The role of oxidative protein modification and the glutathione system in modulation of the redox status of breast epithelial cells

The effects of N-ethylmaleimide (NEM) and 1,4-dithioerythritol (DTE) on the level of oxidative modification of proteins, the state of glutathione and thioredoxin systems and the cellular redox status have been investigated in HBL-100 cells (breast epithelial cells). Breast epithelial cells cultivated in the presence of NEM were characterized by the decreased redox status, increased glutathione reductase activity, and increased concentrations of products of irreversible oxidative modification of proteins and amino acids. Cell cultivation in the presence of DTE shifted the redox status towards reduction processes and increased reversible protein modification by glutathionylation. The proposed model of intracellular redox modulation may be used in the development of new therapeutic approaches to treat diseases accompanied by impaired redox homeostasis (e.g. oncologic, inflammatory, cardiovascular and neurodegenerative disease).     

谷氧还蛋白系统及其对细胞氧化还原态势的调控

细胞内氧化还原调控主要是由谷氧还蛋白系统和硫氧还蛋白系统完成。谷氧还蛋白属于硫氧还蛋白超家族,广泛分布在各种生物体内。作为一种巯基转移酶,它能够催化巯基．二硫键交换反应或者还原蛋白质谷胱甘肽二硫化物,以维持胞内的氧化还原态势。谷氧蛋白系统参与氧化胁迫、蛋白修饰、信号转导、细胞调亡和细胞分化等多种生物过程。对其体内作用靶蛋白的研究,有助于阐明谷氧还蛋白在整个细胞氧化还原网络的重要调控作用。     

The role of protein oxidative modification and the cellular redox status in realization of apoptosis of MCF-7 breast adenocarcinoma cells

The aim of this study was to establish the role of oxidative modification of proteins and redox status in the realization of apoptosis of MCF-7 breast adenocarcinoma cells during cultivation with the SH-group blocker N-ethylmaleimide (NEM) and the SH-group protector 1,4-dithioerythritol (DTE). The activation of apoptosis in MCF-7 breast adenocarcinoma cells was shown to be due to the irreversible modification of redox-sensitive protein molecules. The presence of DTE in the culture medium of cancer cells caused reversible glutathionylation of protein molecules and did not change the number of apoptotic MCF-7 cells.     

Non-reciprocal regulation of the redox state of the glutathione-glutaredoxin and thioredoxin systems

Our studies in yeast show that there is an essential requirement for either an active thioredoxin or an active glutathione (GSH)–glutaredoxin system for cell viability. Glutathione reductase (Glr1) and thioredoxin reductase (Trr1) are key regulatory enzymes that determine the redox state of the GSH–glutaredoxin and thioredoxin systems, respectively. Here we show that Trr1 is required during normal cell growth, whereas there is no apparent requirement for Glr1. Analysis of the redox state of thioredoxins and glutaredoxins in glr1 and trr1 mutants reveals that thioredoxins are maintained independently of the glutathione system. In contrast, there is a strong correlation between the redox state of glutaredoxins and the oxidation state of the GSSG/2GSH redox couple. We suggest that independent redox regulation of thioredoxins enables cells to survive in conditions under which the GSH–glutaredoxin system is oxidized.     

Thioredoxin 1 Is Inactivated Due to Oxidation Induced by Peroxiredoxin under Oxidative Stress and Reactivated by the Glutaredoxin System

The mammalian cytosolic thioredoxin system, comprising thioredoxin (Trx), Trx reductase, and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. Besides the active site thiols, human Trx1 contains three non-active site cysteine residues at positions 62, 69, and 73. A two-disulfide form of Trx1, containing an active site disulfide between Cys-32 and Cys-35 and a non-active site disulfide between Cys-62 and Cys-69, is inactive either as a disulfide reductase or as a substrate for Trx reductase. This could possibly provide a structural switch affecting Trx1 function during oxidative stress and redox signaling. We found that two-disulfide Trx1 was generated in A549 cells under oxidative stress. In vitro data showed that two-disulfide Trx1 was generated from oxidation of Trx1 catalyzed by peroxiredoxin 1 in the presence of H₂O₂. The redox Western blot data indicated that the glutaredoxin system protected Trx1 in HeLa cells from oxidation caused by ebselen, a superfast oxidant for Trx1. Our results also showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced the non-active site disulfide in vitro. This reaction was stimulated by glutaredoxin 1 via the so-called monothiol mechanism. In conclusion, reversible oxidation of the non-active site disulfide of Trx1 is suggested to play an important role in redox regulation and cell signaling via temporal inhibition of its protein-disulfide reductase activity for the transmission of oxidative signals under oxidative stress.     

Regulation of protein function by glutathionylation

The main function of reduced glutathione (GSH) is to protect from oxidative stress as a reactive oxygen scavenger. However, in the context of redox regulation, the ratio between GSH and its oxidized form (GSSG) determines the redox state of redox-sensitive cysteines in some proteins and, thus, acts as a signaling system. While GSH/GSSG can catalyze oxido-reduction of intra- and inter-chain disulfides by thiol-disulfide exchange, this review focuses on the formation of mixed disulfides between glutathione and proteins, also known as glutathionylation. The review discusses the regulatory role of this post-translational modification and the role of protein disulfide oxidoreductases (thioredoxin/thioredoxin reductase, glutaredoxin, protein disulfide isomerase) in the reversibility of this process.     

A hyperthermostable novel protein-disulfide oxidoreductase is reduced by thioredoxin reductase from hyperthermophilic archaeon Pyrococcus horikoshii

A redox protein gene (PH0178) with high sequence homology to a glutaredoxin from Pyrococcus furiosus and a thioredoxin reductase homologue gene (PH1426) were found in the genome sequence of Pyrococcus horikoshii. These two genes were cloned and the corresponding expressed proteins were characterized. The redox protein from PH0178 had strong thioredoxin-like activity, but no glutaredoxin activity. The protein from PH1426 had some reductase activity against thioredoxin from Escherichia coli as well as the redox protein (PH0178). The protein from PH1426 was a typical, homodimeric flavoprotein. These results indicate that the redox protein (PH0178) is not a glutaredoxin but, rather, a new protein-disulfide oxidoreductase that is involved in a thioredoxin-like system with thioredoxin reductase (PH1426) in P. horikoshii. The redox protein and thioredoxin reductase retained their full activities for over 1h at 100 degrees C. The redox potential of the redox protein was similar to that of thioredoxin from E. coli and lower than that of glutathione. Site-directed mutagenesis studies revealed that the active site of the redox protein corresponds to a CPYC sequence, located in the middle of the sequence.     

Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase

Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes.     

Activation of translation via reduction by thioredoxin-thioredoxin reductase in Saccharomyces cerevisiae

Previously we reported that in vitro translation activity in extracts of Saccharomyces cerevisiae was stimulated by dithiothreitol (DTT) and further increased by the addition of thioredoxin (TRX1) [Choi, S.K. (2007) Thioredoxin-mediated regulation of protein synthesis by redox in Saccharomyces cerevisiae. Kor. J. Microbiol. Biotechnol. 35, 36-40]. To identify the pathway affecting translation, we cloned and purified thioredoxin reductase 1 (TRR1), thioredoxin reductase 2 (TRR2), glutaredoxin 1 (GRX1) and glutaredoxin reductase 1 (GLR1) as fusion proteins. Thioredoxin-mediated activation of translation was more effectively stimulated by NADPH or NADH than by DTT. Moreover, addition of TRR1 led to a further increase of translation in the presence of thioredoxin plus NADPH. These findings indicate that redox control via the thioredoxin-thioredoxin reductase system plays an important role in the regulation of translation.     

Snail/beta-catenin signaling protects breast cancer cells from hypoxia attack

The tolerance of cancer cells to hypoxia depends on the combination of different factors – from increase of glycolysis (Warburg Effect) to activation of intracellular growth/apoptotic pathways. Less is known about the influence of epithelial–mesenchymal transition (EMT) and EMT-associated pathways on the cell sensitivity to hypoxia. The aim of this study was to explore the role of Snail signaling, one of the key EMT pathways, in the mediating of hypoxia response and regulation of cell sensitivity to hypoxia, using as a model in vitro cultured breast cancer cells. Earlier we have shown that estrogen-independent HBL-100 breast cancer cells differ from estrogen-dependent MCF-7 cells with increased expression of Snail1, and demonstrated Snail1 involvement into formation of hormone-resistant phenotype. Because Snail1 belongs to hypoxia-activated proteins, here we studied the influence of Snail1 signaling on the cell tolerance to hypoxia. We found that Snail1-enriched HBL-100 cells were less sensitive to hypoxia-induced growth suppression if compared with MCF-7 line (31% MCF-7 vs. 71% HBL-100 cell viability after 1% O₂ atmosphere for 3 days). Snail1 knock-down enhanced the hypoxia-induced inhibition of cell proliferation giving the direct evidence of Snail1 involvement into cell protection from hypoxia attack. The protective effect of Snail1 was shown to be mediated, at least in a part, via beta-catenin which positively regulated expression of HIF-1-dependent genes. Finally, we found that cell tolerance to hypoxia was accompanied with the failure in the phosphorylation of AMPK – the key energy sensor, and demonstrated an inverse relationship between AMPK and Snail/beta-catenin signaling.Totally, our data show that Snail1 and beta-catenin, besides association with loss of hormone dependence, protect cancer cells from hypoxia and may serve as an important target in the treatment of breast cancer. Moreover, we suggest that the level of these proteins as well the level of AMPK phosphorylation may be considered as predictors of the tumor sensitivity to anti-angiogenic drugs.     

Oxidative stress and thioredoxin system

Oxidative stress plays an important role in the modulation of several important physiological functions. On the other side, oxidative stress is accountable for development of many unphysiological changes, which can be deleterious for cells. Consequently, at the present time there is increased interest about study mechanisms and changes evocated by oxidative stress. Despite the highly oxidizing environment (21% oxygen, at sea level), at normal conditions, the cell cytoplasm of all aerobic organisms is reduced and proteins contain free sulfhydryl groups. In the cytoplasm, two major systems were identificated responsible for maintaining a reduced state: thioredoxin and glutathione/glutaredoxin system. Thioredoxin in bacteria, thanks to the low redox potential is the major dithiol reductant in the cytosol, or an advanced equivalent to dithiothreitol of cells (Holmgren 1985). Thioredoxin system acts the dominant role in many physiological processes (see below) and it is also a cell antioxidant.     

Proteomic profiling of acrolein adducts in human lung epithelial cells

Acrolein (2,3-propenal) is a major indoor and outdoor air pollutant originating largely from tobacco smoke or organic combustion. Given its high reactivity, the adverse effects of inhaled acrolein are likely due to direct interactions with the airway epithelium, resulting in altered epithelial function, but only limited information exists to date regarding the primary direct cellular targets for acrolein. Here, we describe a global proteomics approach to characterize the spectrum of airway epithelial protein targets for Michael adduction in acrolein-exposed bronchial epithelial (HBE1) cells, based on biotin hydrazide labeling and avidin purification of biotinylated proteins or peptides for analysis by LC-MS/MS. Identified protein targets included a number of stress proteins, cytoskeletal proteins, and several key proteins involved in redox signaling, including thioredoxin reductase, thioredoxin, peroxiredoxins, and glutathione S-transferase π. Because of the central role of thioredoxin reductase in cellular redox regulation, additional LC-MS/MS characterization was performed on purified mitochondrial thioredoxin reductase to identify the specific site of acrolein adduction, revealing the catalytic selenocysteine residue as the target responsible for enzyme inactivation. Our findings indicate that these approaches are useful in characterizing major protein targets for acrolein, and will enhance mechanistic understanding of the impact of acrolein on cell biology.     

Biomarkers of tumour redox status in response to modulations of glutathione and thioredoxin antioxidant pathways

The ability of certain cancer cells to maintain a highly reduced intracellular environment is correlated with aggressiveness and drug resistance. Since the glutathione (GSH) and thioredoxin (TRX) systems cooperate to a tight regulation of ROS in cell physiology, and to a stimulation of tumour initiation and progression, modulation of the GSH and TRX pathways are emerging as new potential targets in cancer. In vivo methods to assess changes in tumour redox status are critically needed to assess the relevance of redox-targeted agents. The current study assesses in vitro and in vivo biomarkers of tumour redox status in response to treatments targeting the GSH and TRX pathways, by comparing cytosolic and mitochondrial redox nitroxide electron paramagnetic resonance (EPR) probes, and cross-validation with redox dynamic fluorescent measurement. For that purpose, the effect of the GSH modulator buthionine sulfoximine (BSO) and of the TRX reductase inhibitor auranofin were measured in vitro using both cytosolic and mitochondrial EPR and roGFP probes in breast and cervical cancer cells. In vivo, mice bearing breast or cervical cancer xenografts were treated with the GSH or TRX modulators and monitored using the mito-TEMPO spin probe. Our data highlight the importance of using mitochondria-targeted spin probes to assess changes in tumour redox status induced by redox modulators. Further in vivo validation of the mito-tempo spin probe with alternative in vivo methods should be considered, yet the spin probe used in vivo in xenografts demonstrated sensitivity to the redox status modulators.     

Glutathione and Glutaredoxin Act as a Backup of Human Thioredoxin Reductase 1 to Reduce Thioredoxin 1 Preventing Cell Death by Aurothioglucose

Thioredoxin reductase 1 (TrxR1) in cytosol is the only known reductant of oxidized thioredoxin 1 (Trx1) in vivo so far. We and others found that aurothioglucose (ATG), a well known active-site inhibitor of TrxR1, inhibited TrxR1 activity in HeLa cell cytosol but had no effect on the viability of the cells. Using a redox Western blot analysis, no change was observed in redox state of Trx1, which was mainly fully reduced with five sulfhydryl groups. In contrast, auranofin killed cells and oxidized Trx1, also targeting mitochondrial TrxR2 and Trx2. Combining ATG with ebselen gave a strong synergistic effect, leading to Trx1 oxidation, reactive oxygen species accumulation, and cell death. We hypothesized that there should exist a backup system to reduce Trx1 when only TrxR1 activity was lost. Our results showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced Trx1 in vitro and that the reaction was strongly stimulated by glutaredoxin1. Simultaneous depletion of TrxR activity by ATG and glutathione by buthionine sulfoximine led to overoxidation of Trx1 and loss of HeLa cell viability. In conclusion, the glutaredoxin system and glutathione have a backup role to keep Trx1 reduced in cells with loss of TrxR1 activity. Monitoring the redox state of Trx1 shows that cell death occurs when Trx1 is oxidized, followed by general protein oxidation catalyzed by the disulfide form of thioredoxin.     

Characterization of several members of the thiol oxidoreductase family

There is no doubt as to the important role that free radicals and reactive oxygen species play in the cell. Disturbances in intracellular redox proteins are often accompanied by common pathologies, including diabetes, myocardial infarction, neurodegeneration, bronchopulmonary diseases, cancer, etc. Numerous antioxidant enzymes are related to various redox biology systems, the thiol oxidoreductase superfamily playing a key role. The superfamily includes thioredoxin, glutaredoxin, peroxiredoxin, protein disulfide isomerase, and glutathione peroxidase families and a number of other proteins. Apart from their antioxidant function, thiol oxidoreductases are capable of recycling hydroperoxyde to produce specific disulfide bonds within and between proteins, which significantly expands their functional range. In view of this, it is a topical problem of redox biology to characterize the superfamily members biochemically and to study their functional mechanisms.     

Effects of heterologous expression of thioredoxin reductase on the level of reactive oxygen species in COS-7 cells

Thioredoxin reductase (TrxR), a component of the redox control system involving thioredoxin (Trx), is implicated in defense against oxidative stress, control of cell growth and proliferation, and regulation of apoptosis. In the present study a stable transfectant was made by introducing the vector pcDNA3.0 harboring the fission yeast TrxR gene into COS-7 African green monkey kidney fibroblast cells. The exogenous TrxR gene led to an increase in TrxR activity of up to 3.2-fold but did not affect glutathione (GSH) content, or glutaredoxin and caspase-3 activities. Levels of reactive oxygen species (ROS), but not those of nitric oxide (NO), were reduced. Conversely, 1-chloro-2,4-dinitrobezene (CDNB), an irreversible inhibitor of mammalian TrxR, enhanced ROS levels in the COS-7 cells. After treatment with hydrogen peroxide, the level of intracellular ROS was lower in the transfectants than in the vector control cells. These results confirm that TrxR is a crucial determinant of the level of cellular ROS during oxidative stress as well as in the normal state.     

Redox and antioxidant systems of the malaria parasite Plasmodium falciparum

The malaria parasite Plasmodium falciparum is highly adapted to cope with the oxidative stress to which it is exposed during the erythrocytic stages of its life cycle. This includes the defence against oxidative insults arising from the parasite's metabolism of haemoglobin which results in the formation of reactive oxygen species and the release of toxic ferriprotoporphyrin IX. Central to the parasite's defences are superoxide dismutases and thioredoxin-dependent peroxidases; however, they lack catalase and glutathione peroxidases. The vital importance of the thioredoxin redox cycle (comprising NADPH, thioredoxin reductase and thioredoxin) is emphasized by the confirmation that thioredoxin reductase is essential for the survival of intraerythrocytic P. falciparum. The parasites also contain a fully functional glutathione redox system and the low-molecular-weight thiol glutathione is not only an important intracellular thiol redox buffer but also a cofactor for several redox active enzymes such as glutathione S-transferase and glutaredoxin. Recent findings have shown that in addition to these cytosolic redox systems the parasite also has an important mitochondrial antioxidant defence system and it is suggested that lipoic acid plays a pivotal part in defending the organelle from oxidative damage.