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At least 19 recordsLinked to original sources
PubMed2002

Thioredoxin-2 but not thioredoxin-1 is a substrate of thioredoxin peroxidase-1 from Drosophila melanogaster: isolation and characterization of a second thioredoxin in D. Melanogaster and evidence for distinct biological functions of Trx-1 and Trx-2.

As Drosophila melanogaster does not contain glutathione reductase, the thioredoxin system has a key function for glutathione disulfide reduction in insects (Kanzok, S. M., Fechner, A., Bauer, H., Ulschmid, J. K., Müller, H. M., Botella-Munoz, J., Schneuwly, S., Schirmer, R. H., and Becker, K. (2001) Science 291, 643-646). In view of these unique conditions, the protein systems participating in peroxide metabolism and in redox signaling are of special interest. The genes for a second thioredoxin (DmTrx-2) and a thioredoxin peroxidase (DmTPx-1) were cloned and expressed, and the proteins were characterized. In its disulfide form, the 13-kDa protein thioredoxin-2 is a substrate of thioredoxin reductase-1 (K(m) = 5.2 microm, k(cat) = 14.5 s(-1)) and in its dithiol form, an electron donor for TPx-1 (K(m) = 9 microm, k(cat) = 5.4 s(-1)). DmTrx-2 is capable of reducing glutathione disulfide with a second order rate constant of 170 m(-1) s(-1) at pH 7.4 and 25 degrees C. Western blot analysis indicated that this thioredoxin represents up to 1% of the extractable protein of D. melanogaster Schneider cells or whole fruit flies. Recombinant thioredoxin peroxidase-1 (subunit molecular mass = 23 kDa) was found to be a decameric protein that can efficiently use Trx-2 but not Trx-1 as a reducing substrate. The new electron pathway found in D. melanogaster is also representative for insects that serve as vectors of disease. As a first step we have cloned and functionally expressed the gene that is the orthologue of DmTrx-2 in the malaria mosquito Anopheles gambiae.

Amino Acid Sequence↗
PubMed1994

Site-directed mutagenesis of active site cysteines in human thioredoxin produces competitive inhibitors of human thioredoxin reductase and elimination of mitogenic properties of thioredoxin.

Thioredoxin and thioredoxin reductase comprise a redox system ubiquitous in all organisms. To better understand the thiol chemistry of the mammalian thioredoxin-thioredoxin reductase redox system, mutants of human thioredoxin were produced by site-directed mutagenesis in which the two active site cysteines were replaced by serine residues, individually (C32S and C35S) and in combination (C32S/C35S). C35S and C32S/C35S were found to be competitive inhibitors of the reduction of human thioredoxin by human thioredoxin reductase with Ki values of 1.8 and 6.7 microM, respectively. C32S did not inhibit thioredoxin reductase, apparently due to aggregation of the oxidized C32S species. Examination of the three mutant forms of thioredoxin by circular dichroism spectroscopy indicated that there were significant differences in the secondary structures when compared with thioredoxin. There were detectable changes in the circular dichroism spectra when thioredoxin, C35S, and C32S/C35S were bound to thioredoxin reductase, whereas C32S with thioredoxin reductase underwent only a small spectral change. Recombinant human thioredoxin stimulated DNA synthesis and the proliferation of murine fibroblasts. The ability of thioredoxin to stimulate cell proliferation could not be duplicated by either dithiothreitol or glutathione. C32S, C35S, and C32S/C35S failed to stimulate cell proliferation, showing that the redox active form of thioredoxin is necessary for eliciting growth stimulation.

3T3 Cells↗
PubMed1990

The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii: identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components.

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (Mrs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa ("similar") subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa ("variable") subunit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.

Animals↗
PubMed1975

Thioredoxin reductase-mediated hydrogen transfer from Escherichia coli thioredoxin-(SH)2 to phage T4 thioredoxin-S2.

Thioredoxin from Escherichia coli B and phage T4-infected E. coli B are small hydrogen carrier proteins which in their reduced forms are specific hydrogen donors to E. coli and T4-induced ribonucleotide reductase, respectively. The oxidation-reduction active group of both thioredoxins consists of a single cystine residue which is reduced to sulfhydryl form by NADPH in the presence of E. coli thioredoxin reductase. Reduction of T4 thioredoxin-S2 to thioredoxin-(SH)2 led to a 3-fold increase in the quantum yield of tyrosine fluorescence. By using the spectrofluorimetric properties of T4 thioredoxin and E. coli thioredoxin as markers for their oxidized and reduced forms we have shown that E. coli thioredoxin reductase catalyzed the reaction: (see article) whose equilibrium constant favors formation of E. coli thioredoxin-S2 and T4 thioredoxin-(SH)2. This finding suggests that in the T4-infected cell most of the deoxyribonucleotides required for the viral DNA might be synthesized by the T4-induced ribonucleotide reductase while the host ribonucleotide reductase is inactive due to the shortage of reduced E. coli thioredoxin.

Amino Acid Sequence↗
PubMed2002

Cloning of thioredoxin h reductase and characterization of the thioredoxin reductase-thioredoxin h system from wheat.

Thioredoxins h are ubiquitous proteins reduced by NADPH- thioredoxin reductase (NTR). They are able to reduce disulphides in target proteins. In monocots, thioredoxins h accumulate at high level in seeds and show a predominant localization in the nucleus of seed cells. These results suggest that the NTR-thioredoxin h system probably plays an important role in seed physiology. To date, the study of this system in monocots is limited by the lack of information about NTR. In the present study, we describe the cloning of a full-length cDNA encoding NTR from wheat ( Triticum aestivum ). The polypeptide deduced from this cDNA shows close similarity to NTRs from Arabidopsis, contains FAD- and NADPH-binding domains and a disulphide probably interacting with the disulphide at the active site of thioredoxin h. Wheat NTR was expressed in Escherichia coli as a His-tagged protein. The absorption spectrum of the purified recombinant protein is typical of flavoenzymes. Furthermore, it showed NADPH-dependent thioredoxin h reduction activity, thus confirming that the cDNA clone reported in the present study encodes wheat NTR. Using the His-tagged NTR and TRXhA (wheat thioredoxin h ), we successfully reconstituted the wheat NTR-thioredoxin h system in vitro, as shown by the insulin reduction assay. A polyclonal antibody was raised against wheat NTR after immunization of rabbits with the purified His-tagged protein. This antibody efficiently detected a single polypeptide of the corresponding molecular mass in seed extracts and it allowed the analysis of the pattern of accumulation of NTR in different wheat organs and developmental stages. NTR shows a wide distribution in wheat, but, surprisingly, its accumulation in seeds is low, in contrast with the level of thioredoxins h.

Amino Acid Sequence↗
PubMed1996

Histidine patch thioredoxins. Mutant forms of thioredoxin with metal chelating affinity that provide for convenient purifications of thioredoxin fusion proteins.

A cluster of surface amino acid residues on Escherichia coli thioredoxin were systematically mutated in order to provide the molecule with an ability to chelate metal ions. The combined effect of two histidine mutants, E30H and Q62H, gave thioredoxin the capacity to bind to nickel ions immobilized on iminodiacetic acid- and nitrilotriacetic acid-Sepharose resins. Even though these two histidines were more than 30 residues apart in thioredoxin's primary sequence, they were found to satisfy the geometric constraints for metal ion coordination as a result of the thioredoxin tertiary fold. A third histidine mutation, S1H, provided additional metal ion chelation affinity, but the native histidine at position 6 of thioredoxin was found not to participate in binding. All of the histidine mutants exhibited decreased thermal stability as compared with wild-type thioredoxin; however, the introduction of an additional mutation, D26A, increased their melting temperatures beyond that of wild-type thioredoxin. The metal chelating abilities of these histidine mutants of thioredoxin were successfully utilized for convenient purifications of human interleukin-8 and -11 expressed in E. coli as soluble thioredoxin fusion proteins.

Amino Acid Sequence↗
PubMed1999

Oxidation-reduction properties of chloroplast thioredoxins, ferredoxin:thioredoxin reductase, and thioredoxin f-regulated enzymes.

Oxidation-reduction midpoint potentials were determined, as a function of pH, for the disulfide/dithiol couples of spinach and pea thioredoxins f, for spinach and Chlamydomonas reinhardtii thioredoxins m, for spinach ferredoxin:thioredoxin reductase (FTR), and for two enzymes regulated by thioredoxin f, spinach phosphoribulokinase (PRK) and the fructose-1,6-bisphosphatases (FBPase) from pea and spinach. Midpoint oxidation-reduction potential (Em) values at pH 7.0 of -290 mV for both spinach and pea thioredoxin f, -300 mV for both C. reinhardtii and spinach thioredoxin m, -320 mV for spinach FTR, -290 mV for spinach PRK, -315 mV for pea FBPase, and -330 mV for spinach FBPase were obtained. With the exception of spinach FBPase, titrations showed a single two-electron component at all pH values tested. Spinach FBPase exhibited a more complicated behavior, with a single two-electron component being observed at pH values >/= 7.0, but with two components being present at pH values <7.0. The slopes of plots of Em versus pH were close to the -60 mV/pH unit value expected for a process that involves the uptake of two protons per two electrons (i. e., the reduction of a disulfide to two fully protonated thiols) for thioredoxins f and m, for FTR, and for pea FBPase. The slope of the Em versus pH profile for PRK shows three regions, consistent with the presence of pKa values for the two regulatory cysteines in the region between pH 7.5 and 9.0.

Animals↗
PubMed1998

Thioredoxin reductase-thioredoxin fusion enzyme from Mycobacterium leprae: comparison with the separately expressed thioredoxin reductase.

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. A unique gene organization of TrxR and Trx has been found in Mycobacterium leprae, where TrxR and Trx are encoded by a single gene and, therefore, are expressed as a fusion protein (MlTrxR-Trx). This fusion enzyme is able to catalyze the reduction of thioredoxin or 5,5'-dithiobis(2-nitrobenzoic acid) or 1, 4-naphthoquinone by NADPH, though the activity is much lower than that of Escherichia coli TrxR. It has been proposed that a large conformational change is required in catalysis of E. coli TrxR. Because the reductase portion of the enzyme from M. leprae shows significant primary structure similarity with E. coli TrxR, it is possible that MlTrxR-Trx may require a similar conformational change and that the change in conformation may be affected by the tethered Trx. The reductase has been expressed without Trx attached (MlTrxR). As reported here, comparison of the steady-state and pre-steady-state kinetics of MlTrxR-Trx with those of MlTrxR suggests that the low reductase activity of the fusion enzyme is an inherent property of the reductase, and that any steric limitation caused by the attached thioredoxin in the fusion protein makes only a minor contribution to the low activity. Titration of MlTrxR-Trx and MlTrxR with 3-aminopyridine adenine dinucleotide phosphate (AADP+), an NADP(H) analogue, results in only slight quenching of FAD fluorescence, suggesting an enzyme conformation in which the binding site of AADP+ is not close to the FAD, as in one of the conformations of E. coli TrxR.

Adenine Nucleotides↗
PubMed1995

Thioredoxin-linked peroxidase from human red blood cell: evidence for the existence of thioredoxin and thioredoxin reductase in human red blood cell.

A thiol-dependent antioxidant protein (HRPRP) was previously reported as a predominant antioxidant protein in human red blood cell (RBC). The analysis of amino acid sequence of HRPRP with those of human PRP-like gene products indicates that HRPRP is identical to brain PRP (HPRP). This protein act as a peroxidase linked to thioredoxin (Trx)/thioredoxin reductase (TR). Until now, there was no evidence for Trx/TR system in RBC. The existence of the Trx/TR system in RBC was immunologically determined. A 58-kDa protein showing TR activity was partially purified from human RBC and characterized. Our results reveal that HRPRP act as a new type of peroxidase supported by Trx/TR system in human RBC.

Amino Acid Sequence↗
PubMed1999

Localization of thioredoxin reductase and thioredoxin in normal human placenta and their protective effect against oxidative stress.

Recent studies have indicated that oxidative stress is involved in the pathogenesis of pre-eclampsia. Oxidative stress damages systemic tissues, and placental damage may result in intrauterine growth retardation and fetal distress. Thus, this study attempted to elucidate the placental localization of thioredoxin and thioredoxin reductase, substances that may reduce oxidative stress. Furthermore, it studied the defence mechanism of the thioredoxin-thioredoxin reductase system against oxidative stress in mitochondria of normal human placenta where reactive oxygen species are primarily produced. The examination of thioredoxin reductase activity in subcellular fractions of human placenta indicated that thioredoxin reductase was located not only in cytoplasm, but also in mitochondria. The existence of thioredoxin and thioredoxin reductase in human placenta was confirmed immunologically using antibodies raised against thioredoxin and thioredoxin reductase. Thioredoxin and thioredoxin reductase were localized histochemically in cytotrophoblasts, decidua, and stromal cells in the stem villi. The addition of exogenous thioredoxin and thioredoxin reductase to fumarase in mitochondria of human placenta displayed a protective effect against oxidative stress. In conclusion, this study confirmed the intracellular localization and the tissue distribution of thioredoxin and thioredoxin reductase in human placenta. Moreover, the complete thioredoxin-thioredoxin reductase system in human placenta may protect the placenta from damage caused by oxidative stress.

Blotting, Western↗
PubMed1999

Interaction of thioredoxins with target proteins: role of particular structural elements and electrostatic properties of thioredoxins in their interplay with 2-oxoacid dehydrogenase complexes.

The thioredoxin action upon the 2-oxoacid dehydrogenase complexes is investigated by using different thioredoxins, both wild-type and mutated. The attacking cysteine residue of thioredoxin is established to be essential for the thioredoxin-dependent activation of the complexes. Mutation of the buried cysteine residue to serine is not crucial for the activation, but prevents inhibition of the complexes, exhibited by the Clamydomonas reinhardtii thioredoxin m disulfide. Site-directed mutagenesis of D26, W31, F/W12, and Y/A70 (the Escherichia coli thioredoxin numbering is employed for all the thioredoxins studied) indicates that both the active site and remote residues of thioredoxin are involved in its interplay with the 2-oxoacid dehydrogenase complexes. Sequences of 11 thioredoxin species tested biochemically are aligned. The thioredoxin residues at the contact between the alpha3/3(10) and alpha1 helices, the length of the alpha1 helix and the charges in the alpha2-beta3 and beta4-beta5 linkers are found to correlate with the protein influence on the 2-oxoacid dehydrogenase complexes (the secondary structural elements of thioredoxin are defined according to Eklund H et al., 1991, Proteins 11:13-28). The distribution of the charges on the surface of the thioredoxin molecules is analyzed. The analysis reveals the species specific polarization of the thioredoxin active site surroundings, which corresponds to the efficiency of the thioredoxin interplay with the 2-oxoacid dehydrogenase systems. The most effective mitochondrial thioredoxin is characterized by the strongest polarization of this area and the highest value of the electrostatic dipole vector of the molecule. Not only the magnitude, but also the orientation of the dipole vector show correlation with the thioredoxin action. The dipole direction is found to be significantly influenced by the charges of the residues 13/14, 51, and 83/85, which distinguish the activating and inhibiting thioredoxin disulfides.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2000

Physiological functions of thioredoxin and thioredoxin reductase.

Thioredoxin, thioredoxin reductase and NADPH, the thioredoxin system, is ubiquitous from Archea to man. Thioredoxins, with a dithiol/disulfide active site (CGPC) are the major cellular protein disulfide reductases; they therefore also serve as electron donors for enzymes such as ribonucleotide reductases, thioredoxin peroxidases (peroxiredoxins) and methionine sulfoxide reductases. Glutaredoxins catalyze glutathione-disulfide oxidoreductions overlapping the functions of thioredoxins and using electrons from NADPH via glutathione reductase. Thioredoxin isoforms are present in most organisms and mitochondria have a separate thioredoxin system. Plants have chloroplast thioredoxins, which via ferredoxin-thioredoxin reductase regulates photosynthetic enzymes by light. Thioredoxins are critical for redox regulation of protein function and signaling via thiol redox control. A growing number of transcription factors including NF-kappaB or the Ref-1-dependent AP1 require thioredoxin reduction for DNA binding. The cytosolic mammalian thioredoxin, lack of which is embryonically lethal, has numerous functions in defense against oxidative stress, control of growth and apoptosis, but is also secreted and has co-cytokine and chemokine activities. Thioredoxin reductase is a specific dimeric 70-kDa flavoprotein in bacteria, fungi and plants with a redox active site disulfide/dithiol. In contrast, thioredoxin reductases of higher eukaryotes are larger (112-130 kDa), selenium-dependent dimeric flavoproteins with a broad substrate specificity that also reduce nondisulfide substrates such as hydroperoxides, vitamin C or selenite. All mammalian thioredoxin reductase isozymes are homologous to glutathione reductase and contain a conserved C-terminal elongation with a cysteine-selenocysteine sequence forming a redox-active selenenylsulfide/selenolthiol active site and are inhibited by goldthioglucose (aurothioglucose) and other clinically used drugs.

Amino Acid Sequence↗
PubMed1996

Intron position as an evolutionary marker of thioredoxins and thioredoxin domains.

In contrast to prokaryotes, which typically possess one thioredoxin gene per genome, three different thioredoxin types have been described in higher plants. All are encoded by nuclear genes, but thioredoxins m and f are chloroplastic while thioredoxins h have no transit peptide and are probably cytoplasmic. We have cloned and sequenced Arabidopsis thaliana genomic fragments encoding the five previously described thioredoxins h, as well as a sixth gene encoding a new thioredoxin h. In spite of the high divergence of the sequences, five of them possess two introns at positions identical to the previously sequenced tobacco thioredoxin h gene, while a single one has only the first intron. The recently published sequence of Chlamydomonas thioredoxin h shows three introns, two at the same positions as in higher plants. This strongly suggests a common origin for all cytoplasmic thioredoxins of plants and green algae. In addition, we have cloned and sequenced pea DNA genomic fragments encoding thioredoxins m and f. The thioredoxin m sequence shows only one intron between the regions encoding the transit peptide and the mature protein, supporting the prokaryotic origin of this sequence and suggesting that its association with the transit peptide has been facilitated by exon shuffling. In contrast, the thioredoxin f sequence shows two introns, one at the same position as an intron in various plant and animal thioredoxins and the second at the same position as an intron in thioredoxin domains of disulfide isomerases. This strongly supports the hypothesis of a eukaryotic origin for chloroplastic thioredoxin f.

Amino Acid Sequence↗
PubMed1993

Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated.

Amino Acid Sequence↗
PubMedDate not supplied

Thioredoxin and thioredoxin reductase gene expression in human tumors and cell lines, and the effects of serum stimulation and hypoxia.

Thioredoxin and thioredoxin reductase are redox proteins that have been implicated in the control of cell proliferation and transformation. We report the levels and activity of these proteins and their mRNAs in human primary tumors and tumor cell lines. Half of human primary colorectal carcinomas (5/10) examined had increased thioredoxin mRNA, of 3- to over 100-fold, compared to adjacent normal colonic mucosa from the same subject. Thioredoxin reductase protein and activity were increased an average of 2-fold in human colorectal tumors compared to normal mucosa. A number of human hematologic and solid tumor cell lines were studied and showed a 10-fold range of thioredoxin mRNA and a 23-fold range of thioredoxin reductase mRNA. Increased proliferation and hypoxia are factors that might contribute to the increased expression in solid tumors. We found that serum stimulation of growth arrested MCF-7 breast cancer cells caused a 59% increase in thioredoxin mRNA and a 62% increase in thioredoxin reductase mRNA by 24 hours. Exposure of HT-20 colon cancer cells to hypoxia resulted in a 14-fold increase in thioredoxin mRNA by 16 hours, and a transient 4-fold increase in thioredoxin reductase mRNA at 1 hour that had returned to control levels by 8 hours. Cancer cells were found to release thioredoxin into the medium at rates between 1 to 2 pmole/10(6) cells/3 hours. The rate of secretion was not, however, related to cellular-levels of thioredoxin. The results of the study show that the expression of thioredoxin and thioredoxin reductase are increased several fold in some human solid tumors compared to normal tissue. Secretion of thioredoxin, which is known to have a direct growth stimulating activity, by human tumor cells might lead to the stimulation of cancer cell growth.

Cell Hypoxia↗
PubMed1999

Expression of thioredoxin and thioredoxin reductase in placentae of pregnant mice exposed to lipopolysaccharide.

We have previously shown that thioredoxin and thioredoxin reductase were immunohistochemically localized in cytotrophoblasts, decidua and stromal cells in the stem villi of human placenta and that the addition of exogenous thioredoxin and thioredoxin reductase to mitochondrial fractions from human placenta displayed a protective effect on fumarase activity against oxidative stress. In this study, to investigate further the roles of thioredoxin and thioredoxin reductase in protecting pregnancy against oxidative stress, we examined the effect of lipopolysaccharide (LPS), which induces a variety of cytokines and produces radical oxygen species, on the expression of thioredoxin and thioredoxin reductase in mouse placenta. We focused on the placental protective effect in the second trimester, when the onset of placental dysfunction might occasionally lead to a critical state for the fetus. Thus we analysed placentae from mice on day 13 of pregnancy at various time points after they were injected with LPS (50 microg/kg i.p.) or saline as a control. The expressions of thioredoxin and thioredoxin reductase were evaluated by Western blotting and immunohistochemistry. Western blot analysis revealed that LPS approximately quadrupled the expression of both thioredoxin and thioredoxin reductase in the placentae of pregnant mice. When both proteins were localized immunohistochemically, it was found that the decidua and the diploid trophoblasts in the basal zone were intensively stained. Furthermore, the expression of 4-hydroxy-2-nonenal (HNE)-modified proteins, which are markers of oxidative stress, was enhanced in placenta by LPS. Our study suggests that the induced thioredoxin and thioredoxin reductase might protect the placenta from the stress induced by LPS.

Aldehydes↗
PubMed1990

Protein disulfide-isomerase is a substrate for thioredoxin reductase and has thioredoxin-like activity.

We have demonstrated that calf liver protein disulfide-isomerase (Mr 57,000) is a substrate for calf thymus thioredoxin reductase and catalyzes NADPH-dependent insulin disulfide reduction. This reaction can be used as a simple assay for protein disulfide-isomerase during purification in place of the classical method of reactivation of incorrectly oxidized ribonuclease A. Protein disulfide-isomerase contains two redox-active disulfides/molecule which were reduced by NADPH and calf thioredoxin reductase (Km approximately 35 microM). The isomerase was a poor substrate for NADPH and Escherichia coli thioredoxin reductase, but the addition of E. coli thioredoxin resulted in rapid reduction of two disulfides/molecule. Tryptophan fluorescence spectra were shown to monitor the redox state of protein disulfide-isomerase. Fluorescence measurements demonstrated that thioredoxin--(SH)2 reduced the disulfides of the isomerase and allowed the kinetics of the reaction to be followed; the reaction was also catalyzed by calf thioredoxin reductase. Equilibrium measurements showed that the apparent redox potential of the active site disulfide/dithiols of the thioredoxin domains of protein disulfide-isomerase was about 30 mV higher than the disulfide/dithiol of E. coli thioredoxin. Consistent with this, experiments using dithiothreitol or NADPH and thioredoxin reductase-dependent reduction and precipitation of insulin demonstrated differences between protein disulfide-isomerase and thioredoxin, thioredoxin being a better disulfide reductase but less efficient isomerase. Protein disulfide-isomerase is thus a high molecular weight member of the thioredoxin system, able to interact with both mammalian NADPH-thioredoxin reductase and reduced thioredoxin. This may be important for nascent protein disulfide formation and other thiol-dependent redox reactions in cells.

Amino Acid Sequence↗
PubMed1997

Two closely linked genes encoding thioredoxin and thioredoxin reductase in Clostridium litorale.

The genes encoding thioredoxin and thioredoxin reductase of Clostridium litorale were cloned and sequenced. The thioredoxin reductase gene (trxB) encoded a protein of 33.9 kDa, and the deduced amino acid sequence showed 44% identity to the corresponding protein from Escherichia coli. The gene encoding thioredoxin (trxA) was located immediately downstream of trxB. TrxA and TrxB were each encoded by two gene copies, both copies presumably located on the chromosome. Like other thioredoxins from anaerobic, amino-acid-degrading bacteria investigated to date by N-terminal amino acid sequencing, thioredoxin from C. litorale exhibited characteristic deviations from the consensus sequence, e.g., GCVPC instead of WCGPC at the redox-active center. Using heterologous enzyme assays, neither thioredoxin nor thioredoxin reductase were interchangeable with the corresponding proteins of the thioredoxin system from E. coli. To elucidate the molecular basis of that incompatibility, Gly-31 in C. litorale thioredoxin was substituted with Trp (the W in the consensus sequence) by site-directed mutagenesis. The mutant protein was expressed in E. coli and was purified to homogeneity. Enzyme assays using the G31W thioredoxin revealed that Gly-31 was not responsible for the observed incompatibility with the E. coli thioredoxin reductase, but it was essential for activity of the thioredoxin system in C. litorale.

Amino Acid Sequence↗