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Query: UNIPROT:P06889 (Mol)
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A Nicotiana tabacum thioredoxin h gene (EMBL Accession No. Z11803) encoding a new thioredoxin (called h2) was isolated using thioredoxin h1 cDNA (X58527), and represents the first thioredoxin h gene isolated from a higher plant. It encodes a polypeptide of 118 amino acids with the conserved thioredoxin active site Trp-Cys-Gly-Pro-Cys. This gene comprises two introns which have lengths of 1071 and 147 bp respectively, and three exons which encode peptides of 29, 41 and 48 amino acids, respectively. This thioredoxin h shows 66% identity with the amino acid sequence of thioredoxin h1 (X58527) and only around 35% with the choroplastic thioredoxins. The two thioredoxins, h1 and h2, do not have any signal peptides and are most probably cytoplasmic. Using the 3' regions of the mRNAs, two probes specific for thioredoxins h1 and h2 have been prepared. Southern blot analysis shows that thioredoxin sequences are present in only two genomic EcoRI fragments: a 3.3 kb fragment encodes h1 and a 4.5 kb fragment encodes h2. Analysis of the ancestors of the allotetraploid N. tabacum shows that thioredoxin h2 is present in N. sylvestris and N. tomentosiformis but that thioredoxin h1 is absent from both putative ancestors. Thus, the thioredoxin h1 gene has probably been recently introduced in to N. tabacum as a gene of agronomic importance, or linked to such genes. Northern blot analysis shows that both genes are expressed in N. tabacum, mostly in organs or tissues that contain growing cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Gen Genet 1993 Apr
PMID:The Nicotiana tabacum genome encodes two cytoplasmic thioredoxin genes which are differently expressed. 847 34

When we compare the primary structures of the six chloroplast fructose-1,6-biophosphatases (FBPase) so far sequenced, the existence of a poorly conserved fragment in the region just preceeding the redox regulatory cysteines cluster can be observed. This region is a good candidate for binding of FBPase to its physiological modulator thioredoxin (Td), as this association shows clear differences between species. Using a cDNA clone for pea chloroplast FBPase as template, we have amplified by PCR a DNA insert coding for a 19 amino acid fragment (149Pro-167Gly), which was expressed in pGEMEX-1 as a fusion protein. This protein strongly interacts with pea Td m, as shown by ELISA and Superose 12 gel filtration, depending on pH of the medium. Preliminary assays have shown inhibition of FBPase activity in the presence of specific IgG against the 19 amino acid insert. Surprisingly the fusion protein enhances the FBPase activation in competitive inhibition experiments carried out with FBPase and Td. These results show the fundamental role played by this domain in FBPase-Td binding, not only as docking point for Td, but also by inducing some structural modification in the Td molecule. Taking as model the structural data recently published for spinach photosynthetic FBPase, this sequence from a tertiary and quaternary structural point of view appears available for rearrangement.
Plant Mol Biol 1996 Feb
PMID:Binding site on pea chloroplast fructose-1,6-bisphosphatase involved in the interaction with thioredoxin. 860 98

Redox regulation of DNA-binding proteins through the reversible oxidation of key cysteine sulfhydryl groups has been demonstrated to occur in vitro for a range of transcription factors. The direct redox regulation of DNA binding has not been described in vivo, possibly because most protein thiol groups are strongly buffered against oxidation by the highly reduced intracellular environment mediated by glutathione, thioredoxin, and associated pathways. For this reason, only accessible protein thiol groups with high thiol-disulfide oxidation potentials are likely to be responsive to intracellular redox changes. In this article, we demonstrate that zinc finger DNA-binding proteins, in particular members of the Sp-1 family, appear to contain such redox-sensitive -SH groups. These proteins displayed a higher sensitivity to redox regulation than other redox-responsive factors both in vitro and in vivo. This effect was reflected in the hyperoxidative repression of transcription from promoters with essential Sp-1 binding sites, including the simian virus 40 early region, glycolytic enzyme, and dihydrofolate reductase genes. Promoter analyses implicated the Sp-1 sites in this repression. Non-Sp-1-dependent redox-regulated genes including metallothionein and heme oxygenase were induced by the same hyperoxic stress. The studies demonstrate that cellular redox changes can directly regulate gene expression in vivo by determining the level of occupancy of strategically positioned GC-binding sites.
Mol Cell Biol 1996 Mar
PMID:Physical and functional sensitivity of zinc finger transcription factors to redox change. 862 48

The structural gene (trxA) coding for thioredoxin in the photosynthetic bacterium Rhodobacter sphaeroides has been cloned and sequenced previously. In the present study, the role of oxygen in trxA expression in R. sphaeroides Y was investigated using mRNA analyses and plasmid-borne trxA'-lacZ+ translational and transcriptional fusions. Northern analysis revealed a trxA-specific transcript of approximately 420-460 nucleotides, indicating that trxA is transcribed as a single gene. By studying the beta-galactosidase activity in strains harboring various phi(trxA'-lacZ+) fusion constructs, the promoter region of the trxA gene was localized within a 64-bp region located 97 nucleotides upstream of the trxA initiator codon. A single trxA transcription initiation site was mapped by primer extension, 27 bp upstream of the trxA gene. Based on these results and the DNA sequence analysis, we propose that a sigma70 consensus sequence serves as a trxA promoter. Results from oxygen shift experiments, as deduced from both mRNA analysis and fusions of the trxA promoter region to lacZ indicate that transcription of the R. sphaeroides trxA gene is regulated by high oxygen tension. DNA sequences involved in this oxygen regulation were also localized in the 64-bp region containing the trxA promoter. Based on our findings the hypothetical biological function of thioredoxin from R. sphaeroides is discussed.
Mol Gen Genet 1996 Feb 05
PMID:Expression of the thioredoxin gene (trxA) in Rhodobacter sphaeroides Y is regulated by oxygen. 862 18

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.
J Mol Evol 1996 Apr
PMID:Intron position as an evolutionary marker of thioredoxins and thioredoxin domains. 864 11

The flavoenzyme thioredoxin reductase (TrR) catalyzes the reduction of the small redox protein thioredoxin (Tr) by NADPH. It has been proposed that a large conformational change is required in catalysis by TrT in order to visualize a complete pathway for reduction of equivalents. The proposal is based on the comparison of the crystal structures of TrR and glutathione reductase, the latter being a well-understood member of the enzyme family [Waksman, G., et al. (1994) J. Mol. Biol. 236, 800-816]. Bound NADPH is perfectly positioned for electron transfer to the FAD in glutathione reductase, but in TrR, these two components are 17 angstroms apart. In order to provide evidence for the proposed conformational change, a complex between TrR and its substrate Tr involving a mixed disulfide between TrR and Tr was prepared. The redox active disulfide of TrR is composed of Cys135 and Cys138, and the redox active disulfide of Tr is made up of Cys32 and Cys35. The complex C135S-C32S is prepared from forms of TrR and Tr altered by site-directed mutagenesis where Cys138 and Cys35 are remaining in TrR and Tr, respectively. The purified C135S-C32S presents a band on a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis responding to a molecular weight sum of one subunit of TrR and one of Tr. Several observations indicate that C135S-C32S can adopt only one conformation. It was reported previously that TrR C135S can form a charge transfer complex in the presence of ammonium cation in which the donor is the remaining thiolate of Cys138 [Prongay, A.J., et al., (1989) J. Biol. Chem. 264, 2656-2664], while titration of C135S-C32S with NH4Cl does not induce charge transfer, presumably because Cys138 is participating in the mixed dissulfide. Reduction of C135S-C32S with dithiothreitol (DTT) results in a decrease of epsilon454 to a value similar to that of TrR C135S, and subsequent NH4Cl titration leads to charge transfer complex formation in the nascent TrR C135S. Reductive titrations show that approximately 1 equiv of sodium dithionite or NADPH is required to fully reduce C135S-C32S, and treatment with NH4Cl and DTT demonstrates that the mixed disulfide between Cys138 of TrR C135S and Cys35 of TrC32S that locks the structure in a conformation where FAD can be reduced by NADPH, but electrons cannot flow from FADH2 to the mixed disulfide bond.
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PMID:A stable mixed disulfide between thioredoxin reductase and its substrate, thioredoxin: preparation and characterization. 866 71

Nitric oxide (NO) synthase is a hemoprotein containing several cysteinyl residues including thiolate as its proximal heme ligand. Exposure to NO is known to induce S-nitrosylation of protein thiols and modulation of enzyme activities, including the catalytic activity of NO synthase. Because S-nitrosylation of vicinal thiols promotes disulfide formation, we determined whether exposure to NO results in modulation of the catalytic activity of NO synthase and whether disulfide reduction catalyzed by thioredoxin/thioredoxin reductase (T/TR) and/or by glutaredoxin restores the catalytic activity of NO synthase in pulmonary artery endothelial cells (PAEC). Exposure of intact PAEC, isolated total membranes, plasma membranes, or purified NO synthase to NO significantly reduced NO synthase catalytic activity. Similarly, exposure of isolated total membranes or purified NO synthase to potassium ferricyanide (FeCN) also reduced catalytic activity of NO synthase in a concentration-dependent fashion. Although the catalytic activity of NO synthase was significantly reduced following exposure of intact cells to NO, the expression of NO synthase mRNA was unchanged. NO synthase activity in intact cells or isolated membranes exposed to nitrate, nitrite, or 10 ppm nitrogen dioxide gas was comparable to controls. Incubation in the presence of oxyhemoglobin prevented but did not reverse NO-induced inhibition of NO synthase. Incubation in the presence of T/TR but not glutaredoxin reversed NO-induced reduction of NO synthase activity and a purified enzyme preparation exposed directly to NO. Similarly, FeCN-induced reduction of NO synthase activity was also reversed in the presence of T/TR but not by glutaredoxin. These results demonstrate that the interaction of NO with the regulatory domain of NO synthase protein is responsible for post-translational reduction of its catalytic activity. Thioredoxin-regulated reversal of NO-induced modulation of NO synthase protein suggests that an oxidative conformational change in vicinal thiols, resulting in the formation of intramolecular or intermolecular disulfides or both, is involved.
Am J Respir Cell Mol Biol 1996 Sep
PMID:Nitric oxide-induced inhibition of lung endothelial cell nitric oxide synthase via interaction with allosteric thiols: role of thioredoxin in regulation of catalytic activity. 881 Jun 47

The diversity and domain structure of alpha beta T cell receptors (TCR) are similar to immunoglobulins based on sequence homologies, but the three-dimensional structure of the alpha beta-heterodimer has not been solved. To begin structure/function studies, we have compared the properties of a soluble single-chain V alpha V beta TCR (scTCR) expressed in three E. coli systems. The V alpha and V beta regions were expressed with pelB or ompA signal sequences or as a thioredoxin fusion protein. The scTCRs were detected only in the insoluble fraction of the cells and could be solubilized in guanidine and renatured to obtain properly folded scTCR from each system. Only a small fraction (1-5%) of the ompA and pelB scTCRs folded properly. In contrast, the thioredoxin fusion protein exhibited high total yields and a solubility that was ten times higher than the other scTCRs. The thioredoxin fusion protein also bound specifically to the peptide/MHC ligand with a KD of approximately 0.7 microM, as shown by a competitive inhibition assay with Fab fragments that recognize the MHC complex. Furthermore, estimates from saturation binding with antibodies that react with the native TCR indicated that up to 80% of the thioredoxin fusion protein was in the properly folded form. The improved yield, solubility, and binding activity of the thioredoxin-scTCR should make it useful for various structure/ function studies.
Mol Immunol 1996 Jun
PMID:Binding properties and solubility of single-chain T cell receptors expressed in E. coli. 881 Oct 77

A glutathione reductase null mutant of Saccharomyces cerevisiae was isolated in a synthetic lethal genetic screen for mutations which confer a requirement for thioredoxin. Yeast mutants that lack glutathione reductase (glr1 delta) accumulate high levels of oxidized glutathione and have a twofold increase in total glutathione. The disulfide form of glutathione increases 200-fold and represents 63% of the total glutathione in a glr1 delta mutant compared with only 6% in wild type. High levels of oxidized glutathione are also observed in a trx1 delta, trx2 delta double mutant (22% of total), in a glr1 delta, trx1 delta double mutant (71% of total), and in a glr1 delta, trx2 delta double mutant (69% of total). Despite the exceptionally high ratio of oxidized/reduced glutathione, the glr1 delta mutant grows with a normal cell cycle. However, either one of the two thioredoxins is essential for growth. Cells lacking both thioredoxins and glutathione reductase are not viable under aerobic conditions and grow poorly anaerobically. In addition, the glr1 delta mutant shows increased sensitivity to the thiol oxidant diamide. The sensitivity to diamide was suppressed by deletion of the TRX2 gene. The genetic analysis of thioredoxin and glutathione reductase in yeast runs counter to previous studies in Escherichia coli and for the first time links thioredoxin with the redox state of glutathione in vivo.
Mol Biol Cell 1996 Nov
PMID:A glutathione reductase mutant of yeast accumulates high levels of oxidized glutathione and requires thioredoxin for growth. 893 Sep 1

The outer arm dynein of sea urchin sperm axoneme contains three intermediate chains (IC1, IC2, and IC3; M(r) 128,000, 98,000, and 74,000, respectively). IC2 and IC3 are members of the WD family; the WD motif is responsible for a protein-protein interaction. We describe here the molecular cloning of IC1. IC1 has a unique primary structure, the N-terminal part is homologous to the sequence of thioredoxin, the middle part consists of three repetitive sequences homologous to the sequence of nucleoside diphosphate kinase, and the C-terminal part contains a high proportion of negatively charged glutamic acid residues. Thus, IC1 is a novel dynein intermediate chain distinct from IC2 and IC3 and may be a multifunctional protein. The thioredoxin-related part of IC1 is more closely related to those of two redox-active Chlamydomonas light chains than thioredoxin. Antibodies were prepared against the N-terminal and middle domains of IC1 expressed as His-tagged proteins in bacteria. These antibodies cross-reacted with some dynein polypeptides (potential homologues of IC1) from distantly related species. We propose here that the three intermediate chains are the basic core units of sperm outer arm dynein because of their ubiquitous existence. The recombinant thioredoxin-related part of IC1 and outer arm dyneins from sea urchin and distantly related species were specifically bound to and eluted from a phenylarsine oxide affinity column with 2-mercaptoethanol, indicating that they contain vicinal dithiols competent to undergo reversible oxidation/reduction.
Mol Biol Cell 1996 Dec
PMID:Is outer arm dynein intermediate chain 1 multifunctional? 897 Jan 53


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