Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.     

Calcium regulates thioredoxin reductase in human metastatic melanoma

Thioredoxin reductase has been purified from human metastatic melanotic melanoma and amelanotic melanoma tissues. Enzyme from the melanotic melanoma tissue contains bound calcium showing classical sigmoidal allosteric kinetics, whereas enzyme from the amelanotic melanoma yielded normal Michaelis-Menten saturation with substrate. Calcium inhibition can be partially reversed by oxidized thioredoxin. 45Ca has been used to label the amelanotic melanoma enzyme in order to determine the number of calcium-binding sites. These isotope experiments yielded only one calcium-binding site per enzyme molecule. Enzyme labeled with 45Ca was dialyzed for 24 h without loss of radioactivity, but the addition of oxidized thioredoxin to this labeled enzyme caused 60% calcium exchange in 24 h. Comparative studies with Escherichia coli thioredoxin reductase showed similar calcium inhibition as well as partial reactivation with oxidized thioredoxin. The enzyme from E. coli previously sequenced by others, showed considerable homology with the first EF-hands calcium-binding site of calmodulin. Detailed calcium-binding studies indicated that 10(-5) M of this fast exchange ion was sufficient to cause allosteric regulation in 10 min. This strong calcium-binding property could explain the allosteric nature of the thioredoxin reductase purified from human metastatic melanotic melanoma and its role in the regulation of melanin biosynthesis.     

Genomic organisation and alternative splicing of mouse and human thioredoxin reductase 1 genes

Background  

Thioredoxin reductase (TR) is a redox active protein involved in many cellular processes as part of the thioredoxin system. Presently there are three recognised forms of mammalian thioredoxin reductase designated as TR1, TR3 and TGR, that represent the cytosolic, mitochondrial and novel forms respectively. In this study we elucidated the genomic organisation of the mouse (Txnrd1) and human thioredoxin reductase 1 genes (TXNRD1) through library screening, restriction mapping and database mining.     

The structure of human thioredoxin reductase 1 provides insights into C-terminal rearrangements during catalysis

Human thioredoxin reductase (hTrxR) is a homodimeric flavoprotein crucially involved in the regulation of cellular redox reactions, growth and differentiation. The enzyme contains a selenocysteine residue at its C-terminal active site that is essential for catalysis. This redox center is located on a flexible arm, solvent-exposed and reactive towards electrophilic inhibitors, thus representing a target for antitumor drug development. During catalysis reducing equivalents are transferred from the cofactor NADPH to FAD, then to the N-terminal active site cysteine residues and from there to the flexible C-terminal part of the other subunit to be finally delivered to a variety of second substrates at the molecule's surface. Here we report the first crystal structure of hTrxR1 (Sec-->Cys) in complex with FAD and NADP(+) at a resolution of 2.8 A. From the crystals three different conformations of the carboxy-terminal arm could be deduced. The predicted movement of the arm is facilitated by the concerted action of the three side-chain residues of N418, N419 and W407, which act as a guiding bar for the C-terminal sliding process. As supported by previous kinetic data, the three visualized conformations might reflect different stages in enzymatic catalysis. Comparison with other disulfide reductases including human glutathione reductase revealed specific inhibitor binding sites in the intersubunit cavity of hTrxR that can be exploited for structure-based inhibitor development.     

EF-hands calcium binding regulates the thioredoxin reductase/thioredoxin electron transfer in human keratinocytes

Thioredoxin reductases purified from Escherichia coli from human metastatic melanoma tissue and from human keratinocytes are subject to allosteric inhibition by calcium. 45Calcium has been used to show that this enzyme contains a single binding site. Bound calcium does not exchange from thioredoxin reductase upon dialysis for 48 hours or upon exposure to 10(-3) M EGTA. An intelligenetics computer analysis yielded a single EF-hands calcium binding site on E. coli thioredoxin reductase with homology to the first EF-hands site on calmodulin. Calcium exchange from the enzyme requires the addition of the natural electron acceptor oxidized thioredoxin which causes a concentration dependent slow exchange. Due to the large conformational change caused by calcium binding to thioredoxin reductase it has been possible to separate Calcium-free and Calcium-bound enzyme by FPLC chromatography. Human keratinocytes contain 5% thioredoxin reductase in their acidic protein cytosol fraction. The influence of extracellular calcium concentration on the intracellular equilibrium between calcium bound versus calcium free thioredoxin reductase has been assessed. This equilibrium was shown to determine the redox status of keratinocytes via the reduction of thioredoxin. Our results provide the first evidence for calcium dependent regulation of redox conditions in the human epidermis.     

Overexpression of thioredoxin reductase 1 regulates NF-kappa B activation

Thioredoxin reductase (TrxR) is a flavoprotein that contains a C-terminal penultimate selenocysteine (Sec) and has an ability to reduce thioredoxin (Trx), which regulates the activity of NF-kappa B. To date, three TrxR isozymes, TrxR1, TrxR2, and TrxR3, have been identified. In the present study, we found that among these isozymes only TrxR1 was induced by tumor necrosis factor-alpha (TNF alpha) in vascular endothelial cells. Furthermore, the overexpression of TrxR1 enhanced TNF alpha-induced DNA-binding activity of NF-kappa B and NF-kappa B-dependent gene expression. The catalytic Sec residue of TrxR1, which is essential for reducing Trx, was required for this NF-kappa B activation, and aurothiomalate, an inhibitor of TrxR, suppressed TNF alpha-induced activation of NF-kappa B and the expression of NF-kappa B-targeted proinflammatory genes such as E-selectin and cyclooxygenase-2. These results suggest that TrxR1 may act as a positive regulator of NF-kappa B and may play an important role in the cellular inflammatory response.     

Protein modulase appears to be a complex of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin

Protein modulase and ferredoxin/thioredoxin reductase are soluble proteins that have been suggested to catalyze the light-dependent modulation of enzyme activity in the stromal compartment of the chloroplast. Protein modulase is active in vitro without additional ferredoxin and thioredoxin, whereas ferredoxin/thioredoxin reductase requires additional ferredoxin and thioredoxin. We hypothesize that protein modulase is a complex protein composed of ferredoxin/thioredoxin reductase, ferredoxin, and thioredoxin. In reconstituted chloroplast systems, antiserum directed against ferredoxin, at concentrations sufficient to inhibit the photoreduction of NADP, had no effect on light modulation. Antiserum directed against thioredoxin gave variable results: one batch of polyclonal antibodies inhibited light modulation, another was stimulatory, and another was without effect. These results suggest that the ferredoxin and thioredoxin active in light modulation are not free in solution. Furthermore, molecular sieve chromatography of stromal proteins results in the elution of four species that catalyze light modulation. Based on whether or not ferredoxin and/or thioredoxin must be added for activity, these four species have been tentatively identified as protein modulase, a complex of ferredoxin/thioredoxin reductase and ferredoxin, a complex of ferredoxin/thioredoxin reductase and thioredoxin, and ferredoxin/thioredoxin reductase. That is, the four correspond to all the possible combinations of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin. We suggest that buffer ionic strength affects the interactions among these proteins and in part determines the fate of the protein modulase complex in vitro.     

Identification and characterization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1.

We have identified and characterized a thermostable thioredoxin system in the aerobic hyperthermophilic archaeon Aeropyrum pernix K1. The gene (Accession no. APE0641) of A. pernix encoding a 37 kDa protein contains a redox active site motif (CPHC) but its N-terminal extension region (about 200 residues) shows no homology within the genome database. A second gene (Accession no. APE1061) has high homology to thioredoxin reductase and encodes a 37 kDa protein with the active site motif (CSVC), and binding sites for FAD and NADPH. We cloned the two genes and expressed both proteins in E. coli. It was observed that the recombinant proteins could act as an NADPH-dependent protein disulfide reductase system in the insulin reduction. In addition, the APE0641 protein and thioredoxin reductase from E. coli could also catalyze the disulfide reduction. These indicated that APE1061 and APE0641 express thioredoxin (ApTrx) and thioredoxin reductase (ApTR) of A. pernix, respectively. ApTR is expressed as an active homodimeric flavoprotein in the E. coli system. The optimum temperature was above 90 degrees C, and the half-life of heat inactivation was about 4 min at 110 degrees C. The heat stability of ApTR was enhanced in the presence of excess FAD. ApTR could reduce both thioredoxins from A. pernix and E. coli and showed a similar molar specific activity for both proteins. The standard state redox potential of ApTrx was about -262 mV, which was slightly higher than that of Trx from E. coli (-270 mV). These results indicate that a lower redox potential of thioredoxin is not necessary for keeping catalytic disulfide bonds reduced and thereby coping with oxidative stress in an aerobic hyperthermophilic archaea. Furthermore, the thioredoxin system of aerobic hyperthermophilic archaea is biochemically close to that of the bacteria.     

Glutathione reductase and thioredoxin reductase at the crossroad: the structure of Schistosoma mansoni thioredoxin glutathione reductase

Thioredoxin glutathione reductase (TGR) is a key flavoenzyme expressed by schistosomes that bridges two detoxification pathways crucial for the parasite survival in the host's organism. In this article we report the crystal structure (at 2.2 A resolution) of TGR from Schistosoma mansoni (SmTGR), deleted in the last two residues. The structure reveals the peculiar architecture of this chimeric enzyme: the small Glutaredoxin (Grx) domain at the N-terminus is joined to the large thioredoxin reductase (TR) one via an extended complementary surface, involving residues not conserved in the Grx superfamily; the TR domain interacts with an identical partner via its C-terminal domain, forming a dimer with a twisted "W" shape. Although lacking the penultimate Selenocysteine residue (Sec), the enzyme is still able to reduce oxidized glutathione. These data update the interpretation of the interdomain communication in TGR enzymes. The possible function of this enzyme in pathogenic parasites is discussed.     

Effect of thioredoxin reductase 1 on glucocorticoid receptor activity in human outer root sheath cells

Alopecia areata (AA) is a common disease of patchy hair loss on the scalp that can progress to cover the entire scalp and eventually the entire body. Intralesional injection of corticosteroids is the first-line therapy for adult patients, however some patients do not respond to glucocorticoid treatment effectively. To delineate the molecular mechanism underlying glucocorticoid insensitivity, we examined the expression of glucocorticoid receptor (GR) and thioredoxin reductase 1 (TrxR1). In some case of glucocorticoid-resistant AA patients, the expression of TrxR1 was decreased in outer root sheath (ORS). We then investigated the effect of TrxR1 on GR activity using recombinant adenoviruses. Overexpression of TrxR1 markedly increased GR activity in ORS cells cultured in vitro. In addition, TrxR1 protected GR activity against H(2)O(2). Finally, TrxR1-enhanced GR activity was significantly inhibited by the overexpression of dominant negative form of Trx (Trx(C32S/C35S)). These results suggest that decreased TrxR1 may be one putative cause for glucocorticoid resistance in AA, through the impact on intracellular redox system.     

Kinetics of electron transfer from thioredoxin reductase to thioredoxin

The reduction of Escherichia coli thioredoxin by thioredoxin reductase was studied by stopped-flow spectrophotometry. The reaction showed no dependence on thioredoxin concentration, indicating that complex formation was rapid and occurred during the dead time of the instrument. The kobs for the reaction of approximately 20 s-1 probably reflects the rate of electron transfer from thioredoxin reductase to thioredoxin and agrees with the kcat observed by steady-state kinetics. The reaction rate was unaffected by increasing the ionic strength, suggesting a lack of electrostatic stabilization in the interaction of the two proteins. A mutant thioredoxin in which a positively charged lysine in the active-site region was changed to a glutamic acid residue resulted in an electrostatic destabilization. Thioredoxin K36E was still a substrate for the reductase, but binding was impaired so that the rate could be measured by stopped-flow techniques as reflected by a dependence on protein concentration. Raising the ionic strength in this reaction served to shield the negative charge and increased the rate of binding to the reductase.     

Purification of thioredoxin, thioredoxin reductase, and glutathione reductase by affinity chromatography.

A scheme is described for the large scale purification of thioredoxin, thioredoxin reductase, and glutathione reductase. The scheme is based on an initial separation of thioredoxin from the two reductases by affinity chromatography on agarose-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (agarose-2',5'-ADP). The two reductases were then separated by hydrophobic chromatography and purified separately to homogeneity. Thioredoxin was purified to homogeneity by immunoadsorption to agarose containing immobilized goat anti-thioredoxin. Overall yields for thioredoxin, thioredoxin reductase, and glutathione reductase exceeded 80% in each case. Both reductases exhibit an absorption band at approximately 320 nm which appears due to a residual amount of tightly bound NADP. Presence of this absorption band has no apparent effect on the specific activity of either enzyme.     

Terpyridine-platinum(II) complexes are effective inhibitors of mammalian topoisomerases and human thioredoxin reductase 1

Terpyridine-platinum(II) (TP-Pt(II)) complexes are known to possess DNA-intercalating activity and have been regarded as potential antitumor agents. However, their cytotoxic mechanism remains unclear. To investigate the possible mechanism, a series of TP-Pt(II) compounds were prepared and their biological activities assessed. The DNA binding activities of the aromatic thiolato[TP-Pt(II)] complexes were stronger than the aliphatic 2-hydroxylethanethiolato(2,2′:6′,2′′-terpyridine)platinum(II) [TP(HET)]. TP-Pt(II) complexes inhibited topoisomerase IIα or topoisomerase I activity at IC₅₀ values of about 5 μM and 10-20 μM, respectively, whereas the human thioredoxin reductase 1 (hTrxR1) activity was inhibited with IC₅₀ values in the range of 58-78 nM. At the cellular level, they possessed cytotoxicity with IC₅₀ values between 7 and 19 μM against HeLa cells. Additionally, using X-ray crystallography and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, we elucidated that the TP-Pt(II) complexes inhibited hTrxR1 activity by blocking its C-terminal active-site selenocysteine. Therefore, TP-Pt(II) complexes possess inhibitory activities against multiple biological targets, and they may be further studied as anticancer agents.     

Transgenic mouse models for the vital selenoenzymes cytosolic thioredoxin reductase,mitochondrial thioredoxin reductase and glutathione peroxidase 4

Selenium, as an integral part of selenoproteins, is essential for mammals. Unequivocal evidence had been provided more than a decade ago when it was proven that mice incapable of producing any of the 24 selenoproteins failed to develop beyond the gastrulation stage (E6.5). Since then, more specific attempts have been made to unmask novel and essential functions of individual selenoproteins in mice. Genetic disruption of glutathione peroxidase 4 (GPx4; also referred to as phospholipid hydroperoxide glutathione peroxidase, PHGPx) in mice showed for the first time that a specific selenoenzyme is in fact required for early embryonic development. Later on, systemic ablation of cytosolic thioredoxin reductase (Txnrd1) or mitochondrial thioredoxin reductase (Txnrd2) yielded embryonic lethal phenotypes. Beside those three, no other selenoproteins have been found being indispensable for murine development so far. This review aims at summarizing mainly the in vivo findings on these important mammalian selenoenzymes, which have not only common attributes of being required for embryogenesis, but that they are also instrumental in the regulation of cellular redox metabolism.