[Abstract unavailable

We have isolated a 1276-base pair cDNA from a rat heart cDNA library that encodes a novel thioredoxin (Trx2) of 166 amino acid residues with a calculated molecular mass of 18.2 kDa. Trx2 possesses the conserved thioredoxin-active site, Trp-Cys-Gly-Pro-Cys, but lacks structural cysteines present in all mammalian thioredoxins. Trx2 also differs from the previously described rat thioredoxin (Trx1) by the presence of a 60-amino acid extension at the N terminus. This extension has properties characteristic for a mitochondrial translocation signal, and the cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein of 12.2 kDa. Western blot analysis from cytosolic, peroxisomal, and mitochondrial rat liver cell fractions confirmed mitochondrial localization of Trx2. Northern blot and reverse transcriptase-polymerase chain reaction analyses revealed that Trx2 hybridized to a 1.3-kilobase message, and it was expressed in several tissues with the highest expression levels in heart, muscle, kidney, and adrenal gland. N-terminally truncated recombinant protein was expressed in bacteria and characterized biochemically. Trx2 possessed a dithiol-reducing enzymatic activity and, with mammalian thioredoxin reductase and NADPH, was able to reduce the interchain disulfide bridges of insulin. Furthermore, Trx2 was more resistant to oxidation than Trx1.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.

The so-called thioredoxin system, thioredoxin (Trx), thioredoxin reductase (Trr), and NADPH, acts as a disulfide reductase system and can protect cells against oxidative stress. In Saccharomyces cerevisiae, two thioredoxins (Trx1 and Trx2) and one thioredoxin reductase (Trr1) have been characterized, all of them located in the cytoplasm. We have identified and characterized a novel thioredoxin system in S. cerevisiae. The TRX3 gene codes for a 14-kDa protein containing the characteristic thioredoxin active site (WCGPC). The TRR2 gene codes for a protein of 37 kDa with the active-site motif (CAVC) present in prokaryotic thioredoxin reductases and binding sites for NADPH and FAD. We cloned and expressed both proteins in Escherichia coli, and the recombinant Trx3 and Trr2 proteins were active in the insulin reduction assay. Trx3 and Trr2 proteins have N-terminal domain extensions with characteristics of signals for import into mitochondria. By immunoblotting analysis of Saccharomyces subcellular fractions, we provide evidence that these proteins are located in mitochondria. We have also constructed S. cerevisiae strains null in Trx3 and Trr2 proteins and tested them for sensitivity to hydrogen peroxide. The ⌬trr2 mutant was more sensitive to H 2 O 2 , whereas the ⌬trx3 mutant was as sensitive as the wild type. These results suggest an important role of the mitochondrial thioredoxin reductase in protection against oxidative stress in S. cerevisiae. Thioredoxin (Trx)1 is a small protein (M r 12,000) with a conserved sequence (Trp-Cys-Gly-Pro-Cys) in its active site. When thioredoxin is in a reduced state, Trx-(SH) 2 , the two active-site cysteines form a dithiol group that is able to catalyze the reduction of disulfides in a number of proteins. Oxidized thioredoxin (Trx-S 2 ) can be reduced by NADPH through the catalytic action of the flavoenzyme thioredoxin reductase (Trr). Thus, Trx, Trr, and NADPH form a system (the thioredoxin system) that operates as a general disulfide reductase system by the following sequence of reactions (Reactions 1 and 2).

Thioredoxin (Trx) and glutaredoxin (Grxl) are hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis. The viability of a double mutant lacking both Trx and Grxl implies the presence of a third, unknown hydrogen donor. This paper reports the purification and characterization of two proteins with glutaredoxin activity (using hydroxyethyl disulfide as a substrate) from an Escherichia coli mutant lacking Trx and Grxl (AbtrA, grx::kan). Affinity chromatography was used to bind glutaredoxin on a glutathione-on ng thiol-Sepharose column. The molecular weight of Grx2, 27,000, was atypical for glutaredoxins, whereas Grx3 had a molecular weight of 10,000. Amino acid sequence analysis revealed novel structures with putative active sites typical of glutaredoxins: Cys-Pro-Tyr-Cys. The proteins are therefore referred to as Grx2 and Grx3. The low hydrogen donor activity for ribonuceotide reductase in the crude extract was recovered in the purification of Grx3, whereas Grx2 was inactive. As a hydrogen donor for E. coli ribonucleotide reductase, Grx3 showed approximately the same K. value (0.35 pM) as Grxl, whereas its V.. value was only 5% that of Grxl. The combination of the Grx3 hydrogen donor activity and a 25-fold induction of ribonucleotide reductase activity in a AtrxA, gnx double mutant provides an explanation for its viahbility and deoxyribonudeotide biosynthesis. The physiological functions of Grx2 and Grx3 remain to be determined. unless a source of reduced sulfur was added. This indicates that thioredoxin and glutaredoxin (Grxl) are not essential for deoxyribonucleotide biosynthesis but are essential for the reduction of sulfate (7), implying the presence of a third, unknown hydrogen donor system for ribonucleotide reductase in E. coli.The isolation of a Corynebacterium nephridii thioredoxin (8) and a rabbit bone marrow glutaredoxin (9) and thioredoxin (10) that do not react with their homologous ribonucleotide reductases has cast further doubts on the general involvement of thioredoxin and glutaredoxin as the only hydrogen donors for ribonucleotide reductase. Furthermore, immunohistochemical studies show no clear correlation of the distribution of thioredoxin and ribonucleotide reductase in most rat tissues, indicating that thioredoxin is not the physiological hydrogen donor (11).It was observed when E. coli glutaredoxin (Grx1) was purified that this protein had activity as a GSH-disulfide oxidoreductase. But, 98% of such activity in the crude extract was of unknown origin (12). The activity was measured as the enzymatic reduction of the mixed disulfide between the prototype substrate f-hydroxyethyl disulfide (HED) and GSH at the expense of NADPH (recorded at 340 nm) by using the HED assay of Holmgren (12). The HED assay is based on the following reactions (where XSSX is HED):Thioredoxin and glutaredoxin were discovered separately as hydrogen donors for ribonucleotide reductase (1, 2), the enzyme catalyzing deoxyribonucleotide biosynthesis (3). Both thioredoxin ...