IRE1α determines ferroptosis sensitivity through regulation of glutathione synthesis.

Cellular sensitivity to ferroptosis is primarily regulated by mechanisms mediating lipid hydroperoxide detoxification. We show that inositol-requiring enzyme 1 (IRE1α), an endoplasmic reticulum (ER) resident protein critical for the unfolded protein response (UPR), also determines cellular sensitivity to ferroptosis. Cancer and normal cells depleted of IRE1α gain resistance to ferroptosis, while enhanced IRE1α expression promotes sensitivity to ferroptosis. Mechanistically, IRE1α's endoribonuclease activity cleaves and down-regulates the mRNA of key glutathione biosynthesis regulators glutamate-cysteine ligase catalytic subunit (GCLC) and solute carrier family 7 member 11 (SLC7A11). This activity of IRE1α is independent of its role in regulating the UPR and is evolutionarily conserved. Genetic deficiency and pharmacological inhibition of IRE1α have similar effects in inhibiting ferroptosis and reducing renal ischemia-reperfusion injury in mice. Our findings reveal a previously unidentified role of IRE1α to regulate ferroptosis and suggests inhibition of IRE1α as a promising therapeutic strategy to mitigate ferroptosis-associated pathological conditions.

Prevention of age-related truncation of γ-glutamylcysteine ligase catalytic subunit (GCLC) delays cataract formation.

A sharp drop in lenticular glutathione (GSH) plays a pivotal role in age-related cataract (ARC) formation. Despite recognizing GSH's importance in lens defense for decades, its decline with age remains puzzling. Our recent study revealed an age-related truncation affecting the essential GSH biosynthesis enzyme, the γ-glutamylcysteine ligase catalytic subunit (GCLC), at aspartate residue 499. Intriguingly, these truncated GCLC fragments compete with full-length GCLC in forming a heterocomplex with the modifier subunit (GCLM) but exhibit markedly reduced enzymatic activity. Crucially, using an aspartate-to-glutamate mutation knock-in (D499E-KI) mouse model that blocks GCLC truncation, we observed a notable delay in ARC formation compared to WT mice: Nearly 50% of D499E-KI mice remained cataract-free versus ~20% of the WT mice at their age of 20 months. Our findings concerning age-related GCLC truncation might be the key to understanding the profound reduction in lens GSH with age. By halting GCLC truncation, we can rejuvenate lens GSH levels and considerably postpone cataract onset.

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.

Selenium Lessens Osteoarthritis by Protecting Articular Chondrocytes from Oxidative Damage through Nrf2 and NF-κB Pathways.

Osteoarthritis (OA) causes joint pain and disability due to the abnormal production of inflammatory cytokines and reactive oxygen species (ROS) in chondrocytes, leading to cell death and cartilage matrix destruction. Selenium (Se) intake can protect cells against oxidative damage. It is still unknown whether Se supplementation is beneficial for OA. This study investigated the effects of Se on sodium iodoacetate (MIA)-imitated OA progress in human chondrocyte cell line (SW1353 cells) and rats. The results showed that 0.3 µM of Se treatment could protect SW1353 cells from MIA-induced damage by the Nrf2 pathway by promoting the gene expression of glutathione-synthesis-related enzymes such as the glutamate-cysteine ligase catalytic subunit, the glutamate-cysteine ligase modifier subunit, and glutathione synthetase. In addition, glutathione, superoxide dismutase, glutathione peroxidase, and glutathione reductase expressions are also elevated to eliminate excessive ROS production. Moreover, Se could downregulate NF-κB, leading to a decrease in cytokines, matrix proteases, and glycosaminoglycans. In the rats, MIA-induced cartilage loss was lessened after 2 weeks of Se supplementation by oral gavage; meanwhile, glutathione synthesis was increased, and the expressions of pro-inflammatory cytokines were decreased. These results suggest that Se intake is beneficial for OA due to its effects of decreasing cartilage loss by enhancing antioxidant capacity and reducing inflammation.

Contribution of γ-Glutamyl-Cysteine Ligases of Limosilactobacillus reuteri to the Formation of Kokumi-Active γ-Glutamyl Dipeptides in Sourdough Bread.

Kokumi-active γ-glutamyl dipeptides accumulate during sourdough fermentation. γ-Glutamylcysteine ligases (Gcls) of Limosilactobacillus reuteri synthesize γ-glutamyl dipeptides during growth in sourdough. This study aimed to evaluate the contribution of Gcls from strains of L. reuteri in the formation of kokumi-active γ-glutamyl dipeptides in sourdough bread. Among 12 acceptor amino acids, the three Gcls of L. reuteri were the most active to Cys. With the acceptor amino acids Ile, Leu, and Phe, Gcl1 was more active than Gcl2 and Gcl3. Accordingly, Gcl1 contributed to the γ-Glu-Ile synthesis in sourdough fermentation. Proofing and baking strongly influenced the concentration of γ-glutamyl dipeptides in bread. The addition of 10% sourdough increased the content of γ-Glu-Leu and γ-Glu-Phe but not of other γ-glutamyl dipeptides in bread. In conclusion, the accumulation of kokumi γ-glutamyl dipeptides in sourdoughs was attributed to the combined activity of cereal enzymes, γ-glutamyl-cysteine ligases, and other microbial enzymes.

Selenium protects against Pb-induced renal oxidative injury in weaning rats and human renal tubular epithelial cells through activating NRF2.

BACKGROUND: Lead (Pb) poisoning posing a crucial health risk, especially among children, causing devastating damage not only to brain development, but also to kidney function. Thus, an urgent need persists to identify highly effective, safe, and low-toxicity drugs for the treatment of Pb poisoning. The present study focused on exploring the protective effects of Se on Pb-induced nephrotoxicity in weaning rats and human renal tubular epithelial cells, and investigated the possible mechanisms. METHODS: Forty weaning rats were randomly divided into four groups in vivo: control, Pb-exposed, Pb+Se and Se. Serum creatinine (Cr), urea nitrogen (BUN) and hematoxylin and eosin (H&E) staining were performed to evaluate renal function. The activities of antioxidant enzymes in the kidney tissue were determined. In vitro experiments were performed using human renal tubular epithelial cells (HK-2 cells). The cytotoxicity of Pb and Se was detected by 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Inverted fluorescence microscope was used to investigate cell morphological changes and the fluorescence intensity of reactive oxygen species (ROS). The oxidative stress parameters were measured by a multi-detection reader. Nuclear factor-erythroid-2-related factor (NRF2) signaling pathways were measured by Western blot and reverse transcription polymerase chain reaction (RT-PCR) in HK-2 cells. RESULTS: We found that Se alleviated Pb-induced kidney injury by relieving oxidative stress and reducing the inflammatory index. Se significantly increased the activity of the antioxidant enzymes glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), whereas it decreased the excessive release of malondialdehyde (MDA) in the kidneys of weaning rats and HK-2 cells. Additionally, Se enhanced the antioxidant defense systems via activating the NRF2 transcription factor, thereby promoting the to downstream expression of heme oxygenase 1. Furthermore, genes encoding glutamate-cysteine ligase synthetase catalytic (GCLC), glutamate-cysteine ligase synthetase modifier (GCLM) and NADPH quinone oxidoreductase 1 (NQO1), downstream targets of NRF2, formed a positive feedback loop with NRF2 during oxidative stress responses. The MTT assay results revealed a significant decrease in cell viability with Se treatment, and the cytoprotective role of Se was blocked upon knockdown of NRF2 by small interfering RNA (siRNA). MDA activity results also showed that NRF2 knockdown inhibited the NRF2-dependent transcriptional activity of Se. CONCLUSIONS: Our findings demonstrate that Se ameliorated Pb-induced nephrotoxicity by reducing oxidative stress both in vivo and in vitro. The molecular mechanism underlying Se's action in Pb-induced kidney injury is related to the activation of the NRF2 transcription factor and the activity of antioxidant enzymes, ultimately suppressing ROS accumulation.

The glutathione biosynthesis is involved in metamorphosis, antioxidant function, and insecticide resistance in Tribolium castaneum.

BACKGROUND: Reduced glutathione (GSH) synthesis is vital for redox homeostasis, cell-cycle regulation and apoptosis, and immune function. The glutamate-cysteine ligase catalytic subunit (Gclc) is the first and rate-limiting enzyme in GSH synthesis, suggesting the potential use of Gclc as a pesticide target. However, the functional characterization of Gclc, especially its contribution in metamorphosis, antioxidant status and insecticide resistance, is unclear in Tribolium castaneum. RESULTS: In this study, we identified and cloned Gclc from T. castaneum (TcGclc) and found that its expression began to increase significantly from the late larvae (LL) stage (3.491 ± 0.490-fold). Furthermore, RNA interference-mediated knockdown of TcGclc resulted in three types of aberration (100% total aberration rate) caused by the downregulation of genes related to the 20-hydroxyecdysone (20E) pathway. This deficiency was partially rescued by exogenous 20E treatment (53.1% ± 3.2%), but not by antioxidant. Moreover, in the TcGclc knockdown group, GSH content was decreased to 62.3%, and total antioxidant capacity, glutathione peroxidase and total superoxide dismutase activities were reduced by 14.6%, 83.6%, and 82.3%, respectively. In addition, treatment with different insecticides upregulated expression of TcGclc significantly compared with a control group during the late larval stage (P < 0.01). CONCLUSION: Our results indicate that TcGclc has an extensive role in metamorphosis, antioxidant function and insecticide resistance in T. castaneum, thereby expanding our understanding of GSH functions and providing a scientific basis for pest control. © 2024 Society of Chemical Industry.

Nuclear factor erythroid 2-related factor 2 promotes radioresistance by regulating glutamate-cysteine ligase modifier subunit and its unique immunoinvasive pattern.

The enzyme glutamate-cysteine ligase modifier subunit (GCLM) serves as the initial rate-limiting factor in glutathione (GSH) synthesis. GSH is the preferred substrate for glutathione peroxidase 4 (GPX4), directly impacting its activity and stability. This study aims to elucidate the expression of GCLM and its correlation with the nuclear factor erythroid 2-related factor 2 (NFE2L2), commonly referred to as NRF2, in esophageal squamous cell carcinoma (ESCC) and further investigate the potential signaling axis of radiotherapy resistance caused by NRF2-mediated regulation of ferroptosis in ESCC. The expression of NRF2, GCLM, and GPX4 in ESCC was analyzed by bioinformatics, and their relationship with ferroptosis was verified through cell function experiments. Their role in radioresistance was then investigated through multiple validation steps. Bioinformatics analysis was employed to determine the immune infiltration pattern of NRF2 in ESCC. Furthermore, the effect of NRF2-mediated massive macrophage M2 infiltration on radiotherapy and ferroptosis was validated through in vivo experiments. In vitro assays demonstrated that overactivated NRF2 promotes radioresistance by directly binding to the promoter region of GCLM. The Tumor Immune Estimation Resource (TIMER) and quanTIseq analyses revealed NRF2 enrichment in M2 macrophages with a positive correlation. Co-culturing KYSE450 cells with M2 macrophages demonstrated that a significant infiltration of macrophages M2 can render ESCC cells resistant to radiotherapy but restore their sensitivity to ferroptosis in the process. Our study elucidates a link between the NRF2-GCLM-GSH-GPX4 signaling axis in ESCC, highlighting its potential as a therapeutic target for antagonistic biomarkers of resistance in the future. Additionally, it provides a novel treatment avenue for ESCC metastasis and radioresistance.

Development of a mouse model expressing a bifunctional glutathione-synthesizing enzyme to study glutathione limitation in vivo.

Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.

Inhibition of the glutamate-cysteine ligase catalytic subunit with buthionine sulfoximine enhances the cytotoxic effect of doxorubicin and cyclophosphamide in Burkitt lymphoma cells.

Burkitt lymphoma (BL) is a highly aggressive lymphoma that mainly affects children and young adults. Chemotherapy is effective in young BL patients but the outcome in adults is less satisfactory. Therefore, there is a need to enhance the cytotoxic effect of drugs used in BL treatment. Glutathione (GSH) is an important antioxidant involved in processes such as regulation of oxidative stress and drug detoxification. Elevated GSH levels have been observed in many cancers and were associated with chemoresistance. We previously identified GCLC, encoding an enzyme involved in GSH biosynthesis, as an essential gene in BL. We now confirm that knockout of GCLC decreases viability of BL cells and that the GCLC protein is overexpressed in BL tissues. Moreover, we demonstrate that buthionine sulfoximine (BSO), a known inhibitor of GCLC, decreases growth of BL cells but does not affect control B cells. Furthermore, we show for the first time that BSO enhances the cytotoxicity of compounds commonly used in BL treatment, doxorubicin, and cyclophosphamide. Given the fact that BSO itself was not toxic to control cells and well-tolerated in clinical trials, combination of chemotherapy with BSO may allow reduction of the doses of cytotoxic drugs required to obtain effective responses in BL patients.

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.

Increasing glutathione levels by a novel posttranslational mechanism inhibits neuronal hyperexcitability.

Glutathione (GSH) depletion, and impaired redox homeostasis have been observed in experimental animal models and patients with epilepsy. Pleiotropic strategies that elevate GSH levels via transcriptional regulation have been shown to significantly decrease oxidative stress and seizure frequency, increase seizure threshold, and rescue certain cognitive deficits. Whether elevation of GSH per se alters neuronal hyperexcitability remains unanswered. We previously showed that thiols such as dimercaprol (DMP) elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme. Here, we asked if elevation of cellular GSH by DMP altered neuronal hyperexcitability in-vitro and in-vivo. Treatment of primary neuronal-glial cerebrocortical cultures with DMP elevated GSH and inhibited a voltage-gated potassium channel blocker (4-aminopyridine, 4AP) induced neuronal hyperexcitability. DMP increased GSH in wildtype (WT) zebrafish larvae and significantly attenuated convulsant pentylenetetrazol (PTZ)-induced acute 'seizure-like' swim behavior. DMP treatment increased GSH and inhibited convulsive, spontaneous 'seizure-like' swim behavior in the Dravet Syndrome (DS) zebrafish larvae (scn1Lab). Furthermore, DMP treatment significantly decreased spontaneous electrographic seizures and associated seizure parameters in scn1Lab zebrafish larvae. We investigated the role of the redox-sensitive mammalian target of rapamycin (mTOR) pathway due to the presence of several cysteine-rich proteins and their involvement in regulating neuronal excitability. Treatment of primary neuronal-glial cerebrocortical cultures with 4AP or l-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of GSH biosynthesis, significantly increased mTOR complex I (mTORC1) activity which was rescued by pre-treatment with DMP. Furthermore, BSO-mediated GSH depletion oxidatively modified the tuberous sclerosis protein complex (TSC) consisting of hamartin (TSC1), tuberin (TSC2), and TBC1 domain family member 7 (TBC1D7) which are critical negative regulators of mTORC1. In summary, our results suggest that DMP-mediated GSH elevation by a novel post-translational mechanism can inhibit neuronal hyperexcitability both in-vitro and in-vivo and a plausible link is the redox sensitive mTORC1 pathway.

Glutathione and glutathione-dependent enzymes: From biochemistry to gerontology and successful aging.

The tripeptide glutathione (GSH), namely γ-L-glutamyl-L-cysteinyl-glycine, is an ubiquitous low-molecular weight thiol nucleophile and reductant of utmost importance, representing the central redox agent of most aerobic organisms. GSH has vital functions involving also antioxidant protection, detoxification, redox homeostasis, cell signaling, iron metabolism/homeostasis, DNA synthesis, gene expression, cysteine/protein metabolism, and cell proliferation/differentiation or death including apoptosis and ferroptosis. Various functions of GSH are exerted in concert with GSH-dependent enzymes. Indeed, although GSH has direct scavenging antioxidant effects, its antioxidant function is substantially accomplished by glutathione peroxidase-catalyzed reactions with reductive removal of H2O2, organic peroxides such as lipid hydroperoxides, and peroxynitrite; to this antioxidant activity also contribute peroxiredoxins, enzymes further involved in redox signaling and chaperone activity. Moreover, the detoxifying function of GSH is basically exerted in conjunction with glutathione transferases, which have also antioxidant properties. GSH is synthesized in the cytosol by the ATP-dependent enzymes glutamate cysteine ligase (GCL), which catalyzes ligation of cysteine and glutamate forming γ-glutamylcysteine (γ-GC), and glutathione synthase, which adds glycine to γ-GC resulting in GSH formation; GCL is rate-limiting for GSH synthesis, as is the precursor amino acid cysteine, which may be supplemented as N-acetylcysteine (NAC), a therapeutically available compound. After its cell export, GSH is degraded extracellularly by the membrane-anchored ectoenzyme γ-glutamyl transferase, a process occurring, as GSH synthesis and export, in the γ-glutamyl cycle. GSH degradation occurs also intracellularly by the cytoplasmic enzymatic ChaC family of γ-glutamyl cyclotransferase. Synthesis and degradation of GSH, together with its export, translocation to cell organelles, utilization for multiple essential functions, and regeneration from glutathione disulfide by glutathione reductase, are relevant to GSH homeostasis and metabolism. Notably, GSH levels decline during aging, an alteration generally related to impaired GSH biosynthesis and leading to cell dysfunction. However, there is evidence of enhanced GSH levels in elderly subjects with excellent physical and mental health status, suggesting that heightened GSH may be a marker and even a causative factor of increased healthspan and lifespan. Such aspects, and much more including GSH-boosting substances administrable to humans, are considered in this state-of-the-art review, which deals with GSH and GSH-dependent enzymes from biochemistry to gerontology, focusing attention also on lifespan/healthspan extension and successful aging; the significance of GSH levels in aging is considered also in relation to therapeutic possibilities and supplementation strategies, based on the use of various compounds including NAC-glycine, aimed at increasing GSH and related defenses to improve health status and counteract aging processes in humans.

Farnesoid X receptor activation is required for the anti-inflammatory and anti-oxidative stress effects of Alisol B 23-acetate in carbon tetrachloride-induced liver fibrosis in mice.

Previous studies have shown that Alisol B 23-acetate (23ABA) had potent liver-protection effects, however, its roles and potential mechanisms in carbon tetrachloride (CCl4)-induced liver fibrosis remain to be determined. The present study aimed to investigate the effects of 23ABA on CCl4-induced liver fibrosis and tried to elucidate the underlying mechanisms by focusing on regulating of farnesoid X receptor (FXR). In vivo study found that 23ABA alleviated the CCl4-induced liver injury, and showed no obvious systemic toxicity on mice. 23ABA inhibited the collagen production, decreased sera levels of hyaluronic acid (HA) and procollagen type III (PC-III), lowered mRNA expression of α-smooth muscle actin (α-SMA), fibronectin, collagen I and collagen III in livers of mice. 23ABA inhibited the mRNA expressions and the sera levels of interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-α (TNF-α), as well as decreased the expression of cyclooxygenase 2 (COX-2) in fibrotic livers of mice. Besides, 23ABA decreased levels of reactive oxygen species (ROS) and malondialdehyde (MDA), increased glutathione (GSH) level, enhanced activities of superoxide dismutase (SOD) and glutathione reductase (GR) as well as increased mRNA expression of nuclear factor-E2-related factor 2 (Nrf2), glutamate-cysteine ligase, catalytic subunit (GCLC) and glutamate-cysteine ligase, modifier subunit (GCLM). Further study showed that the anti-liver injury and anti-fibrotic effects of 23ABA were abrogated by FXR antagonist guggulsterone (GS) in vivo. In addition, the inhibition effects of 23ABA on liver inflammation and oxidative stress were also weakened by treatment with GS in CCl4-induced fibrotic mice livers. In conclusion, the protective effects of 23ABA against CCl4-induced liver injury and fibrosis, due to FXR-mediated regulation of liver inflammation and oxidative stress.

Characterization of early psychosis patients carrying a genetic vulnerability to redox dysregulation: a computational analysis of mechanism-based gene expression profile in fibroblasts.

In view of its heterogeneity, schizophrenia needs new diagnostic tools based on mechanistic biomarkers that would allow early detection. Complex interaction between genetic and environmental risk factors may lead to NMDAR hypofunction, inflammation and redox dysregulation, all converging on oxidative stress. Using computational analysis, the expression of 76 genes linked to these systems, known to be abnormally regulated in schizophrenia, was studied in skin-fibroblasts from early psychosis patients and age-matched controls (N = 30), under additional pro-oxidant challenge to mimic environmental stress. To evaluate the contribution of a genetic risk related to redox dysregulation, we investigated the GAG trinucleotide polymorphism in the key glutathione (GSH) synthesizing enzyme, glutamate-cysteine-ligase-catalytic-subunit (gclc) gene, known to be associated with the disease. Patients and controls showed different gene expression profiles that were modulated by GAG-gclc genotypes in combination with oxidative challenge. In GAG-gclc low-risk genotype patients, a global gene expression dysregulation was observed, especially in the antioxidant system, potentially induced by other risks. Both controls and patients with GAG-gclc high-risk genotype (gclcGAG-HR) showed similar gene expression profiles. However, under oxidative challenge, a boosting of other antioxidant defense, including the master regulator Nrf2 and TRX systems was observed only in gclcGAG-HR controls, suggesting a protective compensation against the genetic GSH dysregulation. Moreover, RAGE (redox/inflammation interaction) and AGMAT (arginine pathway) were increased in the gclcGAG-HR patients, suggesting some additional risk factors interacting with this genotype. Finally, the use of a machine-learning approach allowed discriminating patients and controls with an accuracy up to 100%, paving the way towards early detection of schizophrenia.

Activation of the Keap1/Nrf2 Pathway as an Adaptive Response to an Electrophilic Metabolite of Morphine.

Morphinone (MO) is an electrophilic metabolite of morphine that covalently binds to protein thiols via its α,ß-unsaturated carbonyl group, resulting in toxicity in vitro and in vivo. Our previous studies identified a variety of redox signaling pathways that are activated during electrophilic stress. Here, we examined in vitro activation of a signaling pathway involving Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) in response to MO. Exposure of HepG2 cells to MO caused covalent modification of Keap1 thiols (evaluated using biotin-PEAC5-maleimide labeling) and nuclear translocation of Nrf2, thereby up-regulating downstream genes encoding ATP binding cassette subfamily C member 2, solute carrier family 7 member 11, glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, glutathione S-transferase alpha 1, and heme oxygenase 1. However, dihydromorphinone, a metabolite of morphine lacking the reactive C7-C8 double bond, had little effect on Nrf2 activation. These results suggest that covalent modification is crucial in the Keap1/Nrf2 pathway activation and that this pathway is a redox signaling-associated adaptive response to MO metabolism.

T Cell Nrf2/Keap1 Gene Editing Using CRISPR/Cas9 and Experimental Kidney Ischemia-Reperfusion Injury.

Aims: T cells play pathophysiologic roles in kidney ischemia-reperfusion injury (IRI), and the nuclear factor erythroid 2-related factor 2/kelch-like ECH-associated protein 1 (Nrf2/Keap1) pathway regulates T cell responses. We hypothesized that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated Keap1-knockout (KO) augments Nrf2 antioxidant potential of CD4+ T cells, and that Keap1-KO CD4+ T cell immunotherapy protects from kidney IRI. Results: CD4+ T cell Keap1-KO resulted in significant increase of Nrf2 target genes NAD(P)H quinone dehydrogenase 1, heme oxygenase 1, glutamate-cysteine ligase catalytic subunit, and glutamate-cysteine ligase modifier subunit. Keap1-KO cells displayed no signs of exhaustion, and had significantly lower levels of interleukin 2 (IL2) and IL6 in normoxic conditions, but increased interferon gamma in hypoxic conditions in vitro. In vivo, adoptive transfer of Keap1-KO CD4+ T cells before IRI improved kidney function in T cell-deficient nu/nu mice compared with mice receiving unedited control CD4+ T cells. Keap1-KO CD4+ T cells isolated from recipient kidneys 24 h post IR were less activated compared with unedited CD4+ T cells, isolated from control kidneys. Innovation: Editing Nrf2/Keap1 pathway in murine T cells using CRISPR/Cas9 is an innovative and promising immunotherapy approach for kidney IRI and possibly other solid organ IRI. Conclusion: CRISPR/Cas9-mediated Keap1-KO increased Nrf2-regulated antioxidant gene expression in murine CD4+ T cells, modified responses to in vitro hypoxia and in vivo kidney IRI. Gene editing targeting the Nrf2/Keap1 pathway in T cells is a promising approach for immune-mediated kidney diseases.

MYB30 integrates light signals with antioxidant biosynthesis to regulate plant responses during postsubmergence recovery.

Submergence is an abiotic stress that limits agricultural production world-wide. Plants sense oxygen levels during submergence and postsubmergence reoxygenation and modulate their responses. Increasing evidence suggests that completely submerged plants are often exposed to low-light stress, owing to the depth and turbidity of the surrounding water; however, how light availability affects submergence tolerance remains largely unknown. Here, we showed that Arabidopsis thaliana MYB DOMAIN PROTEIN30 (MYB30) is an important transcription factor that integrates light signaling and postsubmergence stress responses. MYB DOMAIN PROTEIN30 protein abundance decreased upon submergence and accumulated during reoxygenation. Under submergence conditions, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), a central regulator of light signaling, caused the ubiquitination and degradation of MYB30. In response to desubmergence, however, light-induced MYB30 interacted with MYC2, a master transcription factor involved in jasmonate signaling, and activated the expression of the VITAMIN C DEFECTIVE1 (VTC1) and GLUTATHIONE SYNTHETASE1 (GSH1) gene families to enhance antioxidant biosynthesis. Consistent with this, the myb30 knockout mutant showed increased sensitivity to submergence, which was partially rescued by overexpression of VTC1 or GSH1. Thus, our findings uncover the mechanism by which the COP1-MYB30 module integrates light signals with cellular oxidative homeostasis to coordinate plant responses to postsubmergence stress.

Efficient production of glutathione in Saccharomyces cerevisiae via a synthetic isozyme system.

Glutathione, a tripeptide consisting of cysteine, glutamic acid, and glycine, has multiple beneficial effects on human health. Previous studies have focused on producing glutathione in Saccharomyces cerevisiae by overexpressing γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), which are the rate-limiting enzymes involved in the glutathione biosynthetic pathway. However, the production yield and titer of glutathione remain low due to the feedback inhibition on GSH1. To overcome this limitation, a synthetic isozyme system consisting of a novel bifunctional enzyme (GshF) from Gram-positive bacteria possessing both GSH1 and GSH2 activities, in addition to GSH1/GSH2, was introduced into S. cerevisiae, as GshF is insensitive to feedback inhibition. Given the HSP60 chaperonin system mismatch between bacteria and S. cerevisiae, co-expression of Group-I HSP60 chaperonins (GroEL and GroES) from Escherichia coli was required for functional expression of GshF. Among various strains constructed in this study, the SKSC222 strain capable of synthesizing glutathione with the synthetic isozyme system produced 240 mg L-1 glutathione with glutathione content and yield of 4.3% and 25.6 mgglutathione /gglucose , respectively. These values were 6.6-, 4.9-, and 4.3-fold higher than the corresponding values of the wild-type strain. In a glucose-limited fed-batch fermentation, the SKSC222 strain produced 2.0 g L-1 glutathione in 67 h. Therefore, this study highlights the benefits of the synthetic isozyme system in enhancing the production titer and yield of value-added chemicals by engineered strains of S. cerevisiae.

c-Jun-N Terminal Kinase-Mediated Degradation of γ-Glutamylcysteine Ligase Catalytic Subunit Inhibits GSH Recovery After Acetaminophen Treatment: Role in Sustaining JNK Activation and Liver Injury.

Aims: Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States. Liver glutathione (GSH) depletion and sustained P-JNK (c-Jun-N-terminal kinase) activation are key modulators in the mechanism leading to hepatic necrosis. GSH depletion is directly related to the consumption of GSH by APAP metabolites N-acetyl-p-benzoquinone imine (NAPQI). We previously noticed that the glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in GSH synthesis, rapidly decreased at the same time P-JNK increased. Our aims were to determine if JNK was directly responsible for decreased GCLC causing impaired recovery of GSH and if this was an important factor in determining APAP hepatotoxicity. Results: Immunoprecipitation of JNK after APAP identified binding to GCLC. Expression of a site-directed mutated canonical JNK docking site in GCLC was resistant to degradation and led to rapid restoration of GSH and inhibited sustained JNK activation. The JNK-resistant GCLC markedly protected against necrosis and alanine aminotransferase (ALT) elevation. The proteolytic loss of GCLC was abrogated by inhibition of the proteasome, ubiquitination, or calpain. Innovation: Using mutated-GCLC resistant to JNK-induced degradation, the results allowed us to identify impaired GSH recovery as an important contributor to early progression of APAP toxicity after the metabolism of APAP and initial GSH depletion had occurred. Conclusion: Activated JNK interacts directly with GCLC and leads to proteolytic degradation of GCLC. Degradation of GCLC impairs GSH recovery after APAP allowing the continued activation of JNK. Conversely, rapid recovery of GSH inhibits the sustained activation of the mitogen-activated protein (MAP) kinase cascade and dampens APAP toxicity by suppressing the continued activation of JNK. Antioxid. Redox Signal. 38, 1071-1081.