GSH Homeostasis Research Articles

Copper/iron bimetal phenolic networks boosted apoptosis/ferroptosis/cuproptosis combined tumor therapy through dual glutathione depletion

Cuproptosis, ferroptosis, and reactive oxygen species (ROS)-involved apoptosis are all bound by the overexpressed glutathione (GSH) in tumor microenvironment (TME). Herein, a dual GSH depletor is developed based on copper/iron bimetal gallic acid phenolic networks (CuFeGA MPNs) for combination tumor therapy by GSH depletion boosted apoptosis/ferroptosis/cuproptosis simultaneously. The CuFeGA MPNs are dissociated following exposure to the highly expressed GSH, causing the release of large quantities of GA, Fe2+, and Cu+. The released GA then further consumes the residual GSH. In conjunction with the GSH response-consumption behavior, the GSH depletion is achieved by those dual GSH consumption procedures. Subsequently, the expression of glutathione peroxides 4 (GPX4) is down-regulated by GSH depletion for further accumulation of lipid peroxides (LPO). Moreover, intracellular ROS levels are improved via Fenton reaction with released Cu+ and Fe2+, leading to buildup of LPO concurrently. Eventually, excess ROS can activate mitochondria-associated apoptosis, and the surge of LPO initiates ferroptosis without GSH homeostasis. Meanwhile, the oligomerization of lipoylated dihydrolipoamide S-acetyltransferase (DLAT) and decline of iron-sulfur cluster protein are induced by excessive Cu+ in the absence of GSH chelation, activating cuproptosis. This work provides a feasible approach for the realization of multiple mechanisms combined tumor therapy.

Minimizing Oxidative Stress in the Lens: Alternative Measures for Elevating Glutathione in the Lens to Protect against Cataract.

Oxidative stress plays a major role in the formation of the cataract that is the result of advancing age, diabetes or which follows vitrectomy surgery. Glutathione (GSH) is the principal antioxidant in the lens, and so supplementation with GSH would seem like an intuitive strategy to counteract oxidative stress there. However, the delivery of glutathione to the lens is fraught with difficulties, including the limited bioavailability of GSH caused by its rapid degradation, anatomical barriers of the anterior eye that result in insufficient delivery of GSH to the lens, and intracellular barriers within the lens that limit delivery of GSH to its different regions. Hence, more attention should be focused on alternative methods by which to enhance GSH levels in the lens. In this review, we focus on the following three strategies, which utilize the natural molecular machinery of the lens to enhance GSH and/or antioxidant potential in its different regions: the NRF2 pathway, which regulates the transcription of genes involved in GSH homeostasis; the use of lipid permeable cysteine-based analogues to increase the availability of cysteine for GSH synthesis; and the upregulation of the lens's internal microcirculation system, which is a circulating current of Na+ ions that drives water transport in the lens and with it the potential delivery of cysteine or GSH. The first two strategies have the potential to restore GSH levels in the epithelium and cortex, while the ability to harness the lens's internal microcirculation system offers the exciting potential to deliver and elevate antioxidant levels in its nucleus. This is an important distinction, as the damage phenotypes for age-related (nuclear) and diabetic (cortical) cataract indicate that antioxidant delivery must be targeted to different regions of the lens in order to alleviate oxidative stress. Given our increasing aging and diabetic populations it has become increasingly important to consider how the natural machinery of the lens can be utilized to restore GSH levels in its different regions and to afford protection from cataract.

An all-in-one smartphone-assisted ratiometric fluorescent device for visual and quantitative detection of glutathione

The daily consumption of foods abundant in Glutathione (GSH) can be supplemented to maintain the homeostasis of GSH in human health and alleviate pathologies resulting from abnormal GSH levels. The fluorescence-based visual determination of GSH has gradually attracted the attention of researchers due to its robust performance and versatile implementation. However, the current GSH visual strategy primarily relies on variations in fluorescence intensity at a single emission wavelength, which poses challenges for naked-eye and portable readout, as well as distorted signals caused by complex matrix effects in real samples. Herein, a ratiometric fluorescence sensor based on carbon dots (CDs) combined with an all-in-one 3D-printed smartphone-based device was successfully developed for low-cost, visual and rapid detection of GSH without the need for an external excitation light source. The ratiometric fluorescent materials were synthesized by conjugating blue carbon dots (B-CDs) with yellow carbon dots (Y-CDs) through the utilization of selected Cu2+ ions. The resulting mechanism demonstrated that the coordination interaction between Cu2+ and residual aromatic amino groups in Y-CDs (Y-CDs-Cu2+) contributed to a newly emitted peak at 580 nm, thereby inducing fluorescence resonance energy transfer from B-CDs to Y-CDs-Cu2+. A linear correlation was found between GSH concentrations and R/B values in the range of 10–100 μM, with a limit of detection observed at 4.8 μM. By utilizing this portable device in combination with RGB analysis, the quantitative detection of GSH can be achieved even in complex food matrices such as tomatoes and grapes. The universality of this all-in-one device was further validated by pre-spraying CDs onto a paper strip for visual measurement of GSH. This work offers a portable, visual, and accessible approach to evaluating food safety and nutrition, thereby demonstrating significant economic value and holding profound implications for human health.

Optimized Two-Photon Imaging by Stimuli-Responsive Peptide Self-Assembly Facilitates Self-Assisted Counteraction of Cisplatin-Resistance in Cancer Cells.

Accurate diagnosis and effective treatment of tumors remain significant clinical challenges. While fluorescence imaging is essential for tumor detection, it has limitations in terms of specificity, penetration depth, and emission wavelength. Here, we report a novel glutathione (GSH)-responsive peptide self-assembly excimer probe (pSE) that optimizes two-photon tumor imaging and self-assisted counteraction of the cisplatin resistance in cancer cells. The GSH-responsive self-assembly of pSE induces a monomer-excimer transition of coumarin, promoting a near-infrared redshift of fluorescence emission under two-photon excitation. This process enhances penetration depth and minimizes interference from biological autofluorescence. Moreover, the intracellular self-assembly of pSE impacts GSH homeostasis, modulates relevant signaling pathways, and significantly reduces GSTP1 expression, resulting in decreased cisplatin efflux in cisplatin-resistant cancer cells. The proposed self-assembled excimer probe not only distinguishes cancer cells from normal cells but also enhances the efficacy of cisplatin chemotherapy, offering significant potential in tumor diagnosis and overcoming cisplatin-resistant tumors.

Clioquinol rescues yeast cells from Aβ42 toxicity via the inhibition of oxidative damage.

Alzheimer's disease (AD), the most common form of dementia, has gotten considerable attention. Previous studies have demonstrated that clioquinol (CQ) as a metal chelator is a potential drug for the treatment of AD. However, the mode of action of CQ in AD is still unclear. In our study, the antioxidant effects of CQ on yeast cells expressing Aβ42 were investigated. We found that CQ could reduce Aβ42 toxicity by alleviating reactive oxygen species (ROS) generation and lipid peroxidation level in yeast cells. These alterations were mainly attributable to the increased reduced glutathione (GSH) content and independent of activities of superoxide dismutase (SOD) and/or catalase (CAT). CQ could affect antioxidant enzyme activity by altering the transcription level of related genes. Interestingly, it was noted for the first time that CQ could combine with antioxidant enzymes to reduce their enzymatic activities by molecular docking and circular dichroism spectroscopy. In addition, CQ restored Aβ42-mediated disruption of GSH homeostasis via regulating YAP1 expression to protect cells against oxidative stress. Our findings not only improve the current understanding of the mechanism of CQ as a potential drug for AD treatment but also provide ideas for subsequent drug research and development.

Bag1 protein loss sensitizes mouse embryonic fibroblasts to glutathione depletion.

Bcl2-associated athanogene-1 protein (Bag1) acts as a co-chaperone of heat shock protein 70 and heat shock cognate 70 and regulates multiple cellular processes, including cell proliferation, apoptosis, environmental stress response, and drug resistance. Since Bag1 knockout mice exhibited fetal lethality, the in vivo function of Bag1 remains unclear. In this study, we established a mouse line expressing Bag1 gene missing exon 5, which corresponds to an encoding region for the interface of heat shock protein 70/heat shock cognate 70. Despite mice carrying homoalleles of the Bag1 mutant (Bag1Δex5) expressing undetectable levels of Bag1, Bag1Δex5 homozygous mice developed without abnormalities. Bag1Δex5 protein was found to be highly unstable in cells and in vitro. We found that the growth of mouse embryonic fibroblasts derived from Bag1Δex5-homo mice was attenuated by doxorubicin and a glutathione (GSH) synthesis inhibitor, buthionine sulfoximine. In response to buthionine sulfoximine, Bag1Δex5-mouse embryonic fibroblasts exhibited a higher dropping rate of GSH relative to the oxidized glutathione level. In addition, Bag1 might mitigate cellular hydrogen peroxide levels. Taken together, our results demonstrate that the loss of Bag1 did not affect mouse development and that Bag1 is involved in intracellular GSH homeostasis, namely redox homeostasis.

Development of an Inhibition-Based Colorimetric Method For Glutathione Determination

Objectives: Glutathione (GSH, L-γ-glutamyl-L-cysteinyl-glycine), one of the major cellular antioxidants, is an important non-protein intracellular physiological antioxidant with sulphhydryl groups for detoxification of reactive oxygen species (ROS) in all living organisms. GSH deficiency has been shown to be associated with many human diseases, including cardiovascular, immune and ageing diseases, arthritis and diabetes. Therefore, the development of an accurate, reliable and sensitive method for the determination of GSH in biological fluids is essential for the understanding of GSH homeostasis in medicine and biochemical research Material and Methods: In this study, a very inexpensive, practical, rapid, sensitive, and highly specific colorimetric method for the determination of glutathione (GSH) that can be detected by the naked eye was developed. This method is based on the inhibition of horseradish peroxidase (HRP) by GSH. As the concentration of glutathione increases, a pink coloured compound consisting of 4-chlorophenol, H2O2 and 4-aminoantipyrine (4-AAP) decomposes as a result of the reaction catalyzed by HRP, thus reducing the intensity of the colour. Results: While the linear range of the developed method was found to be between 15.6-1000 mM, the intra- and inter-day repeatability % coefficient of variation values of the method were less than 15%. The effect of potential interfering substances on the developed method was tested, and no interference was found, except for cysteine. Cysteine increased GSH response by 10%. The developed method was used for the determination of GSH in commercial serum samples, and results were obtained between 91-106%. Conclusion: In conclusion, this study has developed a very simple, inexpensive and unique colourimetric method for the determination of GSH.

Time of day differences in the regulation of glutathione levels in the rat lens.

Evidence in non-ocular tissues indicate that the antioxidant glutathione (GSH) may be regulated in a circadian manner leading to the idea that GSH levels in the lens may also be controlled in a circadian manner to anticipate periods of oxidative stress. Male rat Wistar lenses (6 weeks) were collected every 4 hours over a 24-hour period at 6am, 10am, 2pm, 6pm, 10pm and 2am and quantitative-PCR, western blotting and immunohistochemistry performed to examine the expression of core clock genes and proteins (BMAL1, CLOCK, CRY1-2, PER 1-3) and their subcellular localisation over a 24-hour period. Western blotting of lenses was also performed to examine the expression of NRF2, a transcription factor involved in regulating genes involved in GSH homeostasis and GSH related enzymes (GCLC, GS and GR) over the 24-hour period. Finally, HLPC was used to measure GSH levels in the aqueous humour and lenses every 4 hours over a 24-hour period. The rat lens contains the core molecular components of a circadian clock with the expression of core clock proteins, NRF2 and GSH related enzymes fluctuating over a 24-hour period. BMAL1 expression was highest during the day, with BMAL1 localised to the nuclei at 10am. NRF2 expression remained constant over the 24-hour period, although appeared to move in and out of the nuclei every 4 hours. GSH related enzyme expression tended to peak at the start of night which correlated with high levels of GSH in the lens and lower levels of GSH in the aqueous humour. The lens contains the key components of a circadian clock, and time-of-day differences exist in the expression of GSH and GSH related enzymes involved in maintaining GSH homeostasis. GSH levels in the rat lens were highest at the start of night which represents the active phase of the rat when high GSH levels may be required to counteract oxidative stress induced by cellular metabolism. Future work to directly link the clock to regulation of GSH levels in the lens will be important in determining whether the clock can be used to help restore GSH levels in the lens.

Overexpression of gamma-glutamyl cyclotransferase 2;1 (CsGGCT2;1) reduces arsenic toxicity and accumulation in Camelina sativa (L.).

Overexpressing CsGGCT2;1 in Camelina enhances arsenic tolerance, reducing arsenic accumulation by 40-60%. Genetically modified Camelina can potentially thrive on contaminated lands and help safeguard food quality and sustainable food and biofuel production. Environmental arsenic contamination is a serious global issue that adversely affects human health and diminishes the quality of harvested produce. Glutathione (GSH) is known to bind and detoxify arsenic and other toxic metals. A steady level of GSH is maintained within cells via the γ-glutamyl cycle. The γ-glutamyl cyclotransferases (GGCTs) have previously been shown to be involved in GSH degradation and increased tolerance to toxic metals in plants. In this study, we characterized the GGCT2;1 homolog from Camelina sativa for its role in arsenic tolerance and accumulation. Overexpression of CsGGCT2;1 in Camelina under CaMV35S constitutive promoter resulted in strong tolerance to arsenite (AsIII). The overexpression (OE) lines had 2.6-3.5-fold higher shoots and sevenfold to tenfold enhanced root biomass on media supplemented with AsIII, relative to wild-type plants. The CsGGCT2;1 OE lines accumulated 40-60% less arsenic in root and shoot tissues compared to wild-type plants. Further, the OE lines had ~ twofold higher chlorophyll content and 35% lesser levels of malondialdehyde (MDA), an indicator of membrane damage via lipid peroxidation. There was a slight but non-significant increase in 5-oxoproline (5-OP), a product of GSH degradation, in OE lines. However, the transcript levels of Oxoprolinase 1 (OXP1) were upregulated, indicating the accelerated conversion of 5-OP to glutamate, which is further utilized for the resynthesis of GSH to maintain GSH homeostasis. Overall, this research suggests that genetically modified Camelina may have the potential for cultivation on contaminated marginal lands to reduce As accumulation; thereby could help in addressing food safety issues as well as future food and biofuel needs.

Unraveling the In Vitro Toxicity Profile of Psychedelic 2C Phenethylamines and Their N-Benzylphenethylamine (NBOMe) Analogues.

Mescaline derivative (2C phenethylamines) drugs have been modified by the introduction of a N-2-methoxybenzyl group to originate a new series of compounds with recognized and potent psychedelic effects, the NBOMe-drugs. Although they are prevalent in unregulated drug markets, their toxicity profile is still poorly understood, despite several reports highlighting cases of acute intoxication, with brain and liver toxicity. Thus, in this study, mescaline, 2C-N (insertion of a nitro in the para position of the 2C phenethylamines aromatic ring) and 2C-B (insertion of a bromide in the para position of the 2C phenethylamines aromatic ring) and their corresponding NBOMe counterparts, mescaline-NBOMe, 25N-NBOMe and 25B-NBOMe, were synthetized and the in vitro neuro- and hepatocytotoxicity evaluated in differentiated SH-SY5Y and HepG2 cell lines, respectively. Cytotoxicity, oxidative stress, metabolic and energetic studies were performed to evaluate the main pathways involved in their toxicity. Our results demonstrated that the presence of the N-2-methoxybenzyl group significantly increased the in vitro cytotoxicity of 2C phenethylamines drugs in both cell lines, with the NBOMe drugs presenting lower EC50 values when compared to their counterparts. Consistently, our data showed a correlation between the drug's lipophilicity and the EC50 values, except for 2C-B. The 2C-B presented higher cytotoxic effects in both cell lines than mescaline-NBOMe, a result that can be explained by its higher passive permeability. All the NBOMe derivatives were able to cross the blood-brain barrier. Considering metabolic studies, the cytotoxicity of these drugs was shown to be influenced by inhibition of cytochrome P450 (CYP), which suggests a potential role of this enzyme complex, especially CYP3A4 and CYP2D6 isoenzymes in SH-SY5Y cells, in their detoxification or bioactivation. Furthermore, in differentiated SH-SY5Y cells, the drugs were able to induce mitochondrial membrane depolarization, and to disrupt GSH and ATP intracellular levels, these effects being concentration dependent and more pronounced for the NBOMe derivatives. No ROS overproduction was detected for any of the drugs in the tested experimental conditions. A correlation between a drug's lipophilicity and the EC50 values in both cell lines, except for 2C-B, was also obtained. In summary, the introduction of a NBOMe moiety to the parent drugs significantly increases their lipophilicity, brain permeability and cytotoxic effects, with GSH and ATP homeostasis disruption. The inhibition of CYP3A4 and CYP2D6 emphasized that CYP-mediated metabolism impacts the toxicity of these drugs.

A comprehensive assessment of redox balance in small for gestational age newborns and their mothers.

The objective of this study was to assess oxidative stress in small for gestational age (SGA) newborns and their mothers by evaluating intra- and extracellular thiol homeostasis and the quantification of major oxidants and antioxidants. A total of 75 mothers and their 75 newborns (43 SGA) were enrolled in this study. Thiol-disulfide homeostasis, serum myeloperoxidase, catalase, total oxidant, and antioxidant status were analyzed. Additionally, erythrocytic glutathione (GSH) homeostasis was measured. Although native and total thiol levels were decreased, disulfide levels were increased in SGA groups. Additionally, myeloperoxidase activity and total oxidant status levels were significantly elevated whereas total antioxidant status levels and enzymatic antioxidant systems were diminished in SGA groups. Similarly, intra-erythrocytic GSH homeostasis was shifted in favor of oxidants in SGA groups. Our results demonstrate that insufficient antioxidant systems in mothers and a robust source of oxidative stress in SGA might contribute to the pathophysiology of SGA births.

Isotope tracing reveals bacterial catabolism of host-derived glutathione during Helicobacter pylori infection.

Mammalian cells synthesize the antioxidant glutathione (GSH) to shield cellular biomolecules from oxidative damage. Certain bacteria, including the gastric pathogen Helicobacter pylori, can perturb host GSH homeostasis. H. pylori infection significantly decreases GSH levels in host tissues, which has been attributed to the accumulation of reactive oxygen species in infected cells. However, the precise mechanism of H. pylori-induced GSH depletion remains unknown, and tools for studying this process during infection are limited. We developed an isotope-tracing approach to quantitatively monitor host-derived GSH in H. pylori-infected cells by mass spectrometry. Using this method, we determined that H. pylori catabolizes reduced GSH from gastric cells using γ-glutamyl transpeptidase (gGT), an enzyme that hydrolyzes GSH to glutamate and cysteinylglycine (Cys-Gly). gGT is an established virulence factor with immunomodulatory properties that is required for H. pylori colonization in vivo. We found that H. pylori internalizes Cys-Gly in a gGT-dependent manner and that Cys-Gly production during H. pylori infection is coupled to the depletion of intracellular GSH from infected cells. Consistent with bacterial catabolism of host GSH, levels of oxidized GSH did not increase during H. pylori infection, and exogenous antioxidants were unable to restore the GSH content of infected cells. Altogether, our results indicate that H. pylori-induced GSH depletion proceeds via an oxidation-independent mechanism driven by the bacterial enzyme gGT, which fortifies bacterial acquisition of nutrients from the host. Additionally, our work establishes a method for tracking the metabolic fate of host-derived GSH during infection.

A locally activatable sensor for robust quantification of organellar glutathione.

Glutathione (GSH) is the main determinant of intracellular redox potential and participates in multiple cellular signalling pathways. Achieving a detailed understanding of intracellular GSH homeostasis depends on the development of tools to map GSH compartmentalization and intra-organelle fluctuations. Here we present a GSH-sensing platform for live-cell imaging, termed targetable ratiometric quantitative GSH (TRaQ-G). This chemogenetic sensor possesses a unique reactivity turn-on mechanism, ensuring that the small molecule is only sensitive to GSH in a desired location. Furthermore, TRaQ-G can be fused to a fluorescent protein to give a ratiometric response. Using TRaQ-G fused to a redox-insensitive fluorescent protein, we demonstrate that the nuclear and cytosolic GSH pools are independently regulated during cell proliferation. This sensor was used in combination with a redox-sensitive fluorescent protein to quantify redox potential and GSH concentration simultaneously in the endoplasmic reticulum. Finally, by exchanging the fluorescent protein, we created a near-infrared, targetable and quantitative GSH sensor.

Characterization of glutathione-specific gamma glutamyl cyclotransferase (ChaC) in Bombyx mori.

Glutathione (GSH) contributes to redox maintenance and detoxification of various xenobiotic and endogenous substances. γ-glutamyl cyclotransferase (ChaC) is involved in GSH degradation. However, the molecular mechanism underlying GSH degradation in silkworms (Bombyx mori) remains unknown. Silkworms are lepidopteran insects that are considered to be an agricultural pest model. We aimed to examine the metabolic mechanism underlying GSH degradation mediated by B. mori ChaC and successfully identified a novel ChaC gene in silkworms (herein, bmChaC). The amino acid sequence and phylogenetic tree revealed that bmChaC was closely related to mammalian ChaC2. We overexpressed recombinant bmChaC in Escherichia coli, and the purified bmChaC showed specific activity toward GSH. Additionally, we examined the degradation of GSH to 5-oxoproline and cysteinyl glycine via liquid chromatography-tandem mass spectrometry. Quantitative real-time polymerase chain reactionrevealed that bmChaC mRNA expression was observed in various tissues. Our results suggest that bmChaC participates in tissue protection via GSH homeostasis. This study provides new insights into the activities of ChaC and the underlying molecular mechanisms that can aid the development of insecticides to control agricultural pests.

Glutathione in cancer progression and chemoresistance: an update

Glutathione (GSH) is one of the most important components of the cellular antioxidant system, and it is able to exert several pleiotropic functions influencing cell growth, proliferation, adaptation and death. It has been demonstrated that changes in GSH levels underlie the pathogenesis of many human diseases, including cancer. In detail, although on one hand GSH homeostasis plays a protective role from the onset of cancer, on the other, it is involved in cancer progression and in therapy resistance. In this review, after a brief report on the physiological role of GSH, we have focused the attention on its role in cancer and refractoriness to anticancer therapy giving an update on the preclinical and clinical studies relied on the compounds targeting GSH system. Based on these considerations, a deeper knowledge of GSH-dependent network can be crucial to identify new strategies for preventing and/or curing cancer.

Silicon Palliates Chromium Toxicity through the Formation of Root Hairs in Rice (Oryza sativa) Mediated by GSH and IAA.

Along with the rapidly increasing environmental contamination by heavy metals, the exposure of plants to chromium has also magnified, resulting in a declined productivity. Hexavalent chromium [Cr(VI)], the most toxic form of Cr, brings about changes in plant processes at morpho-physiological and biochemical levels. However, silicon (Si) is known to mitigate the impact of abiotic stresses in plants. Here, we demonstrate Si-mediated alleviation of Cr(VI) toxicity and its effects on root hair formation in rice seedlings. Reduced glutathione (GSH) and indole-3 acetic acid (IAA, an important auxin) were assessed for their involvement in root hair formation after the application of Si to Cr(VI)-stressed plants, and our results confirmed their crucial significance in such developmental processes. The expression analysis of genes involved in GSH biosynthesis (OsGS2) and regeneration (OsGR1), and auxin biosynthesis (OsTAA1 and OsYUCCA1) and transport (OsAUX1 and OsPIN1) corroborated their positive role in Si-mediated root hair formation in Cr(VI)-stressed rice seedlings. Moreover, the results indicated that nitric oxide (NO) seems a probable but not fundamental component in Si-mediated formation of roots in rice during exposure to Cr(VI) stress. In this study, the indispensable role of GSH and IAA, redox homeostasis of GSH and IAA biosynthesis and transport are discussed with regard to Si-mediated formation of root hairs in rice under Cr(VI) stress. The results of the study suggest that Si is a protective agent against Cr(VI) stress in rice, and the findings can be used to develop Cr(VI) stress-tolerant varieties of rice with enhanced productivity.

The ChaC family of γ-glutamyl cyclotransferases is required for Leishmania to switch to a slow growth state and for long-term survival of the parasite

The ChaC family of γ-glutamyl cyclotransferases is conserved throughout all Kingdoms and catalyzes the degradation of GSH. So far, the ChaC family proteins in trypanosomal parasites are missing in the literature. Here, we report two members of the ChaC family of γ-glutamyl cyclotransferases (LmChaC2a and LmChaC2b) in the unicellular pathogen Leishmania. Activity measurements suggest that these proteins catalyze degradation of GSH but no other γ-glutamyl peptides. Recombinant LmChaC2a protein shows ∼17-fold lower catalytic efficiency (kcat ∼ 0.9 s−1) than LmChaC2b (kcat ∼ 15 s−1), although they showed comparable Km values (∼1.75 mM for LmChaC2a and ∼2.0 mM for LmChaC2b) toward GSH. qRT-PCR and Western blot analyses suggest that the LmChaC2a protein was found to be constitutively expressed, whereas LmChaC2b was regulated by sulfur stress. To investigate its precise physiological function in Leishmania, we generated overexpressed, knockout, and complement cell lines. Flow cytometric analyses show the presence of a higher intracellular GSH concentration and lower intracellular ROS level, indicative of a more reductive environment in null mutants. We found LmChaC2-expressing cells grow in GSH-containing sulfur-limited media, while the null mutants failed to grow, suggesting that LmChaC2 is crucial for cell growth with GSH as the only sulfur source. Null mutants, although reach the stationary phase rapidly, display impaired long-term survival, indicating that LmChaC2-mediated GSH degradation is necessary for prolonged survival. In vivo studies suggest that LmChaC2-dependent controlled GSH degradation promotes chronic infection by the parasite. Altogether, these data indicate that LmChaC2 plays an important role in GSH homeostasis in Leishmania.

An unexpected discovery toward argon-rich water amelioration of cadmium toxicity in Medicago sativa L.

Argon has organ-protective effects on animals. However, whether or how argon influences plant responses remains elusive. In this study, we discovered that the growth inhibition of hydroponically cultured alfalfa seedlings under 100 μM CdCl2 condition was significantly ameliorated by 100 % saturated argon-rich water (ARW). Less Cd uptake and accumulation were also observed in both root and shoot parts, which could be explained by the modified root cell walls, including the increased cell wall thickness, lignin content, and demethylation degree of covalently bound and ion-bound pectin, as well as the down-regulated expression of natural-resistance-associated-macrophage protein1 (Nramp1) encoding a heavy metal ion transporter in root tissue. The hindered Cd translocation from root to shoot achieved by ARW addition was validated by the decreased expression of heavy metal ATPase 2/4 (HMA2/4) in roots and decreased Cd content in xylem saps. The reestablished glutathione (GSH) homeostasis and redox balance, two important indicators of plant defense against Cd poisoning, were also observed. Further greenhouse experiments demonstrated that the phenotypic and physiological performances of alfalfa plants cultured in Cd-contaminated soil were significantly improved by irrigating with ARW. Above results implied that ARW confers plants tolerance against cadmium toxicity by impairing Cd uptake and accumulation and restoring GSH and redox homeostasis. These findings might open a new window for understanding argon biology in higher plants.

MITOL/MARCH5 determines the susceptibility of cardiomyocytes to doxorubicin-induced ferroptosis by regulating GSH homeostasis

MITOL/MARCH5 is an E3 ubiquitin ligase that plays a crucial role in the control of mitochondrial quality and function. However, the significance of MITOL in cardiomyocytes under physiological and pathological conditions remains unclear.First, to determine the significance of MITOL in unstressed hearts, we assessed the cellular changes with the reduction of MITOL expression by siRNA in neonatal rat primary ventricular cardiomyocytes (NRVMs). MITOL knockdown in NRVMs induced cell death via ferroptosis, a newly defined non-apoptotic programmed cell death, even under no stress conditions. This phenomenon was observed only in NRVMs, not in other cell types. MITOL knockdown markedly reduced mitochondria-localized GPX4, a key enzyme associated with ferroptosis, promoting accumulation of lipid peroxides in mitochondria. In contrast, the activation of GPX4 in MITOL knockdown cells suppressed lipid peroxidation and cell death. MITOL knockdown reduced the glutathione/oxidized glutathione (GSH/GSSG) ratio that regulated GPX4 expression. Indeed, the administration of GSH or N-acetylcysteine improved the expression of GPX4 and viability in MITOL-knockdown NRVMs. MITOL-knockdown increased the expression of the glutathione-degrading enzyme, ChaC glutathione-specific γ-glutamylcyclotransferase 1 (Chac1). The knockdown of Chac1 restored the GSH/GSSG ratio, GPX4 expression, and viability in MITOL-knockdown NRVMs.Further, in cultured cardiomyocytes stressed with DOX, both MITOL and GPX4 were reduced, whereas forced-expression of MITOL suppressed DOX-induced ferroptosis by maintaining GPX4 content. Additionally, MITOL knockdown worsened vulnerability to DOX, which was almost completely rescued by treatment with ferrostatin-1, a ferroptosis inhibitor. In vivo, cardiac-specific depletion of MITOL did not produce obvious abnormality, but enhanced susceptibility to DOX toxicity. Finally, administration of ferrostatin-1 suppressed exacerbation of DOX-induced myocardial damage in MITOL-knockout hearts.The present study demonstrates that MITOL determines the cell fate of cardiomyocytes via the ferroptosis process and plays a key role in regulating vulnerability to DOX treatment.(288/300)

IL-36γ and IL-36Ra Reciprocally Regulate NSCLC Progression by Modulating GSH Homeostasis and Oxidative Stress-Induced Cell Death.

The balance between antioxidants and reactive oxygen species (ROS) critically regulates tumor initiation and progression. However, whether and how the tumor‐favoring redox status is controlled by cytokine networks remain poorly defined. Here, it is shown that IL‐36γ and IL‐36Ra reciprocally regulate the progression of non‐small cell lung cancer (NSCLC) by modulating glutathione metabolism and ROS resolution. Knockout, inhibition, or neutralization of IL‐36γ significantly inhibits NSCLC progression and prolongs survival of the Kras LSL‐G12D/+ Tp53 fl/fl and Kras LSL‐G12D/+ Lkb1 fl/fl mice after tumor induction, whereas knockout of IL‐36Ra exacerbates tumorigenesis in these NSCLC mouse models and accelerates death of mice. Mechanistically, IL‐36γ directly upregulates an array of genes involved in glutathione homeostasis to reduce ROS and prevent oxidative stress‐induced cell death, which is mitigated by IL‐36Ra or IL‐36γ neutralizing antibody. Consistently, IL‐36γ staining is positively and negatively correlated with glutathione biosynthesis and ROS in human NSCLC tumor biopsies, respectively. These findings highlight essential roles of cytokine networks in redox for tumorigenesis and provide potential therapeutic strategy for NSCLC.