Protective Effects of a Jellyfish-Derived Thioredoxin Fused with Cell-Penetrating Peptide TAT-PTD on H2O2-Induced Oxidative Damage.

Thioredoxin (Trx) plays a critical role in maintaining redox balance in various cells and exhibits anti-oxidative, anti-apoptotic, and anti-inflammatory effects. However, whether exogenous Trx can inhibit intracellular oxidative damage has not been investigated. In previous study, we have identified a novel Trx from the jellyfish Cyanea capillata, named CcTrx1, and confirmed its antioxidant activities in vitro. Here, we obtained a recombinant protein, PTD-CcTrx1, which is a fusion of CcTrx1 and protein transduction domain (PTD) of HIV TAT protein. The transmembrane ability and antioxidant activities of PTD-CcTrx1, and its protective effects against H2O2-induced oxidative damage in HaCaT cells were also detected. Our results revealed that PTD-CcTrx1 exhibited specific transmembrane ability and antioxidant activities, and it could significantly attenuate the intracellular oxidative stress, inhibit H2O2-induced apoptosis, and protect HaCaT cells from oxidative damage. The present study provides critical evidence for application of PTD-CcTrx1 as a novel antioxidant to treat skin oxidative damage in the future.

The In Vitro Interaction of 12-Oxophytodienoic Acid and Related Conjugated Carbonyl Compounds with Thiol Antioxidants.

α,ß-unsaturated carbonyls interfere with numerous plant physiological processes. One mechanism of action is their reactivity toward thiols of metabolites like cysteine and glutathione (GSH). This work aimed at better understanding these interactions. Both 12-oxophytodienoic acid (12-OPDA) and abscisic acid (ABA) conjugated with cysteine. It was found that the reactivity of α,ß-unsaturated carbonyls with GSH followed the sequence trans-2-hexenal < 12-OPDA ≈ 12-OPDA-ethylester < 2-cyclopentenone << methyl vinylketone (MVK). Interestingly, GSH, but not ascorbate (vitamin C), supplementation ameliorated the phytotoxic potential of MVK. In addition, 12-OPDA and 12-OPDA-related conjugated carbonyl compounds interacted with proteins, e.g., with members of the thioredoxin (TRX)-fold family. 12-OPDA modified two cysteinyl residues of chloroplast TRX-f1. The OPDAylated TRX-f1 lost its activity to activate the Calvin-Benson-cycle enzyme fructose-1,6-bisphosphatase (FBPase). Finally, we show that 12-OPDA interacts with cyclophilin 20-3 (Cyp20-3) non-covalently and affects its peptidyl-prolyl-cis/trans isomerase activity. The results demonstrate the high potential of 12-OPDA as a diverse interactor and cellular regulator and suggest that OPDAylation may occur in plant cells and should be investigated as novel regulatory mechanism.

Control of protein function through oxidation and reduction of persulfidated states.

Irreversible oxidation of Cys residues to sulfinic/sulfonic forms typically impairs protein function. We found that persulfidation (CysSSH) protects Cys from irreversible oxidative loss of function by the formation of CysSSO1-3H derivatives that can subsequently be reduced back to native thiols. Reductive reactivation of oxidized persulfides by the thioredoxin system was demonstrated in albumin, Prx2, and PTP1B. In cells, this mechanism protects and regulates key proteins of signaling pathways, including Prx2, PTEN, PTP1B, HSP90, and KEAP1. Using quantitative mass spectrometry, we show that (i) CysSSH and CysSSO3H species are abundant in mouse liver and enzymatically regulated by the glutathione and thioredoxin systems and (ii) deletion of the thioredoxin-related protein TRP14 in mice altered CysSSH levels on a subset of proteins, predicting a role for TRP14 in persulfide signaling. Furthermore, selenium supplementation, polysulfide treatment, or knockdown of TRP14 mediated cellular responses to EGF, suggesting a role for TrxR1/TRP14-regulated oxidative persulfidation in growth factor responsiveness.

Quantitative Proteomics Analysis of Barley-Based Liquid Feed and the Effect of Protease Inhibitors and NADPH-Dependent Thioredoxin Reductase/Thioredoxin (NTR/Trx) System.

Liquid feeding strategies have been devised with the aim of enhancing grain nutrient availability for livestock. It is characterized by a steeping/soaking period that softens the grains and initiates mobilization of seed storage reserves. The present study uses 2D gel-based proteomics to investigate the role of proteolysis and reduction by thioredoxins over a 48 h steeping period by monitoring protein abundance dynamics in barley-based liquid feed samples supplemented with either protease inhibitors or NADPH-dependent thioredoxin reductase/thioredoxin (NTR/Trx). Several full-length storage proteins were only identified in the water-extractable fraction of feed containing protease inhibitors, illustrating significant inhibition of proteolytic activities arising during the steeping period. Application of functional NTR/Trx to liquid feed reductively increased the solubility of known and potentially new Trx-target proteins, e.g., outer membrane protein X, and their susceptibility to proteolysis. Thus, the NTR/Trx system exhibits important potential as a feed additive to enhance nutrient digestibility in monogastric animals.

Substrate Metabolism-Driven Assembly of High-Quality CdS xSe1- x Quantum Dots in Escherichia coli: Molecular Mechanisms and Bioimaging Application.

Biosynthesis offers opportunities for cost-effective and sustainable production of semiconductor quantum dots (QDs), but is currently restricted by poor controllability on the synthesis process, resulting from limited knowledge on the assembly mechanisms and the lack of effective control strategies. In this work, we provide molecular-level insights into the formation mechanism of biogenic QDs (Bio-QDs) and its connection with the cellular substrate metabolism in Escherichia coli. Strengthening the substrate metabolism for producing more reducing power was found to stimulate the production of several reduced thiol-containing proteins (including glutaredoxin and thioredoxin) that play key roles in Bio-QDs assembly. This effectively diverted the transformation route of the selenium (Se) and cadmium (Cd) metabolic from Cd3(PO4)2 formation to CdS xSe1- x QDs assembly, yielding fine-sized (2.0 ± 0.4 nm), high-quality Bio-QDs with quantum yield (5.2%) and fluorescence lifetime (99.19 ns) far exceeding the existing counterparts. The underlying mechanisms of Bio-QDs crystallization and development were elucidated by density functional theory calculations and molecular dynamics simulation. The resulting Bio-QDs were successfully used for bioimaging of cancer cells and tumor tissue of mice without extra modification. Our work provides fundamental knowledge on the Bio-QDs assembly mechanisms and proposes an effective, facile regulation strategy, which may inspire advances in controlled synthesis and practical applications of Bio-QDs as well as other bionanomaterials.

Identification of potential inhibitors of Fasciola gigantica thioredoxin1: computational screening, molecular dynamics simulation, and binding free energy studies.

Fasciola gigantica is the causative organism of fascioliasis and is responsible for major economic losses in livestock production globally. F. gigantica thioredoxin1 (FgTrx1) is an important redox-active enzyme involved in maintaining the redox homeostasis in the cell. To identify a potential anti-fasciolid compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (n = 1,67,740) against the FgTrx1 structure. The ligands were docked against FgTrx1 and 309 ligands were found to have better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 30 compounds were found to fit well for re-docking studies. After refinement by molecular docking and drug-likeness analysis, three potential inhibitors (ZINC15970091, ZINC9312362, and ZINC9312661) were identified. These three ligands were further subjected to molecular dynamics simulation (MDS) to compare the dynamics and stability of the protein structure after binding of the ligands. The binding free energy analyses were calculated to determine the intermolecular interactions. The results suggested that the two compounds had a binding free energy of -82.237, and -109.52 kJ.mol-1 for compounds with IDs ZINC9312362 and ZINC9312661, respectively. These predicted compounds displayed considerable pharmacological and structural properties to be drug candidates. We concluded that these two compounds could be potential drug candidates to fight against F. gigantica parasites.

Selenium utilization in thioredoxin and catalytic advantage provided by selenocysteine.

Thioredoxin (Trx) is a major thiol-disulfide reductase that plays a role in many biological processes, including DNA replication and redox signaling. Although selenocysteine (Sec)-containing Trxs have been identified in certain bacteria, their enzymatic properties have not been characterized. In this study, we expressed a selenoprotein Trx from Treponema denticola, an oral spirochete, in Escherichia coli and characterized this selenoenzyme and its natural cysteine (Cys) homologue using E. coli Trx1 as a positive control. (75)Se metabolic labeling and mutation analyses showed that the SECIS (Sec insertion sequence) of T. denticola selenoprotein Trx is functional in the E. coli Sec insertion system with specific selenium incorporation into the Sec residue. The selenoprotein Trx exhibited approximately 10-fold higher catalytic activity than the Sec-to-Cys version and natural Cys homologue and E. coli Trx1, suggesting that Sec confers higher catalytic activity on this thiol-disulfide reductase. Kinetic analysis also showed that the selenoprotein Trx had a 30-fold higher Km than Cys-containing homologues, suggesting that this selenoenzyme is adapted to work efficiently with high concentrations of substrate. Collectively, the results of this study support the hypothesis that selenium utilization in oxidoreductase systems is primarily due to the catalytic advantage provided by the rare amino acid, Sec.

First report of a thioredoxin homologue in jellyfish: molecular cloning, expression and antioxidant activity of CcTrx1 from Cyanea capillata.

Thioredoxins (Trx proteins) are a family of small, highly-conserved and ubiquitous proteins that play significant roles in the resistance of oxidative damage. In this study, a homologue of Trx was identified from the cDNA library of tentacle of the jellyfish Cyanea capillata and named CcTrx1. The full-length cDNA of CcTrx1 was 479 bp with a 312 bp open reading frame encoding 104 amino acids. Bioinformatics analysis revealed that the putative CcTrx1 protein harbored the evolutionarily-conserved Trx active site 31CGPC34 and shared a high similarity with Trx1 proteins from other organisms analyzed, indicating that CcTrx1 is a new member of Trx1 sub-family. CcTrx1 mRNA was found to be constitutively expressed in tentacle, umbrella, oral arm and gonad, indicating a general role of CcTrx1 protein in various physiological processes. The recombinant CcTrx1 (rCcTrx1) protein was expressed in Escherichia coli BL21 (DE3), and then purified by affinity chromatography. The rCcTrx1 protein was demonstrated to possess the expected redox activity in enzymatic analysis and protection against oxidative damage of supercoiled DNA. These results indicate that CcTrx1 may function as an important antioxidant in C. capillata. To our knowledge, this is the first Trx protein characterized from jellyfish species.

Hydration and conformational equilibrium in yeast thioredoxin 1: implication for H(+) exchange.

One of the ancestral features of thioredoxins is the presence of a water cavity. Here, we report that a largely hydrated, conserved, buried aspartic acid in the water cavity modulates the dynamics of the interacting loops of yeast thioredoxin 1 (yTrx1). It is well-established that the aspartic acid, Asp24 for yTrx1, works as a proton acceptor in the reduction of the target protein. We propose a complementary role for Asp24 of coupling hydration and conformational motion of the water cavity and interacting loops. The intimate contact between the water cavity and the interacting loops means that motion at the water cavity will affect the interacting loops and vice versa. The D24N mutation alters the conformational equilibrium for both the oxidized and reduced states, quenching the conformational motion in the water cavity. By measuring the hydration and molecular dynamics simulation of wild-type yTrx1 and the D24N mutant, we showed that Asn24 is more exposed to water than Asp24 and the water cavity is smaller in the mutant, closing the inner part of the water cavity. We discuss how the conformational equilibrium contributes to the mechanism of catalysis and H(+) exchange.

Selenium as an electron acceptor during the catalytic mechanism of thioredoxin reductase.

Mammalian thioredoxin reductase (TR) is a pyridine nucleotide disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys) in the redox-active tetrapeptide Gly-Cys-Sec-Gly motif to catalyze thiol/disulfide exchange reactions. Sec can accelerate the rate of these exchange reactions (i) by being a better nucleophile than Cys, (ii) by being a better electrophile than Cys, (iii) by being a better leaving group than Cys, or (iv) by using a combination of all three of these factors, being more chemically reactive than Cys. The role of the selenolate as a nucleophile in the reaction mechanism was recently demonstrated by creating a mutant of human thioredoxin reductase-1 in which the Cys497-Sec498 dyad of the C-terminal redox center was mutated to either a Ser497-Cys498 dyad or a Cys497-Ser498 dyad. Both mutant enzymes were incubated with human thioredoxin (Trx) to determine which mutant formed a mixed disulfide bond complex. Only the mutant containing the Ser497-Cys498 dyad formed a complex, and this structure has been determined by X-ray crystallography [Fritz-Wolf, K., Kehr, S., Stumpf, M., Rahlfs, S., and Becker, K. (2011) Crystal structure of the human thioredoxin reductase-thioredoxin complex. Nat. Commun. 2, 383]. This experimental observation most likely means that the selenolate is the nucleophile initially attacking the disulfide bond of Trx because a complex resulted only when Cys was present in the second position of the dyad. As a nucleophile, the selenolate of Sec helps to accelerate the rate of this exchange reaction relative to Cys in the Sec → Cys mutant enzyme. Another thiol/disulfide exchange reaction that occurs in the enzymatic cycle of the enzyme is the transfer of electrons from the thiolate of the interchange Cys residue of the N-terminal redox center to the eight-membered selenosulfide ring of the C-terminal redox center. The selenium atom of the selenosulfide could accelerate this exchange reaction by being a good leaving group (attack at the sulfur atom) or by being a good electrophile (attack at the selenium atom). Here we provide strong evidence that the selenium atom is attacked in this exchange step. This was shown by creating a mutant enzyme containing a Gly-Gly-Seccoo- motif that had 0.5% of the activity of the wild-type enzyme. This mutant lacks the adjacent, resolving Cys residue, which acts by attacking the mixed selenosulfide bond that occurs between the enzyme and substrate. A similar result was obtained when Sec was replaced with homocysteine. These results highlight the role of selenium as an electron acceptor in the catalytic mechanism of thioredoxin reductase as well as its established role as a donor of an electron to the substrate.

NTR/NRX define a new thioredoxin system in the nucleus of Arabidopsis thaliana cells.

Thioredoxins (TRX) are key components of cellular redox balance, regulating many target proteins through thiol/disulfide exchange reactions. In higher plants, TRX constitute a complex multigenic family whose members have been found in almost all cellular compartments. Although chloroplastic and cytosolic TRX systems have been largely studied, the presence of a nuclear TRX system has been elusive for a long time. Nucleoredoxins (NRX) are potential nuclear TRX found in most eukaryotic organisms. In contrast to mammals, which harbor a unique NRX, angiosperms generally possess multiple NRX organized in three subfamilies. Here, we show that Arabidopsis thaliana has two NRX genes (AtNRX1 and AtNRX2), respectively, belonging to subgroups I and III. While NRX1 harbors typical TRX active sites (WCG/PPC), NRX2 has atypical active sites (WCRPC and WCPPF). Nevertheless, both NRX1 and NRX2 have disulfide reduction capacities, although NRX1 alone can be reduced by the thioredoxin reductase NTRA. We also show that both NRX1 and NRX2 have a dual nuclear/cytosolic localization. Interestingly, we found that NTRA, previously identified as a cytosolic protein, is also partially localized in the nucleus, suggesting that a complete TRX system is functional in the nucleus. We show that NRX1 is mainly found as a dimer in vivo. nrx1 and nrx2 knockout mutant plants exhibit no phenotypic perturbations under standard growth conditions. However, the nrx1 mutant shows a reduced pollen fertility phenotype, suggesting a specific role of NRX1 at the haploid phase.

Mixed disulfide formation in vitro between a glycoprotein substrate and yeast oligosaccharyltransferase subunits Ost3p and Ost6p.

Oligosaccharyltransferase (OTase) glycosylates selected asparagine residues in secreted and membrane proteins in eukaryotes, and asparagine (N)-glycosylation affects the folding, stability and function of diverse glycoproteins. The range of acceptor protein substrates that are efficiently glycosylated depends on the action of several accessory subunits of OTase, including in yeast the homologous proteins Ost3p and Ost6p. A model of Ost3p and Ost6p function has been proposed in which their thioredoxin-like active site cysteines form transient mixed disulfide bonds with cysteines in substrate proteins to enhance the glycosylation of nearby asparagine residues. We tested aspects of this model with a series of in vitro assays. We developed a whole protein mixed disulfide interaction assay that showed that Ost6p could form mixed disulfide bonds with selected cysteines in pre-reduced yeast Gas1p, a model glycoprotein substrate of Ost3p and Ost6p. A complementary peptide affinity chromatography assay for mixed disulfide bond formation showed that Ost3p could also form mixed disulfide bonds with cysteines in selected reduced tryptic peptides from Gas1p. Together, these assays showed that the thioredoxin-like active sites of Ost3p and Ost6p could form transient mixed disulfide bonds with cysteines in a model substrate glycoprotein, consistent with the function of Ost3p and Ost6p in modulating N-glycosylation substrate selection by OTase in vivo.

Catalytic activity of selenomethionine in removing amino acid, peptide, and protein hydroperoxides.

Selenium is a critical trace element, with deficiency associated with numerous diseases including cardiovascular disease, diabetes, and cancer. Selenomethionine (SeMet; a selenium analogue of the amino acid methionine, Met) is a major form of organic selenium and an important dietary source of selenium for selenoprotein synthesis in vivo. As selenium compounds can be readily oxidized and reduced, and selenocysteine residues play a critical role in the catalytic activity of the key protective enzymes glutathione peroxidase and thioredoxin reductase, we investigated the ability of SeMet (and its sulfur analogue, Met) to scavenge hydroperoxides present on amino acids, peptides, and proteins, which are key intermediates in protein oxidation. We show that SeMet, but not Met, can remove these species both stoichiometrically and catalytically in the presence of glutathione (GSH) or a thioredoxin reductase (TrxR)/thioredoxin (Trx)/NADPH system. Reaction of the hydroperoxide with SeMet results in selenoxide formation as detected by HPLC. Recycling of the selenoxide back to SeMet occurs rapidly with GSH, TrxR/NADPH, or a complete TrxR/Trx/NADPH reducing system, with this resulting in an enhanced rate of peroxide removal. In the complete TrxR/Trx/NADPH system loss of peroxide is essentially stoichiometric with NADPH consumption, indicative of a highly efficient system. Similar reactions do not occur with Met under these conditions. Studies using murine macrophage-like J774A.1 cells demonstrate a greater peroxide-removing capacity in cells supplemented with SeMet, compared to nonsupplemented controls. Overall, these findings demonstrate that SeMet may play an important role in the catalytic removal of damaging peptide and protein oxidation products.

The selenium-independent inherent pro-oxidant NADPH oxidase activity of mammalian thioredoxin reductase and its selenium-dependent direct peroxidase activities.

Mammalian thioredoxin reductase (TrxR) is an NADPH-dependent homodimer with three redox-active centers per subunit: a FAD, an N-terminal domain dithiol (Cys(59)/Cys(64)), and a C-terminal cysteine/selenocysteine motif (Cys(497)/Sec(498)). TrxR has multiple roles in antioxidant defense. Opposing these functions, it may also assume a pro-oxidant role under some conditions. In the absence of its main electron-accepting substrates (e.g. thioredoxin), wild-type TrxR generates superoxide (O ), which was here detected and quantified by ESR spin trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The peroxidase activity of wild-type TrxR efficiently converted the O adduct (DEPMPO/HOO(*)) to the hydroxyl radical adduct (DEPMPO/HO(*)). This peroxidase activity was Sec-dependent, although multiple mutants lacking Sec could still generate O . Variants of TrxR with C59S and/or C64S mutations displayed markedly reduced inherent NADPH oxidase activity, suggesting that the Cys(59)/Cys(64) dithiol is required for O generation and that O is not derived directly from the FAD. Mutations in the Cys(59)/Cys(64) dithiol also blocked the peroxidase and disulfide reductase activities presumably because of an inability to reduce the Cys(497)/Sec(498) active site. Although the bulk of the DEPMPO/HO(*) signal generated by wild-type TrxR was due to its combined NADPH oxidase and Sec-dependent peroxidase activities, additional experiments showed that some free HO(*) could be generated by the enzyme in an H(2)O(2)-dependent and Sec-independent manner. The direct NADPH oxidase and peroxidase activities of TrxR characterized here give insights into the full catalytic potential of this enzyme and may have biological consequences beyond those solely related to its reduction of thioredoxin.

The aza-analogues of 1,4-naphthoquinones are potent substrates and inhibitors of plasmodial thioredoxin and glutathione reductases and of human erythrocyte glutathione reductase.

Various aza-analogues of 1,4-naphthoquinone and menadione were prepared and tested as inhibitors and substrates of the plasmodial thioredoxin and glutathione reductases as well as the human glutathione reductase. The replacement of one to two carbons at the phenyl ring of the 1,4-naphthoquinone core by one to two nitrogen atoms led to an increased oxidant character of the molecules in accordance with both the redox potential values and the substrate efficiencies. Compared to the 1,4-naphthoquinone and menadione, the quinoline-5,8-dione 1 and both quinoxaline-5,8-diones 5 and 6 behaved as the most efficient subversive substrates of the three NADPH-dependent disulfide reductases tested. Modulation of these parameters was observed by alkylation of the aza-naphthoquinone core.

Reduced reliance on the trace element selenium during evolution of mammals.

BACKGROUND: Selenium (Se) is an essential trace element that occurs in proteins in the form of selenocysteine (Sec). It is transported throughout the body in the form of Sec residues in Selenoprotein P (SelP), a plasma protein of unclear origin recently proposed as an experimental marker of dietary Se status. RESULTS: Here, we report that the amino-terminal domain of SelP is distantly related to ancestral bacterial thiol oxidoreductases of the thioredoxin superfamily, and that its carboxy-terminal Se transport domain may have originated in early metazoan evolution by de novo accumulation of Sec residues. Reconstruction of evolutionary changes in the Se transport domain indicates a decrease in Sec content of SelP specifically in the mammalian lineage via replacement of Sec with cysteine (Cys). Sec content of mammalian SelPs varies more than two-fold and is lowest in rodents and primates. Compared to mammals, fish show higher Sec content of SelP, larger selenoproteomes, elevated SelP gene expression, and higher levels of tissue Se. In addition, mammals replaced Sec with Cys in several proteins and lost several selenoproteins altogether, whereas such events are not found in fish. CONCLUSION: These data suggest that evolution from fish to mammals was accompanied by decreased use of Sec and that analyses of SelP, selenoproteomes and Sec/Cys transitions provide a genetic marker of utilization of this trace element in vertebrates. The evolved reduced reliance on Se raises questions regarding the need to maximize selenoprotein expression by Se dietary supplements in situations when pathology is not imminent, a currently accepted practice.

Sulfide : quinone oxidoreductase (SQR) from the lugworm Arenicola marina shows cyanide- and thioredoxin-dependent activity.

The lugworm Arenicola marina inhabits marine sediments in which sulfide concentrations can reach up to 2 mM. Although sulfide is a potent toxin for humans and most animals, because it inhibits mitochondrial cytochrome c oxidase at micromolar concentrations, A. marina can use electrons from sulfide for mitochondrial ATP production. In bacteria, electron transfer from sulfide to quinone is catalyzed by the membrane-bound flavoprotein sulfide : quinone oxidoreductase (SQR). A cDNA from A. marina was isolated and expressed in Saccharomyces cerevisiae, which lacks endogenous SQR. The heterologous enzyme was active in mitochondrial membranes. After affinity purification, Arenicola SQR isolated from yeast mitochondria reduced decyl-ubiquinone (K(m) = 6.4 microm) after the addition of sulfide (K(m) = 23 microm) only in the presence of cyanide (K(m) = 2.6 mM). The end product of the reaction was thiocyanate. When cyanide was substituted by Escherichia coli thioredoxin and sulfite, SQR exhibited one-tenth of the cyanide-dependent activity. Six amino acids known to be essential for bacterial SQR were exchanged by site-directed mutagenesis. None of the mutant enzymes was active after expression in yeast, implicating these amino acids in the catalytic mechanism of the eukaryotic enzyme.

Efficient preparation of an acyclic permutant of kalata B1 from a recombinant fusion protein with thioredoxin.

A new approach to prepare an acyclic permutant of kalata B1, a cysteine-rich plant cyclopeptide with uterotonic activity, is described. The synthetic codon-optimized cDNA sequence encoding this 29-residue peptide was cloned and fused in-frame to the His(6)-tagged thioredoxin gene in the bacterial expression vector pET-32a. The fusion protein was overexpressed in the bacterial host, Escherichia coli strain BL21 (DE3), and isolated by affinity chromatography on a metal-chelating Sepharose column. An enterokinase recognition sequence incorporated immediately upstream of the target peptide allowed the 29-residue peptide to be released without any unwanted residues upon treatment with enterokinase. This peptide was subsequently separated from the larger thioredoxin moiety by ultracentrifugation through a semipermeable membrane. Further purification was achieved using reversed-phase HPLC. Hydrogen peroxide was found to enhance the rate of enterokinase cleavage in a concentration-dependent manner. Thermal stability studies demonstrated that the recombinant acyclic kalata B1 (ac kalata) was exceptionally stable against thermal denaturation. Mass spectrometric analysis revealed that the recombinant ac kalata was obtained in a fully oxidized form, indicating a high reducing potential and a strong tendency of the 29-residue peptide to form a tightly folded structure.

Involvement of FpTRP26, a thioredoxin-related protein, in oxalic acid-resistance of the brown-rot fungus Fomitopsis palustris.

Brown-rot fungus Fomitopsis palustris grows vigorously at high concentrations of oxalic acid (OA), which is fungal metabolite during wood decay. We isolated a cDNA FpTRP26 from F. palustris by functional screening of yeast transformants with cDNAs grown on plates containing OA. FpTRP26 conferred a specific resistance to OA on the transformant. OA-content in transformants grown with 2mM OA decreased by 65% compared to that of the control. The amount of FpTRP26 transcript in F. palustris amplified with increasing OA-accumulation, and was maintained at high levels even in the stationary phase. Its transcription in F. palustris was inducible in response to exogenously added OA. These results suggest that FpTRP26 is involved in the OA-resistance in F. palustris.

Extracellular production of hydrogen selenide accounts for thiol-assisted toxicity of selenite against Saccharomyces cerevisiae.

Administration of selenium in humans has anticarcinogenic effects. However, the boundary between cancer-protecting and toxic levels of selenium is extremely narrow. The mechanisms of selenium toxicity need to be fully understood. In Saccharomyces cerevisiae, selenite in the millimolar range is well tolerated by cells. Here we show that the lethal dose of selenite is reduced to the micromolar range by the presence of thiols in the growth medium. Glutathione and selenite spontaneously react to produce several selenium-containing compounds (selenodiglutathione, glutathioselenol, hydrogen selenide, and elemental selenium) as well as reactive oxygen species. We studied which compounds in the reaction pathway between glutathione and sodium selenite are responsible for this toxicity. Involvement of selenodiglutathione, elemental selenium, or reactive oxygen species could be ruled out. In contrast, extracellular formation of hydrogen selenide can fully explain the exacerbation of selenite toxicity by thiols. Indeed, direct production of hydrogen selenide with D-cysteine desulfhydrase induces high mortality. Selenium uptake by S. cerevisiae is considerably enhanced in the presence of external thiols, most likely through internalization of hydrogen selenide. Finally, we discuss the possibility that selenium exerts its toxicity through consumption of intracellular reduced glutathione, thus leading to severe oxidative stress.