iso-jasmonyl-L-isoleucine increase; however, the strong fertilizer rate-associated increase is alleviated in plants grown at elevated CO2. As well, the changes in ascorbate in response to wounding and wound-induced salicylic acid levels may also contribute to the suppression of the jasmonate burst. Understanding the mechanism underlying the attenuation of the jasmonate burst at elevated CO2 has important implications for fertilization strategies under future predicted climatic conditions.