Structural and biochemical characterization of Arabidopsis alcohol dehydrogenases reveals distinct functional properties but similar redox sensitivity.

Alcohol dehydrogenases (ADHs) are a group of zinc-binding enzymes belonging to the medium-length dehydrogenase/reductase (MDR) protein superfamily. In plants, these enzymes fulfill important functions involving the reduction of toxic aldehydes to the corresponding alcohols (as well as catalyzing the reverse reaction, i.e., alcohol oxidation; ADH1) and the reduction of nitrosoglutathione (GSNO; ADH2/GSNOR). We investigated and compared the structural and biochemical properties of ADH1 and GSNOR from Arabidopsis thaliana. We expressed and purified ADH1 and GSNOR and determined two new structures, NADH-ADH1 and apo-GSNOR, thus completing the structural landscape of Arabidopsis ADHs in both apo- and holo-forms. A structural comparison of these Arabidopsis ADHs revealed a high sequence conservation (59% identity) and a similar fold. In contrast, a striking dissimilarity was observed in the catalytic cavity supporting substrate specificity and accommodation. Consistently, ADH1 and GSNOR showed strict specificity for their substrates (ethanol and GSNO, respectively), although both enzymes had the ability to oxidize long-chain alcohols, with ADH1 performing better than GSNOR. Both enzymes contain a high number of cysteines (12 and 15 out of 379 residues for ADH1 and GSNOR, respectively) and showed a significant and similar responsivity to thiol-oxidizing agents, indicating that redox modifications may constitute a mechanism for controlling enzyme activity under both optimal growth and stress conditions.

Spraying with encapsulated nitric oxide donor reduces weight loss and oxidative damage in papaya fruit.

The combination of nitric oxide (NO) donors with nanomaterials has emerged as a promising approach to reduce postharvest losses. The encapsulation of NO donors provides protection from rapid degradation and controlled release, enhancing the NO effectiveness in postharvest treatments. Moreover, the application method can also influence postharvest responses. In this study, two application methods were evaluated, spraying and immersion, using S-nitrosoglutathione (GSNO, a NO donor) in free and encapsulated forms on papaya fruit. Our hypothesis was that GSNO encapsulated in chitosan nanoparticles would outperform the free form in delaying fruit senescence. In addition, this study marks the pioneering characterization of chitosan nanoparticles containing GSNO within the framework of a postharvest investigation. Overall, our findings indicate that applying encapsulated GSNO (GSNO-NP-S) through spraying preserves the quality of papaya fruit during storage. This method not only minimizes weight loss, ethylene production, and softening, but also stimulates antioxidant responses, thereby mitigating oxidative damage. Consequently, it stands out as the promising technique for delaying papaya fruit senescence. This innovative approach holds the potential to enhance postharvest practices and advance sustainable agriculture.

S-Nitrosoglutathione Attenuates Oxidative Stress and Improves Retention Memory Dysfunctions in Intra-Cerebroventricular-Streptozotocin Rat Model of Sporadic Alzheimer's Disease via Activation of BDNF and Nuclear Factor Erythroid 2-Related Factor-2 Antioxidant Signaling Pathway.

INTRODUCTION: The brain-derived neurotrophic factor (BDNF) and transcription nuclear factor erythroid 2-related factor-2 (NRF-2) play an important role in Alzheimer's disease (AD). However, the interactive involvement of BDNF and NRF-2 in respect to antioxidant mechanisms in different parts of the AD brain is still unclear. Considering the above condition, used S-nitrosoglutathione (GSNO) to examine whether it modulates the BDNF and NRF-2 levels to activate signaling pathway to promote antioxidant levels in AD brains. METHOD: AD was induced by intracerebroventricular infusion of streptozotocin (ICV-STZ, 3 mg/kg) in Wistar rats. The effect of GSNO was analyzed by evaluating the retention of memory in months 1, 2, and 3. After the behavior study, rats were sacrificed and accessed the amyloid beta (Aß)-40, Aß42, glutathione (GSH), BDNF, and NRF-2 levels in the hippocampus, cortex, and amygdala tissue. RESULTS: Pretreatment with GSNO (50 µg/kg/intraperitoneal/day) restored the BDNF, and NRF-2 levels toward normalcy as compared with ICV-STZ + saline-treated animals. Also, GSNO treatment reversed the oxidative stress and increased the GSH levels toward normal levels. Further, reduced Aß levels and neuronal loss in different brain regions. As a result, GSNO treatment improved the cognitive deficits in ICV-STZ-treated rats. CONCLUSION: The results showed that endogenous nitric oxide donor GSNO improved the cognitive deficits and ICV-STZ-induced AD pathological conditions, possibly via attenuating the oxidative stress. Hence, the above finding supported that GSNO treatment may activate BDNF and NRF-2 antioxidant signaling pathways in the AD brain to normalize oxidative stress, which is the main causative factor for ICV-STZ-induced AD pathogenesis.

NIR-activated electrospun nanodetonator dressing enhances infected diabetic wound healing with combined photothermal and nitric oxide-based gas therapy.

Diabetic wounds pose a challenge to healing due to increased bacterial susceptibility and poor vascularization. Effective healing requires simultaneous bacterial and biofilm elimination and angiogenesis stimulation. In this study, we incorporated polyaniline (PANI) and S-Nitrosoglutathione (GSNO) into a polyvinyl alcohol, chitosan, and hydroxypropyltrimethyl ammonium chloride chitosan (PVA/CS/HTCC) matrix, creating a versatile wound dressing membrane through electrospinning. The dressing combines the advantages of photothermal antibacterial therapy and nitric oxide gas therapy, exhibiting enduring and effective bactericidal activity and biofilm disruption against methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Escherichia coli. Furthermore, the membrane's PTT effect and NO release exhibit significant synergistic activation, enabling a nanodetonator-like burst release of NO through NIR irradiation to disintegrate biofilms. Importantly, the nanofiber sustained a uniform release of nitric oxide, thereby catalyzing angiogenesis and advancing cellular migration. Ultimately, the employment of this membrane dressing culminated in the efficacious amelioration of diabetic-infected wounds in Sprague-Dawley rats, achieving wound closure within a concise duration of 14 days. Upon applying NIR irradiation to the PVA-CS-HTCC-PANI-GSNO nanofiber membrane, it swiftly eradicates bacteria and biofilm within 5 min, enhancing its inherent antibacterial and anti-biofilm properties through the powerful synergistic action of PTT and NO therapy. It also promotes angiogenesis, exhibits excellent biocompatibility, and is easy to use, highlighting its potential in treating diabetic wounds.

Computational Insights into the Mechanism of Nitric Oxide Generation from S-Nitrosoglutathione Catalyzed by a Copper Metal-Organic Framework.

The controlled generation of nitric oxide (NO) from endogenous sources, such as S-nitrosoglutathione (GSNO), has significant implications for biomedical implants due to the vasodilatory and other beneficial properties of NO. The water-stable metal-organic framework (MOF) Cu-1,3,5-tris[1H-1,2,3-triazol-5-yl]benzene has been shown to catalyze the production of NO and glutathione disulfide (GSSG) from GSNO in aqueous solution as well as in blood. Previous experimental work provided kinetic data for the catalysis of the 2GSNO → 2NO + GSSG reaction, leading to various proposed mechanisms. Herein, this catalytic process is examined using density functional theory. Minimal functional models of the Cu-MOF cluster and glutathione moieties are established, and three distinct catalytic mechanisms are explored. The most thermodynamically favorable mechanism studied is consistent with prior experimental findings. This mechanism involves coordination of GSNO to copper via sulfur rather than nitrogen and requires a reductive elimination that produces a Cu(I) intermediate, implicating a redox-active copper site. The experimentally observed inhibition of reactivity at high pH values is explained in terms of deprotonation of a triazole linker, which decreases the structural stability of the Cu(I) intermediate. These fundamental mechanistic insights may be generally applicable to other MOF catalysts for NO generation.

Loss of S-nitrosoglutathione reductase disturbs phytohormone homeostasis and regulates shoot side branching and fruit growth in tomato.

S-Nitrosoglutathione plays a central role in nitric oxide (NO) homeostasis, and S-nitrosoglutathione reductase (GSNOR) regulates the cellular levels of S-nitrosoglutathione across kingdoms. Here, we investigated the role of endogenous NO in shaping shoot architecture and controlling fruit set and growth in tomato (Solanum lycopersicum). SlGSNOR silencing promoted shoot side branching and led to reduced fruit size, negatively impacting fruit yield. Greatly intensified in slgsnor knockout plants, these phenotypical changes were virtually unaffected by SlGSNOR overexpression. Silencing or knocking out of SlGSNOR intensified protein tyrosine nitration and S-nitrosation and led to aberrant auxin production and signaling in leaf primordia and fruit-setting ovaries, besides restricting the shoot basipetal polar auxin transport stream. SlGSNOR deficiency triggered extensive transcriptional reprogramming at early fruit development, reducing pericarp cell proliferation due to restrictions on auxin, gibberellin, and cytokinin production and signaling. Abnormal chloroplast development and carbon metabolism were also detected in early-developing NO-overaccumulating fruits, possibly limiting energy supply and building blocks for fruit growth. These findings provide new insights into the mechanisms by which endogenous NO fine-tunes the delicate hormonal network controlling shoot architecture, fruit set, and post-anthesis fruit development, emphasizing the relevance of NO-auxin interaction for plant development and productivity.

Candidate molecules as alternative nitric oxide donors with better antibacterial property against Escherichia coli and Staphylococcus aureus.

AIMS: Four nitric oxide (NO) donors, S-nitrosoglutathione (GSNO), S-nitrosocysteine (CySNO), S-nitroso-N-acetylcysteine (SNAC), and 2-(2-S-nitroso propionamide) acetic acid (GAS) were prepared and their physicochemical characteristics were analyzed. Besides, the antibacterial properties of NO donors were investigated against Escherichia coli and Staphylococcus aureus. METHODS AND RESULTS: UV-visible absorption spectrum and Fourier transform infrared spectrum verified the successful preparation of RSNOs. All NO donors (10 mmol l-1) could release NO continuously, and the amount of NO release was from 80.22 µmol l-1 to 706.63 µmol l-1, in which the release of NO from SNAC was the highest, and the release of NO from NaNO2 was the least. The inhibition zone indicated that all NO donors showed stronger antibacterial activity against E. coli and S. aureus, and the antibacterial ability was in the order of SNAC > GSNO > CySNO > GAS > NaNO2 for both E. coli and S. aureus (P < 0.05). Scanning electron microscopy(SEM) showed that all NO donors could result in varying degrees of damage to cell wall and membrane of both E. coli and S. aureus and the damage of E. coli was more severe. CONCLUSION: Four alternative NO donors were successfully synthesized. All alternative NO donors showed better antibacterial properties against E. coli and S. aureus than NaNO2.

S-Nitrosoglutathione (GSNO)-Mediated Lead Detoxification in Soybean through the Regulation of ROS and Metal-Related Transcripts.

Heavy metal toxicity, including lead (Pb) toxicity, is increasing in soils, and heavy metals are considered to be toxic in small amounts. Pb contamination is mainly caused by industrialization (e.g., smelting and mining), agricultural practices (e.g., sewage sludge and pests), and urban practices (e.g., lead paint). An excessive concentration of Pb can seriously damage and threaten crop growth. Furthermore, Pb adversely affects plant growth and development by affecting the photosystem, cell membrane integrity, and excessive production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide (O2-). Nitric oxide (NO) is produced via enzymatic and non-enzymatic antioxidants to scavenge ROS and lipid peroxidation substrates to protect cells from oxidative damage. Thus, NO improves ion homeostasis and confers resistance to metal stress. In the present study, we investigated the effect of exogenously applied NO and S-nitrosoglutathione in soybean plants Our results demonstrated that exogenously applied NO aids in better growth under lead stress due to its ability in sensing, signaling, and stress tolerance in plants under heavy metal stress along with lead stress. In addition, our results showed that S-nitrosoglutathione (GSNO) has a positive effect on soybean seedling growth under lead-induced toxicity and that NO supplementation helps to reduce chlorophyll maturation and relative water content in leaves and roots following strong bursts under lead stress. GSNO supplementation (200 µM and 100 µM) reduced compaction and approximated the oxidative damage of MDA, proline, and H2O2. Moreover, under plant stress, GSNO application was found to relieve the oxidative damage by reactive oxygen species (ROS) scavenging. Additionally, modulation of NO and phytochelatins (PCS) after prolonged metal reversing GSNO application confirmed detoxification of ROS induced by the toxic metal lead in soybean. In summary, the detoxification of ROS caused by toxic metal concentrations in soybean is confirmed by using NO, PCS, and traditionally sustained concentrations of metal reversing GSNO application.

Inhibition of platelet activation suppresses reactive enteric glia and mitigates intestinal barrier dysfunction during sepsis.

BACKGROUND: Intestinal barrier dysfunction, which is associated with reactive enteric glia cells (EGCs), is not only a result of early sepsis but also a cause of multiple organ dysfunction syndrome. Inhibition of platelet activation has been proposed as a potential treatment for septic patients because of its efficacy in ameliorating the organ damage and barrier dysfunction. During platelet activation, CD40L is translocated from α granules to the platelet surface, serving as a biomarker of platelet activation a reliable predictor of sepsis prognosis. Given that more than 95% of the circulating CD40L originate from activated platelets, the present study aimed to investigate if inhibiting platelet activation mitigates intestinal barrier dysfunction is associated with suppressing reactive EGCs and its underlying mechanism. METHODS: Cecal ligation and puncture (CLP) was performed to establish the sepsis model. 24 h after CLP, the proportion of activated platelets, the level of sCD40L, the expression of tight-junction proteins, the intestinal barrier function and histological damage of septic mice were analyzed. In vitro, primary cultured EGCs were stimulated by CD40L and LPS for 24 h and EGCs-conditioned medium were collected for Caco-2 cells treatment. The expression of tight-junction proteins and transepithelial electrical resistance of Caco-2 cell were evaluated. RESULTS: In vivo, inhibiting platelet activation with cilostazol mitigated the intestinal barrier dysfunction, increased the expression of ZO-1 and occludin and improved the survival rate of septic mice. The efficacy was associated with reduced CD40L+ platelets proportion, decreased sCD40L concentration, and suppressed the activation of EGCs. Comparable results were observed upon treatment with compound 6,877,002, a blocker of CD40L-CD40-TRAF6 signaling pathway. Also, S-nitrosoglutathione supplement reduced intestinal damage both in vivo and in vitro. In addition, CD40L increased release of TNF-α and IL-1ß while suppressed the release of S-nitrosoglutathione from EGCs. These EGCs-conditioned medium reduced the expression of ZO-1 and occludin on Caco-2 cells and their transepithelial electrical resistance, which could be reversed by CD40-siRNA and TRAF6-siRNA transfection on EGCs. CONCLUSIONS: The inhibition of platelet activation is related to the suppression of CD40L-CD40-TRAF6 signaling pathway and the reduction of EGCs activation, which promotes intestinal barrier function and survival in sepsis mice. These results might provide a potential therapeutic strategy and a promising target for sepsis.

Inhibition of platelet activation suppresses reactive enteric glia and mitigates intestinal barrier dysfunction during sepsis.

BACKGROUND: Intestinal barrier dysfunction, which is associated with reactive enteric glia cells (EGCs), is not only a result of early sepsis but also a cause of multiple organ dysfunction syndrome. Inhibition of platelet activation has been proposed as a potential treatment for septic patients because of its efficacy in ameliorating the organ damage and barrier dysfunction. During platelet activation, CD40L is translocated from α granules to the platelet surface, serving as a biomarker of platelet activation a reliable predictor of sepsis prognosis. Given that more than 95% of the circulating CD40L originate from activated platelets, the present study aimed to investigate if inhibiting platelet activation mitigates intestinal barrier dysfunction is associated with suppressing reactive EGCs and its underlying mechanism. METHODS: Cecal ligation and puncture (CLP) was performed to establish the sepsis model. 24 h after CLP, the proportion of activated platelets, the level of sCD40L, the expression of tight-junction proteins, the intestinal barrier function and histological damage of septic mice were analyzed. In vitro, primary cultured EGCs were stimulated by CD40L and LPS for 24 h and EGCs-conditioned medium were collected for Caco-2 cells treatment. The expression of tight-junction proteins and transepithelial electrical resistance of Caco-2 cell were evaluated. RESULTS: In vivo, inhibiting platelet activation with cilostazol mitigated the intestinal barrier dysfunction, increased the expression of ZO-1 and occludin and improved the survival rate of septic mice. The efficacy was associated with reduced CD40L+ platelets proportion, decreased sCD40L concentration, and suppressed the activation of EGCs. Comparable results were observed upon treatment with compound 6877002, a blocker of CD40L-CD40-TRAF6 signaling pathway. Also, S-nitrosoglutathione supplement reduced intestinal damage both in vivo and in vitro. In addition, CD40L increased release of TNF-α and IL-1ß while suppressed the release of S-nitrosoglutathione from EGCs. These EGCs-conditioned medium reduced the expression of ZO-1 and occludin on Caco-2 cells and their transepithelial electrical resistance, which could be reversed by CD40-siRNA and TRAF6-siRNA transfection on EGCs. CONCLUSIONS: The inhibition of platelet activation is related to the suppression of CD40L-CD40-TRAF6 signaling pathway and the reduction of EGCs activation, which promotes intestinal barrier function and survival in sepsis mice. These results might provide a potential therapeutic strategy and a promising target for sepsis.

Ceria Nanoparticles as an Unexpected Catalyst to Generate Nitric Oxide from S-Nitrosoglutathione.

Ceria nanoparticles (NPs) are widely reported to scavenge nitric oxide (NO) radicals. This study reveals evidence that an opposite effect of ceria NPs exists, that is, to induce NO generation. Herein, S-nitrosoglutathione (GSNO), one of the most biologically abundant NO donors, is catalytically decomposed by ceria NPs to produce NO. Ceria NPs maintain a high NO release recovery rate and retain their crystalline structure for at least 4 weeks. Importantly, the mechanism of this newly discovered NO generation capability of ceria NPs from GSNO is deciphered to be attributed to the oxidation of Ce3+ to Ce4+ on their surface, which is supported by X-ray photoelectron spectroscopy and density functional theory analysis. The prospective therapeutic effect of NO-generating ceria NPs is evaluated by the suppression of cancer cells, displaying a significant reduction of 93% in cell viability. Overall, this report is, to the authors' knowledge, the first study to identify the capability of ceria NPs to induce NO generation from GSNO, which overturns the conventional concept of them acting solely as a NO-scavenging agent. This study will deepen our knowledge about the therapeutic effects of ceria NPs and open a new route toward the NO-generating systems for biomedical applications.

Nitric oxide alleviates salt stress through protein S-nitrosylation and transcriptional regulation in tomato seedlings.

MAIN CONCLUSION: NO enhances the resistance of tomato seedlings to salt stress through protein S-nitrosylation and transcriptional regulation, which involves the regulation of MAPK signaling and carbohydrate metabolism. Nitric oxide (NO) regulates various physiological and biochemical processes and stress responses in plants. We found that S-nitrosoglutathione (GSNO) treatment significantly promoted the growth of tomato seedling under NaCl stress, indicating that NO plays a positive role in salt stress resistance. Moreover, GSNO pretreatment resulted in an increase of endogenous NO level, S-nitrosothiol (SNO) content, S-nitrosoglutathione reductase (GSNOR) activity and GSNOR expression under salt stress, implicating that S-nitrosylation might be involved in NO-alleviating salt stress. To further explore whether S-nitrosylation is a key molecular mechanism of NO-alleviating salt stress, the biotin-switch technique and liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) were conducted. A total of 1054 putative S-nitrosylated proteins have been identified, which were mainly enriched in chloroplast, cytoplasm and mitochondrion. Among them, 15 and 22 S-nitrosylated proteins were involved in mitogen-activated protein kinase (MAPK) signal transduction and carbohydrate metabolism, respectively. In MAPK signaling, various S-nitrosylated proteins, SAM1, SAM3, SAM, PP2C and SnRK, were down-regulated and MAPK, MAPKK and MAPKK5 were up-regulated at the transcriptional level by GSNO treatment under salt stress compared to NaCl treatment alone. The GSNO pretreatment could reduce ethylene production and ABA content under NaCl stress. In addition, the activities of enzyme identified in carbohydrate metabolism, their expression at the transcriptional level and the metabolite content were up-regulated by GSNO supplication under salt stress, resulting in the activation of glycolysis and tricarboxylic acid cycle (TCA) cycles. Thus, these results demonstrated that NO might beneficially regulate MAPK signaling at transcriptional levels and activate carbohydrate metabolism at the post-translational and transcriptional level, protecting seedlings from energy deficiency and salinity, thereby alleviating salt stress-induced damage in tomato seedlings. It provides initial insights into the regulatory mechanisms of NO in response to salt stress.

Indole-Based Long-Wavelength Fluorescent Probes for Bioimaging of S-Nitrosylation in Mitochondria.

S-Nitrosylation has been found to play an important role in regulating mitochondrial function. However, probes for detection of protein S-nitrosylation in mitochondria remain unexplored. Herein, a novel 4-(pyridin-4-yl)vinyl-substituted indole was designed, exhibiting a long-wavelength emission and a high fluorescent quantum yield. Functionalization of the 7-position of the indole ring with an arylphosphine ester resulted with probes with efficient mitochondria-targeting ability. Furthermore, the indole-arylphosphine displayed a significant fluorescence enhancement upon exposure to S-nitrosoglutathione (GSNO) at low micromolar concentrations in A431 cells. Taken together, this study provides a new indole-based fluorescent probe with a unique long-wavelength emission for direct detection of S-nitrosylation in mitochondria, which may represent a powerful tool for understanding the critical roles of S-nitrosylation within mitochondria of living organisms.

S-nitrosoglutathione alleviates hyperglycemia-induced neurobehavioral deficits involving nitro-oxidative stress and aberrant monaminergic system.

The present study evaluated the protective role of S-nitrosoglutathione (GSNO) in preventing hyperglycemia-induced nitro-oxidative stress and alterations in monoaminergic system associated with neurobehavioral deficits in mice. Mice were subjected to diabetes by intraperitoneal injection of streptozotocin (40 mg/kg body weight) for 5 days, whereas GSNO (100 µg/kg body weight) was administered daily via oral route for 8 weeks. Diabetic mice showed deficits in neurobehavioral functions associated with memory, learning, anxiety and motor coordination. These neurobehavioral deficits observed in diabetic mice may be attributed to decrease in norepinephrine (NE), dopamine (DA), serotonin (5-HT) and increased monoamine oxidase (MAO) activity in cortex and hippocampus. Further, a significant increase in reactive oxygen species (ROS), protein carbonyls, nitrotyrosine (NT) and lipid peroxidation were observed in brain regions of diabetic animals suggesting increased nitro-oxidative stress. Hyperglycemia induced nitro-oxidative stress appears to involve reduction in redox ratio (GSH/GSSG) and enzymatic antioxidants; catalase (CAT) and superoxide dismutase (SOD) in cortex and hippocampus. However, GSNO supplementation was able to ameliorate alterations in monoaminergic system and nitro-oxidative stress in the brain regions thereby restoring neurobehavioural functions. These findings suggest GSNO as potential therapeutic molecule to prevent diabetic encephalopathy.

Mechanisms of Regulation of the P-Glycoprotein Transporter Protein Functioning under the Action of Nitric Oxide.

Mechanisms of regulation of the P-glycoprotein (Pgp) transporter under the action of nitric oxide (NO) were studied in Caco-2 cells. S-Nitrosoglutathione (GSNO) was used as a NO donor, which was added to the cells at concentrations 1, 10, 50, 100, and 500 µM and incubated for 3, 24, or 72 h. The amount of Pgp was analyzed using Western blotting, activity was determined by monitoring transport of its substrate, fexofenadine. The study showed that a short-term exposure to GSNO for 3 h at 500 µM concentration caused increase in the concentration of peroxynitrite in Caco-2 cells, which reduced the activity, but not the amount of Pgp. Increase in the duration of exposure to 24 h increased the amount and activity of Pgp at GSNO concentrations of 10 and 50 µM, increased the amount without increasing activity at 100 µM concentration, and decreased the amount of the transporter protein at 500 µM. Duration of exposure to GSNO of 72 h at concentration of 10 µM resulted in the increase of the amount and activity of Pgp, while at concentration of 100 and 500 µM it decreased the amount of the transport protein. At the same time, it was shown using specific inhibitors that the increase in the amount of Pgp under the influence of low concentrations of GSNO was realized through the NO-cGMP signaling pathway, and the effect of the higher concentration of GSNO and the respective development of nitrosative stress was realized through Nrf2 and the constitutive androstane receptor.

BR-Mediated Protein S-Nitrosylation Alleviated Low-Temperature Stress in Mini Chinese Cabbage (Brassica rapa ssp. pekinensis).

Brassinosteroids (BRs), a novel plant hormone, are widely involved in plant growth and stress response processes. Nitric oxide (NO), as an important gas signaling molecule, can regulate target protein activity, subcellular localization and function in response to various stresses through post-translational S-nitrosylation modifications. However, the relationship between BR and NO in alleviating low-temperature stress of mini Chinese cabbage remains unclear. The hydroponic experiment combined with the pharmacological and molecular biological method was conducted to study the alleviating mechanism of BR at low temperature in mini Chinese cabbage. The results showed that low temperature inhibited the growth of mini Chinese cabbage seedlings, as evidenced by dwarf plants and yellow leaves. Treatment with 0.05 mg/L BR and 50 µM NO donor S-nitrosoglutathione (GSNO) significantly increased the leaf area, stem diameter, chlorophyll content, dry and fresh weight and proline content. Meanwhile, the malondialdehyde (MDA) content in 0.05 mg/L BR- and 50 µM GSNO-treated leaves were significantly lower than those in other treated leaves under low-temperature conditions. In addition, BR and GSNO applications induced an increase in NO and S-nitrosothiol (SNO) levels in vivo under low-temperature stress. Similarly, spraying BR after the elimination of NO also increased the level of S-nitrosylation in vivo, while spraying GSNO after inhibiting BR biosynthesis decreased the level of NO and SNO in vivo. In contrast, the S-nitrosoglutathione reductase (BrGSNOR) relative expression level and GSNOR enzyme activity were downregulated and inhibited by BR treatment, GSNO treatment and spraying BR after NO clearance, while the relative expression level of BrGSNOR was upregulated and GSNOR enzyme activity was also increased when spraying GSNO after inhibiting BR synthesis. Meanwhile, the biotin switch assay showed that exogenous BR increased the level of total nitrosylated protein in vivo under low-temperature stress. These results suggested that BR might act as an upstream signal of NO, induced the increase of NO content in vivo and then induced the protein S-nitrosylation modification to alleviate the damage of mini Chinese cabbage seedlings under low-temperature stress.

Effect of Nitric Oxide on the Functioning of the P-Glycoprotein Transporter.

We studied the effect of nitric oxide (NO) on the functioning of P-glycoprotein transporter (Pgp) in Caco-2 cells. NO donor S-nitrosoglutathione (GSNO) was used in concentrations of 1, 10, 50, 100, and 500 µM; the duration of exposure was 24 h. The content of Pgp was analyzed by the Western blotting, activity of the transport protein was analyzed by the transport of its substrate fexofenadine. It was shown that GSNO in concentrations of 10 and 50 µM increased the content and activity of Pgp. Increasing the GSNO concentration to 500 µM led to the development of nitrosative stress and a decrease in the content and activity of the transporter protein.

Human sirtuins are differentially sensitive to inhibition by nitrosating agents and other cysteine oxidants.

Sirtuins (e.g. human Sirt1-7) catalyze the removal of acyl groups from lysine residues in proteins in an NAD+-dependent manner, and loss of sirtuin deacylase activity correlates with the development of aging-related diseases. Although multiple reports suggest that sirtuin activity is regulated by oxidative post-translational modifications of cysteines during inflammation and aging, no systematic comparative study of potential direct sirtuin cysteine oxidative modifications has been performed. Here, using IC50 and kinact/KI analyses, we quantified the ability of nitrosothiols (S-nitrosoglutathione and S-nitroso-N-acetyl-d,l-penicillamine), nitric oxide, oxidized GSH, and hydrogen peroxide to post-translationally modify and inhibit the deacylase activity of Sirt1, Sirt2, Sirt3, Sirt5, and Sirt6. The inhibition was correlated with cysteine modification and assessed with chemical-probe and blot-based assays for cysteine S-nitrosation, sulfenylation, and glutathionylation. We show that the primarily nuclear sirtuins Sirt1 and Sirt6, as well as the primarily cytosolic sirtuin Sirt2, are modified and inhibited by cysteine S-nitrosation in response to exposure to both free nitric oxide and nitrosothiols (kinact/KI ≥ 5 m-1 s-1), which is the first report of Sirt2 and Sirt6 inhibition by S-nitrosation. Surprisingly, the mitochondrial sirtuins Sirt3 and Sirt5 were resistant to inhibition by cysteine oxidants. Collectively, these results suggest that nitric oxide-derived oxidants may causatively link nuclear and cytosolic sirtuin inhibition to aging-related inflammatory disease development.

Induction of S-nitrosoglutathione reductase protects root growth from ammonium toxicity by regulating potassium homeostasis in Arabidopsis and rice.

Ammonium (NH4+) is toxic to root growth in most plants already at moderate levels of supply, but mechanisms of root growth tolerance to NH4+ remain poorly understood. Here, we report that high levels of NH4+ induce nitric oxide (NO) accumulation, while inhibiting potassium (K+) acquisition via SNO1 (sensitive to nitric oxide 1)/SOS4 (salt overly sensitive 4), leading to the arrest of primary root growth. High levels of NH4+ also stimulated the accumulation of GSNOR (S-nitrosoglutathione reductase) in roots. GSNOR overexpression improved root tolerance to NH4+. Loss of GSNOR further induced NO accumulation, increased SNO1/SOS4 activity, and reduced K+ levels in root tissue, enhancing root growth sensitivity to NH4+. Moreover, the GSNOR-like gene, OsGSNOR, is also required for NH4+ tolerance in rice. Immunoblotting showed that the NH4+-induced GSNOR protein accumulation was abolished in the VTC1- (vitamin C1) defective mutant vtc1-1, which is hypersensititive to NH4+ toxicity. GSNOR overexpression enhanced vtc1-1 root tolerance to NH4+. Our findings suggest that induction of GSNOR increases NH4+ tolerance in Arabidopsis roots by counteracting NO-mediated suppression of tissue K+, which depends on VTC1 function.

Protein denitrosylation: enzymatic mechanisms and cellular functions.

S-Nitrosylation, the redox-based modification of Cys thiol side chains by nitric oxide, is a common mechanism in signal transduction. Dysregulated S-nitrosylation contributes to a range of human pathologies. New roles for protein denitrosylation in regulating S-nitrosylation are being revealed. Recently, several denitrosylases - the enzymes that mediate Cys denitrosylation - have been discovered, of which two enzyme systems in particular, the S-nitrosoglutathione reductase and thioredoxin systems, have been shown to be physiologically relevant. These highly conserved enzymes regulate signalling through multiple classes of receptors and influence diverse cellular responses. In addition, they protect from nitrosative stress in microorganisms, mammals and plants, thereby exerting profound effects on host-microbe interactions and innate immunity.