S-glutathionylation in hepatocytes is involved in arsenic-induced liver fibrosis through activation of the NLRP3 inflammasome, an effect alleviated by NAC.

Arsenic, a toxicant widely distributed in the environment, is considered as a risk factor for liver fibrosis. At present, the underlying mechanism still needs to be explored. In the present study, we found that, for mice, chronic exposure to arsenic induced liver fibrosis, activated the NLRP3 inflammasome, and increased the levels of reactive oxygen species (ROS). After hepatocytes were co-cultured with hepatic stellate cells (HSCs), we observed the arsenic-activated NLRP3 inflammasome in hepatocytes, and the co-cultured HSCs were activated. Further, we found that, in livers of mice, arsenic disturbed GSH metabolism and promoted protein S-glutathionylation. A 3D molecular docking simulation suggested that NLRP3 binds with GSH, which was confirmed by immunoprecipitation experiments. N-acetylcysteine (NAC) increased the levels of GSH in hepatocytes, which suppressed the S-glutathionylation of NLRP3 and blocked arsenic-induced activation of the NLRP3 inflammasome. Mechanistically, an imbalance of the redox state induced by arsenic promotes the S-glutathionylation of NLRP3, which regulates activation of the NLRP3 inflammasome, leading into the activation of HSCs. Moreover, NAC increases the levels of GSH to block arsenic-induced S-glutathionylation of NLRP3, thereby blocking arsenic-induced liver fibrosis. Thus, via activating HSCs, the S-glutathionylation of NLRP3 in hepatocytes is involved in arsenic-induced liver fibrosis, and, for hepatocytes, NAC alleviates these effects by increasing the levels of GSH. These results reveal a new mechanism and provide a possible therapeutic target for the liver fibrosis induced by environmental factors.

Post-Translational Modifications of Proteins of Malaria Parasites during the Life Cycle.

Post-translational modifications (PTMs) are essential for regulating protein functions, influencing various fundamental processes in eukaryotes. These include, but are not limited to, cell signaling, protein trafficking, the epigenetic control of gene expression, and control of the cell cycle, as well as cell proliferation, differentiation, and interactions between cells. In this review, we discuss protein PTMs that play a key role in the malaria parasite biology and its pathogenesis. Phosphorylation, acetylation, methylation, lipidation and lipoxidation, glycosylation, ubiquitination and sumoylation, nitrosylation and glutathionylation, all of which occur in malarial parasites, are reviewed. We provide information regarding the biological significance of these modifications along all phases of the complex life cycle of Plasmodium spp. Importantly, not only the parasite, but also the host and vector protein PTMs are often crucial for parasite growth and development. In addition to metabolic regulations, protein PTMs can result in epitopes that are able to elicit both innate and adaptive immune responses of the host or vector. We discuss some existing and prospective results from antimalarial drug discovery trials that target various PTM-related processes in the parasite or host.

Glutathione Induces Keap1 S-Glutathionylation and Mitigates Oscillating Glucose-Induced ß-Cell Dysfunction by Activating Nrf2.

Glutathione (GSH), a robust endogenous antioxidant, actively participates in the modulation of the redox status of cysteine residues in proteins. Previous studies have indicated that GSH can prevent ß-cell failure and prediabetes caused by chronic oscillating glucose (OsG) administration. However, the precise mechanism underlying the protective effect is not well understood. Our current research reveals that GSH is capable of reversing the reduction in Nrf2 levels, as well as downstream genes Grx1 and HO-1, in the islet ß-cells of rats induced by chronic OsG. In vitro experiments have further demonstrated that GSH can prevent ß-cell dedifferentiation, apoptosis, and impaired insulin secretion caused by OsG. Additionally, GSH facilitates the translocation of Nrf2 into the nucleus, resulting in an upregulation of Nrf2-targeted genes such as GCLC, Grx1, HO-1, and NQO1. Notably, when the Nrf2 inhibitor ML385 is employed, the effects of GSH on OsG-treated ß-cells are abrogated. Moreover, GSH enhances the S-glutathionylation of Keap1 at Cys273 and Cys288, but not Cys151, in OsG-treated ß-cells, leading to the dissociation of Nrf2 from Keap1 and facilitating Nrf2 nuclear translocation. In conclusion, the protective role of GSH against OsG-induced ß-cell failure can be partially attributed to its capacity to enhance Keap1 S-glutathionylation, thereby activating the Nrf2 signaling pathway. These findings provide novel insights into the prevention and treatment of ß-cell failure in the context of prediabetes/diabetes, highlighting the potential of GSH.

Glutathionylation of a glycolytic enzyme promotes cell death and vigor loss during aging of elm seeds.

Seed deterioration during storage is a major problem in agricultural and forestry production and for germplasm conservation. Our previous studies have shown that a mitochondrial outer membrane protein VOLTAGE-DEPENDENT ANION CHANNEL (VDAC) is involved in programmed cell death (PCD)-like viability loss during the controlled deterioration treatment (CDT) of elm (Ulmus pumila L.) seeds, but its underlying mechanism remains unclear. In this study, we demonstrate that the oxidative modification of GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH) is functioned in the gate regulation of VDAC during the CDT of elm seeds. Through biochemical and cytological methods and observations of transgenic material [Arabidopsis (Arabidopsis thaliana), Nicotiana benthamiana, and yeast (Saccharomyces cerevisiae)], we demonstrate that cysteine S-glutathionylated UpGAPDH1 interacts with UpVDAC3 during seed aging, which leads to a mitochondrial permeability transition and aggravation of cell death, as indicated by the leakage of the mitochondrial pro-apoptotic factor cytochrome c and the emergence of apoptotic nucleus. Physiological assays and inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed that GAPDH glutathionylation is mediated by increased glutathione, which might be caused by increases in the concentrations of free metals, especially Zn. Introduction of the Zn-specific chelator TPEN [(N, N, N', N'-Tetrakis (2-pyridylmethyl)ethylenediamine)] significantly delayed seed aging. We conclude that glutathionylated UpGAPDH1 interacts with UpVDAC3 and serves as a pro-apoptotic protein for VDAC-gating regulation and cell death initiation during seed aging.

GSTP alleviates acute lung injury by S-glutathionylation of KEAP1 and subsequent activation of NRF2 pathway.

Oxidative stress plays an important role in the pathogenesis of acute lung injury (ALI). As a typical post-translational modification triggered by oxidative stress, protein S-glutathionylation (PSSG) is regulated by redox signaling pathways and plays diverse roles in oxidative stress conditions. In this study, we found that GSTP downregulation exacerbated LPS-induced injury in human lung epithelial cells and in mice ALI models, confirming the protective effect of GSTP against ALI both in vitro and in vivo. Additionally, a positive correlation was observed between total PSSG level and GSTP expression level in cells and mice lung tissues. Further results demonstrated that GSTP inhibited KEAP1-NRF2 interaction by promoting PSSG process of KEAP1. By the integration of protein mass spectrometry, molecular docking, and site-mutation validation assays, we identified C434 in KEAP1 as the key PSSG site catalyzed by GSTP, which promoted the dissociation of KEAP1-NRF2 complex and activated the subsequent anti-oxidant genes. In vivo experiments with AAV-GSTP mice confirmed that GSTP inhibited LPS-induced lung inflammation by promoting PSSG of KEAP1 and activating the NRF2 downstream antioxidant pathways. Collectively, this study revealed the novel regulatory mechanism of GSTP in the anti-inflammatory function of lungs by modulating PSSG of KEAP1 and the subsequent KEAP1/NRF2 pathway. Targeting at manipulation of GSTP level or activity might be a promising therapeutic strategy for oxidative stress-induced ALI progression.

Glutaredoxin-1 modulates the NF-κB signaling pathway to activate inducible nitric oxide synthase in experimental necrotizing enterocolitis.

Inducible nitric oxide synthase (iNOS), regulated by nuclear factor kappa B (NF-κB), is crucial for intestinal inflammation and barrier injury in the progression of necrotizing enterocolitis (NEC). The NF-κB pathway is inhibited by S-glutathionylation of inhibitory κB kinase ß (IKKß), which can be restored by glutaredoxin-1 (Grx1). Thus, we aim to explore the role of Grx1 in experimental NEC. Wild-type (WT) and Grx1-knockout (Grx1-/-) mice were treated with an NEC-inducing regimen. Primary intestinal epithelial cells (IECs) were subjected to LPS treatment. The production of iNOS, NO, and inflammation injuries were assessed. NF-κB and involved signaling pathways were also explored. The severity of NEC was attenuated in Grx1-/- mice. Grx1 ablation promoted IKKß glutathionylation, NF-κB inactivation, and decreased iNOS, NO, and O2·- production in NEC mice. Furthermore, Grx1 ablation restrained proinflammatory cytokines and cell apoptosis, ameliorated intestinal barrier damage, and promoted proliferation in NEC mice. Grx1 ablation protected NEC through iNOS and NO inhibition, which related to S-glutathionylation of IKKß to inhibit NF-κB signaling. Grx1-related signaling pathways provide a new therapeutic target for NEC.

Chloroplasts lacking class I glutaredoxins are functional but show a delayed recovery of protein cysteinyl redox state after oxidative challenge.

Redox status of protein cysteinyl residues is mediated via glutathione (GSH)/glutaredoxin (GRX) and thioredoxin (TRX)-dependent redox cascades. An oxidative challenge can induce post-translational protein modifications on thiols, such as protein S-glutathionylation. Class I GRX are small thiol-disulfide oxidoreductases that reversibly catalyse S-glutathionylation and protein disulfide formation. TRX and GSH/GRX redox systems can provide partial backup for each other in several subcellular compartments, but not in the plastid stroma where TRX/light-dependent redox regulation of primary metabolism takes place. While the stromal TRX system has been studied at detail, the role of class I GRX on plastid redox processes is still unknown. We generate knockout lines of GRXC5 as the only chloroplast class I GRX of the moss Physcomitrium patens. While we find that PpGRXC5 has high activities in GSH-dependent oxidoreductase assays using hydroxyethyl disulfide or redox-sensitive GFP2 as substrates in vitro, Δgrxc5 plants show no detectable growth defect or stress sensitivity, in contrast to mutants with a less negative stromal EGSH (Δgr1). Using stroma-targeted roGFP2, we show increased protein Cys steady state oxidation and decreased reduction rates after oxidative challenge in Δgrxc5 plants in vivo, indicating kinetic uncoupling of the protein Cys redox state from EGSH. Compared to wildtype, protein Cys disulfide formation rates and S-glutathionylation levels after H2O2 treatment remained unchanged. Lack of class I GRX function in the stroma did not result in impaired carbon fixation. Our observations suggest specific roles for GRXC5 in the efficient transfer of electrons from GSH to target protein Cys as well as negligible cross-talk with metabolic regulation via the TRX system. We propose a model for stromal class I GRX function in efficient catalysis of protein dithiol/disulfide equilibria upon redox steady state alterations affecting stromal EGSH and highlight the importance of identifying in vivo target proteins of GRXC5.

Macrophage-expressed SRA ameliorates alcohol-induced liver injury by suppressing S-glutathionylation of Notch1 via recruiting thioredoxin.

Scavenger receptor A (SRA) is preferentially expressed in macrophages and implicated as a multifunctional pattern recognition receptor for innate immunity. Hepatic macrophages play a primary role in the pathogenesis of alcoholic liver disease. Herein, we observed that SRA expression was significantly increased in the liver tissues of mice with alcohol-related liver injury. SRA-deficient (SRA-/-) mice developed more severe alcohol-induced liver disease than wild-type mice. Enhanced liver inflammation existed in alcohol-challenged SRA-/- mice and was associated with increased Notch activation in hepatic macrophages compared with wild-type control animals. Mechanistically, SRA directly bound with Notch1 and suppressed its S-glutathionylation, thereby inhibiting Notch pathway activation. Further, we determined that the SRA interacted with thioredoxin-1 (Trx-1), a redox-active protein. SRA inhibited Trx-1 dimerization and facilitated the interaction of Trx-1 with Notch1. Application of a Trx-1-specific inhibitory agent during macrophage stimulation abolished SRA-mediated regulation of the Notch pathway and its downstream targets. In summary, our study revealed that SRA plays a critical role in macrophage inflammatory response by targeting Notch1 for its glutathionylation. SRA-mediated negative regulation of Notch activation might serve as a novel therapeutic strategy for alcohol-induced liver injury.

[Metabolic Stress of Red Blood Cells Induces Hemoglobin Glutathionylation].

Metabolic stress caused by a lack of glucose significantly affects the state of red blood cells, where glycolysis is the main pathway for the production of ATP. Hypoglycemia can be both physiological (occurring during fasting and heavy physical exertion) and pathological (accompanying a number of diseases, such as diabetes mellitus). In this study, we have characterized the state of isolated erythrocytes under metabolic stress caused by the absence of glucose. It was established that 24 h of incubation of the erythrocytes in a glucose-free medium to simulate blood plasma led to a two-fold decrease in the ATP level into them. The cell size, as well as intracellular sodium concentration increased. These findings could be the result of a disruption in ion transporter functioning because of a decrease in the ATP level. The calcium level remained unchanged. With a lack of glucose in the medium of isolated erythrocytes, there was no increase in ROS and a significant change in the level of nitric oxide, while the level of the main low-molecular weight thiol of cells, glutathione (GSH) decreased by almost 2 times. It was found that the metabolic stress of isolated red blood cells induced hemoglobin glutathionylation despite the absence of ROS growth. The cause was the lack of ATP, which led to a decrease in the level of GSH because of the inhibition of its synthesis and, probably, due to a decrease in the NADPH level required for glutathione (GSSG) reduction and protein deglutathionylation. Thus, erythrocyte metabolic stress induced hemoglobin glutathionylation, which is not associated with an increase in ROS. This may have an important physiological significance, since glutathionylation of hemoglobin changes its affinity for oxygen.

Therapeutic Potential for Beta-3 Adrenoreceptor Agonists in Peripheral Arterial Disease and Diabetic Foot Ulcers.

Annually, peripheral arterial disease is estimated to cost over USD 21 billion and diabetic foot disease an estimated at USD 9-13 billion. Mirabegron is a TGA-approved beta-3 adrenoreceptor agonist, shown to be safe and effective in the treatment of overactive bladder syndrome by stimulating bladder smooth muscle relaxation. In this review, we discuss the potential use of beta-3 adrenoreceptor agonists as therapeutic agents repurposed for peripheral arterial disease and diabetic foot ulcers. The development of both conditions is underpinned by the upregulation of oxidative stress pathways and consequential inflammation and hypoxia. In oxidative stress, there is an imbalance of reactive oxygen species and nitric oxide. Endothelial nitric oxide synthase becomes uncoupled in disease states, producing superoxide and worsening oxidative stress. Agonist stimulation of the beta-3 adrenoreceptor recouples and activates endothelial nitric oxide synthase, increasing the production of nitric oxide. This reduces circulating reactive oxygen species, thus decreasing redox modification and dysregulation of cellular proteins, causing downstream smooth muscle relaxation, improved endothelial function and increased angiogenesis. These mechanisms lead to endothelial repair in peripheral arterial disease and an enhanced perfusion in hypoxic tissue, which will likely improve the healing of chronic ulcers.

Reduced GSH Acts as a Metabolic Cue of OPDA Signaling in Coregulating Photosynthesis and Defense Activation under Stress.

12-oxo-phytodienoic acid (OPDA) is a primary precursor of jasmonates, able to trigger autonomous signaling cascades that activate and fine-tune plant defense responses, as well as growth and development. However, its mechanism of actions remains largely elusive. Here we describe a dual-function messenger of OPDA signaling, reduced glutathione (GSH), that cross-regulates photosynthesis machinery and stress protection/adaptation in concert, optimizing plant plasticity and survival potential. Under stress conditions, the rapid induction of OPDA production stimulates GSH accumulation in the chloroplasts, and in turn leads to protein S-glutathionylation in modulating the structure and function of redox-sensitive enzymes such as 2-cysteine (Cys) peroxiredoxin A (2CPA), a recycler in the water-water cycle. GSH exchanges thiol-disulfides with the resolving CysR175, while donating an electron (e-, H+) to the peroxidatic CysP53, of 2CPA, which revives its reductase activity and fosters peroxide detoxification in photosynthesis. The electron flow protects photosynthetic processes (decreased total non-photochemical quenching, NPQ(T)) and maintains its efficiency (increased photosystem II quantum yield, ΦII). On the other hand, GSH also prompts retrograde signaling from the chloroplasts to the nucleus in adjusting OPDA-responsive gene expressions such as Glutathione S-Transferase 6 (GST6) and GST8, and actuating defense responses against various ecological constraints such as salinity, excess oxidants and light, as well as mechanical wounding. We thus propose that OPDA regulates a unique metabolic switch that interfaces light and defense signaling, where it links cellular and environmental cues to a multitude of plant physiological, e.g., growth, development, recovery, and acclimation, processes.

Glutathionyl Hemoglobin and Its Emerging Role as a Clinical Biomarker of Chronic Oxidative Stress.

Hemoglobin is one of the proteins that are more susceptible to S-glutathionylation and the levels of its modified form, glutathionyl hemoglobin (HbSSG), increase in several human pathological conditions. The scope of the present review is to provide knowledge about how hemoglobin is subjected to S-glutathionylation and how this modification affects its functionality. The different diseases that showed increased levels of HbSSG and the methods used for its quantification in clinical investigations will be also outlined. Since there is a growing need for precise and reliable methods for markers of oxidative stress in human blood, this review highlights how HbSSG is emerging more and more as a good indicator of severe oxidative stress but also as a key pathogenic factor in several diseases.

Antioxidant System and Endoplasmic Reticulum Stress in Cataracts.

The primary underlying contributor for cataract, a leading cause of vision impairment and blindness worldwide, is oxidative stress. Oxidative stress triggers protein damage, cell apoptosis, and subsequent cataract formation. The nuclear factor-erythroid 2-related factor 2 (Nrf2) serves as a principal redox transcriptional factor in the lens, offering a line of defense against oxidative stress. In response to oxidative challenges, Nrf2 dissociates from its inhibitor, Kelch-like ECH-associated protein 1 (Keap1), moves to the nucleus, and binds to the antioxidant response element (ARE) to activate the Nrf2-dependent antioxidant system. In parallel, oxidative stress also induces endoplasmic reticulum stress (ERS). Reactive oxygen species (ROS), generated during oxidative stress, can directly damage proteins, causing them to misfold. Initially, the unfolded protein response (UPR) activates to mitigate excessive misfolded proteins. Yet, under persistent or severe stress, the failure to rectify protein misfolding leads to an accumulation of these aberrant proteins, pushing the UPR towards an apoptotic pathway, further contributing to cataractogenesis. Importantly, there is a dynamic interaction between the Nrf2 antioxidant system and the ERS/UPR mechanism in the lens. This interplay, where ERS/UPR can modulate Nrf2 expression and vice versa, holds potential therapeutic implications for cataract prevention and treatment. This review explores the intricate crosstalk between these systems, aiming to illuminate strategies for future advancements in cataract prevention and intervention. The Nrf2-dependent antioxidant system communicates and cross-talks with the ERS/UPR pathway. Both mechanisms are proposed to play pivotal roles in the onset of cataract formation.

Cysteine facilitates the lignocellulolytic response of Trichoderma guizhouense NJAU4742 by indirectly up-regulating membrane sugar transporters.

BACKGROUND: Filamentous fungi possess a rich CAZymes system, which is widely studied and applied in the bio-conversion of plant biomass to alcohol chemicals. Carbon source acquisition is the fundamental driver for CAZymes-producing sustainability and secondary metabolism, therefore, a deeper insight into the regulatory network of sugar transport in filamentous fungi has become urgent. RESULTS: This study reports an important linkage of sulfur assimilation to lignocellulose response of filamentous fungus. Inorganic sulfur addition facilitated biodegradation of rice straw by Trichoderma guizhouense NJAU4742. Cysteine and glutathione were revealed as major intracellular metabolites responsive to sulfur addition by metabolomics, cysteine content was increased in this process and glutathione increased correspondingly. Two membrane sugar transporter genes, Tgmst1 and Tgmst2, were identified as the critical response genes significantly up-regulated when intracellular cysteine increased. Tgmst1 and Tgmst2 were both positively regulated by the glucose regulation-related protein (GRP), up-regulation of both Tgmst1 and Tggrp can cause a significant increase in intracellular glucose. The transcriptional regulatory function of GRP mainly relied on GSH-induced glutathionylation, and the transcription activating efficiency was positively related to the glutathionylation level, furthermore, DTT-induced deglutathionylation resulted in the down-regulation of downstream genes. CONCLUSIONS: Inorganic sulfur addition induces a rise in intracellular Cys content, and the conversion of cysteine to glutathione caused the increase of glutathionylation level of GRP, which in turn up-regulated Tgmst1 and Tgmst2. Subsequently, the sugar transport efficiency of single cells was improved, which facilitated the maintenance of vigorous CAZymes metabolism and the straw-to-biomass conversion.

Clickable Glutathione-Based Identification of Cysteine Glutathionylation.

Clickable glutathione is a glutathione-derived chemical probe designed to identify and analyze protein S-glutathionylation, a major cysteine oxidation in redox signaling. An engineered glutathione synthetase mutant (GS M4) is used to synthesize clickable glutathione in cells or in vitro, which affords utility via click chemistry to detect, identify, and quantify glutathionylation on individual or global proteins in biochemical and mass spectrometric analyses. The clickable glutathione approach is valuable for the unequivocal identification of glutathionylated cysteines, among many reversible cysteine oxoforms, via the direct enrichment and detection of glutathionylated proteins or peptides. Clickable glutathione, in combination with GS M4, has demonstrated utility in the mass-spectrometry-based discovery and profiling of new proteins and cysteines for glutathionylation in cell lines in response to physiologic and oxidative stress. The approach is versatile and applicable to validating the glutathionylation of proteins and cysteines in other biochemical analysis beside mass spectrometry. Here, we describe the applications of clickable glutathione and provide detailed protocols for the identification, profiling, and detection of glutathionylated proteins and cysteines. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identification of glutathionylated cysteine in individual proteins in vitro Basic Protocol 2: Proteomic identification and quantification of glutathionylation Basic Protocol 3: Biochemical validation of glutathionylation in cells.

Robust AMBER Force Field Parameters for Glutathionylated Cysteines.

S-glutathionylation is an oxidative post-translational modification, which is involved in the regulation of many cell signaling pathways. Increasing amounts of studies show that it is crucial in cell homeostasis and deregulated in several pathologies. However, the effect of S-glutathionylation on proteins' structure and activity is poorly understood, and a drastic lack of structural information at the atomic scale remains. Studies based on the use of molecular dynamics simulations, which can provide important information about modification-induced modulation of proteins' structure and function, are also sparse, and there is no benchmarked force field parameters for this modified cysteine. In this contribution, we provide robust AMBER parameters for S-glutathionylation, which we tested extensively against experimental data through a total of 33 µs molecular dynamics simulations. We show that our parameter set efficiently describes the global and local structural properties of S-glutathionylated proteins. These data provide the community with an important tool to foster new investigations into the effect of S-glutathionylation on protein dynamics and function, in a common effort to unravel the structural mechanisms underlying its critical role in cellular processes.

Chemoproteomic strategy identified p120-catenin glutathionylation regulates E-cadherin degradation and cell migration.

Identification of cysteines with high oxidation susceptibility is important for understanding redox-mediated biological processes. In this report, we report a chemical proteomic strategy that finds cysteines with high susceptibility to S-glutathionylation. Our proteomic strategy, named clickable glutathione-based isotope-coded affinity tag (G-ICAT), identified 1,518 glutathionylated cysteines while determining their relative levels of glutathionylated and reduced forms upon adding hydrogen peroxide. Among identified cysteines, we demonstrated that CTNND1 (p120) C692 has high susceptibility to glutathionylation. Also, p120 wild type (WT), compared to C692S, induces its dissociation from E-cadherin under oxidative stress, such as glucose depletion. p120 and E-cadherin dissociation correlated with E-cadherin destabilization via its proteasomal degradation. Lastly, we showed that p120 WT, compared to C692S, increases migration and invasion of MCF7 cells under glucose depletion, supporting a model that p120 C692 glutathionylation increases cell migration and invasion by destabilization of E-cadherin, a core player in cell-cell adhesion.

Changes in Hemoglobin Properties in Complex with Glutathione and after Glutathionylation.

Hemoglobin is the main protein of red blood cells that provides oxygen transport to all cells of the human body. The ability of hemoglobin to bind the main low-molecular-weight thiol of the cell glutathione, both covalently and noncovalently, is not only an important part of the antioxidant protection of red blood cells, but also affects its affinity for oxygen in both cases. In this study, the properties of oxyhemoglobin in complex with reduced glutathione (GSH) and properties of glutathionylated hemoglobin bound to glutathione via an SS bond were characterized. For this purpose, the methods of circular dichroism, Raman spectroscopy, infrared spectroscopy, tryptophan fluorescence, differential scanning fluorimetry, and molecular modeling were used. It was found that the glutathionylation of oxyhemoglobin caused changes in the secondary structure of the protein, reducing the alpha helicity, but did not affect the heme environment, tryptophan fluorescence, and the thermostability of the protein. In the noncovalent complex of oxyhemoglobin with reduced glutathione, the secondary structure of hemoglobin remained almost unchanged; however, changes in the heme environment and the microenvironment of tryptophans, as well as a decrease in the protein's thermal stability, were observed. Thus, the formation of a noncovalent complex of hemoglobin with glutathione makes a more significant effect on the tertiary and quaternary structure of hemoglobin than glutathionylation, which mainly affects the secondary structure of the protein. The obtained data are important for understanding the functioning of glutathionylated hemoglobin, which is a marker of oxidative stress, and hemoglobin in complex with GSH, which appears to deposit GSH and release it during deoxygenation to increase the antioxidant protection of cells.

Nuclear factor kappa B expression in non-small cell lung cancer.

In this mini-review, we discuss the role of NF-κB, a proinflammatory transcription factor, in the expression of genes involved in inflammation, proliferation, and apoptosis pathways, and link it with prognosis of various human cancers, particularly non-small cell lung cancer (NSCLC). We and others have shown that NF-κB activity can be impacted by post-translational S-glutathionylation through reversible formation of a mixed disulfide bond between its cysteine residues and glutathione (GSH). Clinical data analysis showed that high expression of NF-κB correlated with shorter overall survival (OS) in NSCLC patients, suggesting a tumor promotion function for NF-κB. Moreover, NF-κB expression was associated with tumor stage, lymph node metastasis, and 5-year OS in these patients. NF-κB was over-expressed in the cytoplasm of tumor tissue compared to adjacent normal tissues. S-glutathionylation of NF-κB caused negative regulation by interfering with DNA binding activities of NF-κB subunits. In response to oxidants, S-glutathionylation of NF-κB also correlated with enhanced lung inflammation. Thus, S-glutathionylation is an important contributor to NF-κB regulation and clinical results highlight the importance of NF-κB in NSCLC, where NF-κB levels are associated with unfavorable prognosis.

S-Glutathionylation and S-Nitrosylation as Modulators of Redox-Dependent Processes in Cancer Cell.

Development of oxidative/nitrosative stress associated with the activation of oncogenic pathways results from the increase in the generation of reactive oxygen and nitrogen species (ROS/RNS) in tumor cells, where they can have a dual effect. At high concentrations, ROS/RNS cause cell death and limit tumor growth at certain phases of its development, while their low amounts promote oxidative/nitrosative modifications of key redox-dependent residues in regulatory proteins. The reversibility of such modifications as S-glutathionylation and S-nitrosylation that proceed through the electrophilic attack of ROS/RNS on nucleophilic Cys residues ensures the redox-dependent switch in the activity of signaling proteins, as well as the ability of these compounds to control cell proliferation and programmed cell death. The content of S-glutathionylated and S-nitrosylated proteins is controlled by the balance between S-glutathionylation/deglutathionylation and S-nitrosylation/denitrosylation, respectively, and depends on the cellular redox status. The extent of S-glutathionylation and S-nitrosylation of protein targets and their ratio largely determine the status and direction of signaling pathways in cancer cells. The review discusses the features of S-glutathionylation and S-nitrosylation reactions and systems that control them in cancer cells, as well as their relationship with redox-dependent processes and tumor growth.