Γ-glutamylcysteine Synthetase Research Articles

Sulforaphane alleviates membranous nephropathy by inhibiting oxidative stress-associated podocyte pyroptosis.

To investigate the natural product sulforaphane (SFN) in protection of membranous nephropathy (MN) by inhibiting oxidative stress-associated podocyte pyroptosis. A passive Heymann nephritis (PHN) model was established and treated with SFN. Clinical manifestations were examined by testing 24-hr urine protein, albumin, total cholesterol, triglyceride, high-density and low-density lipoprotein levels. Podocyte injury was observed through glomerular ultrastructure and the expression of podocin and desmin. Intrarenal oxidative stress was evaluated through assessment of oxidative markers, including malondialdehyde, 8-isoprostane, and 8-hydroxydeoxyguanosine, and the activities of anti-oxidant enzymes, including total superoxide dismutase, catalase, and γ-glutamylcysteine synthetase. Podocyte and intrarenal pyroptosis were investigated by observing the localization of the GSDMD N-terminus (GSDMD(N)) in podocytes; the expression of pyroptosis signaling pathway, including GSDMD, NF-κB p65, p-NF-κB p65 (Ser536), NLRP3, ASC, caspase-1, IL-1β, and IL-18; and pyroptosis encounter Nrf2 in the glomeruli and kidney. SFN has a protective effect on MN, as reflected by alleviation of nephrotic syndrome, amelioration of podocyte foot process fusion, increased expression and normalization of podocin, and decreased expression of desmin in the glomeruli. Mechanistically, SFN relieved intrarenal oxidative stress, as indicated by decreased renal malondialdehyde, 8-isoprostane, and 8-hydroxydeoxyguanosine and increased activity of total superoxide dismutase, catalase, and γ-glutamylcysteine synthetase. SFN also inhibited podocyte and intrarenal pyroptosis, as revealed by decreased colocalization of GSDMD (N) with synaptopodin and ZO-1, decreased expression of pyroptosis signaling pathway, and increased expression of Nrf2 in the glomeruli and kidney. SFN could alleviate MN by inhibiting oxidative stress-associated podocyte pyroptosis.

Initial Glutathione Depletion During Short-Term Bed Rest: Pinpointing Synthesis and Degradation Checkpoints in the γ-Glutamyl Cycle.

Hypokinesia triggers oxidative stress and accelerates the turnover of the glutathione system via the γ-glutamyl cycle. Our study aimed to identify the regulatory checkpoints controlling intracellular glutathione levels. We measured the intermediate substrates of the γ-glutamyl cycle in erythrocytes from 19 healthy young male volunteers before and during a 10-day experimental bed rest. Additionally, we tracked changes in glutathione levels and specific metabolite ratios up to 21 days of bed rest. Using gas chromatography-mass spectrometry and the internal standard technique, we observed a 9 ± 9% decrease in glutathione levels during the first 5 days of bed rest, followed by an 11 ± 9% increase from the 5th to the 10th day, nearly returning to baseline ambulatory levels. The cysteinyl-glycine-to-glutathione ratio, reflecting γ-glutamyl cyclotransferase activity (a key enzyme in glutathione breakdown), rose by 14 ± 22% in the first 5 days and then fell by 10 ± 14% over the subsequent 5 days, again approaching baseline levels. Additionally, the γ-glutamyl cysteine-to-cysteine ratio, indicative of γ-glutamyl cysteine synthetase activity (crucial for glutathione synthesis), increased by 12 ± 30% on day 5 and by 29 ± 41% on day 10 of bed rest. The results observed on day 21 of bed rest confirm those seen on day 10. By calculating the ratio of product concentration to precursor concentration, we assessed the efficiency of these key enzymes in glutathione turnover. These results were corroborated by directly measuring glutathione synthesis and degradation rates in vivo using stable isotope techniques. Our findings reveal significant changes in glutathione kinetics during the initial days of bed rest and identify potential therapeutic targets for maintaining glutathione levels.

Overexpression of bacterial γ-glutamylcysteine synthetase increases toxic metal(loid)s tolerance and accumulation in Crambe abyssinica.

Transgenic Crambe abyssinica lines overexpressing γ-ECS significantly enhance tolerance to and accumulation of toxic metal(loid)s, improving phytoremediation potential and offering an effective solution for contaminated soil management. Phytoremediation is an attractive environmental-friendly technology to remove metal(loid)s from contaminated soils and water. However, tolerance to toxic metals in plants is a critical limiting factor. Transgenic Crambe abyssinica lines were developed that overexpress the bacterial γ-glutamylcysteine synthetase (γ-ECS) gene to increase the levels of non-protein thiol peptides such as γ-glutamylcysteine (γ-EC), glutathione (GSH), and phytochelatins (PCs) that mediate metal(loid)s detoxification. The present study investigated the effect of γ-ECS overexpression on the tolerance to and accumulation of toxic As, Cd, Pb, Hg, and Cr supplied individually or as a mixture of metals. Compared to wild-type plants, γ-ECS transgenics (γ-ECS1-8 and γ-ECS16-5) exhibited a significantly higher capacity to tolerate and accumulate these elements in aboveground tissues, i.e., 76-154% As, 200-254% Cd, 37-48% Hg, 26-69% Pb, and 39-46% Cr, when supplied individually. This is attributable to enhanced production of GSH (82-159% and 75-87%) and PC2 (27-33% and 37-65%) as compared to WT plants under AsV and Cd exposure, respectively. The levels of Cys and γ-EC were also increased by 56-67% and 450-794% in the overexpression lines compared to WT plants under non-stress conditions, respectively. This likely enhanced the metabolic pathway associated with GSH biosynthesis, leading to the ultimate synthesis of PCs, which detoxify toxic metal(loid)s through chelation. These findings demonstrate that γ-ECS overexpressing Crambe lines can be used for the enhanced phytoremediation of toxic metals and metalloids from contaminated soils.

Shengqing Jiangzhuo capsule ameliorates diabetic nephropathy by improving Keap1/Nrf2 signaling pathway.

Diabetic nephropathy (DN) is a major contributor to end-stage renal failure, and lacking effective treatment options. Shengqing Jiangzhuo capsule (SQJZJN), a traditional Chinese medicine prescription with known efficacy in chronic kidney disease, has not been thoroughly investigated for its potential in DN protection. Eight-week-old male C57BLKS/J db/db, C57BLKS/J db/m mice, and human glomerular mesangial cell (HMC) cells cultured with high glucose were used as experimental models in this study. The in vivo investigation showed that SQJZJN can significantly ameliorate renal pathological damage, reduce serum creatinine, and lower urinary microalbumin levels in db/db mice. In vitro, SQJZJN treatment mitigated advanced glycation end products (AGEs) and reactive oxygen species (ROS), leading to a reduction in renal cell apoptosis. Mechanistically, SQJZJN activated the Keap1/Nrf2/ARE pathway by promoting nuclear factor erythroid-derived 2-related factor 2 (Nrf2), γ-glutamylcysteine synthetase heavy subunit (γ-GCS), and Heme oxygenase-1 (HO-1) expressions, while decreasing Kelch-like ECH-associated protein 1 (KEAP1) expressions. These findings suggest that SQJZJN exerts a protective effect on DN, potentially through the activation of the Keap1/Nrf2/ARE pathway.

A chloroplast sulphate transporter modulates glutathione-mediated redox cycling to regulate cell division.

Glutathione redox cycling is important for cell cycle regulation, but its mechanisms are not well understood. We previously identified a small-sized mutant, suppressor of mat3 15-1 (smt15-1) that has elevated cellular glutathione. Here, we demonstrated that SMT15 is a chloroplast sulphate transporter. Reducing expression of γ-GLUTAMYLCYSTEINE SYNTHETASE, encoding the rate-limiting enzyme required for glutathione biosynthesis, corrected the size defect of smt15-1 cells. Overexpressing GLUTATHIONE SYNTHETASE (GSH2) recapitulated the small-size phenotype of smt15-1 mutant, confirming the role of glutathione in cell division. Hence, SMT15 may regulate chloroplast sulphate concentration to modulate cellular glutathione levels. In wild-type cells, glutathione and/or thiol-containing molecules (GSH/thiol) accumulated in the cytosol at the G1 phase and decreased as cells entered the S/M phase. While the cytosolic GSH/thiol levels in the small-sized mutants, smt15-1 and GSH2 overexpressors, mirrored those of wild-type cells (accumulating during G1 and declining at early S/M phase), GSH/thiol was specifically accumulated in the basal bodies at early S/M phase in the small-sized mutants. Therefore, we propose that GSH/thiol-mediated redox signalling in the basal bodies may regulate mitotic division number in Chlamydomonas reinhardtii. Our findings suggest a new mechanism by which glutathione regulates the multiple fission cell cycle in C. reinhardtii.

Toxicity of antimony to plants: Effects on metabolism of N and S in a rice plant

Excess antimony (Sb) has been shown to damage plant growth. Rice plants readily absorb a large amount of Sb after a long period of flooding, yet the mechanisms underlying Sb toxicity in plants have not been solved. This study was conducted to explore the effects of Sb on the uptake of N and S, and monitor the concentrations of reduced glutathione (GSH) and enzymes associated with these processes. In addition, we analyzed differentially expressed metabolites (DEMs) correlated with amino acids (AAs) and oligopeptides, specifically DEMs containing sulfur (S), GSH and indole–3–acetic acid (IAA). The results showed that antimonite [Sb(III)] inhibited shoot growth whereas antimonate [Sb(V)] stimulated shoot growth. Interestingly, Sb(III)5/10 enhanced shoot concentrations of total nitrogen (N), NH4+–N [only at Sb(III)10] and S; but reduced the shoot concentrations of NO3–N and soluble protein. Sb(III)5/10 addition significantly increased oxidized glutathione (GSSG) concentration and activities of glutathione peroxidase (GSH–Px) and glutathione S-transferase (GST) but non–significantly affected concentration of reduced glutathione (GSH) and activities of γ-glutamylcysteine synthetase (GCL) and glutathione reductase (GR), suggesting Sb(III) restricted GSH recycling. Addition of Sb (1) increased the abundance of DEMs associated with lignins, Ca uptake, toxicity/detoxification, and branched chain AAs; (2) decreased the abundance of AAs inclcuding isoleucine (Ile), leucine (Leu), tryptophan (Trp), tyrosine (Tyr) and histidine (His); (3) increased the abundance of arginine (Arg), putrescine (Put) and spermidine (Spd); and (4) affected methylation and acetylation of many AAs, especially acetylation.

Metformin-induced oxidative stress inhibits LNCaP prostate cancer cell survival.

Preclinical and clinical studies over the past several decades have indicated the potential value of metformin, a widely utilized treatment for Type 2 diabetes, in prostate cancer therapy. Notably, these studies demonstrated metformin's pleiotropic effects on several molecular and metabolic pathways, such as androgen signaling, cell cycle, and cellular bioenergetics. In this study we investigated the role of metformin in regulating intracellular redox status and cell survival in LNCaP prostate cancer cells. The cytotoxic effects of metformin with or without the presence of SBI0206965 (AMPK inhibitor) on LNCaP cells were determined using MTT and trypan blue exclusion assays. Seahorse XP extracellular analysis, Liquid Chromatography/ Mass Spectrophotometry (LC/MS), and 2,7- and Dichlorofluoresin diacetate (DCFDA) assay were used to assess the effects of metformin on cellular bioenergetics, redox status, and redox-related metabolites. mRNA expression and protein concentration of redox-related enzymes were measured using Real Time-qPCR and ELISA assay, respectively. Independently of AMP-activated protein kinase, metformin exhibited a dose- and time-dependent inhibition of LNCaP cell survival, a response mitigated by glutathione or N-acetylcysteine (ROS scavengers) treatment. Notably, these findings were concomitant with a decline in ATP levels and the inhibition of oxidative phosphorylation. The results further indicated metformin's induction of reactive oxygen species, which significantly decreased glutathione levels and the ratio of reduced to oxidized glutathione, as well as the transsulfuration metabolite, cystathionine. Consistent with an induction of oxidative stress condition, metformin increased mRNA levels of the master redox transcription factor Nrf-2 (nuclear factor erythroid-derived 2-like), as well as transsulfuration enzymes cystathionine beta-synthase and cystathionase and GSH synthesis enzymes γ-glutamylcysteine synthetase and glutathione synthetase. Our findings highlight multiple mechanisms by which metformin-induced formation of reactive oxygen species may contribute to its efficacy in prostate cancer treatment, including promotion of oxidative stress, Nrf2 activation, and modulation of redox-related pathways, leading to its anti-survival action.

Dietary glycine supplementation enhances syntheses of creatine and glutathione by tissues of hybrid striped bass (Morone saxatilis ♀ × Morone chrysops ♂) fed soybean meal-based diets

BackgroundWe recently reported that supplementing glycine to soybean meal-based diets is necessary for the optimum growth of 5- to 40-g (Phase-I) and 110- to 240-g (Phase-II) hybrid striped bass (HSB), as well as their intestinal health. Although glycine serves as an essential substrate for syntheses of creatine and glutathione (GSH) in mammals (e.g., pigs), little is known about these metabolic pathways or their nutritional regulation in fish. This study tested the hypothesis that glycine supplementation enhances the activities of creatine- and GSH-forming enzymes as well as creatine and GSH availabilities in tissues of hybrid striped bass (HSB; Morone saxatilis♀ × Morone chrysops♂).MethodsPhase-I and Phase-II HSB were fed a soybean meal-based diet supplemented with 0%, 1%, or 2% glycine for 8 weeks. At the end of the 56-d feeding, tissues (liver, intestine, skeletal muscle, kidneys, and pancreas) were collected for biochemical analyses.ResultsIn contrast to terrestrial mammals and birds, creatine synthesis occurred primarily in skeletal muscle from all HSB. The liver was most active in GSH synthesis among the HSB tissues studied. In Phase-I HSB, supplementation with 1% or 2% glycine increased (P < 0.05) concentrations of intramuscular creatine (15%–19%) and hepatic GSH (8%–11%), while reducing (P < 0.05) hepatic GSH sulfide (GSSG)/GSH ratios by 14%–15%, compared with the 0-glycine group; there were no differences (P > 0.05) in these variables between the 1% and 2% glycine groups. In Phase-II HSB, supplementation with 1% and 2% glycine increased (P < 0.05) concentrations of creatine and GSH in the muscle (15%–27%) and liver (11%–20%) in a dose-dependent manner, with reduced ratios of hepatic GSSG/GSH in the 1% or 2% glycine group. In all HSB, supplementation with 1% and 2% glycine dose-dependently increased (P < 0.05) activities of intramuscular arginine:glycine amidinotransferase (22%–41%) and hepatic γ-glutamylcysteine synthetase (17%–37%), with elevated activities of intramuscular guanidinoacetate methyltransferase and hepatic GSH synthetase and GSH reductase in the 1% or 2% glycine group. Glycine supplementation also increased (P < 0.05) concentrations of creatine and activities of its synthetic enzymes in tail kidneys and pancreas, and concentrations of GSH and activities of its synthetic enzymes in the proximal intestine.ConclusionsSkeletal muscle and liver are the major organs for creatine and GSH syntheses in HSB, respectively. Dietary glycine intake regulates creatine and GSH syntheses by both Phase-I and Phase-II HSB in a tissue-specific manner. Based on the metabolic data, glycine is a conditionally essential amino acid for the growing fish.

Immunocompromisation of wheat host by L-BSO and 2,4-DPA induces susceptibility to the fungal pathogen Fusarium oxysporum

Susceptibility is defined as the disruption of host defence systems that promotes infection or limits pathogenicity. Glutathione (GSH) is a major component of defence signalling pathways that maintain redox status and is synthesised by γ-glutamyl cysteine synthetase (γ-ECS). On the other hand, lignin acts as a barrier in the primary cell wall of vascular bundles (VBs) synthesised by phenylalanine ammonia-lyase (PAL) in the intracellular system of plants. In this study, we used two inhibitors, such as L-Buthionine-sulfoximine (BSO), which irreversibly inhibits γ-ECS, and 2,4-dichlorophenoxyacetic acid (DPA), which reduces PAL activity and leads to the induction of oxidative stress in wheat (Triticum aestivum) seedlings after exposure to Fusarium oxysporum. Seedlings treated with 1 mM L-BSO and 2,4-DPA showed high levels of hydrogen peroxide (H2O2), malondialdehyde (MDA), carbonyl (CO) content, and low activity of antioxidative enzymes [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR)] as compared to wild-type (WT) seedlings under F. oxysporum infection. Further, the content of reduced glutathione (RGSH), ascorbate (ASC), and lignin was decreased in BSO and DPA treated seedlings as compared to WT seedlings during Fusarium infection. Moreover, treatment with BSO and DPA significantly inhibited the relative activity of γ-ECS and PAL (P ≤ 0.001) in WT seedlings during Fusarium infection, which led to disintegrated VBs and, finally, cell death. Our results demonstrate that inhibition of γ-ECS and PAL by BSO and DPA, respectively, disrupts the defence mechanisms of wheat seedlings and induces susceptibility to F. oxysporum.

Electroacupuncture reduces oxidative stress response and improves secondary injury of intracerebral hemorrhage in rats by activating the peroxisome proliferator-activated receptor-γ/nuclear factor erythroid2-related factor 2/γ-glutamylcysteine synthetase pathway.

Intracerebral hemorrhage (ICH) is a severe stroke subtype. Secondary injury is a key factor leading to neurological deficits after ICH. Electroacupuncture (EA) can improve the neurological function after ICH, however, its internal mechanism is still unclear. The aim of this study is to investigate whether EA could ameliorate secondary injury after ICH through antioxidative stress and its potential regulatory mechanism. A rat model of ICH was established by injecting autologous blood into striatum. After the intervention of EA and EA combined with peroxisome proliferator-activated receptor-γ (PPARγ) blocker, Zea-longa scores, modified neurological severity scores and open field tests were used to evaluate the neurological function of the rats. Flow cytometry detected tissue reactive oxygen species (ROS) levels. Tissue tumor necrosis factor-α (TNF-α) levels were analyzed by enzyme-linked immunosorbent assays. The protein expressions of PPAR γ, nuclear factor erythroid2-related factor 2 (Nrf2) and γ-glutamylcysteine synthetase (γ-GCS) were detected by Western blot. Immunohistochemistry was used to observe the activation of microglia. The demyelination degree of axon myelin was observed by transmission electron microscope. Compared with the model group, EA intervention improved neurological function, decreased ROS and TNF-α levels, increased the protein expression of PPARγ, Nrf2 and γ-GCS, and reduced the activation of microglia, it also alleviated axonal myelin sheath damage. In addition, the neuroprotective effect of EA was partially attenuated by PPARγ blocker. EA ameliorated the neurological function of secondary injury after ICH in rats, possibly by activating the PPARγ/Nrf2/γ-GCS signaling pathway, reducing microglia activation, and inhibiting oxidative stress, thus alleviating the extent of axonal demyelination plays a role.

Dietary glycine supplementation enhances glutathione availability in tissues of pigs with intrauterine growth restriction.

This study tested the hypothesis that dietary supplementation with glycine enhances the synthesis and concentrations of glutathione (GSH, a major antioxidant) in tissues of pigs with intrauterine growth restriction (IUGR). At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of two groups, representing supplementation with 1% glycine or 1.19% l-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Blood and other tissues were obtained from the pigs within 1wk after the feeding trial ended at 188 d of age to determine GSH, oxidized GSH (GSSG), and activities of GSH-metabolic enzymes. Results indicated that concentrations of GSH + GSSG or GSH in plasma, liver, and jejunum (P < 0.001) and concentrations of GSH in longissimus lumborum and gastrocnemius muscles (P < 0.05) were lower in IUGR pigs than in NBW pigs. In contrast, IUGR increased GSSG/GSH ratios (an indicator of oxidative stress) in plasma (P < 0.001), jejunum (P < 0.001), both muscles (P < 0.05), and pancreas (P = 0.001), while decreasing activities of γ-glutamylcysteine synthetase and GSH synthetase in liver (P < 0.001) and jejunum (P < 0.01); and GSH reductase in jejunum (P < 0.01), longissimus lumborum muscle (P < 0.01), gastrocnemius muscle (P < 0.05), and pancreas (P < 0.01). In addition, IUGR pigs had greater (P < 0.001) concentrations of thiobarbituric acid reactive substances (TBARS; an indicator of lipid peroxidation) in plasma, jejunum, muscles, and pancreas than NBW pigs. Compared with isonitrogenous controls, dietary glycine supplementation increased concentrations of GSH plus GSSG and GSH in plasma (P < 0.01), liver (P < 0.001), jejunum (P < 0.001), longissimus lumborum muscle (P = 0.001), and gastrocnemius muscle (P < 0.05); activities of GSH-synthetic enzymes in liver (P < 0.01) and jejunum (P < 0.05), while reducing GSSG/GSH ratios in plasma (P < 0.001), jejunum (P < 0.001), longissimus lumborum muscle (P < 0.001), gastrocnemius muscle (P = 0.01), pancreas (P < 0.05), and kidneys (P < 0.01). Concentrations of GSH plus GSSG, GSH, and GSSG/GSH ratios in kidneys were not affected (P > 0.05) by IUGR. Furthermore, glycine supplementation reduced (P < 0.001) TBARS concentrations in plasma, jejunum, muscles, and pancreas. Collectively, IUGR reduced GSH availability and induced oxidative stress in pig tissues, and these abnormalities were prevented by dietary glycine supplementation in a tissue-specific manner.

Human umbilical cord mesenchymal stem cells regulate glutathione metabolism depending on the ERK-Nrf2-HO-1 signal pathway to repair phosphoramide mustard-induced ovarian cancer cells.

The aim of this study was to study the effects of human umbilical cord mesenchymal stem cells (HUC-MSCs) on glutathione (GSH) metabolism in human ovarian cancer cells induced by phosphoramide mustard (PM). The experiment was divided into five groups, namely, the blank group (ovarian cancer cells), the control group (ovarian cancer cells + HUC-MSCs), the model group (ovarian cancer cells + PM), the treatment group (ovarian cancer cells + PM + HUC-MSCs), and the inhibitor group (ovarian cancer cells + PM + HUC-MSCs + extracellular signal-regulated protein kinase inhibitor PD98059). The apoptosis rate of ovarian cancer cells was detected by flow cytometry. Intracellular levels of oxidized glutathione (GSSG), GSH, γ-glutamyl cysteine synthetase (γ-GCS), and intracellular reactive oxygen species (ROS) were detected by enzyme-linked immunosorbent assay. Protein imprinting and real-time fluorescence quantitative PCR were used to detect extracellular regulated protein kinase (ERK), p-ERK heme oxygenase-1 (HO-1), and nuclear factor E2-related factor 2 (Nrf2) protein levels. First, the apoptosis rate in the model group was increased compared with that of the blank group. The levels of γ-GCS, p-ERK, HO-1, and Nrf-2 decreased, while the levels of malondialdehyde, GSSG, and ROS increased. Second, compared with the model group, the apoptosis rate in the treatment group decreased. GSH, γ-GCS, p-ERK, HO-1, and Nrf2 levels increased. Malondialdehyde, GSSG, and ROS levels decreased. Third, after the administration of ERK inhibitor, the apoptosis rate of cells increased. GSH, p-ERK, and HO-1 levels decreased. GSSG and ROS levels increased (P < 0.05), and γ-GCS level had a downward trend compared with the treatment group. To conclude, HUC-MSCs may regulate the ERK-Nrf2-HO-1 pathway to increase γ-GCS expression and GSH production, reduce ROS level and apoptosis of ovarian cancer cells, and improve antioxidant capacity.

Methylglyoxal metabolism is altered during defence response in pigeonpea (Cajanus cajan (L.) Millsp.) against the spotted pod borer (Maruca vitrata).

Pigeonpea (Cajanus cajan ) production can be affected by the spotted pod borer (Maruca vitrata ). Here, we identified biochemical changes in plant parts of pigeonpea after M. vitrata infestation. Two pigeonpea genotypes (AL 1747, moderately resistant; and MN 1, susceptible) were compared for glyoxalase and non-glyoxalase enzyme systems responsible for methylglyoxal (MG) detoxification, γ-glutamylcysteine synthetase (γ-GCS), glutathione-S-transferase (GST) and glutathione content in leaves, flowers and pods under control and insect-infested conditions. MN 1 had major damage due to M. vitrata infestation compared to AL 1747. Lower accumulation of MG in AL 1747 was due to higher activities of enzymes of GSH-dependent (glyoxylase I, glyoxylase II), GSH-independent (glyoxalase III) pathway, and enzyme of non-glyoxalase pathway (methylglyoxal reductase, MGR), which convert MG to lactate. Decreased glyoxylase enzymes and MGR activities in MN 1 resulted in higher accumulation of MG. Higher lactate dehydrogenase (LDH) activity in AL 1747 indicates utilisation of MG detoxification pathway. Higher glutathione content in AL 1747 genotype might be responsible for efficient working of MG detoxification pathway under insect infestation. Higher activity of γ-GCS in AL 1747 maintains the glutathione pool, necessary for the functioning of glyoxylase pathway to carry out the detoxification of MG. Higher activities of GST and GPX in AL 1747 might be responsible for detoxification of toxic products that accumulates following insect infestation, and elevated activities of glyoxylase and non-glyoxylase enzyme systems in AL 1747 after infestation might be responsible for reducing reactive cabanoyl stress. Our investigation will help the future development of resistant cultivars.

A ROS storm generating nanocomposite for enhanced chemodynamic therapy through H2O2 self-supply, GSH depletion and calcium overload.

Catalytic generation of toxic hydroxyl radicals (˙OH) from hydrogen peroxide (H2O2) is an effective strategy for tumor treatment in chemodynamic therapy (CDT). However, the intrinsic features of the microenvironment in solid tumors, characterized by limited H2O2 and overexpressed glutathione (GSH), severely impede the accumulation of intracellular ˙OH, posing significant challenges. To circumvent these critical issues, in this work, a CaO2-based multifunctional nanocomposite with a surface coating of Cu2+ and L-buthionine sulfoximine (BSO) (named CaO2@Cu-BSO) is designed for enhanced CDT. Taking advantage of the weakly acidic environment of the tumor, the nanocomposite gradually disintegrates, and the exposed CaO2 nanoparticles subsequently decompose to produce H2O2, alleviating the insufficient supply of endogenous H2O2 in the tumor microenvironment (TME). Furthermore, Cu2+ detached from the surface of CaO2 is reduced by H2O2 and GSH to Cu+ and ROS. Then, Cu+ catalyzes H2O2 to generate highly cytotoxic ˙OH and Cu2+, forming a cyclic catalysis effect for effective CDT. Meanwhile, GSH is depleted by Cu2+ ions to eliminate possible ˙OH scavenging. In addition, the decomposition of CaO2 by TME releases a large amount of free Ca2+, resulting in the accumulation and overload of Ca2+ and mitochondrial damage in tumor cells, further improving CDT efficacy and accelerating tumor apoptosis. Besides, BSO, a molecular inhibitor, decreases GSH production by blocking γ-glutamyl cysteine synthetase. Together, this strategy allows for enhanced CDT efficiency via a ROS storm generation strategy in tumor therapy. The experimental results confirm and demonstrate the satisfactory tumor inhibition effect both in vitro and in vivo.

Comprehensive Analysis of the Relationship between Cuproptosis-Related Gene GCSH and Prognosis, Tumor Microenvironment Infiltration, and Therapy Response in Endometrial Cancer

Introduction: Cuproptosis is a novel form of cell death regulated by protein lipoylation and implicated in mitochondrial metabolism. However, further research is needed to explore the influence of the cuproptosis-related gene γ-glutamylcysteine synthetase (GCSH) on the prognosis of endometrial cancer (EC), the tumor immune microenvironment, and therapeutic response. Methods: The differential expression of GCSH between EC and normal tissues was analyzed using multiple public databases. Additionally, cancer and adjacent tissues were prospectively collected from 17 EC patients, and immunohistochemical analysis was performed to further investigate GCSH expression differences. The relationship between GCSH and the prognosis and clinicopathological characteristics of EC patients was evaluated, and a nomogram was constructed to predict patient survival based on GCSH expression. Subsequently, Gene set variation analysis was used to explore the potential biological functions of GCSH in EC. The impact of GCSH on the tumor microenvironment (TME) was estimated. Finally, the effect of GCSH on the response to immunotherapy and chemotherapeutic drugs in EC was investigated. Results: The expression of GCSH was significantly upregulated in EC. High GCSH expression was associated with poor prognosis in EC patients. Enrichment analysis revealed an association between high GCSH and immune suppression. Furthermore, high GCSH was found to be associated with a non-inflamed TME, leading to decreased infiltration levels of immune cells. Lastly, it was observed that patients with high GCSH exhibited insensitivity to both immunotherapy and chemotherapeutic drugs. Conclusion: This study revealed the role of GCSH in TME, response to therapy, and clinical prognosis in EC, which provided novel insights for the therapeutic application in EC.

Nitric oxide and AMF-mediated regulation of soil enzymes activities, cysteine-H2S system and thiol metabolites in mitigating chromium (Cr (VI)) toxicity in pigeonpea genotypes.

Cr (VI) hampers plant growth and yield by reducing essential nutrient uptake as it competes for phosphate and sulfate transporters. Nitric oxide (NO) and mycorrhization play important roles in mitigating Cr (VI) toxicity. Present study aimed to compare the potential of AMF (Arbuscular mycorrhizal fungi)-Rhizoglomus intraradices and NO (0.25mM) in alleviating Cr (VI) stress (0, 10 and 20mg/kg) in two differentially tolerant pigeonpea genotypes (Pusa 2001 and AL 201). Cr (VI) toxicity reduced growth, mycorrhizal colonization, nutrient uptake, and overall productivity by inducing reactive oxygen species (ROS) generation, with AL 201 more sensitive than Pusa 2001. NO and AM enhanced activities of soil enzymes, thereby increasing nutrients availability as well as their uptake, with AM more effective than NO. Both amendments reduced oxidative stress and restricted Cr (VI) uptake by increasing the activities of antioxidant and S- assimilatory enzymes, with Pusa 2001 more responsive than AL 201. NO was relatively more efficient in regulating cysteine-H2S system by increasing the activities of biosynthetic enzymes (ATP-sulfurylase (ATPS), O-acetylserine thiol lyase (OASTL), D-cysteine desulfhydrase (DCD) and L-cysteine desulfhydrase (LCD), while AM significantly increased glutathione reductase (GR), γ-glutamylcysteine synthetase (γ-ECS) enzymes activities and resultant glutathione (GSH), phytochelatins (PCs), and non-protein thiols (NP-SH) synthesis. Moreover, co-application of NO and AM proved to be highly beneficial in negating the toxic effects of Cr (VI) due to functional complementarity between them. Study suggested the combined use of NO and AM as a useful strategy in re-establishing pigeonpea plants growing in Cr (VI)-stressed environments.

Extracellular Production of Glutathione by Recombinant Escherichia coli K-12

The goal of this study was to produce a sufficient amount of glutathione in the fermentation medium without the addition of cysteine. This would simplify and reduce the cost of its purification. In addition to reducing the cost of cysteine, it also avoids the inhibition of bacterial growth by cysteine. The gshA, gshB, and cysE genes of Escherichia coli were cloned under the control of the strong T5 promoter of the pQE-80L plasmid and introduced into an E. coli strain knocked out for the genes encoding γ-glutamyltranspeptidase and the GsiABCD glutathione transporter, which are responsible for the recycling of excreted glutathione. The overexpression of the gshA and gshB genes, genes for γ-glutamylcysteine synthetase and glutathione synthetase, and the cysEV95R D96P gene, a gene for serine acetyltransferase with the V95R D96P mutation that makes it insensitive to cysteine, were effective on glutathione production. Na2S2O3 was a good sulfur source for glutathione production, while the addition of Na2SO4 did not affect the glutathione production. With the addition of 50 mM glutamic acid and 75 mM glycine, but without the addition of cysteine, to the simplified SM1 medium, 4.6 mM and 0.56 mM of the reduced and oxidized glutathione, respectively, were accumulated in the extracellular space after 36 h of batch culture. This can eliminate the need to extract glutathione from the bacterial cells for purification.

Proteomic Shift in Mouse Embryonic Fibroblasts Pfa1 during Erastin, ML210, and BSO-Induced Ferroptosis

Ferroptosis is a unique variety of non-apoptotic cell death, driven by massive lipid oxidation in an iron-dependent manner. Since ferroptosis was introduced as a concept in 2012, it has demonstrated its essential role in the pathogenesis in neurodegenerative diseases and an important role in therapy-resistant cancer cells. Thus, detailed molecular understanding of both canonical and alternative ferroptosis pathways is required. There is a set of widely used chemical agents to modulate ferroptosis using different pathway targets: erastin blocks cystine–glutamate antiporter, system xc-; ML210 directly inactivates GPX4; and L-buthionine sulfoximine (BSO) inhibits γ-glutamylcysteine synthetase, an essential enzyme for glutathione synthesis de novo. Most studies have focused on the lipidomic profiling of model systems undergoing death in a ferroptotic modality. In this study, we developed high-quality shotgun proteome sequencing during ferroptosis induction by three widely used chemical agents (erastin, ML210, and BSO) before and after 24 and 48 h of treatment. Chromato-mass spectra were registered in DDA mode and are suitable for further label-free quantification. Both processed and raw files are publicly available and could be a valuable dynamic proteome map for further ferroptosis investigation.

Astaxanthin inhibits oxidative stress and apoptosis in diabetic retinopathy

BackgroundThe pathophysiology of diabetic retinopathy (DR) is thought to be influenced by oxidative stress. Astaxanthin (ASX) is a natural product with antioxidant effect, but it is not clear whether its mechanism of inhibiting the development of DR is related to anti-oxidation. MethodsRats were intraperitoneally injected with streptozotocin (60 mg/kg) to create DR rat models followed by ASX (20 mg/kg) for 45 days. Retinal tissue was examined by Hematoxylin and Eosin staining. By using Enzyme-linked immunosorbent assay (ELISA), 2,7-Dichlorodrhydrofluorescein diace (DCFH-DA) probes, immunohistochemistry and western blot, it was feasible to evaluate the contents of inflammation-related factors (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and macrophage inhibitory cytokine-1 (MIC-1)), oxidative stress-related indicators (glutathione (GSH), malonic dialdehyde (MDA), glutathione peroxidase (GPx), reactive oxygen species (ROS) and Total antioxidant capacity (T-AOC)), antioxidant enzymes (hemoxgenase-1(HO-1) and Quinone Oxidoreductase 1 (NQO1)), and apoptosis-related proteins (Bcl-2, Bcl2 Associated X Protein (BAX), and cleaved-caspase-3). Additionally, antioxidant proteins downstream of the nuclear factor E2 related factors (Nrf-2) pathway, expression levels of Nrf2/ Kelch-like ECH-associated protein 1(Keap 1) pathway-associated proteins, and nuclear and cytoplasmic levels of Nrf2 were assessed using immunohistochemistry, western blot, or quantitative real-time polymerase chain reaction (qRT-PCR). ResultsASX alleviated retinal tissue damage by increasing overall retina thickness and ganglion cell layer (GCL) cell numbers and exerted the anti-inflammatory, anti-oxidative stress, and anti-apoptosis effects in DR rats. Additionally, ASX could inhibit the expression of Keap1, promote the transport of Nrf2 from cytoplasm to nucleus and facilitate the expressions of HO-1, NQO1, γ-glutamylcysteine synthetase, (γ-GCS) and GPx. ConclusionASX exerted antioxidant effects through Nrf2/keap1 pathway, thereby alleviating apoptosis, inflammation, and oxidative stress in retinal tissues of DR rats.

Promising role of selenium in mitigating the negative effects of iron deficiency in soybean leaves

Little is known about the role of selenium (Se) in modulating the nutritional status and the non-enzymatic and enzymatic antioxidant defense system in iron(Fe)-deficient soybean. Hence, this work aimed to evaluate the possible beneficial effect of Se applied in the nutrient solution on photosynthetic pigments, nutritional status, reactive oxygen species accumulation, ascorbate and glutathione metabolites, activity of enzymes involved in the synthesis pathway of these metabolites, and antioxidant defense enzymes, including expression of superoxide dismutase (SOD) isoforms, after exposure to absence, deficiency, and sufficiency of Fe in combination with absence and presence of Se. The experimental design was completely randomized with 5 replications, containing 4 plants per pot. In this work, through physiological, biochemical, and molecular analyses, we observed a beneficial effect of Se in soybean plants with Fe suppression, mainly in the absence of Fe. Thus, in the absence of Fe with Se, there was higher remobilization of Fe and maintenance of foliar N concentration, which resulted in mitigation of leaf chlorosis and decreased loss of photosynthetic pigments. Additionally, under this cultivation condition, the main positive effect of Se was shown to be associated with the modulation of the glutathione cycle, with increases in the reduced glutathione and total glutathione content, reduced glutathione/oxidized glutathione ratio, and in the activity of the γ-glutamylcysteine synthetase, glutathione peroxidase, and glutathione reductase enzymes. The Se supply also reduced the need to replace SOD-Fe with SOD-Cu due to the positive modulation in the crosstalk between Fe and Cu, as well as attenuated the nutritional imbalance of Zn and Mn, mainly in the absence of Fe. These adjustments prevented lipid peroxidation and showed the beneficial role of Se in Fe-depleted plants.