Redox regulation of 3'-phosphoadenylylsulfate reductase from Escherichia coli by glutathione and glutaredoxins

Inorganic sulfate (SO42-, S+VI) is reduced in vivo to sulfite (SO32-, S+IV) via phosphoadenylylsulfate (PAPS) reductase. Escherichia coli lacking glutathione reductase and glutaredoxins (gor-grxA-grxB-grxC-) barely grows on sulfate. We found that incubation of PAPS reductase with oxidized glutathione leads to enzyme inactivation with simultaneous formation of a mixed disulfide between glutathione and the active site Cys-239. A newly developed method based on thiol-specific fluorescent alkylation and gel electrophoresis showed that glutathionylated PAPS reductase is reduced by glutaredoxins via a monothiol mechanism. This glutathionylated species was also observed in poorly growing gor-grxA-grxB-grxC- cells expressing inactive glutaredoxin 2 (Grx2) C9S/C12S. However, it was absent in better growing cells expressing monothiol Grx2 C12S or wild type Grx2. Reversible glutathionylation may thus regulate the activity of PAPS reductase in vivo.     

Interaction of selenite and tellurite with thiol-dependent redox enzymes: Kinetics and mitochondrial implications

The interactions of selenite and tellurite with cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and glutathione reductases (GR) from yeast and mammalian sources were explored. Both TrxR1 and TrxR2 act as selenite and tellurite reductases. Kinetic treatment shows that selenite has a greater affinity than tellurite with both TrxR1 and TrxR2. Considering both k_cat and K_m, selenite shows a better catalytic efficiency than tellurite with TrxR1, whereas with TrxR2, the catalytic efficiency is similar for both chalcogens. Tellurite is a good substrate for GR, whereas selenite is almost completely ineffective. Selenite or tellurite determine a large mitochondrial permeability transition associated with thiol group oxidation. However, with increasing concentrations of both chalcogens, only about 25% of total thiols are oxidized. In isolated mitochondria, selenite or tellurite per se does not stimulate H₂O₂ production, which, however, is increased by the presence of auranofin. They also determine a large oxidation of mitochondrial pyridine nucleotides. In ovarian cancer cells both chalcogens decrease the mitochondrial membrane potential. These results indicate that selenite and tellurite, interacting with the thiol-dependent enzymes, alter the balance connecting pyridine nucleotides and thiol redox state, consequently leading to mitochondrial and cellular alterations essentially referable to a disulfide stress.     

The reducing activity of glutaredoxin 3 toward cytoplasmic substrate proteins is restricted by methionine 43

The reducing proteins glutaredoxin 3 (Grx3) and glutaredoxin 1 (Grx1) are structurally similar but exhibit different specificities toward substrates. Grx1 efficiently reduces ribonucleotide reductase and PAPS reductase, while Grx3 reduces these enzymes inefficiently or not at all. We previously described a selection for Grx3 mutants with increased activity toward substrates of Grx1 in vivo. Remarkably, we repeatedly isolated mutants with changes in only one of the amino acids of Grx3, methionine 43, converting it to either valine, leucine, or isoleucine. In this paper we present additional genetic studies and a biochemical characterization of Grx3-Met43Val, the most efficient mutant. We show that Grx3-Met43Val is able to reduce ribonucleotide reductae and PAPS reductase much more efficiently than the wild-type protein in vitro. The altered protein has an increased Vmax over that of Grx3, nearly the same Vmax as Grx1 while the Km remains high. Molecular dynamics simulations suggest that the Met43Val substitution results in changes in properties of the N-terminal cysteine of the active site leading to a considerably lower pKa. Furthermore, Grx3-Met43Val shows an 11 mV lower redox potential than the wild-type Grx3. These findings provide biochemical and structural explanations for the increased reductive efficiency of the mutant Grx3.     

Mixed effects of effluents from a wastewater treatment plant on river ecosystem metabolism: subsidy or stress?

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Overexpression of glutaredoxin 2 attenuates apoptosis by preventing cytochrome c release

Human mitochondrial glutaredoxin 2 (Grx2) catalyzes glutathione-dependent dithiol reaction mechanisms, reducing protein disulfides, and monothiol reactions, reducing mixed disulfides between proteins and GSH (de-/glutathionylation). Here, we have overexpressed Grx2 in HeLa cells in its mitochondrial form (mGrx2-HeLa) as well as a truncated cytosolic form, lacking the mitochondrial translocation signal (tGrx2-HeLa). The resulting clones were less susceptible to apoptosis induced by 2-deoxy-d-glucose (2-DG) or doxorubicin (Dox). Overexpression of Grx2 inhibited cytochrome c release and caspase activation induced by both agents. In addition, Grx2 prevented 2-DG- and Dox-induced loss of cardiolipin, the phospholipid anchoring cytochrome c to the inner mitochondrial membrane. Overexpression of mGrx2 provided better protection than tGrx2 overexpression, especially after treatment with 2-DG. We propose that Grx2 facilitates the maintenance of cellular redox homeostasis upon treatment with apoptotic agents, thereby preventing cardiolipin oxidation and cytochrome c release.     

Selenite induces apoptosis in sarcomatoid malignant mesothelioma cells through oxidative stress

Malignant mesothelioma cells differentiate into sarcomatoid or epithelioid phenotypes. The sarcomatoid cell type is more resistant to chemotherapy and gives a worse prognosis. We have investigated whether selenite alone and in combination with doxorubicin induced apoptosis in variously differentiated mesothelioma cells. Selenite in concentrations that could potentially be administered to patients strongly inhibited the growth of the sarcomatoid mesothelioma cells (IC50 = 7.5 microM), whereas epithelioid cells were more sensitive to doxorubicin. Benign mesothelial cells remained largely unaffected. Selenite potentiated doxorubicin treatment. Apoptosis was the dominating mode of cell death. The toxicity of selenite was mediated by oxidative stress. Furthermore the activity of the thioredoxin system was directly dependent on the concentration of selenite. This offers a possible mechanism of action of selenite treatment. Our findings suggest that selenite is a promising new drug for the treatment of malignant mesothelioma.     

Methylselenol Formed by Spontaneous Methylation of Selenide Is a Superior Selenium Substrate to the Thioredoxin and Glutaredoxin Systems

Naturally occurring selenium compounds like selenite and selenodiglutathione are metabolized to selenide in plants and animals. This highly reactive form of selenium can undergo methylation and form monomethylated and multimethylated species. These redox active selenium metabolites are of particular biological and pharmacological interest since they are potent inducers of apoptosis in cancer cells. The mammalian thioredoxin and glutaredoxin systems efficiently reduce selenite and selenodiglutathione to selenide. The reactions are non-stoichiometric aerobically due to redox cycling of selenide with oxygen and thiols. Using LDI-MS, we identified that the addition of S-adenosylmethionine (SAM) to the reactions formed methylselenol. This metabolite was a superior substrate to both the thioredoxin and glutaredoxin systems increasing the velocities of the nonstoichiometric redox cycles three-fold. In vitro cell experiments demonstrated that the presence of SAM increased the cytotoxicity of selenite and selenodiglutathione, which could neither be explained by altered selenium uptake nor impaired extra-cellular redox environment, previously shown to be highly important to selenite uptake and cytotoxicity. Our data suggest that selenide and SAM react spontaneously forming methylselenol, a highly nucleophilic and cytotoxic agent, with important physiological and pharmacological implications for the highly interesting anticancer effects of selenium.     

Treatment of human cancer cells with selenite or tellurite in combination with auranofin enhances cell death due to redox shift

Selenium is an essential trace element incorporated as selenocysteine in 25 human selenoproteins. Among them are thioredoxin reductases (TrxR) and glutathione peroxidases, all central proteins in the regulation of the cellular thiol redox state. In this paper the effects of selenite and tellurite treatment in human cancer cells are reported and compared. Our results show that both selenite and tellurite, at relatively low concentrations, are able to increase the expression of mitochondrial and cytosolic TrxR in cisplatin-sensitive (2008) and -resistant (C13) phenotypes. We further investigated the cellular effects induced by selenite or tellurite in combination with the specific TrxR inhibitor auranofin. Selenite pretreatment induced a dramatic increase in auranofin cytotoxicity in both resistant and sensitive cells. Investigation of TrxR activity and expression levels as well as the cellular redox state demonstrated the involvement of TrxR inhibition and redox changes in selenite and auranofin combined action.