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.     

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.     

The flavoprotein Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells

Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. In the unicellular eukaryote yeast, NO may be involved in stress response pathways, but its role is poorly understood due to the lack of mammalian NO synthase (NOS) orthologues. Previously, we have proposed the oxidative stress-induced l-arginine synthesis and its physiological role under stress conditions in yeast Saccharomyces cerevisiae. Here, our experimental results indicated that increased conversion of l-proline into l-arginine led to NO production in response to elevated temperature. We also showed that the flavoprotein Tah18, which was previously reported to transfer electrons to the Fe–S cluster protein Dre2, was involved in NO synthesis in yeast. Gene knockdown analysis demonstrated that Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells. As it appears that such a unique cell protection mechanism is specific to yeasts and fungi, it represents a promising target for antifungal activity.     

S-Nitrosogluthathione reductase activity of amphioxus ADH3: insights into the nitric oxide metabolism

Nitric oxide (NO) is a signalling molecule involved in many physiological functions. An important via of NO action is through the S-nitrosylation of proteins, a post-translational modification that regulates the activity of enzymes, protein-protein interactions and signal transduction pathways. Alcohol dehydrogenase class III (ADH3) recognises S-nitrosoglutathione (GSNO), the main reservoir of non-protein S-nitrosothiol, and functions as an effective GSNO reductase (GSNOR) and as a safeguard against nitrosative stress. To investigate the evolutionary conservation of this metabolic role, we have produced recombinant Branchiostoma floridae ADH3. Pure preparations of ADH3 showed 2-fold higher activity as GSNOR than as formaldehyde dehydrogenase, the previously assumed main role for ADH3. To correlate ADH3 expression in the gut with areas of NO production, we analysed the tissue distribution of the nitric oxide synthase (NOS) enzyme in amphioxus larvae. Immunostaining of the NOS enzyme revealed expression in the gut and in the dorsal region of the club-shaped gland. Co-localization in the gut supports the ADH3 and NOS joint contribution to the NO/SNO homeostasis.     

Detection and quantification of S-nitrosoglutathione (GSNO) in pepper (Capsicum annuum L.) plant organs by LC-ES/MS

Glutathione (GSH) is one of the major, soluble, low molecular weight antioxidants, as well as the major non-protein thiol in plant cells. However, the relevance of this molecule could be even greater considering that it can react with nitric oxide (NO) to generate S-nitrosoglutathione (GSNO) which is considered to function as a mobile reservoir of NO bioactivity in plants. Although this NO-derived molecule has an increased physiological and phytopathological relevance in plants cells, its identification and quantification in plant tissues have not be reported so far. Using liquid chromatography-electrospray/mass spectrometry (LC-ES/MS), a method was set up to detect and quantify simultaneously GSNO as well reduced and oxidized glutathione (GSH and GSSG, respectively) in different pepper plant organs including roots, stems and leaves, and in Arabidopsis leaves. The analysis of NO and GSNO reductase (GSNOR) activity in these pepper organs showed that the content of GSNO was directly related to the content of NO in each organ and oppositely related to the GSNOR activity. This approach opens up new analytical possibilities to understand the relevance of GSNO in plant cells under physiological and stress conditions.     

HIF-NOS信号通路对哺乳动物卵巢NO依赖性功能的调控作用

一氧化氮(NO)作为气体明星分子和信号分子,在哺乳动物体内不同的生理调节过程中具有非常重要的作用,尤其是哺乳动物卵巢功能的调控.一氧化氮合酶(NOS)是NO合成的限速酶,是调节NO合成的关键环节,也是NO依赖性功能调控的重要环节.因此,调节NOS转录/合成的信号通路对哺乳动物卵巢NO依赖性功能具有至关重要的调控作用.最近的研究发现,缺氧诱导因子(HIF)作为转录因子,参与许多与缺氧相关靶基因的转录调控,如NOS和血管内皮生长因子(VEGF)等.本文一方面描述了NO合成及其调控的分子机制,另一方面阐明了HIF作为转录因子对NOS的转录调控,从而揭示HIF在NO依赖性卵巢功能调控中的重要作用,同时为进一步研究哺乳动物卵巢功能的调控提供新的研究方向和理论基础.     

S-Nitrosoglutathion Reductase Activity Modulates the Thermotolerance of Seeds Germination by Controlling ABI5 Stability under High Temperature

Seed germination or dormancy status is strictly controlled by endogenous phytohormone and exogenous environment signals. Abscisic acid (ABA) is the important phytohormone to suppress seed germination. Ambient high temperature (HT) also suppressed seed germination, or called as secondary seed dormancy, through upregulating ABI5, the essential component of ABA signal pathway. Previous result shows that appropriate nitric oxide (NO) breaks seed dormancy through triggering S-nitrosoglutathion reductase (GSNOR1)-dependent S-nitrosylation modification of ABI5 protein, subsequently inducing the degradation of ABI5. Here we found that HT induced the degradation of GSNOR1 protein and reduced its activity, thus accumulated more reactive nitrogen species (RNS) to damage seeds viability. Furthermore, HT increased the S-nitrosylation modification of GSNOR1 protein, and triggered the degradation of GSNOR1, therefore stabilizing ABI5 to suppress seed germination. Consistently, the ABI5 protein abundance was lower in the transgenic line overexpressing GSNOR1, but higher in the gsnor mutant after HT stress. Genetic analysis showed that GSNOR1 affected seeds germination through ABI5 under HT. Taken together, our data reveals a new mechanism by which HT triggers the degradation of GSNOR1, and thus stabilizing ABI5 to suppress seed germination, such mechanism provides the possibility to enhance seed germination tolerance to HT through genetic modification of GNSOR1.     

S‐nitrosylation/denitrosylation as a regulatory mechanism of salt stress sensing in sunflower seedlings

Nitric oxide (NO) and various reactive nitrogen species produced in cells in normal growth conditions, and their enhanced production under stress conditions are responsible for a variety of biochemical aberrations. The present findings demonstrate that sunflower seedling roots exhibit high sensitivity to salt stress in terms of nitrite accumulation. A significant reduction in S‐nitrosoglutathione reductase (GSNOR) activity is evident in response to salt stress. Restoration of GSNOR activity with dithioerythritol shows that the enzyme is reversibly inhibited under conditions of 120 mM NaCl. Salt stress‐mediated S‐nitrosylation of cytosolic proteins was analyzed in roots and cotyledons using biotin‐switch assay. LC‐MS/MS analysis revealed opposite patterns of S‐nitrosylation in seedling cotyledons and roots. Salt stress enhances S‐nitrosylation of proteins in cotyledons, whereas roots exhibit denitrosylation of proteins. Highest number of proteins having undergone S‐nitrosylation belonged to the category of carbohydrate metabolism followed by other metabolic proteins. Of the total 61 proteins observed to be regulated by S‐nitrosylation, 17 are unique to cotyledons, 4 are unique to roots whereas 40 are common to both. Eighteen S‐nitrosylated proteins are being reported for the first time in plant systems, including pectinesterase, phospholipase d ‐alpha and calmodulin. Further physiological analysis of glyceraldehyde‐3‐phosphate dehydrogenase and monodehydroascorbate reductase showed that salt stress leads to a reversible inhibition of both these enzymes in cotyledons. However, seedling roots exhibit enhanced enzyme activity under salinity stress. These observations implicate the role of S‐nitrosylation and denitrosylation in NO signaling thereby regulating various enzyme activities under salinity stress in sunflower seedlings.     

Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock

The current perspective of NO biology is formulated predominantly from studies of NO synthesis. The role of S-nitrosothiol (SNO) formation and turnover in governing NO-related bioactivity remains uncertain. We generated mice with a targeted gene deletion of S-nitrosoglutathione reductase (GSNOR), and show that they exhibit substantial increases in whole-cell S-nitrosylation, tissue damage, and mortality following endotoxic or bacterial challenge. Further, GSNOR(-/-) mice have increased basal levels of SNOs in red blood cells and are hypotensive under anesthesia. Thus, SNOs regulate innate immune and vascular function, and are cleared actively to ameliorate nitrosative stress. Nitrosylation of cysteine thiols is a critical mechanism of NO function in both health and disease.     

Immunolocalization of S-nitrosoglutathione, S-nitrosoglutathione reductase and tyrosine nitration in pea leaf organelles

S-Nitrosoglutathione (GSNO) is a nitrosothiol which plays a major role in the metabolism of NO in higher plants mediating signaling processes. Protein tyrosine nitration (NO₂–Tyr) is a post-translational modification which contributes to protein regulation. The subcellular localization of GSNO, S-nitrosoglutathione reductase (GSNOR), an enzyme which catalyzes its decomposition and protein tyrosine nitration was studied in pea (Pisum sativum L.) leaf plants with the aid of the electron microscopy immunogold-labeling technique. Our findings show that GSNO, GSNOR and nitrated proteins are present in the different subcellular compartments of leaf cells which include chloroplasts, cytosol, mitochondria, and peroxisomes. Given that pea peroxisomes are one of the cell compartments where nitric oxide (NO) has been thoroughly studied, our results provide additional insights into the metabolism of NO in this organelle where NO and GSNO could function as signal molecules in cross talk between the different cell compartments.     

The role of S-nitrosoglutathione reductase (GSNOR) in human disease and therapy

S-nitrosoglutathione reductase (GSNOR), or ADH5, is an enzyme in the alcohol dehydrogenase (ADH) family. It is unique when compared to other ADH enzymes in that primary short-chain alcohols are not its principle substrate. GSNOR metabolizes S-nitrosoglutathione (GSNO), S-hydroxymethylglutathione (the spontaneous adduct of formaldehyde and glutathione), and some alcohols. GSNOR modulates reactive nitric oxide (?NO) availability in the cell by catalyzing the breakdown of GSNO, and indirectly regulates S-nitrosothiols (RSNOs) through GSNO-mediated protein S-nitrosation. The dysregulation of GSNOR can significantly alter cellular homeostasis, leading to disease. GSNOR plays an important regulatory role in smooth muscle relaxation, immune function, inflammation, neuronal development and cancer progression, among many other processes. In recent years, the therapeutic inhibition of GSNOR has been investigated to treat asthma, cystic fibrosis and interstitial lung disease (ILD). The direct action of ?NO on cellular pathways, as well as the important regulatory role of protein S-nitrosation, is closely tied to GSNOR regulation and defines this enzyme as an important therapeutic target.     

Nitric oxide‐mediated regulation of ‐amyloid clearance via alterations of MMP‐9/TIMP‐1

Fibrillar amyloid plaques are largely composed of amyloid‐beta (Aβ) peptides that are metabolized into products, including Aβ1‐16, by proteases including matrix metalloproteinase 9 (MMP‐9). The balance between production and degradation of Aβ proteins is critical to amyloid accumulation and resulting disease. Regulation of MMP‐9 and its endogenous inhibitor tissue inhibitor of metalloproteinase (TIMP)‐1 by nitric oxide (NO) has been shown. We hypothesize that nitric oxide synthase (NOS2) protects against Alzheimer's disease pathology by increasing amyloid clearance through NO regulation of MMP‐9/TIMP‐1 balance. We show NO‐mediated increased MMP‐9/TIMP‐1 ratios enhanced the degradation of fibrillar Aβ in vitro, which was abolished when silenced for MMP‐9 protein translation. The in vivo relationship between MMP‐9, NO and Aβ degradation was examined by comparing an Alzheimer's disease mouse model that expresses NOS2 with a model lacking NOS2. To quantitate MMP‐9 mediated changes, we generated an antibody recognizing the Aβ1‐16 fragment, and used mass spectrometry multi‐reaction monitoring assay for detection of immunoprecipitated Aβ1‐16 peptides. Aβ1‐16 levels decreased in brain lysates lacking NOS2 when compared with strains that express human amyloid precursor protein on the NOS2 background. TIMP‐1 increased in the APPSwDI/NOS2^?/? mice with decreased MMP activity and increased amyloid burden, thereby supporting roles for NO in the regulation of MMP/TIMP balance and plaque clearance.     

Lymphocyte development requires S-nitrosoglutathione reductase

NO is critical to immunity, but its role in the development of the immune system is unknown. In this study, we show that S-nitrosoglutathione reductase (GSNOR), a protein key to the control of protein S-nitrosylation, is important for the development of lymphocytes. Genetic deletion of GSNOR in mice results in significant decrease in both T and B lymphocytes in the periphery. In thymus, GSNOR deficiency causes excessive protein S-nitrosylation, increases apoptosis, and reduces the number of CD4 single-positive thymocytes. Lymphopenia and increase in S-nitrosylation and apoptosis in GSNOR-deficient mice are largely abolished by genetic deletion of inducible NO synthase. Furthermore, the protection of lymphocyte development by GSNOR is apparently intrinsic to hematopoietic cells. Thus, GSNOR, likely through regulation of S-nitrosylation and apoptosis, physiologically plays a protective role in the development of the immune system.     

Development of a dimethylarginine dimethylaminohydrolase (DDAH) assay for high-throughput chemical screening

Nitric oxide (NO) is a potent signaling molecule that needs to be tightly regulated to maintain metabolic and cardiovascular homeostasis. The nitric oxide synthase (NOS)/dimethylarginine dimethylaminohydrolase (DDAH)/asymmetric dimethylarginine (ADMA) pathway is central to this regulation. Specifically, the small-molecule ADMA competitively inhibits NOS, thus lowering NO levels. The majority of ADMA is physiologically metabolized by DDAH, thus maintaining NO levels at a physiological concentration. However, under pathophysiological conditions, DDAH activity is impaired, in part as a result of its sensitivity to oxidative stress. Therefore, the application of high-throughput chemical screening for the discovery of small molecules that could restore or enhance DDAH activity might have significant potential in treating metabolic and vascular diseases characterized by reduced NO levels, including atherosclerosis, hypertension, and insulin resistance. By contrast, excessive generation of NO (primarily driven by inducible NOS) could play a role in idiopathic pulmonary fibrosis, sepsis, migraine headaches, and some types of cancer. In these conditions, small molecules that inhibit DDAH activity might be therapeutically useful. Here, we describe optimization and validation of a highly reproducible and robust assay successfully used in a high-throughput screen for DDAH modulators.     

Nitric oxide production in the hypoxic lung

Nitric oxide (NO) is a potent vasodilator and inhibitor of vascular remodeling. Reduced NO production has been implicated in the pathophysiology of pulmonary hypertension, with endothelial NO synthase (NOS) knockout mice showing an increased risk for pulmonary hypertension. Because molecular oxygen (O2) is an essential substrate for NO synthesis by the NOSs and biochemical studies using purified NOS isoforms have estimated the Michaelis-Menten constant values for O2 to be in the physiological range, it has been suggested that O2 substrate limitation may limit NO production in various pathophysiological conditions including hypoxia. This review summarizes numerous studies of the effects of acute and chronic hypoxia on NO production in the lungs of humans and animals as well as in cultured vascular cells. In addition, the effects of hypoxia on NOS expression and posttranslational regulation of NOS activity by other proteins are also discussed. Most studies found that hypoxia limits NO synthesis even when NOS expression is increased.     

Bacterial nitric-oxide synthases operate without a dedicated redox partner

Bacterial nitric-oxide (NO) synthases (bNOSs) are smaller than their mammalian counterparts. They lack an essential reductase domain that supplies electrons during NO biosynthesis. This and other structural peculiarities have raised doubts about whether bNOSs were capable of producing NO in vivo. Here we demonstrate that bNOS enzymes from Bacillus subtilis and Bacillus anthracis do indeed produce NO in living cells and accomplish this task by hijacking available cellular redox partners that are not normally committed to NO production. These "promiscuous" bacterial reductases also support NO synthesis by the oxygenase domain of mammalian NOS expressed in Escherichia coli. Our results suggest that bNOS is an early precursor of eukaryotic NOS and that it acquired its dedicated reductase domain later in evolution. This work also suggests that alternatively spliced forms of mammalian NOSs lacking their reductase domains could still be functional in vivo. On a practical side, bNOS-containing probiotic bacteria offer a unique advantage over conventional chemical NO donors in generating continuous, readily controllable physiological levels of NO, suggesting a possibility of utilizing such live NO donors for research and clinical needs.     

Nitric oxide alleviates heat stress-induced oxidative damage in Pleurotus eryngii var. tuoliensis

High temperature is one of the major impediments limiting the growth and development of most edible fungi. While many efforts have been made in agricultural practice, the mechanism for resistance to high temperature remains elusive. Nitric oxide (NO) is considered as a signaling molecule involved in regulation of diverse physiological processes and stress responses in animals and plants. However, the role of NO in regulating fungal, particularly edible fungi, response to abiotic stresses, is unknown. The present study demonstrated that NO could effectively alleviate oxidative damage induced by heat stress in mycelia of Pleurotus eryngii var. tuoliensis. Heat stress induced increased thiobarbituric acid reactive substance (TBARS) content in mycelia, and the NO donor sodium nitroprusside (SNP) dramatically decreased TBARS content under high temperature. Moreover, the specific NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxyl-3-oxide (cPTIO), could arrest the SNP action under the stress. Heat stress induced an increase in endogenous NO production in mycelial cells. However, the effect was significantly blocked by the NO synthase (NOS) inhibitor l-N(G)-nitroarginine methyl ester (l-NAME). In contrast, nitrate reductase (NR) activities were not obviously altered during heat stress. The NR suppressor tungstate had no effect on intracellular NO abundance under heat stress. These results suggest that NO can effectively protect mycelia of edible fungi from heat stress-induced oxidative damage and the NOS-dependent NO production may participate in the response to heat stress.