Glutathione-dependent Peroxidase Research Articles

Peroxiredoxin 6 (Prx6) of Penaeus vannamei and effect of phenanthrene on Prx6 and glutathione peroxidase 4 expression, glutathione-dependent peroxidase activity and lipid peroxidation

Polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene (PHE), are common pollutants found in coastal areas where shrimp farming is developed. Even though PAHs can have adverse effects on physiology, shrimp can detoxify and metabolize toxic compounds and neutralize the reactive oxygen species (ROS) produced during this process. This requires the activation of multiple antioxidant enzymes, including peroxiredoxin 6 (Prx6). Prx6 uses glutathione (GSH) to reduce phospholipid hydroperoxides, a function shared with GSH peroxidase 4 (GPx4). Prx6 has been scarcely studied in crustaceans exposed to pollutants. Herein, we report a novel Prx6 from the shrimp Penaeus vannamei that is abundantly expressed in gills and hepatopancreas. To elucidate the involvement of Prx6 in response to PAHs, we analyzed its expression in the hepatopancreas of shrimp sub-lethally exposed to PHE (3.3 μg/L) and acetone (control) for 24, 48, 72, and 96 h, along with GPx4 expression, GSH-dependent peroxidase activity, and lipid peroxidation (indicated by TBARS). We found that GPx4 expression is not affected by PHE, but Prx6 expression and peroxidase activity decreased during the trial. This might contribute to the rise of TBARS found at 48 h of exposure. However, maintaining GPx4 expression could aid to minimize lipid damage during longer periods of exposure to PHE.

Mn(II)-Aloe-Emodin Nanoscale Coordination Polymer Enhances Ferroptosis by Synergistically Enhancing Reactive Oxygen Species Generation via the Nrf2-GPX4 Axis.

Ferroptosis induction is particularly promising for cancer therapy when the apoptosis pathway is compromised. Current strategies in nanomedicine for inducing ferroptosis primarily focus on promoting the accumulation of reactive oxygen species (ROS). However, the presence of intracellular antioxidants, such as nuclear factor erythroid 2-related factor 2 (Nrf2), can limit the effectiveness of such therapy by activating detoxification systems and eliminating ROS. To overcome this challenge, we developed a synergistic ferroptosis-inducing agentby modifying manganese (Mn2+)-1,8-dihydroxy-3-hydroxymethyl-anthraquinone (aloe-emodin, AE) with polyvinyl pyrrolidone (PVP) to create nanoparticles (MAP NPs). In the tumor microenvironment, these NPs degraded and released AE and Mn(II), facilitating the generation of ROS and Mn(IV) through a Fenton-like reaction between hydrogen peroxide (H2O2) and Mn(II). Mn(IV) subsequently interacts with glutathione (GSH) to induce a cyclic catalytic effect, and the depletion of GSH diminished the activation of glutathione-dependent peroxidase 4 (GPX4). Furthermore, AE inhibits the activity of Nrf2 and depleted GSH, thereby synergistically enhancing antitumor efficacy. Here it is demonstrated that MAP NPs effectively generate a robust ROS storm within tumor cells, suggesting that high-performance ferroptosis therapy is effective. Additionally, the inclusion of Mn(II) in the MAP NPs enables real-time monitoring of therapeutic efficacy via magnetic resonance T1-weighted contrast imaging.

High-frequency repetitive transcranial magnetic stimulation protects against cerebral ischemia/reperfusion injury in rats: Involving the mitigation of ferroptosis and inflammation.

Repetitive transcranial magnetic stimulation (rTMS) has been found to attenuate cerebral ischemia/reperfusion (I/R) injury. However, its effects and mechanism of action have not yet been clarified. It has been reported that cerebral I/R injury is closely associated not only with ferroptosis but also with inflammation. Hence, the current study aimed to investigate whether high-frequency rTMS attenuates middle cerebral artery occlusion (MCAO)-induced cerebral I/R injury and further to elucidate the mediatory role of ferroptosis and inflammation. The protective effects of rTMS on experimental cerebral I/R injury were investigated using transient MCAO model rats. Neurological scores and pathological changes of cerebral ischemic cortex were assessed to evaluate the effects of rTMS on cerebral I/R injury. The involvement of ferroptosis and that of inflammation were examined to investigate the mechanism underlying the effects of rTMS. High-frequency rTMS remarkably rescued the MCAO-induced neurological deficits and morphological damage. rTMS treatment also increased the mRNA and protein expression of glutathione-dependent peroxidase 4, decreased the mRNA and protein levels of acyl-CoA synthetase long-chain family member 4 and transferrin receptor in the cortex. Moreover, rTMS administration reduced the cerebrospinal fluid IL-1β, IL-6, and TNF-α concentrations. These findings implicated that high-frequency rTMS alleviates MCAO-induced cerebral I/R injury, and the underlying mechanism could involve the inhibition of ferroptosis and inflammation. Our study identifies rTMS as a promising therapeutic agent for the treatment of cerebral I/R injury. Moreover, the mechanistic insights into ferroptosis and inflammation advance our understanding of it as a potential therapeutic target for diseases beyond cerebral ischemia stroke.

Degradable iron-rich mesoporous dopamine as a dual-glutathione depletion nanoplatform for photothermal-enhanced ferroptosis and chemodynamic therapy

Glutathione (GSH) is a crucial factor in limiting the effects of chemodynamic therapy (CDT) and ferroptosis, an iron-based cell death pathway. Based on this, we constructed iron-rich mesoporous dopamine (MPDA@Fe) nanovehicles with a dual-GSH depletion function by combining MPDA and Fe. Poly (ethylene glycol) (PEG) was further modified to provide desirable stability (PM@Fe) and glucose oxidase (GOx) was grafted onto PM@Fe (GPM@Fe) to address the limitation of hydrogen peroxide (H2O2). After the nanoparticles reached the tumor site, the weakly acidic microenvironment promoted the release of Fe. Then FeII reacted with H2O2 to generate hydroxyl radical (OH) and FeIII. The generated FeIII was reduced to FeII by GSH, which circularly participated in the Fenton reaction and continuously produced tumor inhibitory free radicals. Meanwhile, GOx consumed glucose to provide H2O2 for the reaction. MPDA had also been reported to deplete GSH. Therefore, dual consumption of GSH led to the destruction of intracellular redox balance and inhibition of glutathione-dependent peroxidase 4 (GPX4) expression, resulting in an increase in lipid peroxides (LPO) and further induction of ferroptosis. Additionally, MPDA-mediated photothermal therapy (PTT) raised the temperature of tumor area and produced photothermal-enhanced cascade effects. Hence, the synergistic strategy that combined dual-GSH depletion-induced ferroptosis, enhanced CDT and photothermal cascade enhancement based on MPDA@Fe could provide more directions for designing nanomedicines for cancer treatment.

Polypyrrole-iron phosphate-glucose oxidase-based nanocomposite with cascade catalytic capacity for tumor synergistic apoptosis-ferroptosis therapy

Utilizing the synergistic effect of apoptosis and ferroptosis for tumor elimination is a promising and effective strategy. Herein, a novel PPy-FePO-GOx-PVA nanocomposite with cascade catalytic capacity was designed and fabricated for tumor synergistic apoptosis-ferroptosis therapy. This nanocomposite was consisted of poly(vinyl alcohol)-stabilized polypyrrole (PPy-PVA) core, iron phosphate (FePO) payload, and glucose oxidase (GOx) decoration. The tumor microenvironment (TME) can be reshaped by this nanocomposite through a cascade catalytic process. Abundant gluconic acid and H2O2 are generated in TME during the GOx-mediated glucose depletion process, which could promote the release of iron ions from FePO and elevate the efficiency of the consequent Fenton reaction. A mass of toxic ·OH was produced to oxidize lipids to lipid hydroperoxides, driving tumor cells into ferroptosis pathway. The redox reaction between Fe3+ ions and glutathione can provide Fe2+ ions, consume glutathione, and further inactive glutathione-dependent peroxidase 4, promoting the efficiency of ferroptosis by destroying redox balance. Furthermore, hyperthermia can be generated by PPy component under 1064 nm laser irradiation, causing tumor cell apoptosis. In vivo data reveal that effective tumor elimination is achieved by this PPy-FePO-GOx-PVA nanocomposite under laser irradiation through the synergistic apoptosis-ferroptosis strategy.

Ferroptosis and Its Multifaceted Roles in Cerebral Stroke.

Ferroptosis is a unique regulated cell death defined by the intracellular iron overload and distinct biological features compared with other well-known programmed cell death. Ferroptosis can be triggered by many causes including decreased expression of glutathione (GSH), inhibition of the function of glutathione-dependent peroxidase 4 (GPX4), and system xc–, all of which finally lead to the over-accumulation of lipid peroxides in the cell. Ferroptosis has been reported to play an important role in the pathophysiological process of various cancers. In recent years, much evidence also proved that ferroptosis is involved in the progress of cerebral stroke. In this review, we summarized the characteristics of ferroptosis and the potential relationship between ferroptosis and ischemic and hemorrhagic stroke, to provide new targets and ideas for the therapy of stroke.

PSMA-targeted arsenic nanosheets: a platform for prostate cancer therapy via ferroptosis and ATM deficiency-triggered chemosensitization.

Ferroptosis, a newly recognized form of non-apoptotic cell death, has recently been introduced for effective cancer therapy. The reported ferroptosis-inducing nanomaterials mainly consisted of metal-based components. Herein, we designed an inorganic metal-free nanoplatform, PSMA-targeted arsenic nanosheets (PMANs), which simultaneously increased glutathione (GSH) consumption, suppressed solute carrier family 7 member 11 (SLC7A11) and glutathione-dependent peroxidase 4 (GPX4) expression, and promoted the generation of reactive oxygen species (ROS) and lipid peroxides (LPO). In addition, owing to the large surface area, PMANs efficiently transported doxorubicin (DOX) to prostate cancer for synergistic therapy. Surprisingly, we found that PMANs could sensitize prostate cancer cells to DOX through downregulating the expression of ataxia telangiectasia mutated (ATM), which further augmented the GPX4 downregulation-mediated ferroptotic tumoricidal effect. Given that arsenic trioxide has been routinely and successfully used in the clinical treatment of leukemia for a long time, we anticipate that PMANs will offer a promising strategy for prostate cancer therapy.

Ethanolic extract of Hyptis mutabilis (Rich.) Briq.: An effective sedative and antioxidant agent in fish

The goal of this study was to evaluate the anesthetic potential of an ethanolic extract of Hyptis mutabilis and its effects on behavior, biochemistry, ionic balance, and oxidative stress biomarkers of silver catfish (Rhamdia quelen) after 24-h exposure. Additionally, its chemical composition was determined by high performance liquid chromatography coupled with a diode array detector and tandem mass spectrometry (HPLC-DAD-MS/MS) to identify its bioactive compounds. The concentration range of 50–500 mg L−1 induced only partial loss of equilibrium (14–2 min) in fish. There was quick recovery (about 5 min) with 50 and 100 mg L−1, while not all animals recovered within 30 min exposure to the higher concentrations. Fish exposed to 100 mg L−1 maintained a sedative level during the long-term experiment. There were lower plasma glucose and lactate levels in fish exposed to the extract compared with the control group (water). There were net Na+ and K+ influxes after exposure to the extract, while Cl− effluxes were observed in all groups (treated or not). Ammonia excretion decreased in animals exposed to extract, and creatinine elimination did not change. Fish exposed to 100 mg L−1 showed higher catalase and glutathione S-transferase activities in the kidney, while glutathione-dependent peroxidase activity increased in the liver. Lipid peroxidation in the kidney did not differ among experimental groups, while there were lower levels in the liver of juveniles exposed to the extract. There was no mortality or side effects in any of the experiments. Rosmarinic acid (53.76 ± 0.13 mg g−1 extract) and rutin (5.75 ± 0.28 mg g−1 extract) were identified in the extract based on standards. In conclusion, the ethanolic extract of H. mutabilis is a promising fish sedative at 100 mg L−1. Its use for long-term exposure showed a good safety margin and ability to prevent stress. Hence, it can be proposed for use in the transport of aquatic animals.

Discovery of GPX4 inhibitor by molecular docking simulation as a potential ferroptosis inducer

As one of the most complex diseases in the world, cancer continues as one of the significant public health problems. It was recorded by 2014 that cancer caused 1,551,000 death in Indonesia. One type of programmed cell death (PCD) that played a role in cancer cell treatment is Ferroptosis. Ferroptosis is PCD on iron and characterized by the inactivation of glutathione-dependent peroxidase (GPx4). In this research, a new therapeutic strategy for cancer was developed through the computational approach on synthetic compounds to discover its potential as an inhibitor of GPx4. About 688 compounds derivative from mercaptosuccinic acid acquired from the Zinc15 database. These compounds screened through the Lipinski’s Rule of Three and pharmacological prediction to eliminate ligands with undesired molecular properties. After that, the ligands underwent both rigid and flexible molecular docking simulations to predict their inhibition activity toward GPx4. From molecular docking simulation, (2S)-2-[(Z)-3-phenylprop-2-enyl]sulfanylbutanedioic acid show favorable characteristics as a drug candidate.

Mechanisms of ferroptosis and its applications in cancers

As a regulated cell death, ferroptosis is different from apoptosis, necrosis and autophagy. Its particularity is lipid peroxide accumulation and mitochondrial size reduction. There are many inducers and inhibitors to regulate the ferroptosis process by regulating the production of reactive oxygen species and the reduction of glutathione-dependent peroxidase 4, which play important roles in ferroptosis. In normal tissues, ferroptosis is characterized by aging and degenerative changes, while in cancer tissues, ferroptosis can be inhibited in many ways, thus promoting the growth of tumors. At present, several drugs have been used to promote ferroptosis in cancer cells, and the combination of traditional therapies is expected to become a new treatment mode. Key words: Ferroptosis; Inhibitor; Inducer; Caner therapy

The Potential Role of Ferroptosis in Neonatal Brain Injury.

Ferroptosis is an iron-dependent form of cell death that is characterized by early lipid peroxidation and different from other forms of regulated cell death in terms of its genetic components, specific morphological features, and biochemical mechanisms. Different initiation pathways of ferroptosis have been reported, including inhibition of system Xc-, inactivation of glutathione-dependent peroxidase 4, and reduced glutathione levels, all of which ultimately promote the production of reactive oxygen species, particularly through enhanced lipid peroxidation. Although ferroptosis was first described in cancer cells, emerging evidence now links mechanisms of ferroptosis to many different diseases, including cerebral ischemia and brain hemorrhage. For example, neonatal brain injury is an important cause of developmental impairment and of permanent neurological deficits, and several types of cell death, including iron-dependent pathways, have been detected in the process of neonatal brain damage. Iron chelators and erythropoietin have both shown neuroprotective effects against neonatal brain injury. Here, we have summarized the potential relation between ferroptosis and neonatal brain injury, and according therapeutic intervention strategies.

Ferroptosis as a Novel Therapeutic Target for Friedreich's Ataxia.

Friedreich ataxia (FRDA) is a progressive neuro- and cardio-degenerative disorder characterized by ataxia, sensory loss, and hypertrophic cardiomyopathy. In most cases, the disorder is caused by GAA repeat expansions in the first introns of both alleles of the FXN gene, resulting in decreased expression of the encoded protein, frataxin. Frataxin localizes to the mitochondrial matrix and is required for iron-sulfur-cluster biosynthesis. Decreased expression of frataxin is associated with mitochondrial dysfunction, mitochondrial iron accumulation, and increased oxidative stress. Ferropotosis is a recently identified pathway of regulated, iron-dependent cell death, which is biochemically distinct from apoptosis. We evaluated whether there is evidence for ferroptotic pathway activation in cellular models of FRDA. We found that primary patient-derived fibroblasts, murine fibroblasts with FRDA-associated mutations, and murine fibroblasts in which a repeat expansion had been introduced (knockin/knockout) were more sensitive than normal control cells to erastin, a known ferroptosis inducer. We also found that the ferroptosis inhibitors ethyl 3-(benzylamino)-4-(cyclohexylamino)benzoate (SRS11-92) and ethyl 3-amino-4-(cyclohexylamino)benzoate, used at 500 nM, were efficacious in protecting human and mouse cellular models of FRDA treated with ferric ammonium citrate (FAC) and an inhibitor of glutathione synthesis [L-buthionine (S,R)-sulfoximine (BSO)], whereas caspase-3 inhibitors failed to show significant biologic activity. Cells treated with FAC and BSO consistently showed decreased glutathione-dependent peroxidase activity and increased lipid peroxidation, both hallmarks of ferroptosis. Finally, the ferroptosis inhibitor SRS11-92 decreased the cell death associated with frataxin knockdown in healthy human fibroblasts. Taken together, these data suggest that ferroptosis inhibitors may have therapeutic potential in FRDA.

Gamma-tocopherol ameliorates hyperglycemia-induced hepatic inflammation associated with NLRP3 inflammasome in alloxan-induced diabetic mice.

BACKGROUND/OBJECTIVESHyperglycemia-induced hepatic damage has been recognized as one of the major cause of complications in diabetes. Hepatic complications are associated with inflammation and oxidative stress in diabetes. In this study, we investigated the hypothesis that gamma-tocopherol (GT) supplementation ameliorates NLRP3 inflammasome associated hepatic inflammation in diabetes.MATERIALS/METHODSDiabetes was induced by the intraperitoneal injection of alloxan (150 mg/kg. BW) in ICR mice. All mice were fed with a control diet (AIN-76A). After diabetes was induced (fasting glucose level ≥ 250 mg/dL), the mice were treated with tocopherol-stripped corn oil or GT-supplemented (35 mg/kg) corn oil, respectively, by gavage for 2 weeks.RESULTSGT supplementation reduced fasting blood glucose levels in diabetic mice relative to non-treated diabetic mice. Moreover, GT supplementation ameliorated hyperglycemia-induced hepatic damage by regulation of NOD-like receptor protein 3 (NLRP3)-inflammasome associated inflammation represented by NLRP3, apoptosis-associated speck-like protein containing a caspase-recruitment domain, caspase-1, nuclear factor-κB pathway as well as oxidative stress demonstrated by nuclear factor erythroid 2-related factor 2, NAD(P)H dehydrogenase quinone 1, catalase and glutathione-dependent peroxidase in diabetic mice.CONCLUSIONThe findings suggested that GT supplementation ameliorated hepatic damage by attenuating inflammation and oxidative stress in alloxan-induced diabetic mice. Taken together, GT could be a beneficial nutrient that can ameliorate inflammatory responses associated with NLRP3 inflammasome in hyperglycemia-induced hepatic damage.

Rosmarinic acid protects mice from lipopolysaccharide/d-galactosamine-induced acute liver injury by inhibiting MAPKs/NF-κB and activating Nrf2/HO-1 signaling pathways

Rosmarinic acid (RA) has antioxidation, anticancer, antibacterial, anti-inflammatory and various biological functions. In our study, we aim to evaluate effects of RA on acute liver injury caused by LPS and d-galactosamine (d-GalN) and its underlying molecular mechanism in mice. Our findings showed that RA could protect C57BL/6 mice from LPS/d-GalN-induced acute liver injury, which not only reflected on declining aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of the serum, but also restrained the phosphorylation of nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinase (ERK1/2) and p38 protein expression and the content of tissue myeloperoxidase (MPO) elevation. Moreover, RA could enhance the level of glutathione-dependent peroxidase (GSH-PX). Furthermore, RA promoted that nuclear factor erythroid-2-related factor 2 (Nrf2) transported into nucleus, and then up-regulated heme oxygenase 1 (HO-1), glutamate-cysteine ligase catalytic (GCLC), glutamate cysteine ligase modifier (GCLM) and quinone oxidoreductase (NQO1). These results indicated that RA could protect the mice from acute liver injury induced by LPS/d-GalN.

A combined biochemical and computational studies of the rho-class glutathione s-transferase sll1545 of Synechocystis PCC 6803

Peroxides are one of the most important radicals that cause oxidative stress. Certain Glutathione S-transferases (GSTs) have been reported to show peroxidase activity. We report a novel peroxidase activity of Synechocystis GST- sll1545. The recombinant protein was purified to homogeneity and characterized. Low Km (0.109μM) and high Vmax (0.663μmolmin−1) values suggest a high preference of sll1545 for cumenehydroperoxide. Disc inhibition assay confirmed the ability of the enzyme to protect cells against peroxide-induced damage. sll1545 has very low sequence and structural similarity with theta and alpha class GSTs that exhibit glutathione-dependent peroxidase activity. Recent data from our laboratory shows that sll1545 is also strongly active against dichloroacetate (DCA), which is a characteristic of zeta class GST. Interestingly, sll1545 shows less than 20% sequence identity with zeta class GST. Molecular dynamic simulation results show that sll1545 was much more structurally different from alpha/theta classes. Our results suggest that sll1545 shows structural variation from zeta, theta/alpha classes of GSTs but have related enzymatic activity. Phylogenetic analysis reveal that sll1545 is evolutionally very distinct from the known GSTs. Overall, the data suggest that Synechocystis sll1545 does not belong to any known GST class and represent a novel GST class, which we have named rho.

Functional classification and biochemical characterization of a novel rho class glutathione S-transferase in Synechocystis PCC 6803

We report a novel class of glutathione S-transferase (GST) from the model cyanobacterium Synechocystis PCC 6803 (sll1545) which catalyzes the detoxification of the water pollutant dichloroacetate and also shows strong glutathione-dependent peroxidase activity representing the classical activities of zeta and theta/alpha class respectively. Interestingly, sll1545 has very low sequence and structural similarity with these classes. This is the first report of dichloroacetate degradation activity by any bacterial GST. Based on these results we classify sll1545 to a novel GST class, rho. The present data also indicate potential biotechnological and industrial applications of cyanobacterial GST in dichloroacetate-polluted areas.

Exogenous hydrogen peroxide enhanced the thermotolerance of Festuca arundinacea and Lolium perenne by increasing the antioxidative capacity

Heat stress is one of the most detrimental environment stresses for plants. Hydrogen peroxide (H2O2) is produced quickly in response to various stresses and likely plays a positive role in transmitting stress signal in organisms. This investigation addressed whether an exogenous H2O2 application would affect the heat response of turfgrasses and induce acclimation. Tall fescue (Festuca arundinacea cv. Barlexas) and perennial ryegrass (Lolium perenne cv. Accent), two important cool-season turfgrasses and forages, were sprayed with 10 mM H2O2 before they were treated with heat stress (38/30 °C, day/night) and compared with plants maintained at control temperatures (26/15 °C, day/night). Prior to the initiation of heat stress, H2O2 pretreatment increased the activities of guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and glutathione-dependent peroxidase (GPX) and the ascorbate and glutathione pool, and it decreased the GSH/GSSG ratio. During the heat stress process, pretreated plants from both grasses exhibited higher turfgrass quality and relative water content, and they experienced lower oxidative damage and H2O2 levels. Moreover, the activities of APX, GR, GPX and glutathione-S-transferase increased significantly in response to H2O2 pretreatment under heat stress. These results suggested that H2O2 most likely participated in the transduction of redox signaling and induced the antioxidative defense system, including various enzymatic and nonenzymatic H2O2 scavengers. The scavengers played important roles in improving the thermotolerance of tall fescue and perennial ryegrasses.

Antioxidant system efficiently protects goldfish gills from Ni2+-induced oxidative stress

Fish gills are target organs for waterborne metal ions and this work aimed to investigate the effects of waterborne Ni2+ (10, 25 and 50mgL−1) on goldfish gills. A special focus was on the relationship between Ni uptake and the homeostasis of reactive oxygen species (ROS) in the gills, the tissue, in direct contact with the metal pollutant. Ni-accumulation in the gills occurred as a function of exposure concentration (R2=0.98). The main indices of oxidative stress, namely carbonyl proteins (CP) and lipid peroxides (LOOH), decreased by 21–33% and 21–24%, as well as the activities of principal antioxidant enzymes superoxide dismutase and glutathione-dependent peroxidase, by 29–47% and 41–46%, respectively, in gills of Ni-exposed fish. One of the main players in the antioxidant defense of gills seems to be catalase, which increased by 23–53% in Ni-treated fish, and low molecular mass thiol-containing compounds (L-SH), exceeding untreated controls by 73–105% after fish exposure to 10–50mgL−1 of Ni2+. The increased level of L-SH, mainly represented by reduced glutathione, was supported by enhanced activities of glutathione reductase (by 27–38%), glutathione-S-transferase (56–141%) and glucose-6-phosphate dehydrogenase (by 96–117%) and demonstrates the ability of the antioxidant system of gills to resist Ni-induced oxidative stress.

A novel glutaredoxin domain-containing peroxiredoxin ‘All1541’ protects the N2-fixing cyanobacterium Anabaena PCC 7120 from oxidative stress

Prxs (peroxiredoxins) are ubiquitous thiol-based peroxidases that detoxify toxic peroxides. The Anabaena PCC 7120 genome harbours seven genes/ORFs (open reading frames) which have homology with Prxs. One of these (all1541) was identified to encode a novel Grx (glutaredoxin) domain-containing Prx by bioinformatic analysis. A recombinant N-terminal histidine-tagged All1541 protein was overexpressed in Escherichia coli and purified. Analysis with the protein alkylating agent AMS (4-acetamido-4'-maleimidyl-stilbene-2,2'-disulfonate) showed All1541 to form an intra-molecular disulfide bond. The All1541 protein used glutathione (GSH) more efficiently than Trx (thioredoxin) to detoxify H(2)O(2). Deletion of the Grx domain from All1541 resulted in loss of GSH-dependent peroxidase activity. Employing site-directed mutagenesis, the cysteine residues at positions 50 and 75 were identified as peroxidatic and resolving cysteine residues respectively, whereas both the cysteine residues within the Grx domain (positions 181 and 184) were shown to be essential for GSH-dependent peroxidase activity. On the basis of these data, a reaction mechanism has been proposed for All1541. In vitro All1541 protein protected plasmid DNA from oxidative damage. In Anabaena PCC 7120, all1541 was transcriptionally activated under oxidative stress. Recombinant Anabaena PCC 7120 strain overexpressing All1541 protein showed superior oxidative stress tolerance to H(2)O(2) as compared with the wild-type strain. The results suggest that the glutathione-dependent peroxidase All1541 plays an important role in protecting Anabaena from oxidative stress.

Cloning of a novel glutathione S-transferase 3 ( GST3) gene and expressionanalysis in pearl oyster, Pinctada martensii

Microsomal glutathione S-transferase (MGST) functions in cellular defense against xenobiotics and provides protection against the action of lipid hydroperoxides produced as a consequence of oxidative stress. In this study, a full-length cDNA encoding MGST3 (referred to as PmMGST3) was identified from the pearl oyster, Pinctada martensii by a combination of expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA of PmMGST3 is 971 bp and contains a 5′ UTR of 39 bp, a 3′ UTR of 491 bp with a canonical polyadenylation signal sequence (AATAAA), and an open reading frame (ORF) of 447 bp encoding a polypeptide of 146 residues. The deduced polypeptide contains a conserved motif (FNCx 1QRx 2H) characteristic of the MGST3 subfamily. The PmMGST3 transcript could be detected in all tissues tested, with highest transcript level seen in hepatopancreas. Cadmium treatment significantly increased PmMGST3 mRNA levels in gill and hepatopancreas, while bacterial challenge initially depressed mRNA levels and then increased its level in haemocytes, gill and hepatopancreas in a time-dependent manner. In an assay using cumene hydroperoxide as a substrate, we demonstrated that PmMGST3 possesses glutathione-dependent peroxidase activity. These results suggest that PmMGST3 plays an important role in cellular defense against oxidative stress caused by cadmium and bacteria.