Dihydroxyacetone-induced death is accompanied by advanced glycation endproduct formation in selected proteins of Saccharomyces cerevisiae and Caenorhabditis elegans

Advanced glycation endproduct (AGE) formation is an important mechanism for protein deterioration during diabetic complications and ageing. The effects on AGE formation following dihydroxyacetone (DHA) stress were studied in two model organisms, the yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans. Total protein AGEs, detected using an anti-N(epsilon)-carboxyalkyllysine-specific monoclonal antibody, displayed a strong correlation to DHA-induced yeast cell mortality in the wild-type and hypersensitive as well as resistant mutant strains. During DHA-induced cell death we also detected AGEs as the formation of acidic protein modifications by 2-D PAGE. Furthermore, we confirmed AGE targets immunologically on 2-D gel-separated protein extracted from DHA-treated cells. AGE modification of several metabolic enzymes (Eno2p, Adh1p, Met6 and Pgk1p) and actin (Act1p) displayed a strong correlation to DHA-induced cell death. DHA was toxic to C. elegans even at low concentration and also in this organism AGE formation accompanied death. We propose the use of DHA as a model AGE-generating substance for its apparent lack of a clear oxidative stress connection, and yeast and worm as model organisms to identify genetic determinants of protein AGE formation.     

Glutathione peroxidase 3 of Saccharomyces cerevisiae regulates the activity of methionine sulfoxide reductase in a redox state-dependent way

Glutathione peroxidase (Gpx) is one of the most important anti-oxidant enzymes in yeast. Gpx3 is a ubiquitously expressed isoform that modulates the activities of redox-sensitive thiol proteins, particularly those involved in signal transduction pathways and protein translocation. In order to search for the interaction partners of Gpx3, we carried out immunoprecipitation/2-dimensional gel electrophoresis (IP-2DE), MALDI-TOF mass spectrometry, and a pull down assay. We found that Mxr1, a peptide methionine sulfoxide reductase, interacts with Gpx3. By reducing methionine sulfoxide to methionine, Mxr1 reverses the inactivation of proteins caused by the oxidation of critical methionine residues. Gpx3 can interact with Mxr1 through the formation of an intermolecular disulfide bond. When oxidative stress is induced by H(2)O(2), this interaction is compromised and the free Mxr1 then repairs the oxidized proteins. Our findings imply that this interaction links redox sensing machinery of Gpx3 to protein repair activity of Mxr1. Based on these results, we propose that Gpx3 functions as a redox-dependent exquisite regulator of the protein repair activity of Mxr1.     

Glutathione peroxidase 2 in Saccharomyces cerevisiae is distributed in mitochondria and involved in sporulation

Gpx2, one of three glutathione peroxidase homologs (Gpx1, Gpx2, and Gpx3) in Saccharomyces cerevisiae, is an atypical 2-Cys peroxiredoxin that prefers to use thioredoxin as a reducing agent in vitro. Despite Gpx2 being an antioxidant, no obvious phenotype of gpx2Δ mutant cells in terms of oxidative stress has yet been found. To gain a clue as to Gpx2’s physiological function in vivo, here we identify its intracellular distribution. Gpx2 was found to exist in the cytoplasm and mitochondria. In mitochondria, Gpx2 was associated with the outer membrane of the cytoplasmic-side, as well as the inner membrane of the matrix-side. The redox state of the mitochondrial Gpx2 was regulated by Trx1 and Trx2 (cytoplasmic thioredoxin), and by Trx3 (mitochondrial matrix thioredoxin). In addition, we found that the disruption of GPX2 reduced the sporulation efficiency of diploid cells.     

Interactome analysis of yeast glutathione peroxidase 3

Oxidative stress damages all cellular constituents, and therefore, cell has to possess various defense mechanisms to cope. Saccharomyces cerevisiae, widely used as a model organism for studying cellular responses to oxidative stress, contains three glutathione peroxidase (Gpx) proteins. Among them, Gpx3 plays a major defense role against oxidative stress in S. cerevisiae. In this study, in order to identify the new interaction proteins of Gpx3, we carried out two-dimensional gel electrophoresis after immunoprecipitation (IP-2DE), and MALDI-TOF mass spectrometry. The results showed that several proteins including protein disulfide isomerase, glutaredoxin 2, and SSY protein 3 specifically interact with Gpx3. These findings led us to suggest the possibility that Gpx3, known as a redox sensor and ROS scavenger, has another functional role by interacting with several proteins with various cellular functions.     

Gpx4 protects mitochondrial ATP generation against oxidative damage

Mitochondrial ATP production can be impaired by oxidative stress. Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme found in mitochondria as well as other subcellular organelles that directly detoxifies membrane lipid hydroperoxides. To determine if Gpx4 protects ATP production in vivo, we compared mitochondrial ATP production between wild-type mice and Gpx4 transgenic mice using a diquat model. Diquat (50 mg/kg) significantly decreased mitochondrial ATP synthesis in livers of wild-type mice; however, no decrease in mitochondrial ATP synthesis was detected in Gpx4 transgenic mice after diquat. We observed no differences in activities of mitochondrial respiratory chain complexes between Gpx4 transgenic mice and wild-type mice. However, compared to wild-type mice, diquat-induced loss of mitochondrial membrane potential was attenuated in Gpx4 transgenic mice. Therefore, our results indicate that decreased ATP production under oxidative stress is primarily due to reduced mitochondrial membrane potential and overexpression of Gpx4 maintains mitochondrial membrane potential under oxidative stress.     

Inhibition of CYP2E1 leads to decreased advanced glycated end product formation in high glucose treated ADH and CYP2E1 over-expressing VL-17A cells

Background

In recent years, there has been a growing interest to explore the association between liver injury and diabetes. Advanced glycated end product (AGE) formation which characterizes diabetic complications is formed through hyperglycemia mediated oxidative stress and is itself a source for ROS. Further, in VL-17A cells over-expressing ADH and CYP2E1, greatly increased oxidative stress and decreased viability have been observed with high glucose exposure.

Methods

In VL-17A cells treated with high glucose and pretreated with the different inhibitors of ADH and CYP2E1, the changes in cell viability, oxidative stress parameters and formation of AGE, were studied.

Results

Inhibition of CYP2E1 with 10 μM diallyl sulfide most effectively led to decreases in the oxidative stress and toxicity as compared with ADH inhibition with 2 mM pyrazole or the combined inhibition of ADH and CYP2E1 with 5 mM 4-methyl pyrazole. AGE formation was decreased in VL-17A cells when compared with HepG2 cells devoid of the enzymes. Further, AGE formation was decreased to the greatest extent with the inhibitor for CYP2E1 suggesting that high glucose inducible CYP2E1 and the consequent ROS aid AGE formation.

Conclusions

Thus, CYP2E1 plays a pivotal role in the high glucose induced oxidative stress and toxicity in liver cells as observed through direct evidences obtained utilizing the different inhibitors for ADH and CYP2E1.

General significance

The study demonstrates the role of CYP2E1 mediated oxidative stress in aggravating hyperglycemic insult and suggests that CYP2E1 may be a vital component of hyperglycemia mediated oxidative injury in liver.     

Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice

Exercise is an effective approach for primary and secondary prevention of cardiovascular diseases (CVD) and loss of muscular mass and function. Its benefits are widely documented but incompletely characterized. It has been reported that exercise can induce changes in the expression of antioxidant enzymes including Sod2, Trx1, Prdx3 and Gpx1 and limits the rise in oxidative stress commonly associated with CVD. These enzymes can be subjected to epigenetic regulation, such as DNA methylation, in response to environmental cues. The aim of our study was to determine whether in the early stages of atherogenesis, in young severely dyslipidemic mice lacking LDL receptors and overexpressing human ApoB100 (LDLR^-/-; hApoB^+/+), exercise regulates differentially the expression of antioxidant enzymes by DNA methylation in the skeletal muscles that consume high levels of oxygen and thus generate high levels of reactive oxygen species. Expression of Sod2, Txr1, Prdx3 and Gpx1 was altered by 3 months of exercise and/or severe dyslipidemia in 6-mo dyslipidemic mice. Of these genes, only Gpx1 exhibited changes in DNA methylation associated with dyslipidemia and exercise: we observed both increased DNA methylation with dyslipidemia and a transient decrease in DNA methylation with exercise. These epigenetic alterations are found in the second exon of the Gpx1 gene and occur alongside with inverse changes in mRNA expression. Inhibition of expression by methylation of this specific locus was confirmed in vitro. In conclusion, Gpx1 expression in the mouse skeletal muscle can be altered by both exercise and dyslipidemia through changes in DNA methylation, leading to a fine regulation of free radical metabolism.     

Glutathione peroxidase 3 of Saccharomyces cerevisiae suppresses non-enzymatic proteolysis of glutamine synthetase in an activity-independent manner

Glutathione peroxidase 3 (Gpx3) is ubiquitously expressed and is important antioxidant enzyme in yeast. It modulates the activities of redox-sensitive thiol proteins, particularly those involved in signal transduction pathway and protein translocation. Through immunoprecipitation/two-dimensional gel electrophoresis (IP-2DE), MALDI-TOF mass spectrometry, and a pull down assay, we found glutamine synthetase (GS; EC 6.3.1.2) as a candidate interacting protein with Gpx3. GS is a key enzyme in nitrogen metabolism and ammonium assimilation. It has been known that GS is non-enzymatically cleaved by ROS generated by MFO (thiol/ Fe(3+)/O(2) mixed-function oxidase) system. In this study, it is demonstrated that GS interacts with Gpx3 through its catalytic domain both in vivo and in vitro regardless of redox state. In addition, Gpx3 helps to protect GS from inactivation and degradation via oxidative stress in an activity-independent manner. Based on the results, it is suggested that Gpx3 protects GS from non-enzymatic proteolysis, thereby contributing to cell homeostasis when cell is exposed to oxidative stress.     

Fragmentation of dihydroxyacetone kinase 1 from Saccharomyces cerevisiae indicates a two-domain structure

Global protein expression in Saccharomyces cerevisiae strains either deleted for both yeast dihydroxyacetone kinases (DAK1 and DAK2) or overexpressing DAK1, was characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). We found protein expression in the double deletion strain to be highly similar to wild-type. In the strain overexpressing Dak1p, nine spots representing fragments of the Dak1p protein in the size range 40-20 kDa and amounting to approximately 30% of total Dak1p, were discovered (native size Dak1p migrates at roughly 60 kDa). Fragments were characterized by matrix-assisted laser desorption/ionization mass spectrometry and electrospray mass spectrometry analyses to represent either the N- or the C-terminal part of the DAK1 protein. Cleavage points, predicted from mass spectrometry and 2-D PAGE data, mapped almost exclusively in the middle region showing low sequence conservation between Dak1p and its closest homologues. We hypothesize that observed Dak1p fragments represent stable structural domains shielded from access by native endoproteases. Furthermore, overexpressing Dak1p with the non-native N-terminus (M)A-, resulted in native size Dak1p and N-terminal Dak1p fragments appearing in two major 2-D PAGE forms of approximately equal size and abundance, but with slightly different isoelectric points. However, when overexpressing Dak1p with the native N-terminus (M)S-, only the more acidic 2-D PAGE form appeared. In the N-terminal acetyltransferase mutant nat1delta, (M)A-Dak1p species were converted into the basic form, arguing twin spots to represent forms with acetylated and deacetylated N-termini. Data thus indicated that (M)A-N-termini, in the Dak1p context, were NatA substrates recognized with 50% lower efficiency than (M)S-N-termini.     

Proteomic Analysis of the Effects of Aged Garlic Extract and Its FruArg Component on Lipopolysaccharide-Induced Neuroinflammatory Response in Microglial Cells

Aged garlic extract (AGE) is widely used as a dietary supplement, and is claimed to promote human health through anti-oxidant/anti-inflammatory activities with hypolipidemic, antiplatelet and neuroprotective effects. Prior studies of AGE have mainly focused on its organosulfur compounds, with little attention paid to its carbohydrate derivatives, such as N-α-(1-deoxy-D-fructos-1-yl)-L-arginine (FruArg). The goal of this study is to investigate actions of AGE and FruArg on antioxidative and neuroinflammatory responses in lipopolysaccharide (LPS)-activated murine BV-2 microglial cells using a proteomic approach. Our data show that both AGE and FruArg can significantly inhibit LPS-induced nitric oxide (NO) production in BV-2 cells. Quantitative proteomic analysis by combining two dimensional differential in-gel electrophoresis (2D-DIGE) with mass spectrometry revealed that expressions of 26 proteins were significantly altered upon LPS exposure, while levels of 20 and 21 proteins exhibited significant changes in response to AGE and FruArg treatments, respectively, in LPS-stimulated BV-2 cells. Notably, approximate 78% of the proteins responding to AGE and FruArg treatments are in common, suggesting that FruArg is a major active component of AGE. MULTICOM-PDCN and Ingenuity Pathway Analyses indicate that the proteins differentially affected by treatment with AGE and FruArg are involved in inflammatory responses and the Nrf2-mediated oxidative stress response. Collectively, these results suggest that AGE and FruArg attenuate neuroinflammatory responses and promote resilience in LPS-activated BV-2 cells by suppressing NO production and by regulating expression of multiple protein targets associated with oxidative stress.     

Transgenic mice overexpressing glutathione peroxidase 4 are protected against oxidative stress-induced apoptosis

Glutathione peroxidase 4 (Gpx4) is uniquely involved in the detoxification of oxidative damage to membrane lipids. Our previous studies showed that Gpx4 is essential for mouse survival and that Gpx4 deficiency makes cells vulnerable to oxidative injury. In the present study, we generated two lines of transgenic mice overexpressing Gpx4 (Tg(GPX4) mice) using a genomic clone containing the human GPX4 gene. Both lines of Tg-(GPX4) mice, Tg5 and Tg6, had elevated levels of Gpx4 (mRNA and protein) in all tissues investigated, and overexpression of Gpx4 did not cause alterations in activities of glutathione peroxidase 1, catalase, Cu/Zn superoxide dismutase, and manganese superoxide dismutase. The human GPX4 transgene rescued the lethal phenotype of null mutation of the mouse Gpx4 gene, indicating that the transgene can replace the essential role of mouse Gpx4 in mouse development. Cell death induced by t-butylhydroperoxide and diquat was significantly less in murine embryonic fibroblasts from Tg(GPX4) mice compared with wild type mice. Liver damage and lipid peroxidation induced by diquat were reduced significantly in Tg(GPX4) mice. In addition, diquat-induced apoptosis was decreased in Tg(GPX4) mice, as evidenced by attenuated caspase-3 activation and reduced cytochrome c release from mitochondria. These data demonstrate that Gpx4 plays a role in vivo in the mechanism of apoptosis induced by oxidative stress that most likely occurs through oxidative damage to mitochondrial phospholipids such as cardiolipin.     

The Anopheles gambiae Oxidation Resistance 1 (OXR1) Gene Regulates Expression of Enzymes That Detoxify Reactive Oxygen Species

Background

OXR1 is an ancient gene, present in all eukaryotes examined so far that confers protection from oxidative stress by an unknown mechanism. The most highly conserved region of the gene is the carboxyl-terminal TLDc domain, which has been shown to be sufficient to prevent oxidative damage.

Methodology/Principal Findings

OXR1 has a complex genomic structure in the mosquito A. gambiae, and we confirm that multiple splice forms are expressed in adult females. Our studies revealed that OXR1 regulates the basal levels of catalase (CAT) and glutathione peroxidase (Gpx) expression, two enzymes involved in detoxification of hydrogen peroxide, giving new insight into the mechanism of action of OXR1. Gene silencing experiments indicate that the Jun Kinase (JNK) gene acts upstream of OXR1 and also regulates expression of CAT and GPx. Both OXR1 and JNK genes are required for adult female mosquitoes to survive chronic oxidative stress. OXR1 silencing decreases P. berghei oocyst formation. Unexpectedly, JNK silencing has the opposite effect and enhances Plasmodium infection in the mosquito, suggesting that JNK may also mediate some, yet to be defined, antiparasitic response.

Conclusion

The JNK pathway regulates OXR1 expression and OXR1, in turn, regulates expression of enzymes that detoxify reactive oxygen species (ROS) in Anopheles gambiae. OXR1 silencing decreases Plasmodium infection in the mosquito, while JNK silencing has the opposite effect and enhances infection.     

GPX7 Facilitates BMSCs Osteoblastogenesis via ER Stress and mTOR Pathway

Emerging evidence indicates extensive oxidative stress is a consequence of obesity which impairs bone formation. Glutathione peroxidase 7 (GPX7) is a conserved endoplasmic reticulum (ER) retention protein, lacking of which causes accumulation of reactive oxygen species (ROS) and promotes adipogenesis. Since the imbalance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cell (BMSC) leads to severe bone diseases such as osteoporosis, it is critical to investigate the potential protective role of Gpx7 in osteogenesis. Here, we provide evidence that deficiency of Gpx7 reduces osteogenesis, but increases adipogenesis in both human BMSCs (hBMSCs) and mouse mesenchymal stem cell line. Interestingly, further studies indicate this defect can be alleviated by the ER stress antagonist, but not the ROS inhibitor, unveiling an unexpected finding that, unlike adipogenesis, lacking of Gpx7 inhibits osteogenesis mediating by induced ER stress instead of enhanced ROS. Furthermore, the mTOR signalling pathway is found down-regulation during osteogenic differentiation in Gpx7-deficient condition, which can be rescued by relief of ER stress. Taken together, for the first time we identify a novel function of Gpx7 in BMSCs’ osteogenic differentiation and indicate that Gpx7 may protect against osteoporotic deficits in humans through ER stress and mTOR pathway interplay.     

Multiple deficiencies in antioxidant enzymes in mice result in a compound increase in sensitivity to oxidative stress

To examine the effect of compound deficiencies in antioxidant defense, we have generated mice (Sod2^+/−/Gpx1^−/−) that are deficient in Mn superoxide dismutase (MnSOD) and glutathione peroxidase 1 (Gpx1) by breeding Sod2^+/− and Gpx1^−/− mice together. Although Sod2^+/−/Gpx1^−/− mice showed a 50% reduction in MnSOD and no detectable Gpx1 activity in either mitochondria or cytosol in all tissues, they were viable and appeared normal. Fibroblasts isolated from Sod2^+/−/Gpx1^−/− mice were more sensitive (4- to 6-fold) to oxidative stress (t-butyl hydroperoxide or γ irradiation) than fibroblasts from wild-type mice, and were twice as sensitive as cells from Sod2^+/− or Gpx1^−/− mice. Whole-animal studies demonstrated that survival of the Sod2^+/−/Gpx1^−/− mice in response to whole body γ irradiation or paraquat administration was also reduced compared with that of wild-type, Sod2^+/−, or Gpx1^−/− mice. Similarly, endogenous oxidative stress induced by cardiac ischemia/reperfusion injury led to greater apoptosis in heart tissue from the Sod2^+/−/Gpx1^−/− mice than in that from mice deficient in either MnSOD or Gpx1 alone. These data show that Sod2^+/−/Gpx1^−/− mice, deficient in two mitochondrial antioxidant enzymes, have significantly enhanced sensitivity to oxidative stress induced by exogenous insults and to endogenous oxidative stress compared with either wild-type mice or mice deficient in either MnSOD or Gpx1 alone.     

Lack of the antioxidant glutathione peroxidase-1 does not increase atherosclerosis in C57BL/J6 mice fed a high-fat diet

Oxidative stress is thought to contribute to the initiation and progression of atherosclerosis. As glutathione peroxidase-1 (Gpx1) is an antioxidant enzyme that detoxifies lipid hydroperoxides, we tested the impact of Gpx1 deficiency on atherosclerotic processes and antioxidant enzyme expression in mice fed a high-fat diet (HFD). After 12 weeks of HFD, atherosclerotic lesions at the aortic sinus were of similar size in control and Gpx1-deficient mice. However, after 20 weeks of HFD, lesion size increased further in control but not in Gpx1-deficient mice, even though plasma and aortic wall markers of oxidative damage did not differ between groups. In control mice, the expression of Gpx1 increased and that of Gpx3 decreased at the aortic sinus after 20 weeks of HFD, with no change in the expression of Gpx2, Gpx4, catalase, peroxiredoxin-6, glutaredoxin-1 and -2, or thioredoxin-1 and -2. By comparison, in Gpx1-deficient mice, the expression of antioxidant genes was unaltered except for a decrease in glutaredoxin-1 and an increase in glutaredoxin-2. These changes were associated with increased expression of the proinflammatory marker monocyte chemoattractant protein-1 in control mice but not in Gpx1-deficient mice. In summary, a specific deficiency in Gpx1 was not accompanied by an increase in markers of oxidative damage or increased atherosclerosis in a murine model of HFD-induced atherogenesis.     

Mitochondrial oxidative stress in mice lacking the glutathione peroxidase-1 gene

Oxidative stress resulting from mitochondrially derived reactive oxygen species (ROS) has been hypothesized to damage mitochondrial oxidative phosphorylation (OXPHOS) and to be a factor in aging and degenerative disease. If this hypothesis is correct, then genetically inactivating potential mitochondrial antioxidant enzymes such as glutathione peroxidase-1 (Gpx1; EC 1.11.1.9) should increase mitochondrial ROS production and decrease OXPHOS function. To determine the expression pattern of Gpx1, isoform-specific antibodies were generated and mutant mice were prepared in which the Gpx1 protein was substituted for by beta-galactosidase, driven by the Gpx1 promoter. These experiments revealed that Gpx1 is highly expressed in both the mitochondria and the cytosol of the liver and kidney, but poorly expressed in heart and muscle. To determine the physiological importance of Gpx1, mice lacking Gpx1 were generated by targeted mutagenesis in mouse ES cells. Homozygous mutant Gpx1(tm1Mgr) mice have 20% less body weight than normal animals and increased levels of lipid peroxides in the liver. Moreover, the liver mitochondria were found to release markedly increased hydrogen peroxide, a Gpx1 substrate, and have decreased mitochondrial respiratory control ratio and power output index. Hence, genetic inactivation of Gpx1 resulted in growth retardation, presumably due in part to reduced mitochondrial energy production as a product of increased oxidative stress.