Liver epigenomic signature associated with chronic oxidative stress in a mouse model of glutathione deficiency.

Oxidative stress is intimately involved in the pathogenesis of fatty liver disease (FLD). A major factor contributing to oxidative stress is the depletion of the ubiquitous antioxidant glutathione (GSH). Unexpectedly, chronic GSH deficiency renders glutamate-cysteine ligase modifier subunit (Gclm)-null mice protected from fatty liver injuries. Epigenetic regulation serves as an important cellular mechanism in modulating gene expression and disease outcome in FLD, although it is not well understood how systemic redox imbalance modifies the liver epigenome. In the current study, utilizing the Gclm-null mouse model, we aimed to elucidate redox-associated epigenomic changes and their implications in liver stress response. We performed high-throughput array-based DNA methylation profiling (MeDIP array) in 22,327 gene promoter regions (from -1300 bp to +500 bp of the Transcription Start Sites) in the liver and peripheral blood cells. Results from the MeDIP array demonstrate that, although global methylation enrichment in gene promoters did not change, low GSH resulted in prevalent demethylation at the individual promoter level. Such an effect likely attributed to a declined availability of the methyl donor S-adenosyl methionine (SAM) in Gclm-null liver. Functional enrichment analysis of liver target genes is suggestive of a potential role of epigenetic mechanisms in promoting cellular survival and lipid homeostasis in Gclm-null liver. In comparison with the liver tissue, MeDIP array in peripheral blood cells revealed a panel of 19 gene promoters that are candidate circulating biomarkers for hepatic epigenomic changes associated with chronic GSH deficiency. Collectively, our results provided new insights into the in vivo interplay between liver redox state and DNA methylation status. The current study laid the groundwork for future epigenetic/epigenomic investigations in experimental settings or human populations under conditions of liver oxidative stress induced by environmental or dietary challenges.

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.

Implications of GCLC in prognosis and immunity of lung adenocarcinoma and multi-omics regulation mechanisms.

BACKGROUND: Ferroptosis is an iron-dependent type of regulated cell death, and has been implicated in lung adenocarcinoma (LUAD). Evidence has proved the key role of glutamate-cysteine ligase catalytic subunit (GCLC) in ferroptosis, but its role in LUAD remains unclear. Herein, we explored the implications of GCLC and relevant genes in LUAD prognosis and immunity as well as underlying molecular mechanisms. METHODS: This work gathered mRNA, miRNA, DNA methylation, somatic mutation and copy-number variation data from TCGA-LUAD. WGCNA was utilized for selecting GCLC-relevant genes, and a GCLC-relevant prognostic signature was built by uni- and multivariate-cox regression analyses. Immune compositions were estimated via CIBERSORT, and two immunotherapy cohorts of solid tumors were analyzed. Multi-omics regulatory mechanisms were finally assessed. RESULTS: Our results showed that GCLC was overexpressed in LUAD, and potentially resulted in undesirable survival. A prognostic model was generated, which owned accurate and independent performance in prognostication. GCLC, and relevant genes were notably connected with immune compositions and immune checkpoints. High GCLC expression was linked with better responses to anti-PD-L1 and anti-CTLA-4 treatment. Their possible DNA methylation sites were inferred, e.g., hypomethylation in cg19740353 might contribute to GCLC up-regulation. Frequent genetic mutations also affected their expression. Upstream transcription factors (E2F1/3/4, etc.), post-transcriptional regulation of miRNAs (hsa-mir-30c-1, etc.), lncRNAs (C8orf34-AS1, etc.), and IGF2BP1-mediated m6A modification were identified. It was also found NOP58-mediated SUMOylation post-translational modification. CONCLUSIONS: Together, we show that GCLC and relevant genes exert crucial roles in LUAD prognosis and immunity, and their expression can be controlled by complex multi-omics mechanisms.

Evolution of the pathogenic mold Aspergillus fumigatus on high copper levels identifies novel resistance genes.

Aspergillus fumigatus is the leading cause of severe mold infections in immunocompromised patients. This common fungus possesses innate attributes that allow it to evade the immune system, including its ability to survive the high copper (Cu) levels in phagosomes. Our previous work has revealed that under high Cu levels, the A. fumigatus transcription factor AceA is activated, inducing the expression of the copper exporter CrpA to expel excess Cu. To identify additional elements in Cu resistance, we evolved A. fumigatus wild-type and mutant ΔaceA or ΔcrpA strains under increasing Cu concentrations. Sequencing of the resultant resistant strains identified both shared and unique evolutionary pathways to resistance. Reintroduction of three of the most common mutations in genes encoding Pma1 (plasma membrane H+-ATPase), Gcs1 (glutamate cysteine-ligase), and Cpa1 (carbamoyl-phosphate synthetase), alone and in combination, into wild-type A. fumigatus confirmed their additive role in conferring Cu resistance. Detailed analysis indicated that the pma1 mutation L424I preserves Pma1 H+-ATPase activity under high Cu concentrations and that the cpa1 mutation A37V confers a survival advantage to conidia in the presence of Cu. Interestingly, simultaneous mutations of all three genes did not alter virulence in infected mice. Our work has identified novel Cu-resistance pathways and provides an evolutionary approach for dissecting the molecular basis of A. fumigatus adaptation to diverse environmental challenges.IMPORTANCEAspergillus fumigatus is the most common mold infecting patients with weakened immunity. Infection is caused by the inhalation of mold spores into the lungs and is often fatal. In healthy individuals, spores are engulfed by lung immune cells and destroyed by a combination of enzymes, oxidants, and high levels of copper. However, the mold can protect itself by pumping out excess copper with specific transporters. Here, we evolved A. fumigatus under high copper levels and identified new genetic mutations that help it resist the toxic effects of copper. We studied how these mutations affect the mold's ability to resist copper and how they impact its ability to cause disease. This is the first such study in a pathogenic mold, and it gives us a better understanding of how it manages to bypass our body's defenses during an infection.

Neuronal glutathione depletion elevates the Aß42/Aß40 ratio and tau aggregation in Alzheimer's disease mice.

Alzheimer's disease (AD) involves reduced glutathione levels, causing oxidative stress and contributing to neuronal cell death. Our prior research identified diminished glutamate-cysteine ligase catalytic subunit (GCLC) as linked to cell death. However, the effect of GCLC on AD features such as amyloid and tau pathology remained unclear. To address this, we investigated amyloid pathology and tau pathology in mice by combining neuron-specific conditional GCLC knockout mice with amyloid precursor protein (App) knockin (KI) or microtubule-associated protein tau (MAPT) KI mice. Intriguingly, GCLC knockout resulted in an increased Aß42/40 ratio. Additionally, GCLC deficiency in MAPT KI mice accelerated the oligomerization of tau through intermolecular disulfide bonds. These findings suggest that the decline in glutathione levels, due to aging or AD pathology, may contribute to the progression of AD.

Pharmacological elevation of glutathione inhibits status epilepticus-induced neuroinflammation and oxidative injury.

Glutathione (GSH) is a major endogenous antioxidant, and its depletion has been observed in several brain diseases including epilepsy. Previous studies in our laboratory have shown that dimercaprol (DMP) can elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme and inhibit neuroinflammation in vitro. Here we determined 1) the role of cysteamine as a new mechanism by which DMP increases GSH biosynthesis and 2) its ability to inhibit neuroinflammation and neuronal injury in the rat kainate model of epilepsy. DMP depleted cysteamine in a time- and concentration-dependent manner in a cell free system. To guide the in vivo administration of DMP, its pharmacokinetic profile was determined in the plasma, liver, and brain. The results confirmed DMP's ability to cross the blood-brain-barrier. Treatment of rats with DMP (30 mg/kg) depleted cysteamine in the liver and hippocampus that was associated with increased GCL activity in these tissues. GSH levels were significantly increased (20 %) in the hippocampus 1 h after 30 mg/kg DMP administration. Following DMP (30 mg/kg) administration once daily, a marked attenuation of GSH depletion was seen in the SE model. SE-induced inflammatory markers including cytokine release, microglial activation, and neuronal death were significantly attenuated in the hippocampus with DMP treatment. Taken together, these results highlight the importance of restoring redox status with rescue of GSH depletion by DMP in post epileptogenic insults.

Overexpression of sweetpotato glutamylcysteine synthetase (IbGCS) in Arabidopsis confers tolerance to drought and salt stresses.

Various environmental stresses induce the production of reactive oxygen species (ROS), which have deleterious effects on plant cells. Glutathione (GSH) is an antioxidant used to counteract reactive oxygen species. Glutathione is produced by glutamylcysteine synthetase (GCS) and glutathione synthetase (GS). However, evidence for the GCS gene in sweetpotato remains scarce. In this study, the full-length cDNA sequence of IbGCS isolated from sweetpotato cultivar Xu18 was 1566 bp in length, which encodes 521 amino acids. The qRT-PCR analysis revealed a significantly higher expression of the IbGCS in sweetpotato flowers, and the gene was induced by salinity, abscisic acid (ABA), drought, extreme temperature and heavy metal stresses. The seed germination rate, root elongation and fresh weight were promoted in T3 Arabidopsis IbGCS-overexpressing lines (OEs) in contrast to wild type (WT) plants under mannitol and salt stresses. In addition, the soil drought and salt stress experiment results indicated that IbGCS overexpression in Arabidopsis reduced the malondialdehyde (MDA) content, enhanced the levels of GCS activity, GSH and AsA content, and antioxidant enzyme activity. In summary, overexpressing IbGCS in Arabidopsis showed improved salt and drought tolerance.

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.

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.

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.

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.

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.

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.

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.

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.

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

BACKGROUND: Colorectal 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. METHODS: The 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. RESULTS: Here, 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. CONCLUSIONS: Therefore, 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.

The Emerging Roles of γ-Glutamyl Peptides Produced by γ-Glutamyltransferase and the Glutathione Synthesis System.

L-γ-Glutamyl-L-cysteinyl-glycine is commonly referred to as glutathione (GSH); this ubiquitous thiol plays essential roles in animal life. Conjugation and electron donation to enzymes such as glutathione peroxidase (GPX) are prominent functions of GSH. Cellular glutathione balance is robustly maintained via regulated synthesis, which is catalyzed via the coordination of γ-glutamyl-cysteine synthetase (γ-GCS) and glutathione synthetase, as well as by reductive recycling by glutathione reductase. A prevailing short supply of L-cysteine (Cys) tends to limit glutathione synthesis, which leads to the production of various other γ-glutamyl peptides due to the unique enzymatic properties of γ-GCS. Extracellular degradation of glutathione by γ-glutamyltransferase (GGT) is a dominant source of Cys for some cells. GGT catalyzes the hydrolytic removal of the γ-glutamyl group of glutathione or transfers it to amino acids or to dipeptides outside cells. Such processes depend on an abundance of acceptor substrates. However, the physiological roles of extracellularly preserved γ-glutamyl peptides have long been unclear. The identification of γ-glutamyl peptides, such as glutathione, as allosteric modulators of calcium-sensing receptors (CaSRs) could provide insights into the significance of the preservation of γ-glutamyl peptides. It is conceivable that GGT could generate a new class of intercellular messaging molecules in response to extracellular microenvironments.

Optimized Separation of Carthamin from Safflower by Macroporous Adsorption Resins and Its Protective Effects on PC12 Cells Injured by OGD/R via Nrf2 Signaling Pathway.

The growing demand for safe natural products has reignited people's interest in natural food pigments. Here, we proposed the use of macroporous adsorption resins (MARs) to separate and purify carthamin from safflower. The optimal parameters for carthamin purification with HPD400 MAR were determined as follows: a mass ratio of crude carthamin in sample solution to wet resin of 0.3, a crude carthamin solution concentration of 0.125 g·mL-1, a pH of 6.00, a sample volume flow rate of 0.5 mL·min-1, an ethanol volume fraction of 58%, an elution volume of 4 BV, and an elution volume flow rate of 1.0 mL·min-1. Under the above purification conditions, the recovery rate of carthamin was above 96%. Carthamin dramatically improved the survival rate of PC12 cells damaged by oxygen-glucose deprivation/reoxygenation and protected them from oxidative stress by inhibiting the generation of reactive oxygen species and increasing the total antioxidant capacity and glutathione (GSH) levels. Carthamin promoted extracellularly regulated protein kinase phosphorylation into the nucleus, permitting Nrf2 nuclear translocation and upregulating the gene expression of the rate-limiting enzymes glutamate-cysteine ligase catalytic subunit and glutamate-cysteine ligase regulatory subunit of GSH synthesis to obliterate free radicals and exert antioxidant effects. This study revealed the purification method of carthamin and its antioxidant protective effects, providing important insights into the application of carthamin in functional foods.

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.