We evaluated the effects of heat stress (HS) and methionine supplementation on biological markers of stress and expression of the genes for superoxide dismutase (SOD), thioredoxin (TRx), thioredoxin reductase 1 (TRxR1) and methionine sulfoxide reductase A (MsrA) in broilers aged 1 to 21 or 22 to 42 days. The broilers were divided into two treatments, one with the recommended level of methionine supplementation (MS, supplementation of dl-2-hydroxy-4-methylthiobutanoic acid (dl-HMTBA)) and one without methionine supplementation (MD). The animals were maintained at a temperature of thermal comfort or one of HS (38°C for 24 h). Mortality was only observed in 42-day-old broilers exposed to HS and fed the MD diet, and the rate was 5%. Starter period: we observed an interaction effect between diet and temperature on the gene expression of TRxR1 and MsrA, and expression of these genes was higher in the HS animals that received the MS diet than that in birds with the MD diet. Grower period: the expression of SOD, TRxR1 and MsrA genes, activities of aspartate aminotransferase (AST) and creatine kinase (CK) and content of creatinine were influenced by both study variables. In the HS animals, the expression of these genes, AST activity and creatinine content increased and CK activity decreased. In the animals on the MD diet, the expression of these genes and AST and creatinine values were higher and the CK activity was lower than those for the birds on the MS diet. Our results indicated that under HS conditions, the supplementation with dl-HMTBA could mitigate major damage caused by stress through the action on some genes related to TRx complex activity.

Schistosoma japonicum, a human blood fluke, causes a parasitic disease affecting millions of people in Asia. Thioredoxin–glutathione system of S. japonicum plays a critical role in maintaining the redox balance in parasite, which is a potential target for development of novel antischistosomal agents. Here we cloned the gene of S. japonicum thioredoxin (SjTrx), expressed and purified the recombinant SjTrx in Escherichia coli. Functional assay shows that SjTrx catalyses the dithiothreitol (DTT) reduction of insulin disulphide bonds. The coupling assay of SjTrx with its endogenous reductase, thioredoxin glutathione reductase from S. japonicum (SjTGR), supports its biological function to maintain the redox homeostasis in the cell. Furthermore, the crystal structure of SjTrx in the oxidized state was determined at 2·0 Å resolution, revealing a typical architecture of thioredoxin fold. The structural information of SjTrx provides us important clues for understanding the maintenance function of redox homeostasis in S. japonicum and pathogenesis of this chronic disease.

Uncontrolled inflammation is a contributing factor to many leading causes of human morbidity and mortality including atherosclerosis, cancer and diabetes. Se is an essential nutrient in the mammalian diet that has some anti-inflammatory properties and, at sufficient amounts in the diet, has been shown to be protective in various inflammatory-based disease models. More recently, Se has been shown to alter the expression of eicosanoids that orchestrate the initiation, magnitude and resolution of inflammation. Many of the health benefits of Se are thought to be due to antioxidant and redox-regulating properties of certain selenoproteins. The present review will discuss the existing evidence that supports the concept that optimal Se intake can mitigate dysfunctional inflammatory responses, in part, through the regulation of eicosanoid metabolism. The ability of selenoproteins to alter the biosynthesis of eicosanoids by reducing oxidative stress and/or by modifying redox-regulated signalling pathways also will be discussed. Based on the current literature, however, it is clear that more research is necessary to uncover the specific beneficial mechanisms behind the anti-inflammatory properties of selenoproteins and other Se metabolites, especially as related to eicosanoid biosynthesis. A better understanding of the mechanisms involved in Se-mediated regulation of host inflammatory responses may lead to the development of dietary intervention strategies that take optimal advantage of its biological potency.

Thioredoxins (Trxs) are widely distributed, small proteins that function in redox regulation in a broad spectrum of cellular reactions. Experimental work with barley, wheat and a legume (Medicago truncatula) has established thioredoxin h (Trx h) as a central regulatory protein in seeds, reducing disulphide (S–S) groups of diverse seed proteins, including storage proteins, enzymes and enzyme inhibitors. Trxs appear to be particularly important in plants, as a large number of genes are present compared to mammalian organisms. In Arabidopsis thaliana there are approximately 20 different genes for classical Trxs, and large Trx gene families have also been found in cereals, such as rice, barley, wheat and sorghum. Extensive evidence indicates that adding Trx, reduced nicotinamide adenine dinucleotide phosphate (NADPH) and NADP-thioredoxin reductase (NTR) to cereal flour or seed preparations reduces disulphide (S–S) linkages of storage proteins. The early in vitro studies have been complemented with transgenic barley seed, overexpressing Trx h in protein bodies of the barley endosperm, which showed accelerated germination and early or enhanced expression of associated enzymes, i.e., α-amylase and pullulanase. Overexpression of Trx h levels in wheat was subsequently shown to (1) enhance protein solubility and digestibility, (2) reduce allergenicity of wheat gliadins, and (3) improve dough quality from poor-quality wheat flour. Most recently, we have demonstrated that down-regulation in wheat of Trx h9, a unique thioredoxin, leads to a reduction in the incidence of pre-harvest sprouting, demonstrated in several varieties over multiple generations with field-grown material. Yield and starch content were increased while baking quality in the high-gluten variety remained unchanged. These observations led to the intriguing question of how changes in the endosperm are communicated to the embryo. Studies of Trx h9, a membrane-associated Trx h that can move from cell to cell, provide suggestive evidence for a role of Trx h9 in intercellular communication of redox state.

The ability of the larval forms of Schistosoma mansoni to invade and parasitize their molluscan host, Biomphalaria glabrata, is determined by a multitude of factors. In this study we sought to elucidate the possible mechanisms by which the invading larvae are able to counteract the potentially harmful oxidative environment presented by the host upon initial miracidial infection. This was attempted by examining the gene expression profile of parasite antioxidant enzymes of the linked glutathione-(GSH) thioredoxin (Trx) redox pathway during early intramolluscan larval development. Three such enzymes, the peroxiredoxins (Prx1, Prx2 and Prx3) were examined as to their activity and sites of expression within S. mansoni miracidia and in vitro-cultured mother sporocysts. Results of these studies demonstrated that the H2O2-reducing enzymes Prx1 and 2 are upregulated during early mother sporocyst development compared to miracidia. Immunolocalization studies further indicated that Prx1 and Prx2 proteins are expressed within the apical papillae of miracidia and tegumental syncytium of sporocysts, and are released with parasite excretory-secretory proteins (ESP) during in vitro larval transformation. Removal of Prx1 and Prx2 from larval ESP by immunoabsorption significantly reduced the ability of ESP to breakdown exogenous H2O2, thereby directly linking ESP Prx proteins with H2O2-scavenging activity. Moreover, exposure of live sporocysts to exogenous H2O2 stimulated an upregulation of Prx1 and 2 gene expression suggesting the involvement of H2O2–responsive elements in regulating larval Prx gene expression. These data provide evidence that Prx1 and Prx2 may function in the protection of S. mansoni sporocysts during the early stages of infection.

It has been proposed that certain cell-surface proteins undergo redox reactions, that is, transfer of hydrogens and electrons between closely spaced cysteine thiols that can lead to reduction, formation, or interchange of disulfide bonds. This concept was tested using a membrane-impermeable trivalent arsenical to identify closely spaced thiols in cell-surface proteins. We attached the trivalent arsenical, phenylarsenoxide, to the thiol of reduced glutathione to produce 4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide (GSAO). GSAO bound tightly to synthetic, peptide, and protein dithiols like thioredoxin, but not to monothiols. To identify cell-surface proteins that contain closely spaced thiols, we attached a biotin moiety through a spacer arm to the primary amino group of the γ-glutamyl residue of GSAO (GSAO-B). Incorporation of GSAO-B into proteins was assessed by measuring the biotin using streptavidin-peroxidase. Up to 12 distinct proteins were labeled with GSAO-B on the surface of endothelial and fibrosarcoma cells. The pattern of labeled proteins differed between the different cell types. Protein disulfide isomerase was one of the proteins on the endothelial and fibrosarcoma cell surface that incorporated GSAO-B. These findings demonstrate that the cell-surface environment can support the existence of closely spaced protein thiols and suggest that at least some of these thiols are redox active.

Escherichia coli thioredoxin is a 108 amino acid oxidoreductase and contains a single Met residue at position 37. The protein contains a long α-helical stretch between residues 32 and 49. The central residue of this helix, Pro40, has been replaced by Ser. The stabilities of the oxidized states of two proteins, the single mutant M37L and the double mutant M37L,P40S, have been characterized by differential scanning calorimetry (DSC) and also by a series of isothermal guanidine hydrochloride (GuHCl) melts in the temperature range of 277 to 333 K. The P40S mutation was found to stabilize the protein at all temperatures upto 340 K though both proteins had similar Tm values of about 356 K. At 298 K, the M37L,P40S mutant was found to be more stable than M37L by 1.5 kcal/mol. A combined analysis of GuHCl and calorimetric data was carried out to determine the enthalpy, entropy, and heat capacity change upon unfolding. At 298 K there was a large, stabilizing enthalpic effect in P40S though significant enthalpy-entropy compensation was observed and the two proteins had similar values of ΔCp. Thus, replacement of a Pro in the interior of an α helix can have substantial effects on protein stability.

Although it is usually possible to achieve a favorable yield of a recombinant protein in Escherichia coli, obtaining the protein in a soluble, biologically active form continues to be a major challenge. Sometimes this problem can be overcome by fusing an aggregation-prone polypeptide to a highly soluble partner. To study this phenomenon in greater detail, we compared the ability of three soluble fusion partners—maltose-binding protein (MBP), glutathione S-transferase (GST), and thioredoxin (TRX)—to inhibit the aggregation of six diverse proteins that normally accumulate in an insoluble form. Remarkably, we found that MBP is a far more effective solubilizing agent than the other two fusion partners. Moreover, we demonstrated that in some cases fusion to MBP can promote the proper folding of the attached protein into its biologically active conformation. Thus, MBP seems to be capable of functioning as a general molecular chaperone in the context of a fusion protein. A model is proposed to explain how MBP promotes the solubility and influences the folding of its fusion partners.

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 α3/310 and α1 helices, the length of the α1 helix and the charges in the α2-β3 and β4-β5 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.

Thioredoxins are ubiquitous small-molecular-weight proteins (typically 100–120 amino-acid residues) containing an extremely reactive disulphide bridge with a highly conserved sequence -Cys-Gly(Ala/Pro)-Pro-Cys-. In bacteria and animal cells, thioredoxins participate in multiple reactions which require reduction of disulphide bonds on selected target proteins/enzymes. There is now ample biochemical evidence that thioredoxins exert very specific functions in plants, the best documented being the redox regulation of chloroplast enzymes. Another area in which thioredoxins are believed to play a prominent role is in reserve protein mobilization during the process of germination. It has been discovered that thioredoxins constitute a large multigene family in plants with different subcellular localizations, a unique feature in living cells so far. Evolutionary studies based on these molecules will be discussed, as well as the available biochemical and genetic evidence related to their functions in plant cells. Eukaryotic photosynthetic plant cells are also unique in that they possess two different reducing systems, one extrachloroplastic dependent on NADPH as an electron donor, and the other one chloroplastic, dependent on photoreduced ferredoxin. This review will examine in detail the latest progresses in the area of thioredoxin structural biology in plants, this protein being an excellent model for this purpose. The structural features of the reducing enzymes ferredoxin thioredoxin reductase and NADPH thioredoxin reductase will also be described. The properties of the target enzymes known so far in plants will be detailed with special emphasis on the structural features which make them redox regulatory. Based on sequence analysis, evidence will be presented that redox regulation of enzymes of the biosynthetic pathways first appeared in cyanobacteria possibly as a way to cope with the oxidants produced by oxygenic photosynthesis. It became more elaborate in the chloroplasts of higher plants where a co-ordinated functioning of the chloroplastic and extra chloroplastic metabolisms is required.