A novel ATP-generating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation

Background

It is well-known that elevated amounts of nitric oxide and other reactive nitrogen species (RNS) impact negatively on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. These perturbations severely compromise O₂-dependent energy production. While bacteria are known to adapt to RNS, a key tool employed by macrophages to combat infections, the exact mechanisms are unknown.

Methods

The bacterium was cultured in a defined mineral medium and cell-free extracts obtained at the same growth phase were utilized for various biochemical studies Blue native polyacrylamide gel electrophoresis followed by in-gel activity assays, high performance liquid chromatography and co-immunoprecipitaton are applied to investigate the effects of RNS on the model microbe Pseudomonas fluorescens.

Results

Citrate is channeled away from the tricarboxylic acid cycle using a novel metabolon consisting of citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK). This metabolic engine comprising three disparate enzymes appears to transiently assemble as a supercomplex aimed at ATP synthesis. The up-regulation in the activities of adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK) ensured the efficacy of this ATP-making machine.

Conclusion

Microbes may escape the effects of nitrosative stress by re-engineering metabolic networks in order to generate and store ATP anaerobically when the electron transport chain is defective.

General significance

The molecular configuration described herein provides further understanding of how metabolism plays a key role in the adaptation to nitrosative stress and reveals novel targets that will inform the development of antimicrobial agents to counter RNS-resistant pathogens.     

Fumarate metabolism and ATP production in Pseudomonas fluorescens exposed to nitrosative stress

Although nitrosative stress is known to severely impede the ability of living systems to generate adenosine triphosphate (ATP) via oxidative phosphorylation, there is limited information on how microorganisms fulfill their energy needs in order to survive reactive nitrogen species (RNS). In this study we demonstrate an elaborate strategy involving substrate-level phosphorylation that enables the soil microbe Pseudomonas fluorescens to synthesize ATP in a defined medium with fumarate as the sole carbon source. The enhanced activities of such enzymes as phosphoenolpyruvate carboxylase and pyruvate phosphate dikinase coupled with the increased activities of phospho-transfer enzymes like adenylate kinase and nucleoside diphophate kinase provide an effective strategy to produce high energy nucleosides in an O₂-independent manner. The alternate ATP producing machinery is fuelled by the precursors derived from fumarate with the aid of fumarase C and fumarate reductase. This metabolic reconfiguration is key to the survival of P. fluorescens and reveals potential targets against RNS-resistant organisms.     

Oxidative and nitrosative signaling in plants: Two branches in the same tree?

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) constitute key features underpinning the dynamic nature of cell signaling systems in plants. Despite their importance in many aspects of cell biology, our understanding of oxidative and especially of nitrosative signaling and their regulation remains poorly understood. Early reports have established that ROS and RNS coordinately regulate plant defense responses to biotic stress. In addition, evidence has accumulated demonstrating that there is a strong cross-talk between oxidative and nitrosative signaling upon abiotic stress conditions. The goal of this mini-review is to provide latest findings showing how both ROS and RNS comprise a coordinated oxidative and nitrosative signaling network that modulates cellular responses in response to environmental stimuli.Key words: abiotic stress, nitrosative stress, oxidative stress, reactive nitrogen species, reactive oxygen species, signaling     

Metabolism of reactive nitrogen species in pea plants under abiotic stress conditions

Nitric oxide (*NO) is a key signaling molecule in different physiological processes of animals and plants. However, little is known about the metabolism of endogenous *NO and other reactive nitrogen species (RNS) in plants under abiotic stress conditions. Using pea plants exposed to six different abiotic stress conditions (high light intensity, low and high temperature, continuous light, continuous dark and mechanical wounding), several key components of the metabolism of RNS including the content of *NO, S-nitrosothiols (RSNOs) and nitrite plus nitrate, the enzyme activities of l-arginine-dependent nitric oxide synthase (NOS) and S-nitrosogluthathione reductase (GSNOR), and the profile of protein tyrosine nitration (NO(2)-Tyr) were analyzed in leaves. Low temperature was the stress that produced the highest increase of NOS and GSNOR activities, and this was accompanied by an increase in the content of total *NO and S-nitrosothiols, and an intensification of the immunoreactivity with an antibody against NO(2)-Tyr. Mechanical wounding, high temperature and light also had a clear activating effect on the different indicators of RNS metabolism in pea plants. However, the total content of nitrite and nitrate in leaves was not affected by any of these stresses. Considering that protein tyrosine nitration is a potential marker of nitrosative stress, the results obtained suggest that low and high temperature, continuous light and high light intensity are abiotic stress conditions that can induce nitrosative stress in pea plants.     

Helicobacter pylori proteins response to nitric oxide stress

Helicobacter pylori is a highly pathogenic microorganism with various strategies to evade human immune responses. Nitric oxide (NO) and reactive nitrogen species (RNS) generated via nitric oxide synthase pathway are important effectors during the innate immune response. However, the mechanisms of H. pylori to survive the nitrosative stress are not clear. Here the proteomic approach has been used to define the adaptive response of H. pylori to nitrosative stress. Proteomic analysis showed that 38 protein spots were regulated by NO donor, sodium nitroprusside (SNP). These proteins were involved in protein processing, anti-oxidation, general stress response, and virulence, as well as some unknown functions. Particularly, some of them were participated in iron metabolism, potentially under the control of ferric uptake regulator (Fur). Real time PCR revealed that fur was induced under nitrosative stress, consistent with our deduction. One stress-related protein up-regulated under nitrosative conditions was thioredoxin reductase (TrxR). Inactiva-tion of fur or trxR can lead to increased susceptivity to nitrosative stress respectively. These studies described the adaptive response of H. pylori to nitric oxide stress, and analyzed the relevant role of Fur regulon and TrxR in nitrosative stress management.     

Reactive Oxygen,Nitrogen, Carbonyl and Sulfur Species and Their Roles in Plant Abiotic Stress Responses and Tolerance

Plants being sessile organisms are often exposed to various abiotic stress conditions, which greatly hamper the growth, yields as well as the quality of produce. Plants respond to abiotic stresses in an exceptionally complex and coordinated manner, involving the interactions and crosstalk with many metabolic-molecular pathways. One of the most common responses is generation of reactive chemical species including reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive carbonyl species (RCS) and reactive sulfur species (RSS). ROS and RNS have long attracted attention from the plant researchers for both their damaging as well as protective effects. However, several reports are emerging to confirm similar roles played by the relatively newer 'reactive' members, the RCS and RSS. Plant reactive species are also hailed as vivacious signaling molecules that play regulatory roles in many plant metabolic procedures. Undeniably, these reactive species are involved in virtually all aspects of plant cell functions. Reactive species and the antioxidant machinery maintain a delicate but critical cellular redox-balance which gets disturbed under stress conditions, where their biosynthesis, transportation, scavenging and the overall metabolism gets decisive for plant survival. The current review aims to highlight and discuss the role of ROS, RNS, RCS, and RSS in plants especially under abiotic stresses, cross-talks between them, current approaches and technological advents for their characterization, and a perspective view on exploration/manipulation of the pathways and check-points involved in biosynthesis, transport and scavenging of these reactive species for engineering abiotic stress tolerant crop plants.

     

Involvement of Reactive Nitrogen and Oxygen Species (RNS and ROS) in Sunflower-Mildew Interaction

Nitric oxide (·NO) has been shown to participate in plantresponse against pathogen infection; however, less is knownof the participation of other NO-derived molecules designatedas reactive nitrogen species (RNS). Using two sunflower (Helianthusannuus L.) cultivars with different sensitivity to infectionby the pathogen Plasmopara halstedii, we studied key componentsinvolved in RNS and ROS metabolism. We analyzed the superoxideradical production, hydrogen peroxide content, L-arginine-dependentnitric oxide synthase (NOS) and S-nitrosoglutathione reductase(GSNOR) activities. Furthermore, we examined the location andcontents of ·NO, S-nitrosothiols (RSNOs), S-nitrosoglutathione(GSNO) and protein 3-nitrotyrosine (NO₂-Tyr) by confocal laserscanning microscopy (CLSM) and biochemical analyses. In thesusceptible cultivar, the pathogen induces an increase in proteinsthat undergo tyrosine nitration accompanied by an augmentationin RSNOs. This rise of RSNOs seems to be independent of theenzymatic generation of ·NO because the L-arginine-dependentNOS activity is reduced after infection. These results suggestthat pathogens induce nitrosative stress in susceptible cultivars.In contrast, in the resistant cultivar, no increase of RSNOsor tyrosine nitration of proteins was observed, implying anabsence of nitrosative stress. Therefore, it is proposed thatthe increase of tyrosine nitration of proteins can be considereda general biological marker of nitrosative stress in plantsunder biotic conditions.     

Metabolism of reactive oxygen species and reactive nitrogen species in pepper (Capsicum annuum L.) plants under low temperature stress

Low temperature is an environmental stress that affects crop production and quality and regulates the expression of many genes, and the level of a number of proteins and metabolites. Using leaves from pepper (Capsicum annum L.) plants exposed to low temperature (8 °C) for different time periods (1 to 3 d), several key components of the metabolism of reactive nitrogen and oxygen species (RNS and ROS, respectively) were analysed. After 24 h of exposure at 8 °C, pepper plants exhibited visible symptoms characterized by flaccidity of stems and leaves. This was accompanied by significant changes in the metabolism of RNS and ROS with an increase of both protein tyrosine nitration (NO(2) -Tyr) and lipid peroxidation, indicating that low temperature induces nitrosative and oxidative stress. During the second and third days at low temperature, pepper plants underwent cold acclimation by adjusting their antioxidant metabolism and reverting the observed nitrosative and oxidative stress. In this process, the levels of the soluble non-enzymatic antioxidants ascorbate and glutathione, and the activity of the main NADPH-generating dehydrogenases were significantly induced. This suggests that ascorbate, glutathione and the NADPH-generating dehydrogenases have a role in the process of cold acclimation through their effect on the redox state of the cell.     

Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings

Nitric oxide (NO) and related molecules such as peroxynitrite, S-nitrosoglutathione (GSNO), and nitrotyrosine, among others, are involved in physiological processes as well in the mechanisms of response to stress conditions. In sunflower seedlings exposed to five different adverse environmental conditions (low temperature, mechanical wounding, high light intensity, continuous light, and continuous darkness), key components of the metabolism of reactive nitrogen species (RNS) and reactive oxygen species (ROS), including the enzyme activities L-arginine-dependent nitric oxide synthase (NOS), S-nitrosogluthathione reductase (GSNOR), nitrate reductase (NR), catalase, and superoxide dismutase, the content of lipid hydroperoxide, hydrogen peroxide, S-nitrosothiols (SNOs), the cellular level of NO, GSNO, and GSNOR, and protein tyrosine nitration [nitrotyrosine (NO(2)-Tyr)] were analysed. Among the stress conditions studied, mechanical wounding was the only one that caused a down-regulation of NOS and GSNOR activities, which in turn provoked an accumulation of SNOs. The analyses of the cellular content of NO, GSNO, GSNOR, and NO(2)-Tyr by confocal laser scanning microscopy confirmed these biochemical data. Therefore, it is proposed that mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a down-regulation of GSNOR activity, while NO(2)-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants.     

The response of Paracoccidioides spp. to nitrosative stress

Paracoccidioidomycosis (PCM) is an endemic disease in Latin America caused by species belonging to the genus Paracoccidioides. During infection, immune cells present a variety of defense mechanisms against pathogens. One of these defensive strategies is the production and release of nitric oxide (NO) and S-nitroso thiols (e.g., S-nitrosoglutathione, GSNO), which produce reactive nitrogen species (RNS). This results in damage to DNA and membranes, inhibition of respiration and inactivation of cellular enzymes. In response to nitrosative stress, human pathogenic fungi possess defense mechanisms to prevent the adverse effects of NO, which helps them survive during initial contact with the host immune system. To understand how Paracoccidioides spp. respond to nitrosative stress, we conducted this study to identify genes and proteins that might contribute to this response. The results of proteomic analysis demonstrated that nitrosative stress induced a reduction in the expression of proteins related to the mitochondrial electron transport chain. This hypothesis was supported by the reduced mitochondrial activity observed in the presence of GSNO. Additionally, lipids and branched chain amino acid metabolism enzymes were altered. The role played by enzymes acting in oxidative stress in the RNS response was remarkable. This interface among enzymes acting in both stress responses was confirmed by using a RNA approach to silence the ccp gene in Paracoccidioides. It was observed that mutants with low expression of the ccp gene were more sensitive to nitrosative stress.     

Dynamic determination of Ox-LDL-induced oxidative/nitrosative stress in single macrophage by using fluorescent probes

Increased oxidative/nitrosative stress, resulting from generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears to play an important role in the inflammatory responses to atherosclerosis. By using MitoTracker Orange CM-H(2)TMRos, CM-H(2)DCFDA (DCF-DA), Dihydrorhodamine 123 (DHR123), DAF-FM, Dihydroethidium (DHE) and JC-1 alone or in all combinations of red and green probes, the present study was designed to monitor the ROS and RNS generation in acute exposure of single monocyte U937-derived macrophage to oxidized low density lipoprotein (Ox-LDL). Acute Ox-LDL (100 microg/ml) treatment increased time-dependently production of intracellular nitric oxide (NO), superoxide (O2*-), hydrogen peroxide (H(2)O(2)) and peroxynitrite (ONOO(-)), and decreased mitochondrial membrane potential (Deltapsi) in single cell. Pretreatment of aminoguanidine (an inhibitor of inducible nitric oxide synthase (iNOS), 10 microM) and vitamin C (an antioxidant agent, 100 microM) for 2h, reduced significantly the Ox-LDL-induced increase of NO and O2*-, and vitamin C completely inhibited increase of intracellular NO and O2*-. In contrast to aminoguanidine, Vitamin C pretreatment significantly prevented Ox-LDL-induced overproduction of NO and O2*- (P<0.01), indicating that antioxidant may be more effective in therapeutic application than iNOS inhibitor in dysfunction of ROS/RNS. By demonstrating a complex imbalance of ROS/RNS via fluorescent probes in acute exposure of single cell to Ox-LDL, oxidative/nitrosative stress might be more detected in the early atherosclerotic lesions.     

Peroxisomes sense and respond to environmental cues by regulating ROS and RNS signalling networks

Background Peroxisomes are highly dynamic, metabolically active organelles that used to be regarded as a sink for H₂O₂ generated in different organelles. However, peroxisomes are now considered to have a more complex function, containing different metabolic pathways, and they are an important source of reactive oxygen species (ROS), nitric oxide (NO) and reactive nitrogen species (RNS). Over-accumulation of ROS and RNS can give rise oxidative and nitrosative stress, but when produced at low concentrations they can act as signalling molecules.Scope This review focuses on the production of ROS and RNS in peroxisomes and their regulation by antioxidants. ROS production is associated with metabolic pathways such as photorespiration and fatty acid β-oxidation, and disturbances in any of these processes can be perceived by the cell as an alarm that triggers defence responses. Genetic and pharmacological studies have shown that photorespiratory H₂O₂ can affect nuclear gene expression, regulating the response to pathogen infection and light intensity. Proteomic studies have shown that peroxisomal proteins are targets for oxidative modification, S-nitrosylation and nitration and have highlighted the importance of these modifications in regulating peroxisomal metabolism and signalling networks. The morphology, size, number and speed of movement of peroxisomes can also change in response to oxidative stress, meaning that an ROS/redox receptor is required. Information available on the production and detection of NO/RNS in peroxisomes is more limited. Peroxisomal homeostasis is critical for maintaining the cellular redox balance and is regulated by ROS, peroxisomal proteases and autophagic processes.Conclusions Peroxisomes play a key role in many aspects of plant development and acclimation to stress conditions. These organelles can sense ROS/redox changes in the cell and thus trigger rapid and specific responses to environmental cues involving changes in peroxisomal dynamics as well as ROS- and NO-dependent signalling networks, although the mechanisms involved have not yet been established. Peroxisomes can therefore be regarded as a highly important decision-making platform in the cell, where ROS and RNS play a determining role.     

Nitrosative damage to free and zinc-bound cysteine thiols underlies nitric oxide toxicity in wild-type Borrelia burgdorferi

Borrelia burgdorferi encounters potentially harmful reactive nitrogen species (RNS) throughout its infective cycle. In this study, diethylamine NONOate (DEA/NO) was used to characterize the lethal effects of RNS on B. burgdorferi. RNS produce a variety of DNA lesions in a broad spectrum of microbial pathogens; however, levels of the DNA deamination product, deoxyinosine, and the numbers of apurinic/apyrimidinic (AP) sites were identical in DNA isolated from untreated and DEA/NO-treated B. burgdorferi cells. Strains with mutations in the nucleotide excision repair (NER) pathway genes uvrC or uvrB treated with DEA/NO had significantly higher spontaneous mutation frequencies, increased numbers of AP sites in DNA and reduced survival compared with wild-type controls. Polyunsaturated fatty acids in B. burgdorferi cell membranes, which are susceptible to peroxidation by reactive oxygen species (ROS), were not sensitive to RNS-mediated lipid peroxidation. However, treatment of B. burgdorferi cells with DEA/NO resulted in nitrosative damage to several proteins, including the zinc-dependent glycolytic enzyme fructose-1,6-bisphosphate aldolase (BB0445), the Borrelia oxidative stress regulator (BosR) and neutrophil-activating protein (NapA). Collectively, these data suggested that nitrosative damage to proteins harbouring free or zinc-bound cysteine thiols, rather than DNA or membrane lipids underlies RNS toxicity in wild-type B. burgdorferi.     

Disruption of the Plasmodium falciparum 2-Cys peroxiredoxin gene renders parasites hypersensitive to reactive oxygen and nitrogen species

In parasitism, Plasmodium falciparum is exposed to toxic reactive oxygen species and reactive nitrogen species (RNS). Peroxiredoxins (Prx) are ubiquitously distributed antioxidant enzymes. In bacteria and yeast, Prx have also been implicated in detoxifying RNS. Here, we used a gene targeting strategy to investigate the physiological role of 2-Cys Prx of P. falciparum, PfTPx-1, in living parasite cells. The PfTPx-1-null parasite line was more sensitive to paraquat (a superoxide donor) and sodium nitroprusside (a nitric oxide donor), than wildtype. These findings suggest that PfTPx-1 protects the parasite cells from oxidative and nitrosative stresses.     

Nitric oxide imbalance provokes a nitrosative response in plants under abiotic stress

Nitric oxide (NO), a free radical generated in plant cells, belongs to a family of related molecules designated as reactive nitrogen species (RNS). When an imbalance of RNS takes place for any adverse environmental circumstances, some of these molecules can cause direct or indirect damage at the cellular or molecular level, promoting a phenomenon of nitrosative stress. Thus, this review will emphasize the recent progress in understanding the function of NO and its production under adverse environmental conditions.     

Oxidative stress and autophagy: the clash between damage and metabolic needs

Autophagy is a catabolic process aimed at recycling cellular components and damaged organelles in response to diverse conditions of stress, such as nutrient deprivation, viral infection and genotoxic stress. A growing amount of evidence in recent years argues for oxidative stress acting as the converging point of these stimuli, with reactive oxygen species (ROS) and reactive nitrogen species (RNS) being among the main intracellular signal transducers sustaining autophagy. This review aims at providing novel insight into the regulatory pathways of autophagy in response to glucose and amino acid deprivation, as well as their tight interconnection with metabolic networks and redox homeostasis. The role of oxidative and nitrosative stress in autophagy is also discussed in the light of its being harmful for both cellular biomolecules and signal mediator through reversible posttranslational modifications of thiol-containing proteins. The redox-independent relationship between autophagy and antioxidant response, occurring through the p62/Keap1/Nrf2 pathway, is also addressed in order to provide a wide perspective upon the interconnection between autophagy and oxidative stress. Herein, we also attempt to afford an overview of the complex crosstalk between autophagy and DNA damage response (DDR), focusing on the main pathways activated upon ROS and RNS overproduction. Along these lines, the direct and indirect role of autophagy in DDR is dissected in depth.     

Interplay of Cu,Zn superoxide dismutase and nitric oxide synthase in neurodegenerative processes

Reactive oxygen and nitrogen species (ROS and RNS) have been extensively recognized as important signaling molecules implicated in physiological processes such as gene expression, cell differentiation and immune activation. Nevertheless, continuous production of these species may produce oxidative and/or nitrosative stress resulting in cell damage and ultimately leading to cell death. Due to the high oxygen consumption and relative poor antioxidant defense, the central nervous system is highly susceptible to ROS- and RNS-mediated toxicity. Actually, the oxidative and nitrosative stress have been implicated in the pathogenesis of neurodegeneration of a large variety of neurological disorders. This review will cover some aspects of the involvement of ROS- and RNS-mediated apoptotic processes occurring in cellular models of familial amyotrophic lateral sclerosis (FALS), in particular the cases associated with mutations in SOD1, the gene encoding Cu,Zn superoxide dismutase (Cu,Zn SOD). A possible role for proteasome in the inhibition of neurodegenerative process by balancing ROS and RNS species is envisaged on the basis of evidence provided by results obtained from studies on this experimental model.     

The tandem of free radicals and methylglyoxal

Methylglyoxal is an alpha-oxoaldehyde inevitably produced from triose-phosphate intermediates of phosphorylating glycolysis, and also from amino acids and acetone. Recently, the attention has been focused on the involvement of free radicals in methylglyoxal toxicity. In this review, a summary of the relationship between methylglyoxal metabolism and free radical production is presented, extending discussion from the possible metabolic routes to the toxicological events by reviewing the role of free radicals in both generation and degradation of this 1,2-dicarbonyl as well as in the modification of biological macromolecules, and focusing on the action of methylglyoxal upon cellular glutathione content. Methylglyoxal-provoked free radical generation involving reactive oxygen species (ROS), reactive nitrogen species (RNS) as well as organic radicals like methylglyoxal radial or crosslinked protein radical as potential risk factors to tissue damage propagation, is thoroughly discussed. Special attention is paid to the potential therapeutic interventions. The paper arrives at the conclusion that a tight junction exists between methylglyoxal toxicity and free radical (particularly ROS) generation, though the toxicity of 1,2-dicarbonyl evolves even under anaerobic conditions, too. The events follow a sequence beginning with carbonyl stress essential for the toxicity, leading to free radical formation and finally ending in either apoptosis or necrosis. Both oxidative and nitrosative stress play important but not indispensable role in the development of methylglyoxal toxicity.