RESUMO
Oxidative (or respiratory) burst confers host defense against pathogens by generating reactive species, including reactive nitrogen species (RNS). The microbial infection-induced excessive RNS damages many biological molecules via S-nitrosothiol (SNO) accumulation. However, the mechanism by which the host enables innate immunity activation during oxidative burst remains largely unknown. Here, we demonstrate that S-nitrosoglutathione (GSNO), the main endogenous SNO, attenuates innate immune responses against herpes simplex virus-1 (HSV-1) and Listeria monocytogenes infections. Mechanistically, GSNO induces the S-nitrosylation of stimulator of interferon genes (STING) at Cys257, inhibiting its binding to the second messenger cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Alcohol dehydrogenase 5 (ADH5), the key enzyme that metabolizes GSNO to decrease cellular SNOs, facilitates STING activation by inhibiting S-nitrosylation. Concordantly, Adh5 deficiency show defective STING-dependent immune responses upon microbial challenge and facilitates viral replication. Thus, cellular oxidative burst-induced RNS attenuates the STING-mediated innate immune responses to microbial infection, while ADH5 licenses STING activation by maintaining cellular SNO homeostasis.
Assuntos
Aldeído Oxirredutases , Herpesvirus Humano 1 , S-Nitrosotióis , Proteínas de Membrana/metabolismo , Imunidade Inata , HomeostaseRESUMO
This study evaluated the use of the S-nitrosothiols, S-nitroso-N-acetylcysteine (NAC-SNO) and S-nitroso-N-acetylcysteine ethyl ester (NACET-SNO), at different concentrations (25-300 mg nitrite equivalent - NEq/kg) as sodium nitrite substitutes in restructured cooked hams. The pH value and instrumental cured color were not affected by the type or amount of curing agent used. Products with 25 and 50 mg/kg ingoing nitrite had lower thiobarbituric acid-reactive substance values than those with equimolar amounts of S-nitrosothiols. Products with >150 mg NEq/kg of S-nitrosothiols had residual nitrite similar to 50 mg/kg nitrite, and this resulted in the same volatile compound profile as nitrite added in equimolar amounts. A 300 mg NEq/kg of S-nitrosothiols was required to obtain a similar and minimally stable cured pink color perception as sliced samples with 50-150 mg/kg added nitrite. The results obtained reinforce the great potential of both alternative curing agents in the complete replacement of nitrite by equimolar amounts in restructured cooked products; however, differences in cured color stability should be considered.
Assuntos
Acetilcisteína/análogos & derivados , Produtos da Carne , S-Nitrosotióis , Produtos da Carne/análise , Nitrito de Sódio , S-Nitrosotióis/química , LipídeosRESUMO
S-Nitrosation is a cysteine post-translational modification fundamental to cellular signaling. This modification regulates protein function in numerous biological processes in the nervous, cardiovascular, and immune systems. Small molecule or protein nitrosothiols act as mediators of NO signaling by transferring the NO group (formally NO+) to a free thiol on a target protein through a transnitrosation reaction. The protein targets of specific transnitrosating agents and the extent and functional effects of S-nitrosation on these target proteins have been poorly characterized. S-nitroso-coenzyme A (CoA-SNO) was recently identified as a mediator of endogenous S-nitrosation. Here, we identified direct protein targets of CoA-SNO-mediated transnitrosation using a competitive chemical-proteomic approach that quantified the extent of modification on 789 cysteine residues in response to CoA-SNO. A subset of cysteines displayed high susceptibility to modification by CoA-SNO, including previously uncharacterized sites of S-nitrosation. We further validated and functionally characterized the functional effects of S-nitrosation on the protein targets phosphofructokinase (platelet type), ATP citrate synthase, and ornithine aminotransferase.
Assuntos
Coenzima A , Cisteína , S-Nitrosotióis , Nitrosação , Cisteína/química , Proteômica , Proteínas/metabolismo , S-Nitrosotióis/química , S-Nitrosotióis/metabolismo , Óxido Nítrico/metabolismoRESUMO
Both L- and D-isomers of S-nitrosocysteine (CSNO) can bind to the intracellular domain of voltage-gated potassium channels in vitro. CSNO binding inhibits these channels in the carotid body, leading to increased minute ventilation in vivo. However, only the l-isomer is active in vivo because it requires the l-amino acid transporter (LAT) for transmembrane transport. In rodents and dogs, the esterified D-CSNO precursor-d-cystine dimethyl ester (ATLX-0199)-overcomes opioid- and benzodiazepine-induced respiratory depression while maintaining analgesia. Although ATLX-0199 can enter cells independently of LAT because it is an ester, its stability in plasma is limited by the presence of esterases. Here, we hypothesized that the drug could be sequestered in erythrocytes to avoid de-esterification in circulation. We developed a liquid chromatography-mass spectrometry method for detecting ATLX-0199 and characterized a new metabolite, S-nitroso-d-cysteine monomethyl ester (DNOCE), which is also a D-CSNO precursor. We found that both ATLX-0199 and DNOCE readily enter erythrocytes and neurons and remain stable over 20 min; thus ATLX-0199 can enter cells where the ester is stable, but the thiol is reduced. Depending on hemoglobin conformation, the reduced ester can be S-nitrosylated and enter carotid body neurons, where it then increases minute ventilation. These data may help explain the paradox that ATLX-0199, a dimethyl ester, can avoid de-esterification in plasma and exert its effects at the level of the carotid body.
Assuntos
S-Nitrosotióis , Animais , Cães , S-Nitrosotióis/metabolismo , S-Nitrosotióis/farmacologia , Cisteína/metabolismo , Eritrócitos/metabolismo , Compostos de Sulfidrila , ÉsteresRESUMO
S-Nitrosothiols (SNOs) serve as endogenous carriers and donors of NO within living cells, releasing nitrosonium ions (NO+), NO, or other nitroso derivatives. In this study, we present a bioinspired {Co(NO)2}10 complex 1 that achieved S-nitrosation towards Cys residues. The incorporation of a ferrocenyl group in 1 allowed for fine-tuning of the nitrosation reaction, taking advantage of the redox ability of Cys residues. Complex 1 was synthesized and characterized, demonstrating its NO translation reactivity. Furthermore, complex 1 successfully converted Cys into S-nitrosocysteine (Cys-SNO), as confirmed by UV-Vis, IR, and XAS spectroscopy. This study presents a promising approach for S-nitrosation of Cys residues for further exploration in the modification of Cys-containing peptides.
Assuntos
Cisteína , S-Nitrosotióis , Nitrosação , Cisteína/química , S-Nitrosotióis/química , S-Nitrosotióis/metabolismo , Óxido Nítrico/química , OxirreduçãoRESUMO
Nitrite (O=N-O-, NO2-) and nitrate (O=N(O)-O-, NO3-) are ubiquitous in nature. In aerated aqueous solutions, nitrite is considered the major autoxidation product of nitric oxide (âNO). âNO is an environmental gas but is also endogenously produced from the amino acid L-arginine by the catalytic action of âNO synthases. It is considered that the autoxidation of âNO in aqueous solutions and in O2-containing gas phase proceeds via different neutral (e.g., O=N-O-N=O) and radical (e.g., ONOOâ) intermediates. In aqueous buffers, endogenous S-nitrosothiols (thionitrites, RSNO) from thiols (RSH) such as L-cysteine (i.e., S-nitroso-L-cysteine, CysSNO) and cysteine-containing peptides such as glutathione (GSH) (i.e., S-nitrosoglutathione, GSNO) may be formed during the autoxidation of âNO in the presence of thiols and dioxygen (e.g., GSH + O=N-O-N=O â GSNO + O=N-O- + H+; pKaHONO, 3.24). The reaction products of thionitrites in aerated aqueous solutions may be different from those of âNO. This work describes in vitro GC-MS studies on the reactions of unlabeled (14NO2-) and labeled nitrite (15NO2-) and RSNO (RS15NO, RS15N18O) performed in pH-neutral aqueous buffers of phosphate or tris(hydroxyethylamine) prepared in unlabeled (H216O) or labeled H2O (H218O). Unlabeled and stable-isotope-labeled nitrite and nitrate species were measured by gas chromatography-mass spectrometry (GC-MS) after derivatization with pentafluorobenzyl bromide and negative-ion chemical ionization. The study provides strong indication for the formation of O=N-O-N=O as an intermediate of âNO autoxidation in pH-neutral aqueous buffers. In high molar excess, HgCl2 accelerates and increases RSNO hydrolysis to nitrite, thereby incorporating 18O from H218O into the SNO group. In aqueous buffers prepared in H218O, synthetic peroxynitrite (ONOO-) decomposes to nitrite without 18O incorporation, indicating water-independent decomposition of peroxynitrite to nitrite. Use of RS15NO and H218O in combination with GC-MS allows generation of definite results and elucidation of reaction mechanisms of oxidation of âNO and hydrolysis of RSNO.
Assuntos
Nitritos , S-Nitrosotióis , Nitritos/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Nitratos/química , Óxido Nítrico/química , Hidrólise , Ácido Peroxinitroso/química , Dióxido de Nitrogênio , Isótopos , Glutationa , Concentração de Íons de Hidrogênio , S-Nitrosotióis/química , Compostos de Sulfidrila/análise , ÁguaRESUMO
S-nitrosylation is a post-translational modification in which nitric oxide (NO) binds to the thiol group of cysteine, generating an S-nitrosothiol (SNO) adduct. S-nitrosylation has different physiological roles, and its alteration has also been linked to a growing list of pathologies, including cancer. SNO can affect the function and stability of different proteins, such as the mitochondrial chaperone TRAP1. Interestingly, the SNO site (C501) of TRAP1 is in the proximity of another cysteine (C527). This feature suggests that the S-nitrosylated C501 could engage in a disulfide bridge with C527 in TRAP1, resembling the well-known ability of S-nitrosylated cysteines to resolve in disulfide bridge with vicinal cysteines. We used enhanced sampling simulations and in-vitro biochemical assays to address the structural mechanisms induced by TRAP1 S-nitrosylation. We showed that the SNO site induces conformational changes in the proximal cysteine and favors conformations suitable for disulfide bridge formation. We explored 4172 known S-nitrosylated proteins using high-throughput structural analyses. Furthermore, we used a coarse-grained model for 44 protein targets to account for protein flexibility. This resulted in the identification of up to 1248 proximal cysteines, which could sense the redox state of the SNO site, opening new perspectives on the biological effects of redox switches. In addition, we devised two bioinformatic workflows ( https://github.com/ELELAB/SNO_investigation_pipelines ) to identify proximal or vicinal cysteines for a SNO site with accompanying structural annotations. Finally, we analyzed mutations in tumor suppressors or oncogenes in connection with the conformational switch induced by S-nitrosylation. We classified the variants as neutral, stabilizing, or destabilizing for the propensity to be S-nitrosylated and undergo the population-shift mechanism. The methods applied here provide a comprehensive toolkit for future high-throughput studies of new protein candidates, variant classification, and a rich data source for the research community in the NO field.
Assuntos
Proteínas de Choque Térmico HSP90 , Óxido Nítrico , Proteínas Oncogênicas , S-Nitrosotióis , Cisteína/metabolismo , Óxido Nítrico/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Oxirredução , Processamento de Proteína Pós-Traducional , S-Nitrosotióis/metabolismo , Compostos de Sulfidrila/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismoRESUMO
Conversion of NO to stable S-nitrosothiols is perceived as a biologically important strategy of NO storage and a signal transduction mechanism. Transition-metal ions and metalloproteins are competent electron acceptors that may promote the formation of S-nitrosothiols from NO. We selected N-acetylmicroperoxidase (AcMP-11), a model of protein heme centers, to study NO incorporation to three biologically relevant thiols (glutathione, cysteine, and N-acetylcysteine). The efficient formation of S-nitrosothiols under anaerobic conditions was confirmed with spectrofluorimetric and electrochemical assays. AcMP-11-assisted incorporation of NO to thiols occurs via an intermediate characterized as an N-coordinated S-nitrosothiol, (AcMP-11)Fe2+(N(O)SR), which is efficiently converted to (AcMP-11)Fe2+(NO) in the presence of NO excess. Two possible mechanisms of S-nitrosothiol formation at the heme-iron were considered: a nucleophilic attack on (AcMP-11)Fe2+(NO+) by a thiolate and a reaction of (AcMP-11)Fe3+(RS) with NO. Kinetic studies, performed under anaerobic conditions, revealed that the reversible formation of (AcMP-11)Fe2+(N(O)SR) occurs in a reaction of RS- with (AcMP-11)Fe2+(NO+) and excluded the second mechanism, indicating that the formation of (AcMP-11)Fe3+(RS) is a dead-end equilibrium. Theoretical calculations revealed that N-coordination of RSNO to iron, forming (AcMP-11)Fe2+(N(O)SR), shortens the S-N bond and increases the complex stability compared to S-coordination. Our work unravels the molecular mechanism of heme-iron-assisted interconversion of NO and low-molecular-weight thiols to S-nitrosothiols and recognizes the reversible NO binding in the form of a heme-Fe2+(N(O)SR) motif as an important biological strategy of NO storage.
Assuntos
S-Nitrosotióis , Nitrosação , S-Nitrosotióis/química , Cinética , Compostos de Sulfidrila , Ferro/química , Heme/metabolismo , Óxido Nítrico/químicaRESUMO
Catalytic generation of nitric oxide (NO) from NO donors by nanomaterials has enabled prolonged NO delivery for various biomedical applications, but this approach requires laborious synthesis routes. In this study, a new class of materials, that is, polymeric amines including polyethyleneimine (PEI), poly-L-lysine, and poly(allylamine hydrochloride), is discovered to induce NO generation from S-nitrosothiols (RSNOs) at physiological conditions. Controlled NO generation can be readily achieved by tuning the concentration of the NO donors (RSNOs) and polymers, and the type and molecular weight of the polymers. Importantly, the mechanism of NO generation by these polymers is deciphered to be attributed to the nucleophilic reaction between primary amines on polymers and the SNO groups of RSNOs. The NO-releasing feature of the polymers can be integrated into a suite of materials, for example, simply by embedding PEI into poly(vinyl alcohol) (PVA) hydrogels. The functionality of the PVA/PEI hydrogels is demonstrated for Pseudomonas aeruginosa biofilm prevention with a ≈4 log reduction within 6 h. As NO has potential therapeutic implications in various diseases, the identification of polymeric amines to induce NO release will open new opportunities in NO-generating biomaterials for antibacterial, antiviral, anticancer, antithrombotic, and wound healing applications.
Assuntos
Óxido Nítrico , S-Nitrosotióis , Aminas/farmacologia , Doadores de Óxido Nítrico/farmacologia , Polímeros/farmacologia , Hidrogéis , S-Nitrosotióis/farmacologiaRESUMO
Following pathogen recognition, plant cells produce a nitrosative burst resulting in a striking increase in nitric oxide (NO), altering the redox state of the cell, which subsequently helps orchestrate a plethora of immune responses. NO is a potent redox cue, efficiently relayed between proteins through its co-valent attachment to highly specific, powerfully reactive protein cysteine (Cys) thiols, resulting in formation of protein S-nitrosothiols (SNOs). This process, known as S-nitrosylation, can modulate the function of target proteins, enabling responsiveness to cellular redox changes. Key targets of S-nitrosylation control the production of reactive oxygen species (ROS), the transcription of immune-response genes, the triggering of the hypersensitive response (HR) and the establishment of systemic acquired resistance (SAR). Here, we bring together recent advances in the control of plant immunity by S-nitrosylation, furthering our appreciation of how changes in cellular redox status reprogramme plant immune function.
Assuntos
Imunidade Vegetal , S-Nitrosotióis , Plantas/metabolismo , Óxido Nítrico/metabolismo , Compostos de Sulfidrila/metabolismo , Proteínas/metabolismo , Oxirredução , S-Nitrosotióis/metabolismoRESUMO
Diabetic cardiomyopathy (DCM) is a severe complication of diabetes mellitus that is characterized by aberrant myocardial structure and function and is the primary cause of heart failure and death in diabetic patients. Endothelial dysfunction plays an essential role in diabetes and is associated with an increased risk of cardiovascular events, but its role in DCM is unclear. Previously, we showed that S-nitroso-L-cysteine(CSNO), an endogenous S-nitrosothiol derived from eNOS, inhibited the activity of protein tyrosine phosphatase 1B (PTP1B), a critical negative modulator of insulin signaling. In this study, we reported that CSNO treatment induced cellular insulin-dependent and insulin-independent glucose uptake. In addition, CSNO activated insulin signaling pathway and promoted GLUT4 membrane translocation. CSNO protected cardiomyocytes against high glucose-induced injury by ameliorating excessive autophagy activation, mitochondrial impairment and oxidative stress. Furthermore, nebulized CSNO improved cardiac function and myocardial fibrosis in diabetic mice. These results suggested a potential site for endothelial modulation of insulin sensitivity and energy metabolism in the development of DCM. Data from these studies will not only help us understand the mechanisms of DCM, but also provide new therapeutic options for treatment.
Assuntos
Diabetes Mellitus Experimental , Cardiomiopatias Diabéticas , S-Nitrosotióis , Camundongos , Animais , Cardiomiopatias Diabéticas/tratamento farmacológico , Cardiomiopatias Diabéticas/prevenção & controle , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/induzido quimicamente , S-Nitrosotióis/efeitos adversos , S-Nitrosotióis/metabolismo , Insulina/efeitos adversosRESUMO
S-nitrosothiols exert multiple effects on neural processes in the central and peripheral nervous system. This study shows that intravenous infusion of S-nitroso-L-cysteine (SNO-L-CYS, 1 µmol/kg/min) in anesthetized male Sprague Dawley rats elicits (a) sustained increases in minute ventilation, via increases in frequency of breathing and tidal volume, (b) a decrease in Alveolar-arterial (A-a) gradient, thus improving alveolar gas-exchange, (c) concomitant changes in arterial blood-gas chemistry, such as an increase in pO2 and a decrease in pCO2, (d) a decrease in mean arterial blood pressure (MAP), and (e) an increase in tail-flick (TF) latency (antinociception). Infusion of S-nitroso-D-cysteine (SNO-D-CYS, 1 µmol/kg/min, IV), did not elicit similar responses, except for a sustained decrease in MAP equivalent to that elicited by SNO-L-CYS. A bolus injection of morphine (2 mg/kg, IV) in rats receiving an infusion of vehicle elicited (a) sustained decreases in frequency of breathing tidal volume, and therefore minute ventilation, (b) a sustained decrease in MAP, (c) sustained decreases in pH, pO2 and maximal sO2 with sustained increases in pCO2 and A-a gradient, and (d) a sustained increase in TF latency. In rats receiving SNO-L-CYS infusion, morphine elicited markedly smaller changes in minute ventilation, arterial blood gas chemistry, A-a gradient and MAP. In contrast, the antinociceptive effects of morphine were enhanced in rats receiving the infusion of SNO-L-CYS. The morphine-induced responses in rats receiving SNO-D-CYS infusion were similar to vehicle-infused rats. These data are the first to demonstrate that infusion of an S-nitrosothiol, such as SNO-L-CYS, can stereoselectively ameliorate the adverse effects of morphine on breathing and alveolar gas exchange while promoting antinociception.
Assuntos
Analgesia , Morfina , Animais , Cisteína/análogos & derivados , Cisteína/farmacologia , Masculino , Morfina/farmacologia , Ratos , Ratos Sprague-Dawley , S-NitrosotióisRESUMO
Bacterial infections are a hurdle to the application of medical devices, and in the United States alone, more than one million infection cases are reported annually from indwelling medical devices. Infections not only affect the function of medical devices but also risk the lives and health of patients. Nitric oxide (NO) has been used as an antibacterial therapy that kills bacteria without causing resistance and provides many therapeutic effects such as anti-inflammation, antithrombosis, and angiogenesis. Silicone oils have been widely utilized in manufacturing consumer goods, healthcare products, and medical products. Specifically, liquid silicone oils are used as a medical lubricant that creates lubricated interfaces between medical devices and the exterior physiological environment to improve the performance of medical devices. Herein, we report the first primary S-nitrosothiol-based NO-releasing silicone oil (RSNO-Si) that exhibits proactive antibacterial effects. S-nitrosothiol silicone oils (RSNO-Si) were synthesized and the NO payloads ranged from 34.0 to 603.9 µM. The increased NO payload induced higher-viscosity RSNO-Si oils, as RSNO0.1-Si, RSNO0.5-Si, and RSNO1-Si had viscosities of 12.8 ± 0.1 cP, 32.0 ± 0.2 cP, and 35.1 ± 0.3 cP, respectively. RSNO-Si-SR interfaces were fabricated by infusing silicone rubber (SR) in RSNO-Si oil, and the resulting RSNO-Si-SR disks demonstrated NO release without NO donor leaching. RSNO0.1-Si-SR, RSNO0.5-Si-SR, and RSNO1-Si-SR exhibited maximum NO flux at 0.8, 6.5, and 21.5 × 10 -10 mol cm-2 min-1 in 24 h, respectively. RSNO-Si-SR disks also demonstrated 97.45, 95.40, and 96.08% of inhibition against S. aureus in a 4 h bacterial adhesion assay. Considering the easy synthesis, simple fabrication of non-leaching NO-releasing interfaces, tunable payloads, NO flux levels, and antimicrobial effects, RSNO-Si oils exhibited their potential use as platform chemicals for creating antimicrobial medical device surfaces and other antibacterial materials.
Assuntos
Óxido Nítrico , S-Nitrosotióis , Antibacterianos/farmacologia , Humanos , Óxido Nítrico/farmacologia , Óleos/farmacologia , S-Nitrosotióis/farmacologia , Elastômeros de Silicone/farmacologia , Óleos de Silicone/farmacologia , Staphylococcus aureusRESUMO
Though endogenous S-nitroso-l-cysteine (l-CSNO) signaling at the level of the carotid body increases minute ventilation (vÌE ), neither the background data nor the potential clinical relevance are well-understood by pulmonologists in general, or by pediatric pulmonologists in particular. Here, we first review how regulation of the synthesis, activation, transmembrane transport, target interaction, and degradation of l-CSNO can affect the ventilatory drive. In particular, we review l-CSNO formation by hemoglobin R to T conformational change and by nitric oxide (NO) synthases (NOS), and the downstream effects on vÌE through interaction with voltage-gated K+ (Kv) channel proteins and other targets in the peripheral and central nervous systems. We will review how these effects are independent of-and, in fact may be opposite to-those of NO. Next, we will review evidence that specific elements of this pathway may underlie disorders of respiratory control in childhood. Finally, we will review the potential clinical implications of this pathway in the development of respiratory stimulants, with a particular focus on potential pediatric applications.
Assuntos
Medicamentos para o Sistema Respiratório , S-Nitrosotióis , Criança , Cisteína/análogos & derivados , Cisteína/metabolismo , Hemoglobinas , Humanos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase , S-Nitrosotióis/metabolismoRESUMO
Inducible nitric oxide synthase (NOS2) produces high local concentrations of nitric oxide (NO), and its expression is associated with inflammation, cellular stress signals, and cellular transformation. Additionally, NOS2 expression results in aggressive cancer cell phenotypes and is correlated with poor outcomes in patients with breast cancer. DNA hypomethylation, especially of noncoding repeat elements, is an early event in carcinogenesis and is a common feature of cancer cells. In addition to altered gene expression, DNA hypomethylation results in genomic instability via retrotransposon activation. Here, we show that NOS2 expression and associated NO signaling results in substantial DNA hypomethylation in human cell lines by inducing the degradation of DNA (cytosine-5)methyltransferase 1 (DNMT1) protein. Similarly, NOS2 expression levels were correlated with decreased DNA methylation in human breast tumors. NOS2 expression and NO signaling also resulted in long interspersed noncoding element 1 (LINE-1) retrotransposon hypomethylation, expression, and DNA damage. DNMT1 degradation was mediated by an NO/p38-MAPK/lysine acetyltransferase 5dependent mechanism. Furthermore, we show that this mechanism is required for NO-mediated epithelial transformation. Therefore, we conclude that NOS2 and NO signaling results in DNA damage and malignant cellular transformation via an epigenetic mechanism.
Assuntos
Metilação de DNA , Inflamação , S-Nitrosotióis , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Humanos , Inflamação/genética , Óxido Nítrico , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Retroelementos/genéticaRESUMO
Although reactive oxygen and nitrogen species (ROS/RNS), such as hydrogen peroxide (H2O2), nitric oxide (NO), hydroxyl radicals (OHË), superoxide (O2-) etc., play crucial roles in redox biology and cellular signaling, higher concentrations of these species lead to oxidative and nitrosative stress, which are associated with various pathophysiological conditions like neurodegeneration, cardiovascular diseases and cancer. There is growing evidence that functional impairment of the endothelium is one of the first recognizable signs of the development of atherosclerotic cardiovascular disease. A decreased bioavailability of NO and increased generation of ROS are the two major molecular changes associated with endothelial dysfunction. Therefore, it is a viable strategy to increase the bioavailability of NO while reducing the amount of ROS to prevent the progression of cardiovascular diseases. In this paper, we discuss for the first time that copper vanadate (CuV2O6) can not only release NO from S-nitrosothiols but can also control the ROS levels by functionally mimicking the antioxidant enzyme glutathione peroxidase (GPx) at physiological pH. We used several imaging techniques and spectroscopic measurements to understand the catalysis on the surface of the material during the reactions. The denitrosylation, as well as GPx-like activity, by CuV2O6 can be carried out multiple times without affecting the catalytic activity.
Assuntos
Doenças Cardiovasculares , S-Nitrosotióis , Cobre , Glutationa Peroxidase , Humanos , Peróxido de Hidrogênio , Óxido Nítrico , Espécies Reativas de Nitrogênio , Espécies Reativas de Oxigênio , Vanadatos/farmacologiaRESUMO
S-nitrosylation of proteins is a nitric oxide (NO)-based post-translational modification of cysteine residues. By removing the NO moiety from S-nitrosothiol adducts, denitrosylases restore sulfhydryl protein pool and act as downstream tuners of S-nitrosylation signaling. Alterations in the S-nitrosylation/denitrosylation dynamics are implicated in many pathological states, including cancer ontogenesis and progression, skeletal muscle myogenesis and function. Here, we aim to provide and link different lines of evidence, and elaborate on the possible role of S-nitrosylation/denitrosylation signaling in rhabdomyosarcoma, one of the most common pediatric mesenchymal malignancy.
Assuntos
Rabdomiossarcoma , S-Nitrosotióis , Criança , Humanos , Desenvolvimento Muscular , Óxido Nítrico/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , S-Nitrosotióis/metabolismoRESUMO
BACKGROUND: The level of nitric oxide (NO) is important to protect the heart from ischemic damage in acute coronary syndrome (ACS) patients. S-nitrosothiol (SNO) is a molecule that represents the main form of NO storage in the vascular structure. In addition, dynamic thiol/disulfide homeostasis (TDH) is known to play an important role in maintaining the oxidant-antioxidant balance. In this study, our aim is to evaluate the oxidative/nitrosative stress status according to SNO level and TDH in patients with ACS. METHODS: The study included 124 patients who were admitted to the emergency service and 124 consecutive individuals who applied to the cardiology outpatient clinic with cardiac complaints and underwent coronary angiography (CAG). Blood was drawn from all participants included in the study to determine SNO, nitrite, total thiol, native thiol, and disulfide levels after 12 h of fasting. RESULTS: Serum SNO levels were found to be significantly lower in ACS patients compared to the control group (0.3 ± 0.08 vs. 0.4 ± 0.10 µmol/L, successively, p < 0.001). In addition, while the total thiol, native thiol, and native thiol/total thiol levels were lower in the patient group compared to the control group, nitrite, disulfide/native thiol and disulfide/total thiol levels were higher. As a result of multivariate logistic regression analysis, it was determined that age, gender, smoking, low-density lipoprotein cholesterol, glycosylated haemoglobin, and SNO levels were independent predictors in predicting ACS patients. DISCUSSION: S-nitrosothiol and thiol levels were found to be significantly lower in ACS patients. In addition, SNO molecule was independently associated with the presence of ACS diagnosis.
Assuntos
Síndrome Coronariana Aguda , S-Nitrosotióis , Humanos , Compostos de Sulfidrila , Dissulfetos , Nitritos , Estresse Oxidativo , BiomarcadoresRESUMO
The intracellular concentration of reduced glutathione (GSH) lies in the range of 1-10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis. We also report our findings of two, hitherto unidentified substrates of GSH mediated S-denitrosylation, namely S-nitrosoglutaredoxin 1 (Grx1-SNO) and S-nitrosylated R1 subunit of ribonucleotide reductase (R1-SNO).
Assuntos
Glutarredoxinas/metabolismo , Glutationa/metabolismo , Ribonucleosídeo Difosfato Redutase/metabolismo , S-Nitrosotióis/metabolismo , Óxidos N-Cíclicos/química , Glutarredoxinas/química , Células Hep G2 , Humanos , Ribonucleosídeo Difosfato Redutase/química , S-Nitrosotióis/química , Marcadores de Spin , Detecção de Spin , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMO
Here we show that the fluorescence of fluorescein isothiocyanate (FITC) is not altered by its reaction with primary amines. However, the fluorescence is rapidly quenched upon reaction with small molecular weight thiols including cysteine, glutathione, homocysteine, dithiothreitol, and sulfide. We have taken advantage of the thiol-dependent quenching of FITC to devise a sulfide specific assay by utilizing polydimethylsiloxane (PDMS) membranes that are permeable to hydrogen sulfide but not to larger charged thiols. In addition, we have discovered that the fluorescein dithiocarbamate (FDTC) formed by the reaction with sulfide can specifically react with S-nitrosothiols (RSNO) to regenerate FITC, thus serving as a specific, fluorogenic reagent to detect picomol levels of RSNO. FDTC was tested as an intracellular RSNO-sensor in germinating tomato seedlings (Solanum lycopersicum) via epifluorescence microscopy. Control plant roots exposed to FDTC showed low intracellular fluorescence which increased â¼3-fold upon exposure to extracellular S-nitrosoglutathione and â¼4-fold in the presence of N6022, a S-nitrosoglutathione reductase (GSNOR) inhibitor, demonstrating that FDTC can be used to visualize intracellular RSNO levels.