Fragment-based discovery of a regulatory site in thioredoxin glutathione reductase acting as "doorstop" for NADPH entry

Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectivity inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases

The effect of vitamin C on the erythrocyte antioxidant enzymes in intoxicated-lead rat offsprings

Objective: Lead exposure or lead poisoning is known to cause a large spectrum of physiological, biochemical, and behavioural disorders in animals. This study was aimed at assessing the effect of vitamin C on the erythrocyte superoxide dismutase, glutathione peroxidase and the glutathione reductase activities in intoxicated- lead rat offsprings. Methods: This study was performed on the pups from female Wistar albino rats. The rats were divided into 4 groups and the treatments were administered through drinking water. Group1 (control group) consumed distilled water. Group 2 (lead group) consumed a solution of lead acetate (300mg/L). Group3 (lead + vitamin C) consumed a solution of lead (300mg/L) which was supplemented with vitamin C (2g/L). Group4 (vitamin group) consumed a solution of vitamin C (2g/L). The enzyme activities were determined in all the 4 groups. Results: The administration of lead showed a decrease in the enzyme activities. The superoxide dismutase activity was increased after the administration of lead in combination with vitamin C. The lead treated rats showed significantly lower body weights at birth and at weaning. The vitamin C treatment showed a significant increase in the body weight. The haemoglobin levels were significantly decreased in the lead-treated rats. The addition of vitamin C to the lead treatment and vitamin C alone could elevate the haemoglobin levels significantly. Conclusion: The results of this study showed that lead alterates the erythrocyte antioxidant enzyme activities. There was an increase in the superoxide dismutase activity following the treatment with vitamin C. This study suggests that the treatment with vitamin C during lactation has a therapeutic effect in the treatment of lead intoxication. The administration of vitamin C prevents haemoglobin reduction in the erythrocytes

Assessment of Oxidative Stress in Peste Des Petits Ruminants (Ovine Rinderpest) Affected Goats

The aim of the present investigation was to evaluate oxidative stress in goats affected with peste des petits ruminants (PPR). The experiment was designed to collect blood samples from PPR affected as well as healthy goats during a series of PPR outbreaks which occurred during February to April 2012 in different districts of Rajasthan state (India). Out of total 202 goats of various age groups and of both the sexes, 155 goats were PPR affected and 47 were healthy. Oxidative stress was evaluated by determining various serum biomarkers viz. vitamin A, vitamin C, vitamin E, glutathione, catalase, superoxide dismutase, glutathione reductase and xanthine oxidase, the mean values of which were 1.71±0.09 µmol L-1, 13.02±0.14 µmol L-1, 2.22±0.09 µmol L-1, 3.03±0.07 µmol L-1, 135.12±8.10 kU L-1, 289.13±8.00 kU L-1, 6.11± 0.06 kU L-1 and 98.12±3.12 mU L-1, respectively. Each parameter analysis of variance showed highly significant effect (P=0.0001) of health status and age category. Further interaction between health status and age category was also highly significant (P=0.0001) for each parameter studied. The results indicated that vitamins A, C, E and glutathione levels depressed by 18.95%, 38.67%, 47.64%, and 47.39%, respectively and the serum catalase, superoxide dismutase, glutathione reductase and xanthine oxidase activities increased by 90.79%, 75.11%, 90.34%, and 44.06%, respectively in affected animals as compared to that in healthy ones. On the basis of the altered levels of serum biomarkers of oxidative stress it was concluded that the animals affected with PPR developed oxidative stress

Time course of oxidative damage in different brain regions following transient cerebral ischemia in gerbils

The time course of oxidative damage in different brain regions was investigated in the gerbil model of transient cerebral ischemia. Animals were subjected to both common carotid arteries occlusion for 5 min. After the end of ischemia and at different reperfusion times (2, 6, 12, 24, 48, 72, 96 h and 7 days), markers of lipid peroxidation, reduced and oxidized glutathione levels, glutathione peroxidase, glutathione reductase, manganese-dependent superoxide dismutase (MnSOD) and copper/zinc containing SOD (Cu/ZnSOD) activities were measured in hippocampus, cortex and striatum. Oxidative damage in hippocampus was maximal at late stages after ischemia (48-96 h) coincident with a significant impairment in glutathione homeostasis. MnSOD increased in hippocampus at 24, 48 and 72 h after ischemia, coincident with the marked reduction in the activity of glutathione-related enzymes. The late disturbance in oxidant-antioxidant balance corresponds with the time course of delayed neuronal loss in the hippocampal CA1 sector. Cerebral cortex showed early changes in oxidative damage with no significant impairment in antioxidant capacity. Striatal lipid peroxidation significantly increased as early as 2 h after ischemia and persisted until 48 h with respect to the sham-operated group. These results contribute significant information on the timing and factors that influence free radical formation following ischemic brain injury, an essential step in determining effective antioxidant intervention

Contribution of Fdh3 and Glr1 to Glutathione Redox State, Stress Adaptation and Virulence in Candida albicans

Acknowledgments: We thank Aaron Mitchell and Dominique Sanglard for providing the C. albicans protein kinase and transposon mutant libraries, and Louise Walker for the strain CAMY203.Peer reviewedPublisher PD

Association of oxidative stress with female infertility - a case control study

Objective: To compare stress markers and reproductive hormones in fertile and infertile females, and to relate the markers with age, duration and cause of infertility, and body mass index..Methods: The case-control study was conducted at Aga Khan University Hospital, Karachi, from March 2017 to February 2018. Females aged 16-50 years regardless of ethnic background were recruited from the Australian Concept Infertility Medical Centre, Karachi, and were equally divided into infertile cases group A, and fertile controls group B. Serum follicular stimulating hormone, luteinizing hormone, estradiol, glutathione reductase and cortisol were measured using enzyme-linked innmunosorbent assay. SPSS 19 was used for statistical analysis..Results: There were 328 female subjects divided into two equal groups of 164(50%). Serum luteinizing hormone and cortisol was higher in the group A than in group B (p\u3c0.001). Serum glutathione reductase was low in group A compared to group B (p\u3c0.001). Duration of infertility, serum levels of glutathione reductase and cortisol were also significantly different among infertile females when distributed on the basis of cause of infertility (p\u3c0.05). Serum cortisol had negative correlation with glutathione reductase (p\u3c0.001). Age and body mass index had a positive correlation with serum cortisol (p=0.035; p=0.63), while there was a negative correlation with glutathione reductase (p = -0.732)..Conclusions: Prolonged duration of infertility, age of females and body mass index enhanced the production of stress hormones and decreased antioxidant activity which augmented the risk of infertility

The evolution of glutathione metabolism in phototrophic microorganisms

The low molecular weight thiol composition of a variety of phototropic microorganisms is examined in order to ascertain how evolution of glutathione (GSH) production is related to the evolution of oxygenic photosynthesis. Cells were extracted in the presence of monobromobimane (mBBr) to convert thiols (RSH) to fluorescent derivatives (RSmB) which were analyzed by high performance liquid chromatography (HPLC). Significant levels of GSH were not found in green sulfur bacteria. Substantial levels were present in purple bacteria, cyanobacteria, and eukaryotic algae. Other thiols measured included cysteine, gamma-glutamylcysteine, thiosulfate, coenzyme A, and sulfide. Many of the organisms also exhibited a marked ability to reduce mBBr to syn-(methyl,methyl)bimane, an ability which was quenched by treatment with 2-pyridyl disulfide or 5,5 prime-bisdithio - (2-nitrobenzoic acid) prior to reaction with mBBr. These observations indicate the presence of a reducing system capable of electron transfer to mBBr and reduction of reactive disulfides. The distribution of GSH in phototropic eubacteria indicates that GSH synthesis evolved at or around the time that oxygenic photosynthesis evolved

Reduction of mitochondrial protein mitoNEET [2Fe-2S] clusters by human glutathione reductase

© 2015 Elsevier Inc. All rights reserved. The human mitochondrial outer membrane protein mitoNEET is a newly discovered target of the type 2 diabetes drug pioglitazone. Structurally, mitoNEET is a homodimer with each monomer containing an N-terminal transmembrane α helix tethered to the mitochondrial outer membrane and a C-terminal cytosolic domain hosting a redox-active [2Fe-2S] cluster. Genetic studies have shown that mitoNEET has a central role in regulating energy metabolism in mitochondria. However, the specific function of mitoNEET remains largely elusive. Here we find that the mitoNEET [2Fe-2S] clusters can be efficiently reduced by Escherichia coli thioredoxin reductase and glutathione reductase in an NADPH-dependent reaction. Purified human glutathione reductase has the same activity as E. coli thioredoxin reductase and glutathione reductase to reduce the mitoNEET [2Fe-2S] clusters. However, rat thioredoxin reductase, a human thioredoxin reductase homolog that contains selenocysteine in the catalytic center, has very little or no activity to reduce the mitoNEET [2Fe-2S] clusters. N-ethylmaleimide, a potent thiol modifier, completely inhibits human glutathione reductase from reducing the mitoNEET [2Fe-2S] clusters, indicating that the redox-active disulfide in the catalytic center of human glutathione reductase may be directly involved in reducing the mitoNEET [2Fe-2S] clusters. Additional studies reveal that the reduced mitoNEET [2Fe-2S] clusters in mouse heart cell extracts can be reversibly oxidized by hydrogen peroxide without disruption of the clusters, suggesting that the mitoNEET [2Fe-2S] clusters may undergo redox transition to regulate energy metabolism in mitochondria in response to oxidative signals

Reduced antioxidant enzyme activity in brains of mice transgenic for human presenilin-1 with single or multiple mutations

Alzheimer's disease-related mutations in the presenilin-1 gene (PS1) are leading to an elevated production of neurotoxic beta-amyloid 1-42 and may additionally enhance oxidative stress. Here, we provide in vivo evidence indicating that brains of transgenic mice expressing different human Alzheimer-linked PS1 mutations exhibit a reduced activity of two antioxidant enzymes. For this purpose, mice transgenic for human PS1 and for single and multiple PS1 mutations were generated. Mice with multiple PS1 mutations showed a significantly decreased activity of the antioxidant enzymes Cu/Zn superoxide dismutase and glutathione reductase already at an age of 3-4 months. As expected, this effect was less pronounced for the mice with a single PS1 mutation. By contrast, animals bearing normal human PS1 showed significantly elevated enzyme activities relative to non-transgenic littermate controls