Inhibition of the human thioredoxin system. A molecular mechanism of mercury toxicity

Mercury toxicity mediated by different forms of mercury is a major health problem; however, the molecular mechanisms underlying toxicity remain elusive. We analyzed the effects of mercuric chloride (HgCl(2)) and monomethylmercury (MeHg) on the proteins of the mammalian thioredoxin system, thioredoxin reductase (TrxR) and thioredoxin (Trx), and of the glutaredoxin system, glutathione reductase (GR) and glutaredoxin (Grx). HgCl(2) and MeHg inhibited recombinant rat TrxR with IC(50) values of 7.2 and 19.7 nm, respectively. Fully reduced human Trx1 bound mercury and lost all five free thiols and activity after incubation with HgCl(2) or MeHg, but only HgCl(2) generated dimers. Mass spectra analysis demonstrated binding of 2.5 mol of Hg(2+) and 5 mol of MeHg(+)/mol of Trx1 with the very strong Hg(2+) complexes involving active site and structural disulfides. Inhibition of both TrxR and Trx activity was observed in HeLa and HEK 293 cells treated with HgCl(2) or MeHg. GR was inhibited by HgCl(2) and MeHg in vitro, but no decrease in GR activity was detected in cell extracts treated with mercurials. Human Grx1 showed similar reactivity as Trx1 with both mercurial compounds, with the loss of all free thiols and Grx dimerization in the presence of HgCl(2), but no inhibition of Grx activity was observed in lysates of HeLa cells exposed to mercury. Overall, mercury inhibition was selective toward the thioredoxin system. In particular, the remarkable potency of the mercury compounds to bind to the selenol-thiol in the active site of TrxR should be a major molecular mechanism of mercury toxicity.     

Thioredoxin alters the matrix metalloproteinase/tissue inhibitors of metalloproteinase balance and stimulates human SK-N-SH neuroblastoma cell invasion.

Thioredoxin (Trx) inhibited tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 activity with an approximate IC50 of 0.3 microM, matrix metalloproteinase (MMP)-2 activity with an approximate IC50 of 2 microM but did not inhibit MMP-9 activity. This differential capacity of Trx to inhibit TIMP and MMP activity resulted in the promotion of MMP-2 and MMP-9 activity in the presence of molar TIMP excess. Inhibition of TIMP and MMP-2 activity by Trx was dependent upon thioredoxin reductase (TrxR), was abolished by Trx catalytic site mutation and did not result from TIMP or MMP-2 degradation. HepG2 hepatocellular carcinoma cells induced to secrete Trx inhibited TIMP activity in the presence of TrxR. SK-N-SH neuroblastoma cells secreted TrxR, which inhibited TIMP and MMP-2 activity in the presence of Trx. Trx stimulated SK-N-SH invasive capacity in vitro in the absence of exogenous TrxR. This study therefore identifies a novel extracellular role for the thioredoxin/thioredoxin reductase redox system in the differential inhibition of TIMP and MMP activity and provides a novel mechanism for altering the TIMP/MMP balance that is of potential relevance to tumor invasion.     

A novel antioxidant mechanism of ebselen involving ebselen diselenide,a substrate of mammalian thioredoxin and thioredoxin reductase

The antioxidant mechanism of ebselen involves recently discovered reductions by mammalian thioredoxin reductase (TrxR) and thioredoxin (Trx) forming ebselen selenol. Here we describe a previously unknown reaction; ebselen reacts with its selenol forming an ebselen diselenide with a rate constant of 372 m(-1)s(-1). The diselenide also was a substrate of TrxR forming the selenol with K(m) of 40 microm and k(cat) of 79 min(-1) (k(cat)/K(m) of 3.3 x 10(4) m(-1)s(-1)). Trx increased the reduction because of its fast reaction with diselenide (rate constant 1.7 x 10(3) m(-1)s(-1)). Diselenide stimulated the H2O2 reductase activity of TrxR, even more efficiently with Trx present. Because the mechanism of ebselen as an antioxidant has been assumed to involve glutathione peroxidase-like activity, we compared the H2O2 reductase activity of ebselen with the GSH and Trx systems. TrxR at 50 nm, far below the estimated physiological level, gave 8-fold higher activity compared with 1 mm GSH; addition of 5 microm Trx increased this difference to 13-fold. The rate constant of ebselen selenol reacting with H2O2 was estimated to be faster than 350 m(-1)s(-1). We propose novel mechanisms for ebselen antioxidant action involving ebselen selenol and diselenide formation, with the thioredoxin system rather than glutathione as the predominant effector and target.     

Motexafin gadolinium, a tumor-selective drug targeting thioredoxin reductase and ribonucleotide reductase

Motexafin gadolinium (MGd) is a chemotherapeutic drug that selectively targets tumor cells and mediates redox reactions generating reactive oxygen species. Thioredoxin (Trx), NADPH, and thioredoxin reductase (TrxR) of the cytosol/nucleus or mitochondria are major thiol-dependent reductases with many functions in cell growth, defense against oxidative stress, and apoptosis. Mammalian TrxRs are selenocysteine-containing flavoenzymes; MGd was an NADPH-oxidizing substrate for human or rat TrxR1 with a Km value of 8.65 microM (kcat/Km of 4.86 x 10(4) M(-1) s(-1)). The reaction involved redox cycling of MGd by oxygen producing superoxide and hydrogen peroxide. MGd acted as a non-competitive inhibitor (IC50 of 6 microM) for rat TrxR. In contrast, direct reaction between MGd and reduced human Trx was negligible. The corresponding reaction with reduced Escherichia coli Trx was also negligible, but MGd was a better substrate (kcat/Km of 2.23 x 10(5) M(-1) s(-1)) for TrxR from E. coli and a strong inhibitor of Trx-dependent protein disulfide reduction. Ribonucleotide reductase (RNR), a 1:1 complex of the non-identical R1- and R2-subunits, catalyzes the essential de novo synthesis of deoxyribonucleotides for DNA synthesis using electrons from Trx and TrxR. MGd inhibited recombinant mouse RNR activity with either 3 microM reduced human Trx (IC50 2 microM) or 4 mM dithiothreitol (IC50 6 microM) as electron donors. Our results demonstrate MGd-induced enzymatic generation of reactive oxygen species by TrxR plus a powerful inhibition of RNR. This may explain the effects of the drug on cancer cells, which often overproduce TrxR and have induced RNR for replication and repair.     

Reactive oxygen species, antioxidants, and the mammalian thioredoxin system.

Reactive oxygen species (ROS) are known mediators of intracellular signaling cascades. Excessive production of ROS may, however, lead to oxidative stress, loss of cell function, and ultimately apoptosis or necrosis. A balance between oxidant and antioxidant intracellular systems is hence vital for cell function, regulation, and adaptation to diverse growth conditions. Thioredoxin reductase (TrxR) in conjunction with thioredoxin (Trx) is a ubiquitous oxidoreductase system with antioxidant and redox regulatory roles. In mammals, extracellular forms of Trx also have cytokine-like effects. Mammalian TrxR has a highly reactive active site selenocysteine residue resulting in a profound reductive capacity, reducing several substrates in addition to Trx. Due to the reactivity of TrxR, the enzyme is inhibited by many clinically used electrophilic compounds including nitrosoureas, aurothioglucose, platinum compounds, and retinoic acid derivatives. The properties of TrxR in combination with the functions of Trx position this system at the core of cellular thiol redox control and antioxidant defense. In this review, we focus on the reactions of the Trx system with ROS molecules and different cellular antioxidant enzymes. We summarize the TrxR-catalyzed regeneration of several antioxidant compounds, including ascorbic acid (vitamin C), selenium-containing substances, lipoic acid, and ubiquinone (Q10). We also discuss the general cellular effects of TrxR inhibition. Dinitrohalobenzenes constitute a unique class of immunostimulatory TrxR inhibitors and we consider the immunomodulatory effects of dinitrohalobenzene compounds in view of their reactions with the Trx system.     

Biochemical and thermodynamic comparison of the selenocysteine containing and non-containing thioredoxin glutathione reductase of Fasciola gigantica

The thiol-disulfide redox metabolism in platyhelminth parasites depends entirely on a single selenocysteine (Sec) containing flavoenzyme, thioredoxin glutathione reductase (TGR) that links the classical thioredoxin (Trx) and glutathione (GSH) systems. In the present study, we investigated the catalytic and structural properties of different variants of Fasciola gigantica TGR to understand the role of Sec. The recombinant full-length Sec containing TGR (FgTGRsec), TGR without Sec (FgTGR) and TGRsec without the N-terminal glutaredoxin (Grx) domain (?NTD-FgTGRsec) were purified to homogeneity. Biochemical studies revealed that Sec597 is responsible for higher thioredoxin reductase (TrxR) and glutathione reductase (GR) activity of FgTGRsec. The N-terminal Grx domain was found to positively regulate the DTNB-based TrxR activity of FgTGRsec. The FgTGRsec was highly sensitive to inhibition by auranofin (AF). The structure of FgTGR was modeled, and the inhibitor AF was docked, and binding sites were identified. Unfolding studies suggest that all three proteins are highly cooperative molecules since during GdnHCl-induced denaturation, a monophasic unfolding of the proteins without stabilization of any intermediate is observed. The Cm for GdnHCl induced unfolding of FgTGR was higher than FgTGRsec and ?NTD-FgTGRsec suggesting that FgTGR without Sec was more stable in solution than the other protein variants. The free energy of stabilization for the proteins was also determined. To our knowledge, this is also the first report on unfolding and stability analysis of any TGR.     

Substrate and inhibitor specificities differ between human cytosolic and mitochondrial thioredoxin reductases: Implications for development of specific inhibitors

The cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and thioredoxins (Trx1 and Trx2) are key components of the mammalian thioredoxin system, which is important for antioxidant defense and redox regulation of cell function. TrxR1 and TrxR2 are selenoproteins generally considered to have comparable properties, but to be functionally separated by their different compartments. To compare their properties we expressed recombinant human TrxR1 and TrxR2 and determined their substrate specificities and inhibition by metal compounds. TrxR2 preferred its endogenous substrate Trx2 over Trx1, whereas TrxR1 efficiently reduced both Trx1 and Trx2. TrxR2 displayed strikingly lower activity with dithionitrobenzoic acid (DTNB), lipoamide, and the quinone substrate juglone compared to TrxR1, and TrxR2 could not reduce lipoic acid. However, Sec-deficient two-amino-acid-truncated TrxR2 was almost as efficient as full-length TrxR2 in the reduction of DTNB. We found that the gold(I) compound auranofin efficiently inhibited both full-length TrxR1 and TrxR2 and truncated TrxR2. In contrast, some newly synthesized gold(I) compounds and cisplatin inhibited only full-length TrxR1 or TrxR2 and not truncated TrxR2. Surprisingly, one gold(I) compound, [Au(d2pype)(2)]Cl, was a better inhibitor of TrxR1, whereas another, [(iPr(2)Im)(2)Au]Cl, mainly inhibited TrxR2. These compounds also inhibited TrxR activity in the cytoplasm and mitochondria of cells, but their cytotoxicity was not always dependent on the proapoptotic proteins Bax and Bak. In conclusion, this study reveals significant differences between human TrxR1 and TrxR2 in substrate specificity and metal compound inhibition in vitro and in cells, which may be exploited for development of specific TrxR1- or TrxR2-targeting drugs.     

New thioredoxins and glutaredoxins as electron donors of 3'-phosphoadenylylsulfate reductase

Reduction of inorganic sulfate to sulfite in prototrophic bacteria occurs with 3'-phosphoadenylylsulfate (PAPS) as substrate for PAPS reductase and is the first step leading to reduced sulfur for cellular biosynthetic reactions. The relative efficiency as reductants of homogeneous highly active PAPS reductase of the newly identified second thioredoxin (Trx2) and glutaredoxins (Grx1, Grx2, Grx3, and a mutant Grx1C14S) was compared with the well known thioredoxin (Trx1) from Escherichia coli. Trx1, Trx2, and Grx1 supported virtually identical rates of sulfite formation with a Vmax ranging from 6.6 units mg-1 (Trx1) to 5.1 units mg-1 (Grx1), whereas Grx1C14S was only marginally active, and Grx2 and Grx3 had no activity. The structural difference between active reductants had no effect upon Km PAPS (22.5 microM). Grx1 effectively replaced Trx1 with essentially identical Km-values: Km trx1 (13.7 microM), Km grx1 (14.9 microM), whereas the Km trx2 was considerably higher (34.2 microM). The results agree with previous in vivo data suggesting that Trx1 or Grx1 is essential for sulfate reduction but not for ribonucleotide reduction in E. coli.     

The thioredoxin reductase-glutaredoxins-ferredoxin crossroad pathway for selenate tolerance in Synechocystis PCC6803

Most organisms use two systems to maintain the redox homeostasis of cellular thiols. In the thioredoxin (Trx) system, NADPH sequentially reduces thioredoxin reductases (NTR), Trxs and protein disulfides. In the glutaredoxin (Grx) system, NADPH reduces the glutathione reductase enzyme occurring in most organisms, glutathione, Grxs, and protein disulfides or glutathione-protein mixed disulfides. As little is known concerning these enzymes in cyanobacteria, we have undertaken their analysis in the model strain Synechocystis PCC6803. We found that Grx1 and Grx2 are active, and that Grx2 but not Grx1 is crucial to tolerance to hydrogen peroxide and selenate. We also found that Synechocystis has no genuine glutathione reductase and uses NTR as a Grx electron donor, in a novel integrative pathway NADPH-NTR-Grx1-Grx2-Fed7 (ferredoxin 7), which operates in protection against selenate, the predominant form of selenium in the environment. This is the first report on the occurrence of a physical interaction between a Grx and a Fed, and of an electron transfer between two Grxs. These findings are discussed in terms of the (i) selectivity of Grxs and Feds ( Synechocystis possesses nine Feds), (ii) crucial importance of NTR for cell fitness and (iii) resistance to selenate, in absence of a Thauera selenatis -like selenate reductase.     

Redox atlas of the mouse : Immunohistochemical detection of glutaredoxin-, peroxiredoxin-, and thioredoxin-family proteins in various tissues of the laboratory mouse

Background

Oxidoreductases of the thioredoxin family of proteins have been thoroughly studied in numerous cellular and animal models mimicking human diseases. Despite of their well documented role in various disease conditions, no systematic information on the presence of these proteins is available.

Methods

Here, we have systematically analyzed the presence of some of the major constituents of the glutaredoxin (Grx)-, peroxiredoxin (Prx)-, and thioredoxin (Trx)-systems, i.e. Grx1, Grx2, Grx3 (TXNL-2/PICOT), Grx5, nucleoredoxin (Nrx), Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, Trx1, thioredoxin reductase 1 (TrxR1), Trx2, TrxR2, and γ-glutamyl cysteine synthetase (γ-GCS) in various tissues of the mouse using immunohistochemistry.

Results

The identification of the Trx family proteins in the central nervous system, sensory organs, digestive system, lymphatic system, reproductive system, urinary system, respiratory system, endocrine system, skin, heart, and muscle revealed a number of significant differences between these proteins with respect to their distribution in these tissues.

Conclusion

Our results imply more specific functions and interactions between the proteins of this family than previously assumed.

General significance

Crucial functions of Trx family proteins have been demonstrated in various disease conditions. A detailed overview on their distribution in various tissues will be helpful to fully comprehend their potential role and the interactions of these proteins in the most thoroughly studied model for human diseases—the laboratory mouse.This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.     

Activity assays of mammalian thioredoxin and thioredoxin reductase: Fluorescent disulfide substrates,mechanisms, and use with tissue samples

Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin–glutathione disulfide (Di-E–GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC–insulin), which displayed higher fluorescence on disulfide reduction. Di-E–GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC–insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.     

Identification and characterization of TRP14, a thioredoxin-related protein of 14 kDa. New insights into the specificity of thioredoxin function

We have identified and characterized a 14-kDa human thioredoxin (Trx)-related protein designated TRP14. This cytosolic protein was expressed in all tissues and cell types examined, generally in smaller amounts than Trx1. Although TRP14 contains five cysteines, only the two Cys residues in its WCPDC motif were exposed and redox sensitive. Unlike Trx1, which was an equally good substrate for both Trx reductase 1 (TrxR1) and TrxR2, oxidized TRP14 was reduced by TrxR1 but not by TrxR2. Biochemical characterization of TRP14 suggested that, like Trx1, TRP14 is a disulfide reductase; its active site cysteine is sufficiently nucleophilic with the pK(a) value of 6.1; and its redox potential (-257 mV) is similar to those of other cellular thiol reductants. However, although TRP14 reduced small disulfide-containing peptides, it did not reduce the disulfides of known Trx1 substrates, ribonucleotide reductase, peroxiredoxin, and methionine sulfoxide reductase. These results suggest that TRP14 and Trx1 might act on distinct substrate proteins.     

Inhibition of the thioredoxin system is a basis for the antileukemic potential of 13-hydroxy-15-oxo-zoapatlin

The mammalian thioredoxin (Trx) system, composed of Trx, Trx reductase (TrxR), and NADPH, is the most important thiol system involved in the redox control of signaling and regulatory proteins in apoptosis and cell proliferation. Here we addressed the inhibition of the Trx system by 13-hydroxy-15-oxo-zoapatlin (OZ), a nor-kaurane diterpene previously shown to possess proapoptotic potential and to cause cell cycle arrest in leukemia cells. OZ was found, by both biochemical and mass spectrometry-based approaches, to target Trx1 and TrxR in a cell-free system. In particular, the formation of reversible OZ adducts to Trx1 Cys35, Cys62, and Cys73 was demonstrated. We next showed that OZ efficiently inhibited Trx and TrxR catalytic activity in Molt4 cells. The occurrence of oxidative modifications of Trx molecules was assessed by "redox Western blot" analyses. OZ-mediated Trx oxidation resulted in apoptosis signaling kinase-1 release and activation of downstream JNK and p38 pathways. By means of specific inhibitors of these two stress-activated protein kinases, we demonstrated that the JNK pathway plays a major role in determining the apoptotic fate of OZ-exposed cells, whereas p38 activation seems to be involved mainly in OZ-induced G2/M block.     

Regulation of the catalytic activity and structure of human thioredoxin 1 via oxidation and S-nitrosylation of cysteine residues

The mammalian cytosolic/nuclear thioredoxin system, comprising thioredoxin (Trx), selenoenzyme thioredoxin reductase (TrxR), and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. The active site of reduced Trx comprises Cys(32)-Gly-Pro-Cys(35) thiols that catalyze target disulfide reduction, generating a disulfide. Human Trx1 has also three structural Cys residues in positions 62, 69, and 73 that upon diamide oxidation induce a second Cys(62)-Cys(69) disulfide as well as dimers and multimers. We have discovered that after incubation with H(2)O(2) only monomeric two-disulfide molecules are generated, and they are inactive but able to regain full activity in an autocatalytic process in the presence of NADPH and TrxR. There are conflicting results regarding the effects of S-nitrosylation on Trx antioxidant functions and which residues are involved. We found that S-nitrosoglutathione-mediated S-nitrosylation at physiological pH is critically dependent on the redox state of Trx. Starting from fully reduced human Trx, both Cys(69) and Cys(73) were nitrosylated, and the active site formed a disulfide; the nitrosylated Trx was not a substrate for TrxR but regained activity after a lag phase consistent with autoactivation. Treatment of a two-disulfide form of Trx1 with S-nitrosoglutathione resulted in nitrosylation of Cys(73), which can act as a trans-nitrosylating agent as observed by others to control caspase 3 activity (Mitchell, D. A., and Marletta, M. A. (2005) Nat. Chem. Biol. 1, 154-158). The reversible inhibition of human Trx1 activity by H(2)O(2) and NO donors is suggested to act in cell signaling via temporal control of reduction for the transmission of oxidative and/or nitrosative signals in thiol redox control.     

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.     

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.