Monitoring disulfide bond formation in the eukaryotic cytosol

Glutathione is the most abundant low molecular weight thiol in the eukaryotic cytosol. The compartment-specific ratio and absolute concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) are, however, not easily determined. Here, we present a glutathione-specific green fluorescent protein-based redox probe termed redox sensitive YFP (rxYFP). Using yeast with genetically manipulated GSSG levels, we find that rxYFP equilibrates with the cytosolic glutathione redox buffer. Furthermore, in vivo and in vitro data show the equilibration to be catalyzed by glutaredoxins and that conditions of high intracellular GSSG confer to these a new role as dithiol oxidases. For the first time a genetically encoded probe is used to determine the redox potential specifically of cytosolic glutathione. We find it to be -289 mV, indicating that the glutathione redox status is highly reducing and corresponds to a cytosolic GSSG level in the low micromolar range. Even under these conditions a significant fraction of rxYFP is oxidized.     

Nrf2‐Mediated Resistance to Oxidant‐Induced Redox Disruption in Embryos

Events that control developmental changes occur during specific windows of gestation and if disrupted, can lead to dysmorphogenesis or embryolethality. One largely understudied aspect of developmental control is redox regulation, where the untimely disruption of intracellular redox potentials (E_h) may alter development, suggesting that tight control of developmental‐stage–specific redox states is necessary to support normal development. In this study, mouse gestational day 8.5 embryos in whole embryo culture were treated with 10 μM dithiole‐3‐thione (D3T), an inducer of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2). After 14 hr, D3T‐treated and ‐untreated conceptuses were challenged with 200 μM hydrogen peroxide (H₂O₂) to induce oxidant‐induced change to intracellular E_hs. Redox potentials of glutathione (GSH), thioredoxin‐1 (Trx1), and mitochondrial thioredoxin‐2 (Trx2) were then measured over a 2‐hr rebounding period following H₂O₂ treatment. D3T treatment increased embryonic expression of known Nrf2‐regulated genes, including those responsible for redox regulation of major intracellular redox couples. Exposure to H₂O₂ without prior D3T treatment produced significant oxidation of GSH, Trx1, and Trx2, based on E_h values, where GSH and Trx2 E_h recovered, reaching to pre‐H₂O₂ E_h ranges, but Trx1 E_h remained oxidized. Following H₂O₂ addition in culture to embryos that received D3T pretreatments, GSH, Trx1, and Trx2 were insulated from significant oxidation. These data show that Nrf2 activation may serve as a means to protect the embryo from chemically induced oxidative stress through the preservation of intracellular redox states during development, allowing normal morphogenesis to ensue.     

A unique thioredoxin of the parasitic nematode Haemonchus contortus with glutaredoxin activity

The dependency of parasites on the cellular redox systems has led to their investigation as novel drug targets. Defence against oxidative damage is through the thioredoxin and glutathione systems. The classic thioredoxin is identified by the active site Cys-Gly-Pro-Cys (CGPC). Here we describe the identification of a unique thioredoxin in the parasitic nematode, Haemonchus contortus. This thioredoxin-related protein, termed HcTrx5, has an arginine in its active site (Cys-Arg-Ser-Cys; CRSC) that is not found in any other organism. Recombinant HcTrx5 was able to reduce the disulfide bond in insulin, and be regenerated by mammalian thioredoxin reductase with a K_m 2.19 ± 1.5 μM, similar to the classic thioredoxins. However, it was also able to reduce insulin when glutathione and glutathione reductase replaced the thioredoxin reductase. When coupled with H. contortus peroxiredoxin, HcTrx5 was active using either the thioredoxin reductase or the glutathione and glutathione reductase. HcTrx5 is expressed through the life cycle, with highest expression in the adult stage. The unique activity of this thioredoxin makes it a potential drug target for the control of this parasite.     

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.     

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.     

Differential oxidation of thioredoxin-1, thioredoxin-2, and glutathione by metal ions

Metal toxicity often includes the generation of reactive oxygen species (ROS) and subsequent oxidative stress, but whether metals have different effects on the major thiol antioxidant systems is unknown. Here, we examine the effects of arsenic, cadmium, cesium, copper, iron, mercury, nickel, and zinc on glutathione (GSH), cytoplasmic thioredoxin-1 (Trx1), and mitochondrial thioredoxin-2 (Trx2) redox states. GSH/GSSG redox states were determined by HPLC, and Trx1 and Trx2 redox states were determined by Redox Western blot methods. Copper, iron, and nickel showed significant oxidation of GSH but relatively little oxidation of either Trx1 or Trx2. Arsenic, cadmium, and mercury showed little oxidation of GSH but significantly oxidized both Trx1 and Trx2. The magnitude of effects of arsenic, cadmium, and mercury was greater for the mitochondrial Trx2 (>60 mV) compared to the cytoplasmic Trx1 (20 to 40 mV). Apoptosis signal-regulating kinase 1 (ASK1) may be activated by two different pathways, one dependent upon GSH and glutaredoxin and the other independent of GSH and dependent upon thioredoxin. ASK1 activation and cell death were observed with metals that oxidized thioredoxins but not with metals that oxidized GSH. These findings show that metals have differential oxidative effects on the major thiol antioxidant systems and that activation of apoptosis may be associated with metal ions that oxidize thioredoxin and activate ASK1. The differential oxidation of the major thiol antioxidant systems by metal ions suggest that the distinct thiol/disulfide redox couples represented by GSH/GSSG and the thioredoxins may convey different levels of control in apoptotic and toxic signaling pathways.     

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 as a subcellular biomarker of redox imbalance in human prostate cancer progression

We determined protein levels and subcellular distribution of thioredoxin 1 (Trx1) in human prostate tissues using tissue microarrays and analyzed redox changes in Trx1 in the nucleus and cytoplasm in cell culture models with a redox Western blot technique. We demonstrated increased nuclear Trx1 levels in high- versus low-grade human prostate cancers. Despite increased protein levels, the oxidized forms of nuclear Trx1 were higher in prostate cancer cell lines compared to their benign counterparts, suggesting that nuclear redox imbalance occurred selectively in cancer cells. A growth-stimulating dose of androgen caused transient oxidation of Trx1 in androgen-responsive prostate cancer cells only, suggesting a loss of both androgen- and redox-signaling mechanisms during cancer progression. Androgen-independent PC3 cells showed a significant increase in nuclear and cytoplasmic Trx1 protein levels, but a significant decrease in total Trx activity. Trx1 redox state and activity correlated with the sensitivity of prostate cancer cells to pro-oxidant agents, and downregulation of Trx1 sensitized cancer cells to these agents. Our findings suggest that loss of Trx function because of oxidation and corresponding redox imbalance may play important roles in prostate cancer progression and response to therapies; and Trx1 may serve as a biomarker of subcellular redox imbalance in prostate cancer.     

Thioredoxin 1 as a subcellular biomarker of redox imbalance in human prostate cancer progression

We determined protein levels and subcellular distribution of thioredoxin 1 (Trx1) in human prostate tissues using tissue microarrays and analyzed redox changes in Trx1 in the nucleus and cytoplasm in cell culture models with a redox Western blot technique. We demonstrated increased nuclear Trx1 levels in high- versus low-grade human prostate cancers. Despite increased protein levels, the oxidized forms of nuclear Trx1 were higher in prostate cancer cell lines compared to their benign counterparts, suggesting that nuclear redox imbalance occurred selectively in cancer cells. A growth-stimulating dose of androgen caused transient oxidation of Trx1 in androgen-responsive prostate cancer cells only, suggesting a loss of both androgen- and redox-signaling mechanisms during cancer progression. Androgen-independent PC3 cells showed a significant increase in nuclear and cytoplasmic Trx1 protein levels, but a significant decrease in total Trx activity. Trx1 redox state and activity correlated with the sensitivity of prostate cancer cells to pro-oxidant agents, and downregulation of Trx1 sensitized cancer cells to these agents. Our findings suggest that loss of Trx function because of oxidation and corresponding redox imbalance may play important roles in prostate cancer progression and response to therapies; and Trx1 may serve as a biomarker of subcellular redox imbalance in prostate cancer.     

Oxidation of nuclear thioredoxin during oxidative stress

Thioredoxin 1 (Trx1) is a key redox control system within the nucleus, yet little is known about the sensitivity of nuclear Trx1 to oxidative stress. The present study compared oxidant-induced changes in the redox states of nuclear Trx1, cytoplasmic Trx1, and cellular glutathione (GSH). Nuclear Trx1 was more reducing than cytoplasmic Trx1 and cellular GSH in proliferating cells. tert-Butylhydroperoxide caused an increase in the total amount of nuclear Trx1, but this was accompanied by a 60 mV oxidation. Thus, the increase in nuclear Trx1 levels did not correspond to an increase in the overall reducing capacity of Trx1 in the nucleus.     

Cadmium induced mitochondrial redox changes in germinating pea seed

Mitochondria play an essential role in producing the energy required for seedling growth following imbibition. Heavy metals, such as cadmium impair mitochondrial functioning in part by altering redox regulation. The activities of two protein redox systems present in mitochondria, thioredoxin (Trx) and glutaredoxin (Grx), were analysed in the cotyledons and embryo of pea (Pisum sativum L.) germinating seeds exposed to toxic Cd concentration. Compared to controls, Cd-treated germinating seeds showed a decrease in total soluble protein content, but an increase in –SH content. Under Cd stress conditions, Grx and glutathione reductase (GR) activities as well as glutathione (GSH) concentrations decreased both in cotyledons and the embryo. Similar results were obtained with the Trx system: Trx and NADPH-dependent thioredoxin reductase (NTR) activities were not stimulated, whereas total NAD(P) contents diminished in the embryo. However, Cd enhanced the levels of all components of the Trx system in the cotyledons. On the other hand, Cd caused a significant increase in oxidative stress parameters such as the redox ratio of coenzymes (oxidized to reduced forms) and NAD(P)H oxidase activities. These results indicate that Cd induces differential redox responses on different seed tissues. We suggest that neither Grx system nor Trx one may improve the redox status of mitochondrial thiols in the embryo of germinating pea seeds exposed to Cd toxicity, but in the cotyledons the contribution of Trx/NTR/NADPH can be established in despite the vulnerability of the coenzyme pools due to enzymatic oxidation.     

Redox biology of the intestine

Abstract

The intestinal tract, known for its capability for self-renew, represents the first barrier of defence between the organism and its luminal environment. The thiol/disulfide redox systems comprising the glutathione/glutathione disulfide (GSH/GSSG), cysteine/cystine (Cys/CySS) and reduced and oxidized thioredoxin (Trx/TrxSS) redox couples play important roles in preserving tissue redox homeostasis, metabolic functions, and cellular integrity. Control of the thiol-disulfide status at the luminal surface is essential for maintaining mucus fluidity and absorption of nutrients, and protection against chemical-induced oxidant injury. Within intestinal cells, these redox couples preserve an environment that supports physiological processes and orchestrates networks of enzymatic reactions against oxidative stress. In this review, we focus on the intestinal redox and antioxidant systems, their subcellular compartmentation, redox signalling and epithelial turnover, and contribution of luminal microbiota, key aspects that are relevant to understanding redox-dependent processes in gut biology with implications for degenerative digestive disorders, such as inflammation and cancer.     

Glutathione and thioredoxin redox during differentiation in human colon epithelial (Caco-2) cells

Cellular redox, maintained by the glutathione (GSH)- and thioredoxin (Trx)-dependent systems, has been implicated in the regulation of a variety of biological processes. The redox state of the GSH system becomes oxidized when cells are induced to differentiate by chemical agents. The aim of this study was to determine the redox state of cellular GSH/glutathione disulfide (GSH/GSSG) and Trx as a consequence of progression from proliferation to contact inhibition and spontaneous differentiation in colon carcinoma (Caco-2) cells. Results showed a significant decrease in GSH concentration, accompanied by a 40-mV oxidation of the cellular GSH/GSSG redox state and a 28-mV oxidation of the extracellular cysteine/cystine redox state in association with confluency and increase in differentiation markers. The redox state of Trx did not change. Thus the two central cellular antioxidant and redox-regulating systems (GSH and Trx) were independently controlled. According to the Nernst equation, a 30-mV oxidation is associated with a 10-fold change in the reduced/oxidized ratio of a redox-sensitive dithiol motif. Therefore, the measured 40-mV oxidation of the cellular GSH/GSSG couple or the 28-mV oxidation of the extracellular cysteine/cystine couple should be sufficient to function in signaling or regulation of differentiation in Caco-2 cells.     

Susceptibility of Human Head and Neck Cancer Cells to Combined Inhibition of Glutathione and Thioredoxin Metabolism

Increased glutathione (GSH) and thioredoxin (Trx) metabolism are mechanisms that are widely implicated in resistance of cancer cells to chemotherapy. The current study determined if simultaneous inhibition of GSH and Trx metabolism enhanced cell killing of human head and neck squamous cell carcinoma (HNSCC) cells by a mechanism involving oxidative stress. Inhibition of GSH and Trx metabolism with buthionine sulfoximine (BSO) and auranofin (AUR), respectively, induced significant decreases in clonogenic survival compared to either drug alone in FaDu, Cal-27 and SCC-25 HNSCC cells in vitro and in vivo in Cal-27 xenografts. BSO+AUR significantly increased glutathione and thioredoxin oxidation and suppressed peroxiredoxin activity in vitro. Pre-treatment with N-acetylcysteine completely reversed BSO+AUR-induced cell killing in FaDu and Cal-27 cells, while catalase and selenium supplementation only inhibited BSO+AUR-induced cell killing in FaDu cells. BSO+AUR decreased caspase 3/7 activity in HNSCC cells and significantly reduced the viability of both Bax/Bak double knockout (DKO) and DKO-Bax reconstituted hematopoietic cells suggesting that necrosis was involved. BSO+AUR also significantly sensitized FaDu, Cal-27, SCC-25 and SQ20B cells to cell killing induced by the EGFR inhibitor Erlotinib in vitro. These results support the conclusion that simultaneous inhibition of GSH and Trx metabolism pathways induces oxidative stress and clonogenic killing in HNSCCs and this strategy may be useful in sensitizing HNSCCs to EGFR inhibitors.     

Linked thioredoxin-glutathione systems in platyhelminth parasites: alternative pathways for glutathione reduction and deglutathionylation

In most organisms, thioredoxin (Trx) and/or glutathione (GSH) systems are essential for redox homeostasis and deoxyribonucleotide synthesis. Platyhelminth parasites have a unique and simplified thiol-based redox system, in which the selenoprotein thioredoxin-glutathione reductase (TGR), a fusion of a glutaredoxin (Grx) domain to canonical thioredoxin reductase domains, is the sole enzyme supplying electrons to oxidized glutathione (GSSG) and Trx. This enzyme has recently been validated as a key drug target for flatworm infections. In this study, we show that TGR possesses GSH-independent deglutathionylase activity on a glutathionylated peptide. Furthermore, we demonstrate that deglutathionylation and GSSG reduction are mediated by the Grx domain by a monothiolic mechanism and that the glutathionylated TGR intermediate is resolved by selenocysteine. Deglutathionylation and GSSG reduction via Grx domain, but not Trx reduction, are inhibited at high [GSSG]/[GSH] ratios. We found that Trxs (cytosolic and mitochondrial) provide alternative pathways for deglutathionylation and GSSG reduction. These pathways are operative at high [GSSG]/[GSH] and function in a complementary manner to the Grx domain-dependent one. Despite the existence of alternative pathways, the thioredoxin reductase domains of TGR are an obligate electron route for both the Grx domain- and the Trx-dependent pathways. Overall, our results provide an explanation for the unique array of thiol-dependent redox pathways present in parasitic platyhelminths. Finally, we found that TGR is inhibited by 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7), giving further evidence for NO donation as a mechanism of action for oxadiazole N-oxide TGR inhibitors. Thus, NO donors aimed at TGR could disrupt the entire redox homeostasis of parasitic flatworms.     

Selective protection of nuclear thioredoxin-1 and glutathione redox systems against oxidation during glucose and glutamine deficiency in human colonic epithelial cells

Little is known about the relative sensitivities of antioxidant systems in nuclei, mitochondria, and cytoplasm. The present study examined the oxidation of the thiol-dependent antioxidant systems in these subcellular compartments under conditions of limited energy supply of human colonic epithelial HT-29 cells induced by depletion of glucose (Glc) and glutamine (Gln) from the culture medium. Increased oxidation of dichlorofluoroscein (DCF) indicated an increased level of reactive oxygen species (ROS). Redox Western blot analysis showed oxidation of cytosolic thioredoxin-1 (Trx1) and mitochondrial thioredoxin-2 (Trx2) by 24 h, but little oxidation of nuclear Trx1. The Trx1 substrate, redox factor-1 (Ref-1), was also oxidized in cytosol but was reduced in nuclei. Protein S-glutathionylation (PrSSG), expressed as a ratio of protein thiol (PrSH), was also increased in the cytosol, while nuclear PrSSG/PrSH was not. Taken together, the data show that oxidative stress induced by depletion of Glc and Gln affects the redox states of proteins in the cytoplasm and mitochondria more than those in the nucleus. These results indicate that the nuclear compartment has better protection against oxidative stress than cytoplasm or mitochondria. These results further suggest that energy and/or substrate supply may contribute to sensitivity of mitochondrial and cytoplasmic systems to oxidative damage.     

Intestinal redox biology and oxidative stress

The intestinal epithelium sits at the interface between an organism and its luminal environment, and as such is prone to oxidative damage induced by luminal oxidants. Mucosal integrity is maintained by the luminal redox status of the glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) couples which also support luminal nutrient absorption, mucus fluidity, and a diverse microbiota. The epithelial layer is uniquely organized for rapid self-renewal that is achieved by the well-regulated processes of crypt stem cell proliferation and crypt-to-villus cell differentiation. The GSH/GSSG and Cys/CySS redox couples, known to modulate intestinal cell transition through proliferation, differentiation or apoptosis, could govern the regenerative potential of the mucosa. These two couples, together with that of the thioredoxin/thioredoxin disulfide (Trx/TrxSS) couple are the major intracellular redox systems, and it is proposed that they each function as distinctive redox control nodes or circuitry in the control of metabolic processes and networks of enzymatic reactions. Specificity of redox signaling is accomplished in part by subcellular compartmentation of the individual redox systems within the mitochondria, nucleus, endoplasmic reticulum, and cytosol wherein each defined redox environment is suited to the specific metabolic function within that compartment. Mucosal oxidative stress would result from the disruption of these unique redox control nodes, and the subsequent alteration in redox signaling can contribute to the development of degenerative pathologies of the intestine, such as inflammation and cancer.