Glutathione (GSH) and GSH‐related enzymes constitute the most important defense system that protects cells from free radical, radiotherapy, and chemotherapy attacks. In this study, we aim to explore ...the potential role and regulatory mechanism of the GSH redox cycle in drug resistance in glioblastoma multiforme (GBM) cells. We found that temozolomide (TMZ)‐resistant glioma cells displayed lower levels of endogenous reactive oxygen species and higher levels of total antioxidant capacity and GSH than sensitive cells. Moreover, the expression of glutathione reductase (GSR), the key enzyme of the GSH redox cycle, was higher in TMZ‐resistant cells than in sensitive cells. Furthermore, silencing GSR in drug‐resistant cells improved the sensitivity of cells to TMZ or cisplatin. Conversely, the over‐expression of GSR in sensitive cells resulted in resistance to chemotherapy. In addition, the GSR enzyme partially prevented the oxidative stress caused by pro‐oxidant L‐buthionine ‐sulfoximine. The modulation of redox state by GSH or L‐buthionine –sulfoximine regulated GSR‐mediated drug resistance, suggesting that the action of GSR in drug resistance is associated with the modulation of redox homeostasis. Intriguingly, a trend toward shorter progress‐free survival was observed among GBM patients with high GSR expression. These results indicated that GSR is involved in mediating drug resistance and is a potential target for improving GBM treatment.
Glioblastoma multiforme is the most aggressive type of brain tumor. We show that glutathione reductase (GSR), the key enzyme of the glutathione (GSH) redox cycle, mediates drug resistance by regulating redox homeostasis and is a potential target for improving GBM treatment. These findings provide new ideas and strategies for reversing drug resistance.
In this review article we examine the role of glutathione reductase in the regulation, modulation and maintenance of cellular redox homoeostasis. Glutathione reductase is responsible for maintaining ...the supply of reduced glutathione; one of the most abundant reducing thiols in the majority of cells. In its reduced form, glutathione plays key roles in the cellular control of reactive oxygen species. Reactive oxygen species act as intracellular and extracellular signalling molecules and complex cross talk between levels of reactive oxygen species, levels of oxidised and reduced glutathione and other thiols, and antioxidant enzymes such as glutathione reductase determine the most suitable conditions for redox control within a cell or for activation of programmed cell death. Additionally, we discuss the translation and expression of glutathione reductase in a number of organisms including yeast and humans. In yeast and human cells, a single gene expresses more than one form of glutathione reductase, destined for residence in the cytoplasm or for translocation to different organelles; in plants, however, two genes encoding this protein have been described. In general, insects and kinetoplastids (a group of protozoa, including Plasmodia and Trypanosoma) do not express glutathione reductase or glutathione biosynthetic enzymes. Instead, they express either the thioredoxin system or the trypanothione system. The thioredoxin system is also present in organisms that have the glutathione system and there may be overlapping functions with cross-talk between the two systems. Finally we evaluate therapeutic targets to overcome oxidative stress associated cellular disorders.
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•Most aerobic organisms use glutathione as a major cellular antioxidant.•Reduction of the oxidised form helps to reduced glutathione pools.•The thioredoxin and glutathione systems usually act cooperatively.•Organisms lacking glutathione reductase utilise other, similar thiol systems.•Many pathogens have unusual thiol redox systems, providing potential drug targets.
Cardiac aging is an important contributing factor for heart failure, which affects a large population but remains poorly understood.
The purpose of this study is to investigate whether Klotho plays a ...role in cardiac aging.
Heart function declined in old mice (24 months), as evidenced by decreases in fractional shortening, ejection fraction, and cardiac output. Heart size and weight, cardiomyocyte size, and cardiac fibrosis were increased in old mice, indicating that aging causes cardiac hypertrophy and remodeling. Circulating Klotho levels were dramatically decreased in old mice, which prompted us to investigate whether the Klotho decline may cause heart aging. We found that
gene mutation (KL-/-) largely decreased serum klotho levels and impaired heart function. Interestingly, supplement of exogenous secreted Klotho prevented heart failure, hypertrophy, and remodeling in both old mice and KL (-/-) mice. Secreted Klotho treatment inhibited excessive cardiac oxidative stress, senescence and apoptosis in old mice and KL (-/-) mice. Serum phosphate levels in KL (-/-) mice were kept in the normal range, suggesting that Klotho deficiency-induced heart aging is independent of phosphate metabolism. Mechanistically, Klotho deficiency suppressed GR (glutathione reductase) expression and activity in the heart via inhibition of transcription factor Nrf2 (nuclear factor-erythroid 2 p45-related factor 2). Furthermore, cardiac-specific overexpression of GR prevented excessive oxidative stress, apoptosis, and heart failure in both old and KL (-/-) mice.
Klotho deficiency causes cardiac aging via impairing the Nrf2-GR pathway. Supplement of exogenous secreted Klotho represents a promising therapeutic strategy for aging-associated cardiomyopathy and heart failure.
Sorafenib, a first-line drug for advanced hepatocellular carcinoma (HCC), shows a favorable anti-tumor effect while resistance is a barrier impeding patients from benefiting from it. Thus, more ...efforts are needed to lift this restriction. Herein, we first find that solute carrier family 27 member 5 (SLC27A5/FATP5), an enzyme involved in the metabolism of fatty acid and bile acid, is downregulated in sorafenib-resistant HCC. SLC27A5 deficiency facilitates the resistance towards sorafenib in HCC cells, which is mediated by suppressing ferroptosis. Further mechanism studies reveal that the loss of SLC27A5 enhances the glutathione reductase (GSR) expression in a nuclear factor erythroid 2-related factor 2 (NRF2)-dependent manner, which maintains glutathione (GSH) homeostasis and renders insensitive to sorafenib-induced ferroptosis. Notably, SLC27A5 negatively correlates with GSR, and genetic or pharmacological inhibition of GSR strengthens the efficacy of sorafenib through GSH depletion and the accumulation of lipid peroxide products in SLC27A5-knockout and sorafenib-resistant HCC cells. Based on our results, the combination of sorafenib and carmustine (BCNU), a selective inhibitor of GSR, remarkably hamper tumor growth by enhancing ferroptotic cell death in vivo. In conclusion, we describe that SLC27A5 serves as a suppressor in sorafenib resistance and promotes sorafenib-triggered ferroptosis via restraining the NRF2/GSR pathway in HCC, providing a potential therapeutic strategy for overcoming sorafenib resistance.
Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since ...the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.
The ascorbate–glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this ...cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO⁻) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO⁻ and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO⁻. The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADP-binding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO⁻. These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO⁻ or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150 mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate–glutathione cycle by nitric oxide (NO)-PTMs, thus indicating the close involvement of NO and reactive oxygen species metabolism in antioxidant defence against nitro-oxidative stress situations in plants.
The redox-based protein S-nitrosylation is a conserved mechanism modulating nitric oxide (NO) signaling and has been considered mainly as a non-enzymatic reaction. S-nitrosylation is regulated by the ...intracellular NO level that is tightly controlled by S-nitrosoglutathione reductase (GSNOR). However, the molecular mechanisms regulating S-nitrosylation selectivity remain elusive. Here, we characterize an Arabidopsis “repressor of” gsnor1 (rog1) mutation that specifically suppresses the gsnor1 mutant phenotype. ROG1, identical to the non-canonical catalase, CAT3, is a transnitrosylase that specifically modifies GSNOR1 at Cys-10. The transnitrosylase activity of ROG1 is regulated by a unique and highly conserved Cys-343 residue. A ROG1C343T mutant displays increased catalase but decreased transnitrosylase activities. Consistent with these results, the rog1 mutation compromises responses to NO under both normal and stress conditions. We propose that ROG1 functions as a transnitrosylase to regulate the NO-based redox signaling in plants.
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•ROG1 or CAT3 acts as a transnitrosylase to regulate NO signaling in plants•Cysteine-343 is vital for ROG1 activity•ROG1 transnitrosylates GSNOR1 to regulate its stability•rog1 mutants have reduced sensitivity to NO under normal and stress conditions
Chen et al. identify and characterize the non-canonical catalase ROG1/CAT3 as a transnitrosylase in Arabidopsis. ROG1 contains a highly conserved cysteine-343, which is crucial for its activity, and ROG1 transnitrosylates GSNOR1 to regulate its stability. ROG1 regulates NO signaling under both normal and stress conditions.
Tight control of cellular redox homeostasis is essential for protection against oxidative damage and for maintenance of normal metabolism as well as redox signaling events. Under oxidative stress ...conditions, the tripeptide glutathione can switch from its reduced form (GSH) to oxidized glutathione disulfide (GSSG), and thus, forms an important cellular redox buffer. GSSG is normally reduced to GSH by 2 glutathione reductase (GR) isoforms encoded in the Arabidopsis genome, cytosolic GR1 and GR2 dual-targeted to chloroplasts and mitochondria. Measurements of total GR activity in leaf extracts of wild-type and 2 gr1 deletion mutants revealed that almost equal to65% of the total GR activity is attributed to GR1, whereas almost equal to35% is contributed by GR2. Despite the lack of a large share in total GR activity, gr1 mutants do not show any informative phenotype, even under stress conditions, and thus, the physiological impact of GR1 remains obscure. To elucidate its role in plants, glutathione-specific redox-sensitive GFP was used to dynamically measure the glutathione redox potential (EGSH) in the cytosol. Using this tool, it is shown that EGSH in gr1 mutants is significantly shifted toward more oxidizing conditions. Surprisingly, dynamic reduction of GSSG formed during induced oxidative stress in gr1 mutants is still possible, although significantly delayed compared with wild-type plants. We infer that there is functional redundancy in this critical pathway. Integrated biochemical and genetic assays identify the NADPH-dependent thioredoxin system as a backup system for GR1. Deletion of both, NADPH-dependent thioredoxin reductase A and GR1, prevents survival due to a pollen lethal phenotype.
•2,6-bis(2-aminophenylthio)pyridine was synthesized and identified by X-ray diffraction method.•Four new Schiff bases derived from 2,6-bis(2-aminophenylthio)pyridine were synthesized and ...characterized by spectrophotometric method.•Glutathione reductases inhibitory activities of all compounds were evaluated.•IFD docking analysis of the new Schiff bases was carried out for hGR (PDB:1GRA)•ADME/T properties of the compounds were predicted by Qikprop program.
2,6-bis(2-aminophenylthio)pyridine was synthesized and identified by x-ray diffraction method. Its new Schiff bases (H2L1, H2L2, L3 and L4) were synthesized and characterized by elemental analysis, FT-IR, LC-MS, 1H NMR and 13C NMR techniques. in vitro glutathione reductase activities of the compounds were tested on yeast and human glutathione reductase. L4 enhanced both glutathione reductase activities, resulting in AC50 values of 15.06 µM and 15.89 µM, respectively. H2L1, H2L2 and L3 were found to be the inhibitors in the range of 50.09 – 55.23 µM for yeast glutathione reductase, and in the range of 56.12–66.87 µM for human glutathione reductase. According to molecular docking analysis at the xanthine binding site in human glutathione reductase (PDB: 1XAN), 2,6-bis(2-aminophenylthio)pyridine is predicted to have antimalarial property due to having a higher XP docking score than the malaria drug Chloroquine. Also, five binding pockets at the human glutathione reductase (PDB:1GRA) were identified using Sitemap analysis for Schiff bases which are non-competitive inhibitors. IFD-Docking scores were found to correlate with experimental IC50 value of H2L1, H2L2 and L3. Based on the fact that Schiff bases have higher IFD docking scores at binding pocket-1 than at the active site, it can be predicted that Schiff bases prefer to bind to binding pocket-1 in the presence of natural substrate. The difference in glutathione reductase activities of Schiff bases was attributed to the fact that the conformation of the activator L4-human GR complex was different from that of other inhibitor Schiff bases-human glutathione reductase complexes. ADMET calculations predicted that synthesized ligands obey the Lipinski Rule (rule of five) and Jorgensen Rule (rule of three).
Summary
Thiol‐based redox‐regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin‐dependent processes. In parallel, ...glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione‐mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo‐lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (EGSH) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal EGSH dynamics, we show that stromal EGSH is highly reducing in wild‐type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.
Significance Statement
Chloroplast physiology in the light and in the dark is underpinned by various redox cascades, modulating scavenging of reactive oxygen species, damage repair and enzymatic functions. We find that the glutathione redox potential (EGSH) dynamically reacts to light and that a permanently shifted EGSH causes light‐sensitivity, disturbing non‐photochemical quenching and protein homeostasis. Our data suggest that redox processes downstream of stromal EGSH play a fundamental role in regulating photosynthetic efficiency.
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