The Glutathione Metabolite γ-Glutamyl-Glutamate Partially Activates Glutamate NMDA Receptors in Central Neurons With Higher Efficacy for GluN2B-Containing Receptors

Gamma-L-glutamyl-L-glutamate (γ-Glu-Glu) was synthetized and further characterized for its activity on cultured neurons. We observed that γ-Glu-Glu elicited excitatory effects on neurons likely by activating mainly the N-methyl-D-aspartate (NMDA) receptors. These effects were dependent on the integrity of synaptic transmission as they were blocked by tetrodotoxin (TTX). We next evaluated its activity on NMDA receptors by testing it on cells expressing these receptors. We observed that γ-Glu-Glu partially activated NMDA receptors and exhibited better efficacy for NMDA receptors containing the GluN2B subunit. Moreover, at low concentration, γ-Glu-Glu potentiated the responses of glutamate on NMDA receptors. Finally, the endogenous production of γ-Glu-Glu was measured by LC-MS on the extracellular medium of C6 rat astroglioma cells. We found that extracellular γ-Glu-Glu concentration was, to some extent, directly linked to GSH metabolism as γ-Glu-Glu can be a by-product of glutathione (GSH) breakdown after γ-glutamyl transferase action. Therefore, γ-Glu-Glu could exert excitatory effects by activating neuronal NMDA receptors when GSH production is enhanced.

Glutathione Is Required for the Early Alert Response and Subsequent Acclimation in Cadmium-Exposed Arabidopsis thaliana Plants

Pollution by cadmium (Cd) is a worldwide problem, posing risks to human health and impacting crop yield and quality. Cadmium-induced phytotoxicity arises from an imbalance between antioxidants and pro-oxidants in favour of the latter. The Cd-induced depletion of the major antioxidant glutathione (GSH) strongly contributes to this imbalance. Rather than being merely an adverse effect of Cd exposure, the rapid depletion of root GSH levels was proposed to serve as an alert response. This alarm phase is crucial for an optimal stress response, which defines acclimation later on. To obtain a better understanding on the importance of GSH in the course of these responses and how these are defined by the rapid GSH depletion, analyses were performed in the GSH-deficient cadmium-sensitive 2-1 (cad2-1) mutant. Cadmium-induced root and leaf responses related to oxidative challenge, hydrogen peroxide (H2O2), GSH, ethylene, and aminocyclopropane-1-carboxylic acid (ACC) were compared between wild-type (WT) and mutant Arabidopsis thaliana plants. Although the cad2-1 mutant has significantly lower GSH levels, root GSH depletion still occurred, suggesting that the chelating capacity of GSH is prioritised over its antioxidative function. We demonstrated that responses related to GSH metabolism and ACC production were accelerated in mutant roots and that stress persisted due to suboptimal acclimation. In general, the redox imbalance in cad2-1 mutant plants and the lack of proper transient ethylene signalling contributed to this suboptimal acclimation, resulting in a more pronounced Cd effect.

Phytochelatin Synthase in Heavy Metal Detoxification and Xenobiotic Metabolism

Phytochelatin synthase (PCS) is well-known for its role in heavy metal detoxification in plants, yeasts and non-vertebrate animals. It is a protease-like enzyme that catalyzes glutathione (GSH) to form phytochelatins (PCs), a group of Cys-rich and non-translational polypeptides with a high affinity to heavy metals. In addition, PCS also functions in xenobiotic metabolism by processing GS-conjugates in the cytosol. Because PCS is involved in GSH metabolism and the degradation of GS-conjugates, it is one of the important components in GSH homeostasis and GSH-mediated biodegradation. This chapter reviews the biochemical mechanism of PCS, how the enzyme activity is regulated, and its roles in heavy metal detoxification as well as GS-S-conjugate metabolism. This chapter also highlights the potential applications of PCS in the improvement of plant performance under combined stresses.

Physiological And Proteomic Analyses Reveals That Brassinosteroids Application Improves The Chilling Stress Tolerance of Pepper Seedlings

Brassinosteroids (BRs) are important in plant resistance to chilling stress. However, limited information is available regarding the specific mechanisms involved at proteomic level. We utilized iTRAQ proteomic approach, physiological assays and information obtained from cellular ultrastructure to clarify the underlying molecular mechanism of BRs to alleviate chilling stress in pepper (Capsicum annuum L.). Foliar application of 24-epibrassinolide (EBR) improved photosynthesis and improved cell structure by presenting a distinct mesophyll cell and chloroplast with well-developed thylakoid membranes in the leaves of pepper seedlings. We identified 346 differentially expressed proteins (DEPs), including 217 up-regulated proteins and 129 down-regulated proteins in plants under chilling (Chill) and Chill + EBR treated plants. Most of the DEPs were related to multiple pathways, including photosynthesis, carbohydrates metabolism, energy metabolism, protein biosynthesis, amino acids synthesis, redox and stress defence (ascorbate peroxidase, glutathione peroxidase, and superoxide dismutase). Up-regulated DEPs were associated with photosynthetic electron transfer chain, oxidative phosphorylation, GSH metabolism pathway, Calvin cycle and signaling pathway. The physiochemical analysis showed that EBR treatment improved the tolerance of pepper seedlings to chilling stress.

Tris(methylthio)methane Produced by Mortierella hyalina Affects Sulfur Homeostasis in Arabidopsis

Microbial volatiles are important factors in symbiotic interactions with plants. Mortierella hyalina is a beneficial root-colonizing fungus with a garlic-like smell, and promotes growth of Arabidopsis seedlings. GC-MS analysis of the M. hyalina headspace and NMR analysis of the extracted essential oil identified the sulfur-containing volatile tris(methylthio)methane (TMTM) as the major compound. Its incorporation in seedlings was shown by 34S labeling experiment. Under sulfur deficiency, TMTM downregulated sulfur deficiency-responsive genes, prevented glucosinolate (GSL) and glutathione (GSH) diminishment, and sustained plant growth. However, excess TMTM led to accumulation of GSH and GSL and reduced plant growth. Since TMTM is not directly incorporated into cysteine, we propose that the volatile from M. hyalina influences the plant sulfur metabolism by interfering with the GSH metabolism, and alleviates sulfur imbalances under sulfur stress.

Cystathionine-β-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation

Hyperactivation of oncogenic pathways downstream of RAS and PI3K/AKT in normal cells induces a senescence-like phenotype that acts as a tumor-suppressive mechanism that must be overcome during transformation. We previously demonstrated that AKT-induced senescence (AIS) is associated with profound transcriptional and metabolic changes. Here, we demonstrate that human fibroblasts undergoing AIS display increased Cystathionine-β-synthase (CBS) expression and consequent activation of the transsulfuration pathway controlling hydrogen sulfide (H2S) and glutathione (GSH) metabolism. Activated transsulfuration pathway during AIS maintenance enhances the antioxidant capacity, protecting senescent cells from ROS-induced cell death via GSH and H2S. Importantly, CBS depletion allows cells that have undergone AIS to escape senescence and re-enter the cell cycle, indicating the importance of CBS activity in maintaining AIS. Mechanistically, we show this restoration of proliferation is mediated through suppressing mitochondrial respiration and reactive oxygen species (ROS) production and increasing GSH metabolism. These findings implicate a potential tumor-suppressive role for CBS in cells with inappropriately activated PI3K/AKT signaling. Consistent with this concept, in human gastric cancer cells with activated PI3K/AKT signaling, we demonstrate that CBS expression is suppressed due to promoter hypermethylation. CBS loss cooperates with activated PI3K/AKT signaling in promoting anchorage-independent growth of gastric epithelial cells, while CBS restoration suppresses the growth of gastric tumors in vivo. Taken together, we find that CBS is a novel regulator of AIS and a potential tumor suppressor in PI3K/AKT-driven gastric cancers, providing a new exploitable metabolic vulnerability in these cancers.

Glutathione in the Nervous System as a Potential Therapeutic Target to Control the Development and Progression of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a rare neurological disorder that affects the motor neurons responsible for regulating muscle movement. However, the molecular pathogenic mechanisms of ALS remain poorly understood. A deficiency in the antioxidant tripeptide glutathione (GSH) in the nervous system appears to be involved in several neurodegenerative diseases characterized by the loss of neuronal cells. Impaired antioxidant defense systems, and the accumulation of oxidative damage due to increased dysfunction in GSH homeostasis are known to be involved in the development and progression of ALS. Aberrant GSH metabolism and redox status following oxidative damage are also associated with various cellular organelles, including the mitochondria and nucleus, and are crucial factors in neuronal toxicity induced by ALS. In this review, we provide an overview of the implications of imbalanced GSH homeostasis and its molecular characteristics in various experimental models of ALS.

Essential role for Ggct in erythrocyte antioxidant defense

GGCT encodes gamma-glutamyl cyclotransferase enzyme activity, and its expression is up-regulated in various human cancers. gamma-glutamyl cyclotransferase enzyme activity was originally purified from human red blood cells (RBCs), however physiological function of GGCT in RBCs is still not clear. Here we reported that Ggct deletion in mouse leads to splenomegaly and progressive anemia phenotypes, due to elevated oxidative damage and shortened life span of Ggct-/- RBCs. Ggct-/- RBCs have increased reactive oxygen species (ROS), and are more sensitive to H2O2 induced damage compared to control RBCs. Glutathione (GSH) and GSH synthesis precursor L-cysteine are decreased in Ggct-/- RBCs. Our study suggests a critical function of Ggct in RBC redox balance and life span maintenance through regulating GSH metabolism.

Tris(methylthio)methane Produced by Mortierella hyalina Affects Sulfur Homeostasis in Arabidopsis

Microbial volatiles are important factors in symbiotic interactions with plants. Mortierella hyalina is a beneficial root-colonizing fungus with a garlic-like smell, and promotes growth of Arabidopsis seedlings. GC-MS analysis of the M. hyalina headspace and NMR analysis of the extracted essential oil identified the sulfur-containing volatile tris(methylthio)methane (TMTM) as the major compound. Its incorporation in seedlings was shown by 34S labeling experiment. Under sulfur deficiency, TMTM downregulated sulfur deficiency-responsive genes, prevented glucosinolate (GSL) and glutathione (GSH) diminishment, and sustained plant growth. However, excess TMTM led to accumulation of GSH and GSL and reduced plant growth. Since TMTM is not directly incorporated into cysteine, we propose that the volatile from M. hyalina influences the plant sulfur metabolism by interfering with the GSH metabolism, and alleviates sulfur imbalances under sulfur stress.

The DUG Pathway Governs Degradation of Intracellular Glutathione in Aspergillus nidulans

ABSTRACT Glutathione (GSH) is an abundant tripeptide that plays a crucial role in shielding cellular macromolecules from various reactive oxygen and nitrogen species in fungi. Understanding GSH metabolism is of vital importance for deciphering redox regulation in these microorganisms. In the present study, to better understand the GSH metabolism in filamentous fungi, we investigated functions of the dugB and dugC genes in the model fungus Aspergillus nidulans. These genes are orthologues of dug2 and dug3, which are involved in cytosolic GSH degradation in Saccharomyces cerevisiae. The deletion of dugB, dugC, or both resulted in a moderate increase in the GSH content in mycelia grown on glucose, reduced conidium production, and disturbed sexual development. In agreement with these observations, transcriptome data showed that genes encoding mitogen-activated protein (MAP) kinase pathway elements (e.g., steC, sskB, hogA, and mkkA) or regulatory proteins of conidiogenesis and sexual differentiation (e.g., flbA, flbC, flbE, nosA, rosA, nsdC, and nsdD) were downregulated in the ΔdugB ΔdugC mutant. Deletion of dugB and/or dugC slowed the depletion of GSH pools during carbon starvation. It also reduced accumulation of reactive oxygen species and decreased autolytic cell wall degradation and enzyme secretion but increased sterigmatocystin formation. Transcriptome data demonstrated that enzyme secretions—in contrast to mycotoxin production—were controlled at the posttranscriptional level. We suggest that GSH connects starvation and redox regulation to each other: cells utilize GSH as a stored carbon source during starvation. The reduction of GSH content alters the redox state, activating regulatory pathways responsible for carbon starvation stress responses. IMPORTANCE Glutathione (GSH) is a widely distributed tripeptide in both eukaryotes and prokaryotes. Owing to its very low redox potential, antioxidative character, and high intracellular concentration, GSH profoundly shapes the redox status of cells. Our observations suggest that GSH metabolism and/or the redox status of cells plays a determinative role in several important aspects of fungal life, including oxidative stress defense, protein secretion, and secondary metabolite production (including mycotoxin formation), as well as sexual and asexual differentiations. We demonstrated that even a slightly elevated GSH level can substantially disturb the homeostasis of fungi. This information could be important for development of new GSH-producing strains or for any biotechnologically relevant processes where the GSH content, antioxidant capacity, or oxidative stress tolerance of a fungal strain is manipulated.