Subunit Of Glutamate-cysteine Ligase Research Articles

SNORA56-mediated pseudouridylation of 28 S rRNA inhibits ferroptosis and promotes colorectal cancer proliferation by enhancing GCLC translation

BackgroundColorectal cancer (CRC) is one of the most common malignancies and is characterized by reprogrammed metabolism. Ferroptosis, a programmed cell death dependent on iron, has emerged as a promising strategy for CRC treatment. Although small nucleolar RNAs are extensively involved in carcinogenesis, it is unclear if they regulate ferroptosis during CRC pathogenesis.MethodsThe dysregulated snoRNAs were identified using published sequencing data of CRC tissues. The expression of the candidate snoRNAs, host gene and target gene were assessed by real-time quantitative PCR (RT-qPCR), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and western blots. The biological function of critical molecules was investigated using in vitro and in vivo strategies including Cell Counting Kit-8 (CCK8), colony formation assay, flow cytometry, Fe2+/Fe3+, GSH/GSSG and the xenograft mice models. The ribosomal activities were determined by polysome profiling and O-propargyl-puromycin (OP-Puro) assay. The proteomics was conducted to clarify the downstream targets and the underlying mechanisms were validated by IHC, Pearson correlation analysis, protein stability and rescue assays. The clinical significance of the snoRNA was explored using the Cox proportional hazard model, receiver operating characteristic (ROC) and survival analysis.ResultsHere, we investigated the SNORA56, which was elevated in CRC tissues and plasma, and correlated with CRC prognosis. SNORA56 deficiency in CRC impaired proliferation and triggered ferroptosis, resulting in reduced tumorigenesis. Mechanistically, SNORA56 mediated the pseudouridylation of 28 S rRNA at the U1664 site and promoted the translation of the catalytic subunit of glutamate cysteine ligase (GCLC), an indispensable rate-limiting enzyme in the biosynthesis of glutathione, which can inhibit ferroptosis by suppressing lipid peroxidation.ConclusionsTherefore, the SNORA56/28S rRNA/GCLC axis stimulates CRC progression by inhibiting the accumulation of cellular peroxides, and it may provide biomarker and therapeutic applications in CRC.

Covalent Targeting of Glutamate Cysteine Ligase to Inhibit Glutathione Synthesis.

Dysregulated oxidative stress plays a major role in cancer pathogenesis and some types of cancer cells are particularly vulnerable to inhibition of their cellular antioxidant capacity. Glutamate-cysteine ligase (GCL) is the first and rate-limiting step in the synthesis of the major cellular antioxidant glutathione (GSH). Developing a GCL inhibitor may be an attractive therapeutic strategy for certain cancer types that are particularly sensitive to oxidative stress. In this study, we reveal a cysteine-reactive ligand, EN25, that covalently targets an allosteric cysteine C114 on GCLM, the modifier subunit of GCL, and leads to inhibition of GCL activity. This interaction also leads to reduced cellular GSH levels and impaired cell viability in ARID1A-deficient cancer cells, which are particularly vulnerable to glutathione depletion, but not in ARID1A-positive cancer cells. Our studies uncover a novel potential ligandable site within GCLM that can be targeted to inhibit GSH synthesis in vulnerable cancer cell types.

Polymorphisms of the GCLC Gene Are Novel Genetic Markers for Susceptibility to Psoriasis Associated with Alcohol Abuse and Cigarette Smoking.

The aim of this pilot study was to investigate whether single nucleotide polymorphisms (SNP) in the gene encoding the catalytic subunit of glutamate cysteine ligase (GCLC) are associated with the risk and clinical features of psoriasis. A total of 944 unrelated individuals, including 474 patients with a diagnosis of psoriasis and 470 healthy controls, were recruited for the study. Six common SNPs in the GCLC gene were genotyped using the MassArray-4 system. Polymorphisms rs648595 (OR = 0.56, 95% CI 0.35-0.90; Pperm = 0.017) and rs2397147 (OR = 0.54, 95% CI 0.30-0.98; Pperm = 0.05) were associated with susceptibility to psoriasis in males. In the male group, diplotype rs2397147-C/C × rs17883901-G/G was associated with a decreased risk of psoriasis (FDR-adjusted p = 0.014), whereas diplotype rs6933870-G/G × rs17883901-G/G (FDR-adjusted p = 0.045) showed an association with an increased disease risk in females. The joint effects of SNPs with tobacco smoking (rs648595 and rs17883901) and alcohol abuse (rs648595 and rs542914) on psoriasis risk were observed (Pperm ≤ 0.05). We also found multiple sex-independent associations between GCLC gene polymorphisms and various clinical features such as earlier disease onset, the psoriatic triad, and specific localizations of skin lesions. The present study is the first to show that polymorphisms of the GCLC gene are significantly associated with the risk of psoriasis and related to its clinical features.

EGR1 is crucial for the chlorogenic acid-provided promotion on liver regeneration and repair after APAP-induced liver injury.

Improper use of acetaminophen (APAP) will induce acute liver failure. This study is designed to investigate whether early growth response-1 (EGR1) participated in the promotion on liver repair and regeneration after APAP-induced hepatotoxicity provided by natural compound chlorogenic acid (CGA). APAP induced the nuclear accumulation of EGR1 in hepatocytes regulated by extracellular-regulated protein kinase (ERK)1/2. In Egr1 knockout (KO) mice, the liver damage caused by APAP (300 mg/kg) was more severe than in wild-type (WT) mice. Results of chromatin immunoprecipitation and sequencing (ChIP-Seq) manifested that EGR1 could bind to the promoter region in Becn1, Ccnd1, and Sqstm1 (p62) or the catalytic/modify subunit of glutamate-cysteine ligase (Gclc/Gclm). Autophagy formation and APAP-cysteine adduct (APAP-CYS) clearance were decreased in Egr1 KO mice administered with APAP. The EGR1 deletion reduced hepatic cyclin D1 expression at 6, 12, or 18 h post APAP administration. Meanwhile, the EGR1 deletion also decreased hepatic p62, Gclc and Gclm expression, GCL enzymatic activity, and glutathione (GSH) content and decreased nuclear factor erythroid 2-related factor 2 (Nrf2) activation and thus aggravated oxidative liver injury induced by APAP. CGA increased EGR1 nuclear accumulation; enhanced hepatic Ccnd1, p62, Gclc, and Gclm expression; and accelerated the liver regeneration and repair in APAP-intoxicated mice. In conclusion, EGR1 deficiency aggravated liver injury and obviously delayed liver regeneration post APAP-induced hepatotoxicity through inhibiting autophagy, enhancing liver oxidative injury, and retarding cell cycle progression, but CGA promoted the liver regeneration and repair in APAP-intoxicated mice via inducing EGR1 transcriptional activation.

The Impact of Gclm on Efferocytosis and Atherosclerotic Progression

Atherosclerosis is the leading cause of coronary artery disease (CAD) and stroke which are the primary contributors to cardiovascular disease related deaths in the world. This is an inflammatory disease characterized by the accumulation of lipid and fibrous elements in large arteries resulting in the development of a plaque. Progression of the disease leads to the accumulation of apoptotic cells and formation of a necrotic core making the plaque unstable and prone to rupture. Rupture can lead to artery occlusion resulting in stroke or myocardial infarction (MI). One of the main contributors to a large necrotic core is macrophages that are unable to clear the accumulated apoptotic cells and as a result undergo secondary necrosis. This clearance process, referred to as efferocytosis, is impaired in advanced atherosclerotic lesions. It has been shown that the process that leads to a thin fibrous cap (characteristic of an unstable plaque) and a large necrotic core are accelerated by a decrease in glutathione (GSH) availability due to a deletion on the modifier subunit of Glutamate Cysteine Ligase (GCLM). The objective of this study is to identify the role and mechanism of GSH during the formation of the necrotic core in atherosclerosis. We hypothesize that availability of GSH is important in macrophages for adequate efferocytosis of apoptotic cells during atherosclerosis progression.MethodsTriglycerides, total cholesterol, and HDL levels were measured on PCSK9 virus driven atherosclerosis in wild type and heterozygous GCLM mice. Sections of the aortic root from these mice were stained with Movat Pentachrome to measure plaque area and percent necrosis. An efferocytic index was measured using PHK26 labelled apoptotic cells (AC). AC were added to cultured bone marrow derived macrophages (BMDM) allowing for their uptake. Percent positive macrophages containing a fluorescently labelled AC were quantified. BMDM from WT, Het, and KO GCLM mice were collected for total protein, surface receptor expression, and mRNA analysis.ResultsEfferocytosis was affected in KO mice as compared to WT and Het BMDM. RNA analysis has showed a decrease in efferocytosis receptors such as MerTK, confirmed by cell surface expression.ConclusionThese results suggest that a loss of GCLM and GSH during efferocytosis impairs adequate efferocytosis.

Abstract 453: Investigating The Role Of Glutathione During Macrophage Apoptotic Cell Clearance

Atherosclerosis (ASVD) is characterized by the buildup of lipids and fibrous elements in large arteries creating a plaque. As it progresses, apoptotic cells (AC) accumulate forming a necrotic core making the plaque unstable and prone to rupture. Rupture can lead to artery occlusion and moreover to stroke or myocardial infarction (MI). A main contributor to a large necrotic core is the inability of macrophages to clear accumulated AC and as a result undergo secondary necrosis. This clearance process, referred to as efferocytosis, is impaired in advanced atherosclerotic lesions. It has been shown that the process that leads to a thin fibrous cap and a large necrotic core are accelerated by a decrease in glutathione (GSH) availability by a deletion of the modifier subunit of Glutamate Cysteine Ligase (GCLM) in mice. Additionally, it has also been shown that a single nucleotide polymorphism (SNP) in the promoter region of human GCLM (-588 C/T) has an increased risk for MI. Our objective is to identify the role and mechanism of the GCLM SNP during the formation of the necrotic core in atherosclerosis. We hypothesize that proper function of GCLM and GSH is important in macrophages for adequate function and clearance of AC in ASVD. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from blood donors, genotyped to test for the presence of SNP, and used for RNA sequencing. Efferocytic index was measured using labelled AC in GCLM WT, Het, and KO murine bone marrow derived macrophages (BMDM) and PBMCs. Percent positive macrophages containing fluorescently labelled AC were quantified for each sample. Cells were collected for total protein, surface receptor expression, and mRNA analysis. Results: Efferocytosis was affected in macrophages with defective GCLM as compared to control. RNA analysis has showed a decrease in efferocytosis receptors such as MerTK, confirmed by cell surface expression. Conclusion: These results suggest that loss of GCLM and GSH in macrophages impairs adequate efferocytosis.

The Impact of Genetic Polymorphisms in Glutamate-Cysteine Ligase, a Key Enzyme of Glutathione Biosynthesis, on Ischemic Stroke Risk and Brain Infarct Size.

The purpose of this pilot study was to explore whether polymorphisms in genes encoding the catalytic (GCLC) and modifier (GCLM) subunits of glutamate-cysteine ligase, a rate-limiting enzyme in glutathione synthesis, play a role in the development of ischemic stroke (IS) and the extent of brain damage. A total of 1288 unrelated Russians, including 600 IS patients and 688 age- and sex-matched healthy subjects, were enrolled for the study. Nine common single nucleotide polymorphisms (SNPs) of the GCLC and GCLM genes were genotyped using the MassArray-4 system. SNP rs2301022 of GCLM was strongly associated with a decreased risk of ischemic stroke regardless of sex and age (OR = 0.39, 95%CI 0.24–0.62, p < 0.0001). Two common haplotypes of GCLM possessed protective effects against ischemic stroke risk (p < 0.01), but exclusively in nonsmoker patients. Infarct size was increased by polymorphisms rs636933 and rs761142 of GCLC. The mbmdr method enabled identifying epistatic interactions of GCLC and GCLM gene polymorphisms with known IS susceptibility genes that, along with environmental risk factors, jointly contribute to the disease risk and brain infarct size. Understanding the impact of genes and environmental factors on glutathione metabolism will allow the development of effective strategies for the treatment of ischemic stroke and disease prevention.

Genetic variation at the catalytic subunit of glutamate cysteine ligase contributes to the susceptibility to sporadic colorectal cancer: a pilot study.

Glutathione is a tripeptide detoxifying a variety of exogenous and endogenous free radicals and carcinogens, and a deficiency of glutathione is associated with an increased host susceptibility to oxidative stress, a pathological condition implicated in the development and progression of cancer. The catalytic subunit of glutamate-cysteine ligase (GCLC) is an enzyme responsible for the initial and rate-limiting step of glutathione biosynthesis. The aim of this pilot study was to investigate whether genetic variation at the GCLC gene contributes to the risk of colorectal cancer (CRC). DNA samples from 681 unrelated Russian individuals (283 patients with CRC and 398 age- and sex-matched healthy controls) were genotyped for six common functional SNPs of the GCLC gene (SNPs) such as rs12524494, rs17883901, rs606548, rs636933, rs648595 and rs761142 of the GCLC gene using the MassARRAY-4 system. We found that genotype rs606548-C/T is significantly associated with increased risk of CRC regardless of sex and age (OR 2.24; 95% CI 1.24-4.03; P = 0.007, FDR = 0.04). Moreover, ten GCLC genotype combinations showed association with the risk of CRC (P < 0.05). Functional SNP annotation enabled establishing the CRC-associated polymorphisms are associated with a decreased GCLC expression that may be attributed to epigenetic effects of histone modifications operating in a colon-specific manner. The present study was the first to show that genetic variation at the catalytic subunit of glutamate-cysteine ligase may contribute to the risk of colorectal cancer risk. However, further genetic association studies with a larger sample size are required to substantiate the role of GCLC gene polymorphisms in the development of sporadic colorectal cancer.

Glutathione-dependent redox balance characterizes the distinct metabolic properties of follicular and marginal zone B cells

The metabolic principles underlying the differences between follicular and marginal zone B cells (FoB and MZB, respectively) are not well understood. Here we show, by studying mice with B cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that glutathione synthesis affects homeostasis and differentiation of MZB to a larger extent than FoB, while glutathione-dependent redox control contributes to the metabolic dependencies of FoB. Specifically, Gclc ablation in FoB induces metabolic features of wild-type MZB such as increased ATP levels, glucose metabolism, mTOR activation, and protein synthesis. Furthermore, Gclc-deficient FoB have a block in the mitochondrial electron transport chain (ETC) due to diminished complex I and II activity and thereby accumulate the tricarboxylic acid cycle metabolite succinate. Finally, Gclc deficiency hampers FoB activation and antibody responses in vitro and in vivo, and induces susceptibility to viral infections. Our results thus suggest that Gclc is required to ensure the development of MZB, the mitochondrial ETC integrity in FoB, and the efficacy of antiviral humoral immunity.

GSH and Zinc Supplementation Attenuate Cadmium-Induced Cellular Stress and Stimulation of Choline Uptake in Cultured Neonatal Rat Choroid Plexus Epithelia.

Choroid plexus (CP) sequesters cadmium and other metals, protecting the brain from these neurotoxins. These metals can induce cellular stress and modulate homeostatic functions of CP, such as solute transport. We previously showed in primary cultured neonatal rat CP epithelial cells (CPECs) that cadmium induced cellular stress and stimulated choline uptake at the apical membrane, which interfaces with cerebrospinal fluid in situ. Here, in CPECs, we characterized the roles of glutathione (GSH) and Zinc supplementation in the adaptive stress response to cadmium. Cadmium increased GSH and decreased the reduced GSH-to-oxidized GSH (GSSG) ratio. Heat shock protein-70 (Hsp70), heme oxygenase (HO-1), and metallothionein (Mt-1) were induced along with the catalytic and modifier subunits of glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis. Inhibition of GCL by l-buthionine sulfoximine (BSO) enhanced stress protein induction and stimulation of choline uptake by cadmium. Zinc alone did not induce Hsp70, HO-1, or GCL subunits, or modulate choline uptake. Zinc supplementation during cadmium exposure attenuated stress protein induction and stimulation of choline uptake; this effect persisted despite inhibition of GSH synthesis. These data indicated up-regulation of GSH synthesis promotes adaptation to cadmium-induced cellular stress in CP, but Zinc may confer cytoprotection independent of GSH.

Compromised glutathione synthesis results in high susceptibility to acetaminophen hepatotoxicity in acatalasemic mice

Acatalasemia is caused by genetic defect in the catalase gene. Human achatalasemia patients are able to scavenge physiological hydrogen peroxide but are vulnerable to exogenous oxidative stress. In the present study, we used an acetaminophen-induced hepatotoxicity model in acatalasemic mice to explore this vulnerability. Interestingly, the acetaminophen-induced decrease in total glutathione levels was more prolonged in acatalasemic mice. While the subunits of glutamate-cysteine ligase, a glutathione synthase enzyme, were increased by acetaminophen in the liver of wild-type mice, their expression was lower and was further reduced by acetaminophen in acatalasemic mice. This feature was also observed in immortalized hepatocytes derived from the livers of these mice. However, when catalase was knocked down in HepG2 cells, a cultured human liver cell line, the expression of glutamate-cysteine ligase subunits was increased, suggesting that the low expression of glutamate-cysteine ligase subunits in acatalasemia may be due to other mechanism than catalase deficiency. Therefore, when other factors were investigated, it was found that transforming growth factor-β1 was up-regulated by acetaminophen in the liver of acatalasemic mice, which may inhibit the expression of glutamate-cysteine ligase subunits. The results of this study suggest a new toxic mechanism of acetaminophen-induced liver injury in patients with acatalasemia.

Melatonin Modulates the Antioxidant Defenses and the Expression of Proinflammatory Mediators in Pancreatic Stellate Cells Subjected to Hypoxia.

Pancreatic stellate cells (PSC) play a major role in the formation of fibrotic tissue in pancreatic tumors. On its side, melatonin is a putative therapeutic agent for pancreatic cancer and inflammation. In this work, the actions of melatonin on PSC subjected to hypoxia were evaluated. Reactive oxygen species (ROS) generation reduced (GSH) and oxidized (GSSG) levels of glutathione, and protein and lipid oxidation were analyzed. The phosphorylation of nuclear factor erythroid 2-related factor (Nrf2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), and the regulatory protein nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (IκBα) was studied. The expression of Nrf2-regulated antioxidant enzymes, superoxide dismutase (SOD) enzymes, cyclooxygenase 2 (COX-2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also studied. Total antioxidant capacity (TAC) was assayed. Finally, cell viability was studied. Under hypoxia and in the presence of melatonin generation of ROS was observed. No increases in the oxidation of proteins or lipids were detected. The phosphorylation of Nrf2 and the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1, heme oxygenase-1, SOD1, and of SOD2 were augmented. The TAC was increased. Protein kinase C was involved in the effects of melatonin. Melatonin decreased the GSH/GSSG ratio at the highest concentration tested. Cell viability dropped in the presence of melatonin. Finally, melatonin diminished the phosphorylation of NF-kB and the expression of COX-2, IL-6, and TNF-α. Our results indicate that melatonin, at pharmacological concentrations, modulates the red-ox state, viability, and the expression of proinflammatory mediators in PSC subjected to hypoxia.

Tolerable upper intake level of iron damages the liver of weaned piglets.

Iron is one of the essential trace elements, which is often supplemented as an additive to meet the growing needs of toddlers and young animals. Recommended nutrient intake (RNI) and tolerable upper intake levels (UL) are always set when the iron is supplemented. The purpose of this study was to evaluate the subacute (28days) toxicity of UL iron to weaned piglet liver. Thirty 23-day-old weaned piglets were divided into three groups and, respectively, supplemented with 100, 300 or 3000 (UL) mg/kg iron. UL iron caused significant weight loss in 4th week (p<0.05). Divalent metal transporter 1(DMT1) decreased significantly, ferroportin 1 and ferritin increased significantly in the liver of UL iron group (p<0.05). Although there was no significant effect on liver morphology, UL iron significantly increased hepatic iron, reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (p<0.05). UL iron significantly reduced glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and total anti-oxidation capacity (T-AOC) in the liver (p<0.05). Nuclear factor erythroid 2-related factor 2 (Nrf2) activated subunits of glutamate cysteine ligase (Gclc) and glutathione S-transferase A1 (Gsta1) upregulation in the UL iron group liver, thereby increasing resistance to oxidative stress. In conclusion, UL iron supplementation altered iron metabolism, generated free radicals, reduced antioxidant enzyme activity and activated Nrf2 signalling pathway in the weaned piglet liver.

Pre-treatment twice with liposomal clodronate protects against acetaminophen hepatotoxicity through a pre-conditioning effect

Background and aimAcetaminophen (APAP) overdose is a major cause of acute liver injury, but the role of macrophages in the propagation of the hepatotoxicity is controversial. Early research revealed that macrophage inhibitors protect against APAP injury. However, later work demonstrated that macrophage ablation by acute pre-treatment with liposomal clodronate (LC) exacerbates the toxicity. To our surprise, during other studies, we observed that pre-treatment twice with LC seemed to protect against APAP hepatotoxicity, in contrast to acute pre-treatment. The aim of this study was to confirm that observation and to explore the mechanisms. MethodsWe treated mice with empty liposomes (LE) or LC twice per week for 1 week before APAP overdose and collected blood and liver tissue at 0, 2, and 6 h post-APAP. We then measured liver injury (serum alanine aminotransferase activity, histology), APAP bioactivation (total glutathione, APAP-protein adducts), oxidative stress (oxidized glutathione (GSSG)), glutamate-cysteine ligase subunit c (Gclc) mRNA, and nuclear factor erythroid 2-related factor (Nrf2) immunofluorescence. We also confirmed the ablation of macrophages by F4/80 immunohistochemistry. ResultsPre-treatment twice with LC dramatically reduced F4/80 staining, protected against liver injury, and reduced oxidative stress at 6 h post-APAP, without affecting APAP bioactivation. Importantly, Gclc mRNA was higher in the LC group at 0 h and total glutathione was higher at 2 h, indicating accelerated glutathione re-synthesis after APAP overdose due to greater basal glutamate-cysteine ligase. Oxidative stress was lower in the LC groups at both time points. Finally, total Nrf2 immunofluorescence was higher in the LC group. ConclusionsWe conclude that multiple pre-treatments with LC protect against APAP by accelerating glutathione re-synthesis through glutamate-cysteine ligase. Investigators using twice or possibly more LC pre-treatments to deplete macrophages, including peritoneal macrophages, should be aware of this possible confounder.

Genetic variants in glutamate cysteine ligase confer protection against type 2 diabetes.

Oxidative stress contributes to the pathogenesis of type 2 diabetes (T2D). This study investigated whether single nucleotide polymorphisms (SNPs) at genes encoding glutamate cysteine ligase catalytic (rs12524494, rs17883901, rs606548, rs636933, rs648595, rs761142 at GCLC) and modifier (rs2301022, rs3827715, rs7517826, rs41303970 at GCLM) subunits are associated with susceptibility to type 2 diabetes. 2096 unrelated Russian subjects were enrolled for the study. Genotyping was done with the use of the MassArray System. Plasma levels of reactive oxygen species (ROS) and glutathione in the study subjects were analyzed by fluorometric and colorimetric assays, respectively.The present study found, for the first time, an association of SNP rs41303970 in the GCLM gene with a decreased risk of T2D (P = 0.034, Q = 0.17). Minor alleles such as rs12524494-G GCLC gene (P = 0.026, Q = 0.17) and rs3827715-C GCLM gene (P = 0.03, Q = 0.17) were also associated with reduced risk for T2D. Protective effects of variant alleles such as rs12524494-G at GCLC (P = 0.02, Q = 0.26) and rs41303970-A GCLM (P = 0.013, Q = 0.25) against the risk of T2D were seen solely in nonsmokers. As compared with healthy controls, diabetic patients had markedly increased levels of ROS and decreased levels of total GSH in plasma. Interestingly, fasting blood glucose level positively correlated with oxidized glutathione concentration (rs = 0.208, P = 0.01). Three SNPs rs17883901, rs636933, rs648595 at GCLC and one rs2301022 at GCLM were associated with decreased levels of ROS, while SNPs rs7517826, rs41303970 at GCLM were associated with increased levels of total GSH in plasma. Single nucleotide polymorphisms in genes encoding glutamate cysteine ligase subunits confer protection against type 2 diabetes and their effects are mediated through increased levels of glutathione.

Down regulation of glutathione and glutamate cysteine ligase in the inflammatory response of macrophages

Glutathione (GSH) plays critical roles in the inflammatory response by acting as the master substrate for antioxidant enzymes and an important anti-inflammatory agent. In the early phase of the inflammatory response of macrophages, GSH content is decreased due to the down regulation of the catalytic subunit of glutamate cysteine ligase (GCLC). In the current study we investigated the underlying mechanism for this phenomenon. In human THP1-differentiated macrophages, GCLC mRNA had a half-life of 4 h under basal conditions, and it was significantly reduced to less than 2 h upon exposure to lipopolysaccharide (LPS), suggesting an increased decay of GCLC mRNA in the inflammatory response. The half-life of GCLC protein was >10 h under basal conditions, and upon LPS exposure the degradation rate of GCLC protein was significantly increased. The pan-caspase inhibitor Z-VAD-FMK but not the proteasome inhibitor MG132, prevented the down regulation of GCLC protein caused by LPS. Both caspase inhibitor Z-LEVD-FMK and siRNA of caspase-5 abrogated LPS-induced degradation of GCLC protein. In addition, supplement with γ-GC, the GCLC product, efficiently restored GSH content and suppressed the induction of NF-κB activity by LPS. In conclusion, these data suggest that GCLC down-regulation in the inflammatory response of macrophages is mediated through both increased mRNA decay and caspase-5-mediated GCLC protein degradation, and γ-GC is an efficient agent to restore GSH and regulate the inflammatory response.

Inhibition of the glutathione biosynthetic pathway increases phytochemical toxicity to Spodoptera litura and Nilaparvata lugens

Phytochemicals are toxic to insects, but their insecticidal efficiencies are usually low compared to synthetic insecticides. Understanding the mechanism of insect adaptation to phytochemicals will provide guidance for increasing their efficacy. Reduced glutathione (GSH) is a scavenger of reactive oxygen species (ROS) induced by phytochemicals. However, in insects, the pathway of GSH biosynthesis in response to phytochemicals is unclear. We found that exposure to 0.5% indole-3-methanol (I3C), xanthotoxin, and rotenone (ROT) significantly retarded the growth of Spodoptera litura larvae. The oxidative stress in S. litura larvae exposed to phytochemicals was increased. The up-regulation of glutamate cysteine ligase but not glutathione reductase revealed that the de novo synthesis pathway is responsible for GSH synthesis in phytochemical-treated larvae. Treatment with the inhibitor (BSO) of γ-glutamylcysteine synthetase (gclc), a subunit of glutamate cysteine ligase, resulted in decreases of GSH levels and GST activities, increases of ROS levels in I3C-treated larvae, which finally caused midgut necrosis and larval death. Treatment with BSO or I3C alone did not cause larval death. The addition of GSH could partly reduce the influence of I3C and BSO on S. litura growth. Nilaparvata lugens gclc RNAi confirmed the result of BSO treatment in S. litura. N. lugens gclc RNAi significantly increased the mortality of ROT-sprayed N. lugens, in which ROS levels were significantly increased. All data indicate that gclc is involved in insect response to phytochemical treatment. Treatment with dsgclc will increase the insecticidal efficacy of plant-derived compounds.

Glutathione Restricts Serine Metabolism to Preserve Regulatory T Cell Function

Regulatory Tcells (Tregs) maintain immune homeostasis and prevent autoimmunity. Serine stimulates glutathione (GSH) synthesis and feeds into the one-carbon metabolic network (1CMet) essential for effector Tcell (Teff) responses. However, serine's functions, linkage to GSH, and role in stress responses in Tregs are unknown. Here, we show, using mice with Treg-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that GSH loss in Tregs alters serine import and synthesis and that the integrity of this feedback loop is critical for Treg suppressive capacity. Although Gclc ablation does not impair Treg differentiation, mutant mice exhibit severe autoimmunity and enhanced anti-tumor responses. Gclc-deficient Tregs show increased serine metabolism, mTOR activation, and proliferation but downregulated FoxP3. Limitation of cellular serine invitro and invivo restores FoxP3 expression and suppressive capacity of Gclc-deficient Tregs. Our work reveals an unexpected role for GSH in restricting serine availability to preserve Treg functionality.

Antioxidant supplementation partially rescues accelerated ovarian follicle loss, but not oocyte quality, of glutathione-deficient mice†.

The tripeptide thiol antioxidant glutathione (GSH) has multiple physiological functions. Female mice lacking the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in GSH synthesis, have decreased GSH concentrations, ovarian oxidative stress, preimplantation embryonic mortality, and accelerated age-related decline in ovarian follicles. We hypothesized that supplementation with thiol antioxidants, N-acetyl cysteine (NAC), or α-lipoic acid (ALA) will rescue this phenotype. Gclm-/- and Gclm+/+ females received 0 or 80mM NAC in drinking water from postnatal day (PND) 21-30; follicle growth was induced with equine chorionic gonadotropin (eCG) on PND 27, followed by an ovulatory dose of human CG and mating with a wild type male on PND 29 and zygote harvest 20h after hCG. N-acetyl cysteine supplementation failed to rescue the low rate of second pronucleus formation in zygotes from Gclm-/- versus Gclm+/+ females. In the second study, Gclm-/- and Gclm+/+ females received diet containing 0, 150, or 600mg/kg ALA beginning at weaning and were mated with wild type males from 8 to 20 weeks of age. α-Lipoic acid failed to rescue the decreased offspring production of Gclm-/- females. However, 150mg/kg diet ALA partially rescued the accelerated decline in primordial follicles, as well as the increased recruitment of follicles into the growing pool and the increased percentages of follicles with γH2AX positive oocytes or granulosa cells of Gclm-/- females. We conclude that ovarian oxidative stress is the cause of accelerated primordial follicle decline, while GSH deficiency per se may be responsible for preimplantation embryonic mortality in Gclm-/- females.

Chitosan Oligosaccharides Induce Apoptosis in Human Renal Carcinoma via Reactive-Oxygen-Species-Dependent Endoplasmic Reticulum Stress.

In recent years, various studies have confirmed the role of natural products as effective cancer prevention and treatment drugs. The present study demonstrated that chitosan oligosaccharide (COS) from shells of shrimp and crab caused an inhibitory effect on the proliferation of human renal carcinoma in vitro and in vivo. First, the in vivo biodistribution of COS was investigated by the synthesis of cyanine-7-labeled COS (COS-Cy7) following tail vein injection. The kidney was found to be a major target organ. Then, the impacts on renal carcinoma cell proliferation, apoptosis, and reactive oxygen species (ROS) production were observed in vitro, and an orthotopic xenograft tumor model was designed to evaluate the antitumor efficacy of COS in vivo. In renal carcinoma cells, COS induced G2/M phase arrest and apoptosis in a ROS-dependent fashion. COS significantly promoted mRNA expression of nuclear factor erythroid 2-related factor (Nrf2) and Nrf2 target genes, such as heme oxygenase 1, modifier subunit of glutamate cysteine ligase, and solute carrier family 7 member 11. Additionally, COS significantly upregulated the protein expression of glucose-regulated protein 78, protein RNA-like endoplasmic reticulum (ER) kinase, eukaryotic initiation factor 2α, activating transcription factor 4, C/EBP homologous protein, and cytochrome c, which justified the activation of the ER stress signaling pathway. In vivo, COS repressed tumor growth and induced apoptosis and ROS accumulation, consistent with the in vitro results. Taken together, COS repressed human renal carcinoma growth and induced apoptosis both in vitro and in vivo, mainly via ROS-dependent ER stress pathways.