Function Of Glutathione Peroxidase Research Articles

Selenium-SelK-GPX4 axis protects nucleus pulposus cells against mechanical overloading-induced ferroptosis and attenuates senescence of intervertebral disc

Intervertebral disc degeneration (IVDD) is one of the most prevalent spinal degenerative disorders and imposes places heavy medical and economic burdens on individuals and society. Mechanical overloading applied to the intervertebral disc (IVD) has been widely recognized as an important cause of IVDD. Mechanical overloading-induced chondrocyte ferroptosis was reported, but the potential association between ferroptosis and mechanical overloading remains to be illustrated in nucleus pulposus (NP) cells. In this study, we discovered that excessive mechanical loading induced ferroptosis and endoplasmic reticulum (ER) stress, which were detected by mitochondria and associated markers, by increasing the intracellular free Ca2+ level through the Piezo1 ion channel localized on the plasma membrane and ER membrane in NP cells. Besides, we proposed that intracellular free Ca2+ level elevation and the activation of ER stress are positive feedback processes that promote each other, consistent with the results that the level of ER stress in coccygeal discs of aged Piezo1-CKO mice were significantly lower than that of aged WT mice. Then, we confirmed that selenium supplementation decreased intracellular free Ca2+ level by mitigating ER stress through upregulating Selenoprotein K (SelK) expression. Besides, ferroptosis caused by the impaired production and function of Glutathione peroxidase 4 (GPX4) due to mechanical overloading-induced calcium overload could be improved by selenium supplementation through Se-GPX4 axis and Se-SelK axis in vivo and in vitro, eventually presenting the stabilization of the extracellular matrix (ECM). Our findings reveal the important role of ferroptosis in mechanical overloading-induced IVDD, and selenium supplementation promotes significance to attenuate ferroptosis and thus alleviates IVDD, which might provide insights into potential therapeutic interventions for IVDD.

The functions of glutathione peroxidase in ROS homeostasis and fruiting body development in Hypsizygus marmoreus.

Glutathione peroxidase (GPX) is one of the most important antioxidant enzymes for maintaining reactive oxygen species (ROS) homeostasis. Although studies on fungi have suggested many important physiological functions of GPX, few studies have examined the role of this enzyme in Basidiomycetes, particularly its functions in fruiting body developmental processes. In the present study, GPX-silenced (GPxi) strains were obtained by using RNA interference. The GPxi strains of Hypsizygus marmoreus showed defects in mycelial growth and fruiting body development. In addition, the results indicated essential roles of GPX in controlling ROS homeostasis by regulating intracellular H2O2 levels, maintaining GSH/GSSG balance, and promoting antioxidant enzyme activity. Furthermore, lignocellulose enzyme activity levels were reduced and the mitochondrial phenotype and mitochondrial complex activity levels were changed in the H. marmoreus GPxi strains, possibly in response to impediments to mycelial growth and fruiting body development. These findings indicate that ROS homeostasis has a complex influence on growth, fruiting body development, GSH/GSSG balance, and carbon metabolism in H. marmoreus.Key points• ROS balance, energy metabolism, fruiting development.

GPx6 is involved in the in vitro induced capacitation and acrosome reaction in porcine sperm

Glutathione peroxidases (GPxs) are regarded as important protectors against oxidative stress. Some members of this protein family were reported to play key roles in protecting sperm against oxidative stress. Whether GPx6 a member of the GPx family also plays a role in protection against oxidative stress is not known to date. The objective of the present study was to evaluate the localization and function of glutathione peroxidase 6 (GPx6) in boar accessory sex glands, seminal plasma, and sperm, as well as the effect of GPx6 on vitality and capacitation in boar sperm. qPCR and Western blot analysis demonstrated the presence of GPx6 in testis, epididymis, bulbourethral glands, prostate, seminal vesicle, sperm and seminal plasma. Incubation of sperm with an GPx6 antibody had no significant effect on the viability of boar sperm prior to capacitation. Surprisingly, when capacitated sperm was incubated with the GPx6 antibody for 240 min, sperm vitality was significantly improved. Western blotting showed that in capacitated sperm without prior pretreatment, GPx6 protein content was reduced compared to sperm before capacitation. To further confirm a role for GPx6 in sperm capacitation, we tested sperm acrosome reaction by ACR.2 and FITC-PSA. The results showed that treatment of sperm with the GPx6 antibody significantly increased sperm capacitation and acrosome reaction. Furthermore, we examined the concentration of cAMP in sperm after capacitation. ELISA demonstrated that the cAMP concentration in the sperm exposed to the GPx6 antibody was significantly higher than that of the control group. In addition, the exposure of sperm to the GPx6 antibody significantly increased the concentration of H2O2, while the expression of SOD3 and CAT were decreased. Based on these observations we would like to postulate that in the boar reproductive tract the GPx6 protein becomes attached to the sperm head preventing the sperm to undergo premature capacitation by affecting components of the antioxidant pathway. How GPx6 expression following ejaculation becomes suppressed to allow sperm capacitation to take place needs further investigation.

Lipid Peroxidation and GPX4 Inhibition Are Common Causes for Myofibroblast Differentiation and Ferroptosis.

Ferroptosis is a new form of regulated cell death. Fibroblast-to-myofibroblast differentiation is known to be involved in the pathogenesis of idiopathic pulmonary fibrosis. Utilizing HFL1 cell line treated with transforming growth factor-β1 (TGF-β1), we investigated the relationship between ferroptosis and pulmonary fibrosis, and the function of glutathione peroxidase 4 (GPX4) in them. The results indicated that α-smooth muscle actin and collagen I (COL I) mRNA expression levels increased significantly from 24 h after TGF-β1-treatment, and further rose after TGF-β1+erastin treatment. The levels of reactive oxygen species (ROS), malondialdehyde were increased, and the levels of GPX4 mRNA and protein were reduced after treatment with TGF-β1, and all these were magnified after TGF-β1+erastin treatment. All these changes induced by TGF-β1 and erastin can be recovered by Fer-1 treatment. The cell viability rate was decreased significantly when treated with TGF-β1+erastin, but no obvious variation of cell viability was found in TGF-β1-treated group and in other groups, suggesting that ROS, lipid peroxidation, and GPX4 inhibition are not sufficient conditions for ferroptosis. Collectively, our study reveals that ROS, lipid peroxidation, and GPX4 play important roles in pulmonary fibrosis and ferroptosis induced by erastin. Erastin promoted fibroblast-to-myofibroblast differentiation by increasing lipid peroxidation and inhibiting the expression of GPX4. Fer-1 may inhibit pulmonary fibrosis and ferroptosis through suppressing lipid peroxidation and enhancing GPX4 expression.

Loss of glutathione peroxidase 7 promotes TNF-α-induced NF-κB activation in Barrett’s carcinogenesis

Esophageal adenocarcinoma (EAC) is a classic example of inflammation-associated cancer, which develops through GERD (gastroesophageal reflux disease)-Barrett's esophagus (BE)-dysplasia-adenocarcinoma sequence. The incidence of EAC has been rising rapidly in the USA and Western countries during the last few decades. The functions of glutathione peroxidase 7 (GPX7), an antioxidant enzyme frequently silenced during Barrett's tumorigenesis, remain largely uncharacterized. In this study, we investigated the potential role of GPX7 in regulating nuclear factor-kappaB (NF-κB) activity in esophageal cells. Western blot analysis, immunofluorescence and luciferase reporter assay data indicated that reconstitution of GPX7 expression in CP-A (non-dysplastic BE cells) and FLO-1 (EAC cells) abrogated tumor necrosis factor-α (TNF-α)-induced NF-κB transcriptional activity (P < 0.01) and nuclear translocation of NF-κB-p65 (P = 0.01). In addition, we detected a marked reduction in phosphorylation levels of components of NF-κB signaling pathway, p-p65 (S536), p-IκB-α (S32) and p-IKKα/β (S176/180), as well as significant suppression in induction of NF-κB target genes [TNF-α, interleukin (IL)-6, IL-8, IL-1β, CXCL-1 and CXCL-2] following treatment with TNF-α in GPX7-expressing FLO-1 cells as compared with control cells. We validated these effects by knockdown of GPX7 expression in HET1A (normal esophageal squamous cells). We found that GPX7-mediated suppression of NF-κB is independent of reactive oxygen species level and GPX7 antioxidant function. Further mechanistic investigations demonstrated that GPX7 promotes protein degradation of TNF-receptor 1 (TNFR1) and TNF receptor-associated factor 2 (TRAF2), suggesting that GPX7 modulates critical upstream regulators of NF-κB. We concluded that the loss of GPX7 expression is a critical step in promoting the TNF-α-induced activation of proinflammatory NF-κB signaling, a major player in GERD-associated Barrett's carcinogenesis.

Differential expression and anti‐oxidant function of glutathione peroxidase 3 in mouse uterus during decidualization

Glutathione peroxidase 3 (GPX3) is an important member of antioxidant enzymes for reducing reactive oxygen species and maintaining the oxygen balance. Gpx3 mRNA is strongly expressed in decidual cells from days 5 to 8 of pregnancy. After pregnant mice are treated with GPX inhibitor for 3 days, pregnancy rate is significantly reduced. Progesterone stimulates Gpx3 expression through PR/HIF1α in mouse endometrial stromal cells. In the decidua, the high level of GPX3 expression is closely associated with the reduction of hydrogen peroxide (H2O2). Based on our data, GPX3 may play a major role in reducing H2O2 during decidualization.

Transcriptional regulation of selenium-dependent glutathione peroxidase from Venerupis philippinarum in response to pathogen and contaminants challenge

Glutathione peroxidases (GPx) are key enzymes in the antioxidant systems of living organisms by catalyzing the reduction of peroxides to non-reactive products. In the present study, the full-length cDNA encoding a selenium-dependent GPx was identified from Venerupis philippinarum (designated as VpSe-GPx), and the spatial and temporal expression patterns post- Vibrio anguillarum, heavy metals and benzo[a]pyrene (B[a]P) challenge were also investigated. VpSe-GPx possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase. The VpSe-GPx mRNA was found to be most abundantly expressed in hepatopancreas. Vibrio challenge could significantly up-regulate the mRNA expression of VpSe-GPx, and the highest expression level was detected at 24 h post-infection with 6.5-fold increase compared with that in the control group. For heavy metals exposure, the expression of VpSe-GPx was significantly induced by 20, 40 μg L −1 Cd and 10, 20 μg L −1 Cu but depressed by 10 μg L −1 Cd and 40 μg L −1 Cu. With regards to B[a]P exposure, the expression of VpSe-GPx mRNA was significantly induced at 48 and 96 h post challenge. All these results suggested that VpSe-GPx was potentially involved in mediating the immune response and antioxidant defense in V. Philippinarum.

Multiple roles of microsomal glutathione transferase 1 in cellular protection: A mechanistic study

The aim of this study was to investigate the involvement of membrane-bound microsomal glutathione transferase 1 (MGST1) in cellular resistance against oxidative stress as well as its mechanism of protection. MGST1 is ubiquitously expressed and predominantly located in the endoplasmic reticulum and outer mitochondrial membrane. Utilizing MCF7 cells overexpressing MGST1 we show significant protection against agents that are known to induce lipid peroxidation (e.g., cumene hydroperoxide and tert-butylhydroperoxide) and an end-product of lipid peroxidation (e.g., 4-hydroxy-2-nonenal). Furthermore, our results demonstrate that MGST1 protection can be enhanced by vitamin E when toxicity depends on oxidative stress, but not when direct alkylation is the dominant mechanism. Mitochondria in MGST1-overexpressing cells were shown to be protected from oxidative insult as measured by calcium loading capacity and respiration. MGST1 induces cellular resistance against cisplatin. Here we used vitamin E to elucidate whether oxidative stress caused by cisplatin is significant for cell toxicity. The results indicate that oxidative stress and induction of lipid peroxidation are not the most prominent toxic mechanism of cisplatin in our cell system. We thus conclude that MGST1 protects cells (and mitochondria) by both conjugation and glutathione peroxidase functions. A new protective mechanism against cisplatin is also indicated.

CDNA cloning and mRNA expression of a selenium-dependent glutathione peroxidase from Zhikong scallop Chlamys farreri

The glutathione peroxidases are essential enzymes of the cellular antioxidant defence system. In the present study, the full-length cDNA sequence encoding an extracellular glutathione peroxidase (designated CfGPx3) was isolated from Zhikong scallop Chlamys farreri. The complete cDNA was of 1194 bp, containing a 5' untranslated region (UTR) of 50 bp, a 3' UTR of 490 bp and an open reading frame (ORF) of 654 bp encoding a polypeptide of 217 amino acids. CfGPx3 possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase, such as the selenocysteine encoded by stop codon UGA, the GPx signature motif (96LGVPCNQF103) and the active site motif (179WNFEKF184). The high similarity of CfGPx3 with GPx from other organisms indicated that CfGPx3 should be a new member of the glutathione peroxidase family. By fluorescent quantitative real-time PCR, the CfGPx3 mRNA was universally detected in the tissues of haemocytes, gill, gonad, muscle and hepatopancreas with the highest expression in hepatopancreas. After scallops were challenged by Listonella anguillarum, the expression level of CfGPx3 transcript in haemocytes was significantly up-regulated (P<0.05) at 8h post challenge. These results suggested that CfGPx3 was potentially involved in the immune response of scallops and perhaps contributed to the protective effects against oxidative stress.

Structure, synthesis and function of glutathione peroxidase.

Structure, synthesis and function of glutathione peroxidase.

Function of Glutathione Peroxidase in Endothelial Cell Vitality

The two human umbilical vein endothelial cell-derived lines, ECRF24 and ECV304, differ in responsiveness to oxidative stress. In confluent monolayers of ECRF24, but not in ECV304, peroxides induce stress responses such as plasma membrane blebbing and nuclear condensation. The peroxide effect on ECRF24 was preceded by oxidation of reduced glutathione (GSH) and of NAD(P)H, and by oxidation of the redox-sensitive probe, chloromethyl 2′,7′-dichlorofluorescin (DCFH). In monolayers of ECV304, peroxides induced only minimal oxidation of GSH, NAD(P)H and DCFH, which was associated with a greatly reduced GSH peroxidase activity in these cells. However, in spite of the absence of a blebbing response, ECV304 were more susceptible than ECRF24 to membrane lipid peroxidation and peroxide-induced necrosis. Only for ECV304, the culturing with high levels of polyunsaturated fatty acids increased lipid peroxidation and cellular death. Treatment of these cells with the GSH peroxidase mimic ebselen effectively reversed their decreased vitality. We conclude that, in peroxide-treated endothelial cells, cell death (necrosis) can result from lipid peroxidation by peroxide that has not been removed by GSH peroxidases, whereas extensive peroxidase activity may cause a stress response (blebbing). The data further identify ECV304 as a stress-sensitive cell line, where peroxides exert their effects independently of GSH oxidation.

Blood Superoxide Dismutase, Catalase and Glutathione Peroxidase Activities in Familial and Sporadic Amyotrophic Lateral Sclerosis

Recent studies have implicated free radicals in the pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal, paralytic disorder of motor neurons. Herein we report on measurements of erythrocyte activity of the three main free radical scavenging enzymes: copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase, and glutathione peroxidase. We studied 31 patients with sporadic ALS, 18 with familial ALS, and 24 controls, Mean Cu/Zn-SOD activity was reduced in eight familial ALS patients with mutations of Cu/Zn-SOD but was normal in patients with both familial ALS without identified Cu/Zn-SOD mutations and sporadic ALS. Glutathione peroxidase activity was significantly reduced only in sporadic ALS patients treated with insulin-like growth factor I (100 μg/kg). Catalase activity was normal in sporadic and familial ALS. Neither glutathione peroxidase nor catalase activities correlated significantly with duration of symptoms or age at onset. Vitamin E, vitamin C, and β-carotene did not affect any of the three enzyme activities. These observations indicate that disturbances of catalase and glutathione peroxidase function are not likely to be central factors in the pathogenesis of ALS.

Metabolism of hydrogen peroxide in isolated hepatocytes: Relative contributions of catalase and glutathione peroxidase in decomposition of endogenously generated H 2O 2

The compartmentation of hydrogen peroxide catabolism was studied in isolated hepatocytes. Hydrogen peroxide generation in the peroxisomal compartment was stimulated by addition of glycolate and in the endoplasmic reticular compartment (cytosolic compartment) by ethylmorphine. The rate of catabolism by catalase was estimated from the concentration of methanol required to decrease the steady-state concentration of catalase Compound I to the half-maximal value. The rate of catabolism by glutathione peroxidase was assessed in a semiquantitative manner by the rate of GSSG efflux. The relationship of GSSG efflux to catalase-dependent metabolism of H 2O 2 in the presence of increasing concentrations of glycolate was sigmoidal. This indicates that the function of glutathione peroxidase is small relative to that of catalase at low rates of H 2O 2 production in the peroxisomal fraction, but that the contribution of the former system increases as the peroxisomal H 2O 2 production rate is enhanced, and suggests that the accumulation of a steady-state concentration of H 2O 2 in the nanomolar range in the peroxisomes is sufficient to allow diffusion of H 2O 2 into the cytosol. Following pretreatment of animals with aminotriazole to inhibit catalase, glycolate caused GSSG release at rates nearly double those in control cells. This indicates that even incomplete inhibition of catalase in cells can result in enhanced release of H 2O 2 into the cytosol and demonstrates the relationship of GSSG release to H 2O 2 production under these conditions. An estimate of the rate of H 2O 2 diffusion to catalase during ethylmorphine metabolism was made from the steady-state level of Compound I and measured formate concentrations. This rate increased threefold as the rate of GSH loss increased from 1 to 2 nmol/10 6 cells per min, indicating that as the rate of H 2O 2 production in the endoplasmic reticulum becomes maximally stimulated in the presence of ethylmorphine, the rate of H 2O 2 metabolism by catalase becomes larger. A comparison of ethylmorphine-stimulated rates of GSSG efflux from cells of control and aminotriazole-treated rats shows that, unlike experiments with glycolate, no difference in the rate of efflux is observed. These results support the conclusion that in hepatocytes catalase has a relatively minor role in catabolism of H 2O 2 at low rates of H 2O 2 generation in the endoplasmic reticulum, but that the catalase function increases as the rate of H 2O 2 production is enhanced.