Exogenous salicylic acid (SA) significantly improved abiotic tolerance in higher plants, and ascorbate (ASA) and glutathione (GSH) play important roles in abiotic tolerance. In this study, SA (0.5mM) ...markedly increased the contents of ASA and GSH in SA-treated plants during salt stress (250mM NaCl). The transcript levels of the genes encoding ASA and GSH cycle enzymes were measured using quantitative real-time PCR. The results indicated that, during salt stress, exogenous SA significantly enhanced the transcripts of glutathione peroxidase (GPX1), phospholipid hydroperoxide glutathione peroxidase (GPX2) and dehydroascorbate reductase (DHAR) genes at 12h, glutathione reductase (GR) at 24h, 48h and 72h, glutathione-S-transferase 1 (GST1), 2 (GST2), monodehydroascorbate reductase (MDHAR) and glutathione synthetase (GS) at the 48h and 72h after salt stress, respectively. The results implied that SA temporally regulated the transcript levels of the genes encoding ASA–GSH cycle enzymes, resulting in the increased contents of GSH and ASA and enhanced salt tolerance.
•Salicylic acid (SA) enhanced the salt tolerance of wheat seedlings.•SA increased the contents of ASA and GSH in salt-stressed wheat seedlings.•SA temporally regulated the transcript levels of eight genes encoding ASA and GSH.•SA-induced salt tolerance may be associated with the ASA–GSH biosynthesis.
Glutathione-dependent catalysis is a metabolic adaptation to chemical challenges encountered by all life forms. In the course of evolution, nature optimized numerous mechanisms to use glutathione as ...the most versatile nucleophile for the conversion of a plethora of sulfur-, oxygen- or carbon-containing electrophilic substances.
This comprehensive review summarizes fundamental principles of glutathione catalysis and compares the structures and mechanisms of glutathione-dependent enzymes, including glutathione reductase, glutaredoxins, glutathione peroxidases, peroxiredoxins, glyoxalases 1 and 2, glutathione transferases and MAPEG. Moreover, open mechanistic questions, evolutionary aspects and the physiological relevance of glutathione catalysis are discussed for each enzyme family.
It is surprising how little is known about many glutathione-dependent enzymes, how often reaction geometries and acid–base catalysts are neglected, and how many mechanistic puzzles remain unsolved despite almost a century of research. On the one hand, several enzyme families with non-related protein folds recognize the glutathione moiety of their substrates. On the other hand, the thioredoxin fold is often used for glutathione catalysis. Ancient as well as recent structural changes of this fold did not only significantly alter the reaction mechanism, but also resulted in completely different protein functions.
Glutathione-dependent enzymes are excellent study objects for structure–function relationships and molecular evolution. Notably, in times of systems biology, the outcome of models on glutathione metabolism and redox regulation is more than questionable as long as fundamental enzyme properties are neither studied nor understood. Furthermore, several of the presented mechanisms could have implications for drug development. This article is part of a Special Issue entitled Cellular functions of glutathione.
► Fundamental principles of glutathione catalysis are summarized. ► Mechanisms of enzymes with five non-related protein folds are compared. ► Evolutionary aspects and open mechanistic questions are discussed. ► The physiological relevance of glutathione catalysis is highlighted.
Despite the fact that iron represents a crucial element for the catalysis of many metabolic reactions, its accumulation in the cell leads to the production of reactive oxygen species (ROS), provoking ...pathological conditions such as cancer, cardiovascular diseases, diabetes, neurodegenerative diseases, and fertility. Thus, ROS are neutralized by the enzymatic antioxidant system for the purpose of protecting cells against any damage. Iron is a potential risk factor for male fertility. However, the mechanism of action of iron on the testicular antioxidant system at the gene and protein levels is not fully understood. Thus, the purpose of the current research was to ensure a better understanding of how the long-term iron treatment influences both gene expression and enzyme activities of the testicular antioxidant system in rat testis. The data of our study showed that a significant dose-dependent increase occurred in the iron level in rat testis. A reduction occurred in reduced glutathione (GSH) levels, which represent a marker of oxidative stress, along with long-term iron overload. The expression and activity of glucose 6-phosphate dehydrogenase (
G6pd
), glutathione reductase (
Gr
), glutathione peroxidase (
Gpx
), and glutathione S-transferases (
Gst
) were significantly affected by the presence of iron. The findings of the current research demonstrate that the long-term toxic dietary iron overload influences the gene expression and enzyme activity of the testicular antioxidant defense system, but the actual effect occurs at the protein level. This may modify the sperm function and dysfunction of the male reproductive system.
The acclimation of reduced glutathione (GSH) biosynthesis and GSH-utilizing enzymes to salt stress was studied in two tomato species that differ in stress tolerance. Salt increased GSH content and ...GSH:GSSG (oxidized glutathione) ratio in oxidative stress-tolerant
Lycopersicon pennellii (Lpa) but not in
Lycopersicon esculentum (Lem). These changes were associated with salt-induced upregulation of γ-glutamylcysteine synthetase protein, an effect which was prevented by preincubation with buthionine sulfoximine. Salt treatment induced glutathione peroxidase and glutathione-
S-transferase but not glutathione reductase activities in Lpa. These results suggest a mechanism of coordinate upregulation of synthesis and metabolism of GSH in Lpa, that is absent from Lem.
The glutathione (GSH) biosynthetic pathway in brain tumors. (A) Most brain tumors arise from 3 main cell types or their progenitors: neurons (green), oligodendrocytes (cyan), and astrocytes ...(magenta). (B) GSH biosynthesis consists of two ATP-dependent reactions: (1) glutamate cysteine ligase (GCL)-mediated formation of γ-glutamyl-cysteine (γGluCys) from glutamate (Glu) and cysteine (Cys) and (2) GSH synthetase (GS)-catalyzed formation of GSH from γGluCys and glycine (Gly). Buthionine sulfoximine (BSO) is an irreversible inhibitor of GCL. Detoxification of chemotherapeutic agents proceeds via the GSH-S-transferase (GST)-mediated formation of GSH-drug conjugates (GSH-X) followed by efflux by multidrug resistance protein (Mrp) transporters. γ-Glutamyl transpeptidase (γGT) can break GSH down into Glu and cysteinylglycine (CysGly). CysGly is further hydrolyzed by cellular dipeptidases (DP) followed by the transporter-mediated uptake of the constituent amino acids that can serve as substrates for either cellular GSH production or protein synthesis. Additional Cys may also be obtained via the glutamate-cystine antiporter (Xc-)-mediated uptake of extracellular cystine (CysCys).
Chemotherapy is central to the current treatment modality for primary human brain tumors, but despite high-dose and intensive treatment regimens there has been little improvement in patient outcome. The development of tumor chemoresistance has been proposed as a major contributor to this lack of response. While there have been some improvements in our understanding of the molecular mechanisms underlying brain tumor drug resistance over the past decade, the contribution of glutathione (GSH) and the GSH-related enzymes to drug resistance in brain tumors have been largely overlooked. GSH constitutes a major antioxidant defense system in the brain and together with the GSH-related enzymes plays an important role in protecting cells against free radical damage and dictating tumor cell response to adjuvant cancer therapies, including irradiation and chemotherapy. Glutamate cysteine ligase (GCL), glutathione synthetase (GS), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferases (GST), and GSH complex export transporters (GS-X pumps) are major components of the GSH-dependent enzyme system that function in a dynamic cascade to maintain redox homeostasis. In many tumors, the GSH system is often dysregulated, resulting in a more drug resistant phenotype. This is commonly associated with GST-mediated GSH conjugation of various anticancer agents leading to the formation of less toxic GSH–drug complexes, which can be readily exported from the cell. Advances in our understanding of the mechanisms of drug resistance and patient selection based on biomarker profiles will be crucial to adapt therapeutic strategies and improve outcomes for patients with primary malignant brain tumors.
Reduced glutathione (GSH) is an essential non-enzymatic antioxidant in mammalian cells. GSH can act directly as an antioxidant to protect cells against free radicals and pro-oxidants, and as a ...cofactor for antioxidant and detoxification enzymes such as glutathione peroxidases, glutathione S-transferases, and glyoxalases. Glutathione peroxidases detoxify peroxides by a reaction that is coupled to GSH oxidation to glutathione disulfide (GSSG). GSSG is converted back to GSH by glutathione reductase and cofactor NADPH. GSH can regenerate vitamin E following detoxification reactions of vitamin E with lipid peroxyl radicals (LOO). GSH is a cofactor for GST during detoxification of electrophilic substances and xenobiotics. Dicarbonyl stress induced by methylglyoxal and glyoxal is alleviated by glyoxalase enzymes and GSH. GSH regulates redox signaling through reversible oxidation of critical protein cysteine residues by S-glutathionylation. GSH is involved in other cellular processes such as protein folding, protecting protein thiols from oxidation and crosslinking, degradation of proteins with disulfide bonds, cell cycle regulation and proliferation, ascorbate metabolism, apoptosis and ferroptosis.
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.
•GSH artificially oxidizes during sample handling producing GSSG.•Most of GSH to GSSG ratios reported in the literature are erroneous.•Immediate SH alkylation with N-ethylmaleimide is mandatory.
...Glutathione (GSH) is the most abundant low-molecular-mass thiol within cells and one of the major antioxidant compounds in body fluids. Under pro-oxidant conditions, two GSH molecules donate one electron each and are converted into glutathione disulfide (GSSG). The GSH/GSSG molar ratio is considered a powerful index of oxidative stress and disease risk. Despite high interest in GSH/GSSG titration as measures of thiol redox balance, no broad agreement has yet been reached as to the best pre-analytical and analytical methods for the quantitation of these molecules in biological samples. Consequently, measured concentrations of GSH and GSSG and calculated GSH/GSSG molar ratios vary widely among laboratories. Here, we describe in detail the main analytical and pre-analytical problems related to the artificial oxidation of the sulfhydryl (SH) group of GSH that occur during sample manipulation. We underline how this aspect has been neglected for long time after its first description more than fifty years ago. Finally, selected reliable procedures and methods to measure GSH and GSSG in biological samples are discussed.
We present two different methods for determining levels of glutathione in complex biological samples and plasma. The DTNB/GR enzyme recycling method is sensitive and requires no specialized ...equipment. The HPLC method is particularly useful for situations in which sample amounts are limited. Detailed instructions for performing each method as well as the advantages and disadvantages of each are discussed in this chapter.
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