Glutathione-dependent Oxidoreductase Research Articles

A Disruption in Iron-Sulfur Center BiogenesisviaInhibition of Mitochondrial Dithiol Glutaredoxin 2 May Contribute to Mitochondrial and Cellular Iron Dysregulation in Mammalian Glutathione-Depleted Dopaminergic Cells: Implications for Parkinson's Disease

Parkinson's disease (PD) is characterized by early glutathione depletion in the substantia nigra (SN). Among its various functions in the cell, glutathione acts as a substrate for the mitochondrial enzyme glutaredoxin 2 (Grx2). Grx2 is involved in glutathionylation of protein cysteine sulfhydryl residues in the mitochondria. Although monothiol glutathione-dependent oxidoreductases (Grxs) have previously been demonstrated to be involved in iron-sulfur (Fe-S) center biogenesis, including that in yeast, here we report data suggesting the involvement of mitochondrial Grx2, a dithiol Grx, in iron-sulfur biogenesis in a mammalian dopaminergic cell line. Given that mitochondrial dysfunction and increased cellular iron levels are two important hallmarks of PD, this suggests a novel potential mechanism by which glutathione depletion may affect these processes in dopaminergic neurons. We report that depletion of glutathione as substrate results in a dose-dependent Grx2 inhibition and decreased iron incorporation into a mitochondrial complex I (CI) and aconitase (m-aconitase). Mitochondrial Grx2 inhibition through siRNA results in a corresponding decrease in CI and m-aconitase activities. It also results in significant increases in iron-regulatory protein (IRP) binding, likely as a consequence of conversion of Fe-S-containing cellular aconitase to its non-Fe-S-containing IRP1 form. This is accompanied by increased transferrin receptor, decreased ferritin, and subsequent increases in mitochondrial iron levels. This suggests that glutathione depletion may affect important pathologic cellular events associated with PD through its effects on Grx2 activity and mitochondrial Fe-S biogenesis.

Expression of Glutaredoxin-1 in Nasal Polyps and Airway Epithelial Cells

Glutaredoxins (GRX)-1 is glutathione-dependent oxidoreductase. However, the role of these enzymes remains unknown in airway inflammatory diseases. Therefore, we aimed to establish the expression pattern of GRX-1 in the nasal polyps (NPs) and to assess the regulatory mechanisms associated with GRX-1 expression in interleukin (IL)-1 beta-treated airway epithelial cells. The expression of GRX-1 in NPs and normal nasal mucosa were analyzed by reverse-transcription polymerase chain reaction and immunohistochemical staining. IL-1 beta-induced reactive oxygen species (ROS) formation and GRX-1 expression in the airway epithelial cells was determined by flow cytometry and immunoassay. The expression level of GRX-1 in NPs was significantly higher than in the normal nasal mucosa (p < 0.05). GRX-1 was highly expressed in the surface epithelial cells and the submucosal glandular cells in the NPs. IL-1 beta increased the intracellular ROS formation and GRX-1 expression in airway epithelial cells. The inhibition of IL-1 beta-induced ROS production by N-acetyl-cystein, an ROS scavenger, reduced GRX-1 expression. Diphenyleneiodonium and apocynin, NADPH oxidase inhibitors, did not abolish IL-1 beta-induced ROS formation and GRX-1 expression, whereas budesonide attenuated it. High GRX-1 expression in NPs might be a primary defense against chronic inflammatory oxidative stress in nasal mucosa. IL-1 beta-induced up-regulation of GRX-1 in airway epithelial cells is probably mediated by ROS. Glucocorticoids can regulate IL-1 beta-induced ROS formation and GRX-1 expression.

Origin and Diversification of Land Plant CC-Type Glutaredoxins

Glutaredoxins (GRXs) are ubiquitous glutathione-dependent oxidoreductase enzymes implicated in redox homeostasis, particularly oxidative stress response. Three major classes of GRX genes exist, the CPYC, CGFS classes are present in all pro- and eukaryote species, whereas the CC-type class GRXs are specific to land plants. In the basal land plant Physcomitrella patens, only two CC-type GRXs are present, compared with 21 in Arabidopsis. In contrast, sizes of the CPYC and CGFS classes remained rather similar throughout plant evolution, raising the interesting question as to when the CC-type GRXs first originated and how and why they expanded during land plant evolution. Recent evidence suggests that CC-type GRXs may have been recruited during evolution into diverse plant-specific functions of flower development (ROXY1, ROXY2) and pathogenesis response (ROXY19/GRX480). In the present study, GRX genes from the genomes of a range of green algae and evolutionarily diverse land plant species were identified; Ostreococcus, Micromonas, Volvox, Selaginella, Vitis, Sorghum, and Brachypodium. Previously identified sequences from Chlamydomonas, Physcomitrella, Oryza, Arabidopsis, and Populus were integrated to generate a more comprehensive understanding of the forces behind the evolution of various GRX classes. The analysis indicates that the CC-type GRXs probably arose by diversification from the CPYC class, at a time coinciding with colonization of land by plants. This strong differential expansion of the CC-type class occurred exclusively in the angiosperms, mainly through paleopolyploidy duplication events shortly after the monocot–eudicot split, and more recently through multiple tandem duplications that occurred independently in five investigated angiosperm lineages. The presented data suggest that following duplications, subfunctionalization, and subsequent neofunctionalization likely facilitated the sequestration of land plant-specific CC-type GRXs into novel functions such as development and pathogenesis response.

Identification, Expression Pattern, and Characterization of Mouse Glutaredoxin 2 Isoforms

Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase involved in the maintenance of mitochondrial redox homeostasis. Grx2 was first characterized as mitochondrial protein, but alternative mRNA variants lacking the transit peptide-encoding first exon were demonstrated for human and proposed for mouse. We systematically screened for alternative transcript variants of mouse Grx2. We identified a total of six exons, three constitutive (II, III, and IV), two alternative first exons (exons Ia and Ic), and one single-cassette exon (exon IIIb) located between exons III and IV. Exons Ic and IIIb are not present in the human genome; mice lack human exon Ib. The six exons give rise to five transcript variants that encode three protein isoforms: mitochondrial Grx2a, a cytosolic isoform that is homologous to the cytosolic/nuclear human Grx2c and present in specific cells of many tissues and the testis-specific isoform Grx2d that is unique to mice. Mouse Grx2c can form an iron/sulfur cluster-bridged dimer, is enzymatically active as a monomer, and can donate electrons to ribonucleotide reductase. Testicular cells lack mitochondrial Grx2a but contain cytosolic Grx2. Prominent immunostaining was detected in spermatogonia and spermatids. These results provide evidence for additional functions of Grx2 in the cytosol, in cell proliferation, and in cellular differentiation.

Redox properties and evolution of human glutaredoxins

Glutaredoxins (Grxs) are glutathione-dependent oxidoreductases that belong to the thioredoxin superfamily catalyzing thiol-disulfide exchange reactions via active site cysteine residues. Focusing on the human dithiol glutaredoxins having a C-X-Y-C active site sequence motif, the redox potentials of hGrx1 and hGrx2 were determined to be -232 and -221 mV, respectively, using a combination of redox buffers, protein-protein equilibrium and thermodynamic linkage. In addition, a nonactive site disulfide was identified between Cys28 and Cys113 in hGrx2 using redox buffers and chemical digestion. This disulfide confers nearly five kcal mol(-1) additional stability by linking the C-terminal helix to the bulk of the protein. The redox potential of this nonactive site disulfide was determined to be -317 mV and is thus expected to be present in all but the most reducing conditions in vivo. As all human glutaredoxins contain additional nonactive site cysteine residues, a full phylogenetic analysis was performed to help elucidate their structural and functional roles. Three distinct groups were found: Grx1, Grx2, and Grx5, the latter representing a highly conserved group of monothiol glutaredoxins having a C-G-F-S active site sequence, with clear homologs from bacteria to human. Grx1 and Grx2 diverged from a common ancestor before the origin of vertebrates, possibly even earlier in animal evolution. The highly stabilizing nonactive site disulfide observed in hGrx2 is found to be a conserved feature within the deuterostomes and appears to be the only additional conserved intramolecular disulfide within the glutaredoxins.

Reduced glutathione levels affect the culmination and cell fate decision in Dictyostelium discoideum

Glutaredoxins have been known to be glutathione-dependent oxidoreductases that participate in the redox regulation of various cellular processes. To understand the role of glutaredoxins in the development, we examined glutaredoxin 1 (Grx1) of Dictyostelium discoideum. Its mRNA was highly accumulated at the mound and the culmination stages. When Grx1-overexpressing cells were developed, their culmination was delayed, and the expression of marker genes for prespore and spore decreased. Interestingly, they had about 1.5-fold higher amount of reduced glutathione (GSH) compared with parental cells and their prolonged migration was repressed by the oxidant such as hydrogen peroxide. To confirm the effect of GSH on the culmination, glutathione reductase (Gsr) was overexpressed or underexpressed. Similar to Grx1-overexpressing cells, Gsr-overexpressing cells contained about 1.5-fold higher amount of GSH and exhibited the delayed culmination. In contrast, the knockdown mutant of Gsr had nearly 50% lower amount of GSH and showed accelerated culmination. Taken together, these data suggest that the culmination of Dictyostelium is controlled by GSH. In addition, the cells having higher GSH levels showed a prestalk tendency in the chimeric slugs with parental cells, indicating that the difference in the amount of GSH may affect the determination of cell fate.

Characterization of human glutaredoxin 2 as iron–sulfur protein: A possible role as redox sensor

Human mitochondrial glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase (active site: Cys-Ser-Tyr-Cys) that facilitates the maintenance of mitochondrial redox homeostasis upon induction of apoptosis by oxidative stress. Here, we have characterized Grx2 as an iron-sulfur center-containing member of the thioredoxin fold protein family. Mossbauer spectroscopy revealed the presence of a four cysteine-coordinated nonoxidizable [2Fe-2S]2+ cluster that bridges two Grx2 molecules via two structural Cys residues to form dimeric holo Grx2. Coimmunoprecipitation of radiolabeled iron with Grx2 from human cell lines indicated the presence of the cluster in vivo. The [2Fe-2S]-bridged dimer was enzymatically inactive, but degradation of the cluster and the resulting monomerization of Grx2 activated the protein. Slow degradation under aerobic conditions was prevented by the presence of glutathione, whereas glutathione disulfide as well as one-electron oxidants or reductants promoted monomerization of Grx2. We propose that the iron-sulfur cluster serves as a redox sensor for the activation of Grx2 during conditions of oxidative stress when free radicals are formed and the glutathione pool becomes oxidized.

The Yeast Glutaredoxins Are Active as Glutathione Peroxidases

The yeast Saccharomyces cerevisiae contains two glutaredoxins, encoded by GRX1 and GRX2, which are active as glutathione-dependent oxidoreductases. Our studies show that changes in the levels of glutaredoxins affect the resistance of yeast cells to oxidative stress induced by hydroperoxides. Elevating the gene dosage of GRX1 or GRX2 increases resistance to hydroperoxides including hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide. The glutaredoxin-mediated resistance to hydroperoxides is dependent on the presence of an intact glutathione system, but does not require the activity of phospholipid hydroperoxide glutathione peroxidases (GPX1-3). Rather, the mechanism appears to be mediated via glutathione conjugation and removal from the cell because it is absent in strains lacking glutathione-S-transferases (GTT1, GTT2) or the GS-X pump (YCF1). We show that the yeast glutaredoxins can directly reduce hydroperoxides in a catalytic manner, using reducing power provided by NADPH, GSH, and glutathione reductase. With cumene hydroperoxide, high pressure liquid chromatography analysis confirmed the formation of the corresponding cumyl alcohol. We propose a model in which the glutathione peroxidase activity of glutaredoxins converts hydroperoxides to their corresponding alcohols; these can then be conjugated to GSH by glutathione-S-transferases and transported into the vacuole by Ycf1.

Differential regulation of glutaredoxin gene expression in response to stress conditions in the yeast Saccharomyces cerevisiae

Glutaredoxins are small heat-stable proteins that are active as glutathione-dependent oxidoreductases and are encoded by two genes, designated GRX1 and GRX2, in the yeast Saccharomyces cerevisiae. We report here that the expression of both genes is induced in response to various stress conditions including oxidative, osmotic, and heat stress and in response to stationary phase growth and growth on non-fermentable carbon sources. Furthermore, both genes are activated by the high-osmolarity glycerol pathway and negatively regulated by the Ras-protein kinase A pathway via stress-responsive STRE elements. GRX1 contains a single STRE element and is induced to significantly higher levels compared to GRX2 following heat and osmotic shock. GRX2 contains two STRE elements, and is rapidly induced in response to reactive oxygen species and upon entry into stationary phase growth. Thus, these data support the idea that the two glutaredoxin isoforms in yeast play distinct roles during normal cellular growth and in response to stress conditions.

Glutathione, glutathione-dependent and antioxidant enzymes in mussel, Mytilus galloprovincialis, exposed to metals under field and laboratory conditions: implications for the use of biochemical biomarkers

The effects of exposure to metals under and laboratory conditions were investigated in the Mediterranean mussel Mytilus galloprovincialis. The examined biological responses included the concentrations of heavy metals, the level of glutathione, and the activity of several enzymes selected among glutathione-dependent oxidoreductases and hydrolases: glutathione reductase. EC1.6.4.2; glyoxalase I, EC4.4.1.5; glyoxalase II, EC3.1.2.6; glutathione S-transferases, EC2.5.1.18; Se-dependent, EC1 11.1.9 and Se-independent, EC2.5.1.18 glutathione peroxidases; catalase, EC1.11.1.6; superoxide dismutase, EC1.15.1.1; alkaline phosphatase, EC3.1.3.1; cholinesterase, EC3.1.1.8; arylesterases, EC3.1.1.2. The analyses were carried out on digestive gland and gills of mussels from two populations, respectively from a polluted and a clean site. The same parameters were measured in control mussels transplanted to the polluted environment, and in molluscs exposed to copper under laboratory conditions. The comparison between different field and laboratory exposures was expected to give useful indications for a proper use of biochemical responses as biomarkers in monitoring trace metal pollution. Compared to control mussels, the polluted ones (native, transplanted and copper-exposed) showed significantly lower levels of glutathione and higher activities of the glyoxalases (which detoxify reactive α-ketoaldehydes formed in cellular oxidative processes). On the other hand, native mussels from both the polluted and control populations exhibited similar enzymatic activities of glutathione reductase, glutathione peroxidases, catalase, superoxide dismutase and alkaline phosphatase, whereas, in both transplanted and copper-exposed mussels, these enzymes showed significant variations. This finding could suggest the occurrence of some adaptation or compensatory mechanism in chronically polluted organisms. No clear results were obtained with glutathione S-transferases, whereas arylesterases and cholinesterases appeared not to be affected by metal pollution. From the results, three different kind of biological responses were identified and the implications for ecotoxicological studies discussed.