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.     

Synthesis of peptide substrates for mammalian thioredoxin reductase.

Mammalian thioredoxin reductase (TR) catalyzes the reduction of the redox-active disulfide bond of thioredoxin (Trx) and is similar in structure and mechanism to glutathione reductase except for a C-terminal 16-amino acid extension containing a rare vicinal selenylsulfide bond. This vicinal selenylsulfide bond is essentially a substrate for the enzyme's N-terminal redox center. Here we report the synthesis of peptide substrates for the truncated enzyme missing the C-terminal redox center. We developed a procedure for the synthesis of peptides containing cyclic vicinal disulfide/selenylsulfide bonds as well as their corresponding acyclic heterodimers. Vicinal disulfide bonds form eight-membered ring structures and are difficult to synthesize owing to their propensity to dimerize during oxidation. Our procedure makes use of two key improvements for on-resin disulfide bond formation presented previously by Galande and coworkers (Galande AK, Weissleder R, Tung C-H. An effective method of on-resin disulfide bond formation in peptides. J. Comb. Chem. 2005; 7: 174-177). First, the addition of an amine base to the deprotection solution allows the complete removal of the StBu group, allowing it to be replaced with a 5-Npys group. The second enhancement is the direct use of a Cys(Mob) or Sec(Mob) derivative as the nucleophilic partner instead of utilizing a naked sulfhydryl or selenol. These improvements result in the formation of a vicinal disulfide (or selenylsulfide) bond in high purity and yield. A direct comparison with the Galande procedure is presented. We also present a novel strategy for the formation of an acyclic, interchain selenylsulfide-linked peptide (linking H-PTVTGC-OH and H-UG-OH). Cysteine analogs of the cyclic and acyclic peptides were also synthesized. The results show that the ring structure contributes a factor of 52 to the rate, but the presence of selenium in the peptide is more important to catalysis than the presence of the ring.     

A novel ubiquinone reductase activity in rat cytosol.

Ubiquinone (UQ) reductase activity which reduces UQ to ubiquinol (UQH2) in rat tissues was roughly proportional to the UQH2/total UQ ratio in respective tissues. The highest activity was found in the liver, showing the highest UQH2/total UQ ratio. A greater part of liver UQ reductase activity was located in the cytosol. Within a week, the liver UQ reductase activity decreased by 80% even at -20 degrees C. The DT-diaphorase activity was stable. UQ reductase required NADPH as the hydrogen donor and was not inhibited by a less than 1 microM concentration of dicoumarol. There was no stimulation of UQ reductase in the presence of bovine serum albumin nor in Triton X-100. Yet, both stimulated DT-diaphorase. As a result, UQ reductase appeared to be a novel NADPH-UQ oxidoreductase and responsible for the UQ redox state in liver.     

Intracellular accumulation of bilirubin as a defense mechanism against increased oxidative stress

Antioxidant, anti-inflammatory and anti-atherogenic effects have been associated with elevations of unconjugated bilirubin (UCB) in serum and with the induction of heme oxygenase-1 (HO-1), the rate-limiting enzyme in UCB synthesis. The aim of this study was to investigate the intracellular metabolism and antioxidant properties of UCB in human hepatoblastoma HepG2 cells and tissues of Wistar rats exposed to oxidative stressors and lipopolysaccharide (LPS), respectively. Intracellular UCB concentrations in HepG2 cells correlated with its levels in culture media (p < 0.001) and diminished lipid peroxidation in a dose-dependent manner (p < 0.001). Moreover, induction of HO-1 with sodium arsenite led to 2.4-fold (p = 0.01) accumulation of intracellular UCB over basal level while sodium azide-derived oxidative stress resulted in a 60% drop (p < 0.001). This decrease was ameliorated by UCB elevation in media or by simultaneous induction of HO-1. In addition, hyperbilirubinemia and liver HO-1 induction in LPS-treated rats resulted in a 2-fold accumulation of tissue UCB (p = 0.01) associated with enhanced protection against lipid peroxidation (p = 0.02). In conclusion, hyperbilirubinemia and HO-1 induction associated with inflammation and oxidative stress increase intracellular concentrations of UCB, thus enhancing the protection of cellular lipids against peroxidation. Therefore, the previously reported protective effects of hyperbilirubinemia and HO-1 induction are at least in part due to intracellular accumulation of UCB.     

Selenodiglutathione is a highly efficient oxidant of reduced thioredoxin and a substrate for mammalian thioredoxin reductase.

Selenium compounds like selenite (SeO3(2-) may form a covalent adduct with glutathione (GSH) in the form of selenodiglutathione (GS-Se-SG), which is assumed to be important in the metabolism of selenium. We have isolated GS-Se-SG and studied its reactions with NADPH and thioredoxin reductase from calf thymus or with thioredoxin reductase and thioredoxin from Escherichia coli. Incubation of 0.1 microM calf thymus thioredoxin reductase or 0.1 microM thioredoxin reductase and 1 microM thioredoxin from E. coli with 5, 10, or 20 microM GS-Se-SG resulted in a fast initial reaction, followed by a large and continued oxidation of NADPH. However, anaerobic incubation of 0.1 microM calf thymus thioredoxin reductase and 20 microM GS-Se-SG resulted only in oxidation of a stoichiometric amount of NADPH; admission of oxygen started continuous NADPH oxidation. Contrary to the mammalian enzyme, GS-Se-SG was not a substrate for thioredoxin reductase from E. coli. The rate of the oxygen-dependent reaction between calf thymus thioredoxin reductase and GS-Se-SG was increased 2-fold in the presence of 4 mM GSH, indicating that HSe- was the reactive intermediate. Glutathione reductase from rat liver reduced GS-Se-SG with a very slow continued oxidation of NADPH, and the presence of the enzyme did not affect the oxygen-dependent nonstoichiometric oxidation of NADPH by GS-Se-SG and thioredoxin reductase. Fluorescence spectroscopy showed GS-Se-SG to be a very efficient oxidant of reduced thioredoxin from E. coli and kinetically superior to insulin disulfides. Thioredoxin-dependent reduction of CDP to dCDP by ribonucleotide reductase was effectively inhibited by GS-Se-SG.     

A novel role for thioredoxin reductase in the iron metabolism of S. cerevisiae

Intracellular levels of iron are tightly regulated. Saccharomyces cerevisiae uses well-defined pathways to extract iron molecules from the environment. Once inside the cell, the iron molecules must be transferred to target sites via an intracellular iron transporter. Although analogous carriers have been described for other metals, such as copper, an iron transporter has yet to be identified. We used two-dimensional gel electrophoresis and mass spectrometry techniques to attempt to identify the iron transporter from cytosolic fraction of S. cerevisiae. In this study, we identified the iron-binding activity of thioredoxin reductase, and our data suggest a potential role for this enzyme in intracellular iron transport.     

The role of corneal crystallins in the cellular defense mechanisms against oxidative stress

The refracton hypothesis describes the lens and cornea together as a functional unit that provides the proper ocular transparent and refractive properties for the basis of normal vision. Similarities between the lens and corneal crystallins also suggest that both elements of the refracton may also contribute to the antioxidant defenses of the entire eye. The cornea is the primary physical barrier against environmental assault to the eye and functions as a dominant filter of UV radiation. It is routinely exposed to reactive oxygen species (ROS)-generating UV light and molecular O(2) making it a target vulnerable to UV-induced damage. The cornea is equipped with several defensive mechanisms to counteract the deleterious effects of UV-induced oxidative damage. These comprise both non-enzymatic elements that include proteins and low molecular weight compounds (ferritin, glutathione, NAD(P)H, ascorbate and alpha-tocopherol) as well as various enzymes (catalase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione reductase, and superoxide dismutase). Several proteins accumulate in the cornea at unusually high concentrations and have been classified as corneal crystallins based on the analogy of these proteins with the abundant taxon-specific lens crystallins. In addition to performing a structural role related to ocular transparency, corneal crystallins may also contribute to the corneal antioxidant systems through a variety of mechanisms including the direct scavenging of free radicals, the production of NAD(P)H, the metabolism and/or detoxification of toxic compounds (i.e. reactive aldehydes), and the direct absorption of UV radiation. In this review, we extend the discussion of the antioxidant defenses of the cornea to include these highly expressed corneal crystallins and address their specific capacities to minimize oxidative damage.     

The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress

The serine protease HtrA (DegP), which is indispensable for cell survival at elevated temperatures, is a peripheral membrane protein, localized on the periplasmic side of the inner membrane in Escherichia coli, and the biochemical and genetic evidence indicates that the physiological role of HtrA is to degrade denatured proteins formed in the cellular envelope during heat shock. The aim of this study was to find out if the HtrA protease contributes to protection of the cell against oxidative stress. We compared the influence of various oxidizing agents on htrA mutant cells with their effects on wild-type bacteria, and found that the htrA mutation did not increase sensitivity to hydrogen peroxide or paraquat but made the cell extremely sensitive to ferrous [Fe(II)] ions, which are known to enhance oxidation of proteins. Treatment with ferrous ions caused a larger increase in the level of protein carbonyl groups in the membrane fraction of the cell than in the periplasm and cytoplasm. Iron-induced oxidation of membrane proteins was enhanced in the htrA mutant relative to wild-type cells. Inhibition of the growth of the htrA mutant by iron could be alleviated more efficiently by a nitroxide antioxidant that localizes in the membranes (A-TEMPO) than by a derivative (4OH-TEMPO) that acts mainly in the soluble fraction of the cell. Inhibition of the growth of the htrA mutant was more pronounced following treatment with cumene hydroperoxide, which partitions into membranes, than with t-butyl hydroperoxide, which forms radical mainly in the cytosol. Both ferrous ions and cumene hydroperoxide, but not hydrogen peroxide, paraquat or t-butyl hydroperoxide, induced synthesis of HtrA. Our results show that HtrA plays a role in defense against oxidative shock and support the hypothesis that HtrA participates in the degradation of oxidatively damaged proteins localized in the cell envelope, especially those associated with the membranes.     

Three distinct-type glutathione S-transferases from Escherichia coli important for defense against oxidative stress

Previously, we characterized glutathione S-transferase (GST) B1-1 from Escherichia coli enzymologically and structurally. Besides GST B1-1, E. coli has seven genes that encode GST-like proteins, for which, except SspA, neither biological roles nor biochemical properties are known. Here we show that the GST-like YfcF and YfcG proteins have low but significant GSH-conjugating activity toward 1-chloro-2,4-dinitorobenzene and GSH-dependent peroxidase activity toward cumene hydroperoxide. Analysis involving site-directed mutagenesis suggested that Ser16 and Asn11 were important for the activities of YfcF and YfcG, respectively. On the contrary, no residue around the catalytic site of GST B1-1 has been demonstrated to be essential for catalytic activity. Deletions of the gst, yfcF, and yfcG genes each decreased the resistibility of the bacteria to hydrogen peroxide, which was recovered by transformation with the expression plasmid for the deleted enzyme. The inactive YfcF(S16G) and YfcG(N11A) mutants, however, could not rescue the knockout bacteria. Thus, E. coli has at least three GSTs of distinct classes, all of which are important for defense against oxidative stress in spite of the structural diversity. This seems consistent with the hypothesis that GSTs constitute a protein superfamily that has evolved from a thioredoxin-like ancestor in response to the development of oxidative stress.     

The polypeptide composition of the mitochondrial NADH: ubiquinone reductase complex from several mammalian species.

The polypeptide composition of isolated mitochondrial NADH:ubiquinone reductase (NADH dehydrogenase) is very similar to that of material immunoprecipitated from detergent-solubilized bovine heart submitochondrial particles by antisera to the holoenzyme. The specificity of the antisera for dehydrogenase polypeptides was determined by immunoblotting, which showed that antisera reacting with only a few proteins were able to immunoprecipitate all others in parallel. The polypeptide compositions of rat, rabbit and human NADH dehydrogenase were determined by immunoprecipitation of the enzyme from solubilized submitochondrial particles and proved to be very similar to that of the bovine heart enzyme, particularly in the high-Mr region. Further homologies in these and other species were explored by immunoblotting with antisera to the holoenzyme and monospecific antisera raised against iron-sulphur-protein subunits of the enzyme.     

Methaneseleninic acid is a substrate for truncated mammalian thioredoxin reductase: implications for the catalytic mechanism and redox signaling

Mammalian thioredoxin reductase is a homodimeric pyridine nucleotide disulfide oxidoreductase that contains the rare amino acid selenocysteine (Sec) on a C-terminal extension. We previously have shown that a truncated version of mouse mitochondrial thioredoxin reductase missing this C-terminal tail will catalyze the reduction of a number of small molecules. Here we show that the truncated thioredoxin reductase will catalyze the reduction of methaneseleninic acid. This reduction is fast at pH 6.1 and is only 4-fold slower than that of the full-length enzyme containing Sec. This finding suggested to us that if the C-terminal Sec residue in the holoenzyme became oxidized to the seleninic acid form (Sec-SeO(2)(-)) that it would be quickly reduced back to an active state by enzymic thiols and further suggested to us that the enzyme would be very resistant to irreversible inactivation by oxidation. We tested this hypothesis by reducing the enzyme with NADPH and subjecting it to high concentrations of H(2)O(2) (up to 50 mM). The results show that the enzyme strongly resisted inactivation by 50 mM H(2)O(2). To determine the redox state of the C-terminal Sec residue, we attempted to inhibit the enzyme with dimedone. Dimedone alkylates protein sulfenic acid residues and presumably will alkylate selenenic acid (Sec-SeOH) residues as well. The enzyme was not inhibited by dimedone even when a 150-fold excess was added to the reaction mixture containing the enzyme and H(2)O(2). We also tested the ability of the truncated enzyme to resist inactivation by oxidation as well and found that it also was resistant to high concentrations of H(2)O(2). One assumption for the use of Sec in enzymes is that it is catalytically superior to the use of cysteine. We and others have previously suggested that there are reasons for the use of Sec in enzymes that are unrelated to the conversion of substrate to product. The data presented here support this assertion. The results also imply that the redox signaling function of the thioredoxin system can remain active under oxidative stress.     

Metformin-ROS-Nrf2 connection in the host defense mechanism against oxidative stress,apoptosis, cancers,and ageing

Reactive oxygen species (ROS) acts as a second messenger to trigger biological responses in low concentrations, while it is implicated to be toxic to biomolecules in high concentrations. Mild inhibition of respiratory chain Complex I by metformin at physiologically relevant concentrations stimulates production of low-level mitochondrial ROS. The ROS seems to induce anti-oxidative stress response via activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione peroxidase (GPx), which results in not only elimination of ROS but also activation of cellular responses including resistance to apoptosis, metabolic changes, cell proliferation, senescence prevention, lifespan extension, and immune T cell activation against cancers, regardless of its effect controlling blood glucose level and T2DM. Although metformin's effect against T2DM, cancers, and ageing, are believed mostly attributed to the activation of AMP-activated protein kinase (AMPK), the cellular responses involving metformin-ROS-Nrf2 axis might be another natural asset to improve healthspan and lifespan.     

Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: A review

Effects of aerobic conditions on strictly anaerobic microorganisms belonging to diverse taxa (clostridia, acetogenic bacteria, lactic acid bacteria, bacteroids, sulfate-reducing bacteria, and methanogenic archaea) and differing considerably in their oxygen resistance have been reviewed, with emphasis on the role of aerotolerance in the ecology of anaerobes. Consideration is given to components of nutritive media for anaerobe culturing, which decrease the toxic effects of oxygen and there by contribute significantly to maintenance and storage of industrial cultures of strictly anaerobic microorganisms. Physiological and biochemical factors are described, accounting for the relative resistance of many strict anaerobes to oxygen and products of incomplete reduction thereof. Specific attention is given to regulation of enzymes of antioxidative defense, operating in the cells of strict anaerobes under the conditions of oxidative stress caused by oxygen, superoxide anion, or hydrogen peroxide.     

A proposed reaction mechanism for rice NADPH thioredoxin reductase C, an enzyme with protein disulfide reductase activity

NADPH thioredoxin reductase C (NTRC) is an interesting NTR with a thioredoxin (Trx) domain at the C-terminus, able to conjugate both activities for 2-Cys peroxiredoxin (Prx) reduction. NTRC is dimeric in the presence of NADPH and interacted with dimeric 2-Cys Prx through the Trx module by a mixed disulfide between Cys377 of NTRC and Cys61 of the 2-Cys Prx. NTRC variants of both NTR and Trx active sites were inactive, but 1:1 mixtures of both variants allowed partial recovery of activity suggesting inter-subunit transfer of electrons during catalysis. Based on these results we propose a model for the reaction mechanism of NTRC.

Structured summary

MINT-7017333: 2cys Prx (uniprotkb:Q6ER94) and 2cys Prx (uniprotkb:Q6ER94) bind (MI:0407) by molecular sieving (MI:0071)MINT-7017101, MINT-7017183: NTRC (uniprotkb:Q70G58) and 2cys Prx (uniprotkb:Q6ER94) bind (MI:0407) by enzymatic studies (MI:0415)     

The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress

The serine protease HtrA (DegP), which is indispensable for cell survival at elevated temperatures, is a peripheral membrane protein, localized on the periplasmic side of the inner membrane in Escherichia coli, and the biochemical and genetic evidence indicates that the physiological role of HtrA is to degrade denatured proteins formed in the cellular envelope during heat shock. The aim of this study was to find out if the HtrA protease contributes to protection of the cell against oxidative stress. We compared the influence of various oxidizing agents on htrA mutant cells with their effects on wild-type bacteria, and found that the htrA mutation did not increase sensitivity to hydrogen peroxide or paraquat but made the cell extremely sensitive to ferrous [Fe(II)] ions, which are known to enhance oxidation of proteins. Treatment with ferrous ions caused a larger increase in the level of protein carbonyl groups in the membrane fraction of the cell than in the periplasm and cytoplasm. Iron-induced oxidation of membrane proteins was enhanced in the htrA mutant relative to wild-type cells. Inhibition of the growth of the htrA mutant by iron could be alleviated more efficiently by a nitroxide antioxidant that localizes in the membranes (A-TEMPO) than by a derivative (4OH-TEMPO) that acts mainly in the soluble fraction of the cell. Inhibition of the growth of the htrA mutant was more pronounced following treatment with cumene hydroperoxide, which partitions into membranes, than with t-butyl hydroperoxide, which forms radical mainly in the cytosol. Both ferrous ions and cumene hydroperoxide, but not hydrogen peroxide, paraquat or t-butyl hydroperoxide, induced synthesis of HtrA. Our results show that HtrA plays a role in defense against oxidative shock and support the hypothesis that HtrA participates in the degradation of oxidatively damaged proteins localized in the cell envelope, especially those associated with the membranes. Received: 9 March 1999 / Accepted: 31 May 1999     

The role of the natural polyamine putrescine in defense against oxidative stress in Escherichia coli

Putrescine up-regulated, in a concentration-dependent manner, the expression levels of the oxyR and katG genes of Escherichia coli cells exposed to hydrogen peroxide. Its stimulatory effect was more pronounced under conditions of strong oxidative stress. 1,4-Diamino-2-butanone, a specific inhibitor of putrescine synthesis, also inhibited oxyR expression under oxidative stress. When added to inhibited cells, putrescine relieved this inhibitory effect. Addition of putrescine to E. coli cultures exposed to oxidative stress led to increased cell survival.