Overlapping roles of the cytoplasmic and mitochondrial redox regulatory systems in the yeast Saccharomyces cerevisiae

Thioredoxins are small, highly conserved oxidoreductases which are required to maintain the redox homeostasis of the cell. Saccharomyces cerevisiae contains a cytoplasmic thioredoxin system (TRX1, TRX2, and TRR1) as well as a complete mitochondrial thioredoxin system, comprising a thioredoxin (TRX3) and a thioredoxin reductase (TRR2). In the present study we have analyzed the functional overlap between the two systems. By constructing mutant strains with deletions of both the mitochondrial and cytoplasmic systems (trr1 trr2 and trx1 trx2 trx3), we show that cells can survive in the absence of both systems. Analysis of the redox state of the cytoplasmic thioredoxins reveals that they are maintained independently of the mitochondrial system. Similarly, analysis of the redox state of Trx3 reveals that it is maintained in the reduced form in wild-type cells and in mutants lacking components of the cytoplasmic thioredoxin system (trx1 trx2 or trr1). Surprisingly, the redox state of Trx3 is also unaffected by the loss of the mitochondrial thioredoxin reductase (trr2) and is largely maintained in the reduced form unless cells are exposed to an oxidative stress. Since glutathione reductase (Glr1) has been shown to colocalize to the cytoplasm and mitochondria, we examined whether loss of GLR1 influences the redox state of Trx3. During normal growth conditions, deletion of TRR2 and GLR1 was found to result in partial oxidation of Trx3, indicating that both Trr2 and Glr1 are required to maintain the redox state of Trx3. The oxidation of Trx3 in this double mutant is even more pronounced during oxidative stress or respiratory growth conditions. Taken together, these data indicate that Glr1 and Trr2 have an overlapping function in the mitochondria.     

Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes

Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast Saccharomyces cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allowed us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments under a given condition. We observed that the nuclear GSH redox environment is highly reducing and similar to the cytosol under steady-state conditions. Furthermore, these sensors are able to detect redox variations specific for their respective compartments in glutathione reductase (Glr1) and thioredoxin pathway (Trr1, Trx1, Trx2) mutants that have altered subcellular redox environments. Our mutant redox data provide in vivo evidence that glutathione and the thioredoxin redox systems have distinct but overlapping functions in controlling subcellular redox environments. We also monitored the dynamic response of nucleus-rxYFP and cytosol-rxYFP to GSH depletion and to exogenous low and high doses of H₂O₂ bursts. These observations indicate a rapid and almost simultaneous oxidation of both nucleus-rxYFP and cytosol-rxYFP, highlighting the robustness of the rxYFP sensors in measuring real-time compartmental redox changes. Taken together, our data suggest that the highly reduced yeast nuclear and cytosolic redox states are maintained independently to some extent and under distinct but subtle redox regulation. Nucleus- and cytosol-rxYFP register compartment-specific localized redox fluctuations that may involve exchange of reduced and/or oxidized glutathione between these two compartments. Finally, we confirmed that GSH depletion has profound effects on mitochondrial genome stability but little effect on nuclear genome stability, thereby emphasizing that the critical requirement for GSH during growth is linked to a mitochondria-dependent process.     

The Thioredoxin-Thioredoxin Reductase System Can Function in Vivo as an Alternative System to Reduce Oxidized Glutathione in Saccharomyces cerevisiae

Cellular mechanisms that maintain redox homeostasis are crucial, providing buffering against oxidative stress. Glutathione, the most abundant low molecular weight thiol, is considered the major cellular redox buffer in most cells. To better understand how cells maintain glutathione redox homeostasis, cells of Saccharomyces cerevisiae were treated with extracellular oxidized glutathione (GSSG), and the effect on intracellular reduced glutathione (GSH) and GSSG were monitored over time. Intriguingly cells lacking GLR1 encoding the GSSG reductase in S. cerevisiae accumulated increased levels of GSH via a mechanism independent of the GSH biosynthetic pathway. Furthermore, residual NADPH-dependent GSSG reductase activity was found in lysate derived from glr1 cell. The cytosolic thioredoxin-thioredoxin reductase system and not the glutaredoxins (Grx1p, Grx2p, Grx6p, and Grx7p) contributes to the reduction of GSSG. Overexpression of the thioredoxins TRX1 or TRX2 in glr1 cells reduced GSSG accumulation, increased GSH levels, and reduced cellular glutathione E_h′. Conversely, deletion of TRX1 or TRX2 in the glr1 strain led to increased accumulation of GSSG, reduced GSH levels, and increased cellular E_h′. Furthermore, it was found that purified thioredoxins can reduce GSSG to GSH in the presence of thioredoxin reductase and NADPH in a reconstituted in vitro system. Collectively, these data indicate that the thioredoxin-thioredoxin reductase system can function as an alternative system to reduce GSSG in S. cerevisiae in vivo.     

Thioredoxins are required for protection against a reductive stress in the yeast Saccharomyces cerevisiae

Thioredoxins are small, highly conserved oxidoreductases that are required to maintain the redox homeostasis of the cell. They have been best characterized for their role as antioxidants in protection against reactive oxygen species. We show here that thioredoxins (TRX1, TRX2) and thioredoxin reductase (TRR1) are also required for protection against a reductive stress induced by exposure to dithiothreitol (DTT). This sensitivity to reducing conditions is not a general property of mutants affected in redox control, as mutants lacking components of the glutathione/glutaredoxin system are unaffected. Furthermore, TRX2 gene expression is induced in response to DTT treatment, indicating that thioredoxins form part of the cellular response to a reductive challenge. Our data indicate that the sensitivity of thioredoxin mutants to reducing stress appears to be a consequence of elevated glutathione levels, which is present predominantly in the reduced form (GSH). The elevated GSH levels also result in a constitutively high unfolded protein response (UPR), indicative of an accumulation of unfolded proteins in the endoplasmic reticulum (ER). However, there does not appear to be a general defect in ER function in thioredoxin mutants, as oxidative protein folding of the model protein carboxypeptidase Y occurs with similar kinetics to the wild-type strain, and trx1 trx2 mutants are unaffected in sensitivity to the glycosylation inhibitor tunicamycin. Furthermore, trr1 mutants are resistant to tunicamycin, consistent with their high UPR. The high UPR seen in trr1 mutants can be abrogated by the GSH-specific reagent 1-chloro-2,4-dinitrobenzene. In summary, thioredoxins are required to maintain redox homeostasis in response to both oxidative and reductive stress conditions.     

Glutathionylation of the Active Site Cysteines of Peroxiredoxin 2 and Recycling by Glutaredoxin

Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations, and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with glutathione (GSH). Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 was able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH and when the reduced protein was treated with H₂O₂ and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 m⁻¹ s⁻¹) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knock-out mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling.     

Molecular mapping of functionalities in the solution structure of reduced Grx4, a monothiol glutaredoxin from Escherichia coli

The ubiquitous glutaredoxin protein family is present in both prokaryotes and eukaryotes, and is closely related to the thioredoxins, which reduce their substrates using a dithiol mechanism as part of the cellular defense against oxidative stress. Recently identified monothiol glutaredoxins, which must use a different functional mechanism, appear to be essential in both Escherichia coli and yeast and are well conserved in higher order genomes. We have employed high resolution NMR to determine the three-dimensional solution structure of a monothiol glutaredoxin, the reduced E. coli Grx4. The Grx4 structure comprises a glutaredoxin-like alpha-beta fold, founded on a limited set of strictly conserved and structurally critical residues. A tight hydrophobic core, together with a stringent set of secondary structure elements, is thus likely to be present in all monothiol glutaredoxins. A set of exposed and conserved residues form a surface region, implied in glutathione binding from a known structure of E. coli Grx3. The absence of glutaredoxin activity in E. coli Grx4 can be understood based on small but significant differences in the glutathione binding region, and through the lack of a conserved second GSH binding site. MALDI experiments suggest that disulfide formation on glutathionylation is accompanied by significant structural changes, in contrast with dithiol thioredoxins and glutaredoxins, where differences between oxidized and reduced forms are subtle and local. Structural and functional implications are discussed with particular emphasis on identifying common monothiol glutaredoxin properties in substrate specificity and ligand binding events, linking the thioredoxin and glutaredoxin systems.     

Depletion of thiol reducing capacity impairs cytosolic but not mitochondrial iron-sulfur protein assembly machineries

Iron‑sulfur (Fe/S) clusters are versatile inorganic cofactors that play central roles in essential cellular functions, from respiration to genome stability. >30 proteins involved in Fe/S protein biogenesis in eukaryotes are known, many of which bind clusters via cysteine residues. This opens up the possibility that the thiol-reducing glutaredoxin and thioredoxin systems are required at both the Fe/S biogenesis and target protein level to counteract thiol oxidation. To address the possible interplay of thiol redox chemistry and Fe/S protein biogenesis, we have characterized the status of the mitochondrial (ISC) and cytosolic (CIA) Fe/S protein assembly machineries in Saccharomyces cerevisiae mutants in which the three partially redundant glutathione (Glr1) and thioredoxin (Trr1 and Trr2) oxidoreductases have been inactivated in either mitochondria, cytosol, or both compartments. Cells devoid of mitochondrial oxidoreductases maintained a functional mitochondrial ISC machinery and showed no altered iron homeostasis despite a non-functional complex II of the respiratory chain due to redox-specific defects. In cells that lack either cytosolic or total cellular thiol reducing capacity, both the ISC system and iron homeostasis were normal, yet cytosolic and nuclear Fe/S target proteins were not matured. This dysfunction could be attributed to a failure in the assembly of [4Fe‑4S] clusters in the CIA factor Nar1, even though Nar1 maintained robust protein levels and stable interactions with later-acting CIA components. Overall, our analysis has uncovered a hitherto unknown thiol-dependence of the CIA machinery and has demonstrated the surprisingly varying sensitivity of Fe/S proteins to thiol oxidation.     

谷氧还蛋白系统及其对细胞氧化还原态势的调控

细胞内氧化还原调控主要是由谷氧还蛋白系统和硫氧还蛋白系统完成。谷氧还蛋白属于硫氧还蛋白超家族,广泛分布在各种生物体内。作为一种巯基转移酶,它能够催化巯基．二硫键交换反应或者还原蛋白质谷胱甘肽二硫化物,以维持胞内的氧化还原态势。谷氧蛋白系统参与氧化胁迫、蛋白修饰、信号转导、细胞调亡和细胞分化等多种生物过程。对其体内作用靶蛋白的研究,有助于阐明谷氧还蛋白在整个细胞氧化还原网络的重要调控作用。     

Evolution of redoxin genes in the green lineage

The availability of the Arabidopsis genome revealed the complexity of the gene families implicated in dithiol disulfide exchanges. Most non-green organisms present less dithiol oxidoreductase genes. The availability of the almost complete genome sequence of rice now allows a systematic search for thioredoxins, glutaredoxins and their reducers. This shows that all redoxin families previously defined for Arabidopsis have members in the rice genome and that all the deduced rice redoxins fall within these families. This establishes that the redoxin classification applies both to dicots and monocots. Nevertheless, within each redoxin type the number of members is not the same in these two higher plants and it is not always possible to define orthologues between rice and Arabidopsis. The sequencing of two unicellular algae (Chlamydomonas and Ostreococcus) genomes are almost finished. This allowed us to follow the origin of the different gene families in the green lineage. It appears that most thioredoxin and glutaredoxin types, their chloroplastic, mitochondrial and cytosolic reducers are always present in these unicellular organisms. Nevertheless, striking differences appear in comparison to higher plant redoxins. Some thioredoxin types are not present in these algal genomes including thioredoxins o, clot and glutaredoxins CCxC. Numerous redoxins, including the cytosolic thioredoxins, do not fit with the corresponding higher plant classification. In addition both algae present a NADPH-dependent thioredoxin reductase with a selenocysteine which is highly similar to the animal thioredoxin reductases, a type of thioredoxin reductase not present in higher plants. An erratum to this article can be found at     

Glutathione and Glutaredoxin Act as a Backup of Human Thioredoxin Reductase 1 to Reduce Thioredoxin 1 Preventing Cell Death by Aurothioglucose

Thioredoxin reductase 1 (TrxR1) in cytosol is the only known reductant of oxidized thioredoxin 1 (Trx1) in vivo so far. We and others found that aurothioglucose (ATG), a well known active-site inhibitor of TrxR1, inhibited TrxR1 activity in HeLa cell cytosol but had no effect on the viability of the cells. Using a redox Western blot analysis, no change was observed in redox state of Trx1, which was mainly fully reduced with five sulfhydryl groups. In contrast, auranofin killed cells and oxidized Trx1, also targeting mitochondrial TrxR2 and Trx2. Combining ATG with ebselen gave a strong synergistic effect, leading to Trx1 oxidation, reactive oxygen species accumulation, and cell death. We hypothesized that there should exist a backup system to reduce Trx1 when only TrxR1 activity was lost. Our results showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced Trx1 in vitro and that the reaction was strongly stimulated by glutaredoxin1. Simultaneous depletion of TrxR activity by ATG and glutathione by buthionine sulfoximine led to overoxidation of Trx1 and loss of HeLa cell viability. In conclusion, the glutaredoxin system and glutathione have a backup role to keep Trx1 reduced in cells with loss of TrxR1 activity. Monitoring the redox state of Trx1 shows that cell death occurs when Trx1 is oxidized, followed by general protein oxidation catalyzed by the disulfide form of thioredoxin.     

Regulation of protein function by glutathionylation

The main function of reduced glutathione (GSH) is to protect from oxidative stress as a reactive oxygen scavenger. However, in the context of redox regulation, the ratio between GSH and its oxidized form (GSSG) determines the redox state of redox-sensitive cysteines in some proteins and, thus, acts as a signaling system. While GSH/GSSG can catalyze oxido-reduction of intra- and inter-chain disulfides by thiol-disulfide exchange, this review focuses on the formation of mixed disulfides between glutathione and proteins, also known as glutathionylation. The review discusses the regulatory role of this post-translational modification and the role of protein disulfide oxidoreductases (thioredoxin/thioredoxin reductase, glutaredoxin, protein disulfide isomerase) in the reversibility of this process.     

Mammalian selenoprotein thioredoxin-glutathione reductase. Roles in disulfide bond formation and sperm maturation

Thioredoxin reductases (TRs) are important redox regulatory enzymes, which control the redox state of thioredoxins. Mammals have cytosolic and mitochondrial TRs, which contain an essential selenocysteine residue and reduce cytosolic and mitochondrial thioredoxins. In addition, thioredoxin/glutathione reductase (TGR) was identified, which is a fusion of an N-terminal glutaredoxin domain and the TR module. Here we show that TGR is expressed at low levels in various tissues but accumulates in testes after puberty. The protein is particularly abundant in elongating spermatids at the site of mitochondrial sheath formation but is absent in mature sperm. We found that TGR can catalyze isomerization of protein and interprotein disulfide bonds and localized this function to its thiol domain. TGR targets include proteins that form structural components of the sperm, including glutathione peroxidase GPx4/PHGPx. Together, TGR and GPx4 can serve as a novel disulfide bond formation system. Both enzymes contain a catalytic selenocysteine consistent with the role of selenium in male reproduction.     

Thioredoxin 1 Is Inactivated Due to Oxidation Induced by Peroxiredoxin under Oxidative Stress and Reactivated by the Glutaredoxin System

The mammalian cytosolic thioredoxin system, comprising thioredoxin (Trx), Trx reductase, and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. Besides the active site thiols, human Trx1 contains three non-active site cysteine residues at positions 62, 69, and 73. A two-disulfide form of Trx1, containing an active site disulfide between Cys-32 and Cys-35 and a non-active site disulfide between Cys-62 and Cys-69, is inactive either as a disulfide reductase or as a substrate for Trx reductase. This could possibly provide a structural switch affecting Trx1 function during oxidative stress and redox signaling. We found that two-disulfide Trx1 was generated in A549 cells under oxidative stress. In vitro data showed that two-disulfide Trx1 was generated from oxidation of Trx1 catalyzed by peroxiredoxin 1 in the presence of H₂O₂. The redox Western blot data indicated that the glutaredoxin system protected Trx1 in HeLa cells from oxidation caused by ebselen, a superfast oxidant for Trx1. Our results also showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced the non-active site disulfide in vitro. This reaction was stimulated by glutaredoxin 1 via the so-called monothiol mechanism. In conclusion, reversible oxidation of the non-active site disulfide of Trx1 is suggested to play an important role in redox regulation and cell signaling via temporal inhibition of its protein-disulfide reductase activity for the transmission of oxidative signals under oxidative stress.     

A novel monothiol glutaredoxin (Grx4) from Escherichia coli can serve as a substrate for thioredoxin reductase

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750-2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3',5'-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.     

Structural basis for the thioredoxin-like activity profile of the glutaredoxin-like NrdH-redoxin from Escherichia coli.

NrdH-redoxin is a representative of a class of small redox proteins that contain a conserved CXXC motif and are characterized by a glutaredoxin-like amino acid sequence and thioredoxin-like activity profile. The crystal structure of recombinant Escherichia coli NrdH-redoxin in the oxidized state has been determined at 1.7 A resolution by multiwavelength anomalous diffraction. NrdH-redoxin belongs to the thioredoxin superfamily and is structurally most similar to E. coli glutaredoxin 3 and phage T4 glutaredoxin. The angle between the C-terminal helix alpha3 and strand beta4, which differs between thioredoxin and glutaredoxin, has an intermediate value in NrdH-redoxin. The orientation of this helix is to a large extent determined by an extended hydrogen-bond network involving the highly conserved sequence motif (61)WSGFRP(D/E)(67), which is unique to this subclass of the thioredoxin superfamily. Residues that bind glutathione in glutaredoxins are in general not conserved in NrdH-redoxin, and no glutathione-binding cleft is present. Instead, NrdH-redoxin contains a wide hydrophobic pocket at the surface, similar to thioredoxin. Modeling studies suggest that NrdH-redoxin can interact with E. coli thioredoxin reductase at this pocket and also via a loop that is complementary to a crevice in the reductase in a similar manner as observed in the E. coli thioredoxin-thioredoxin reductase complex.     

Characterization of Escherichia coli null mutants for glutaredoxin 2.

Three Escherichia coli glutaredoxins catalyze GSH-disulfide oxidoreductions, but the atypical 24-kDa glutaredoxin 2 (Grx2, grxB gene), in contrast to the 9-kDa glutaredoxin 1 (Grx1, grxA gene) and glutaredoxin 3 (Grx3, grxC gene), is not a hydrogen donor for ribonucleotide reductase. To improve the understanding of glutaredoxin function, a null mutant for grxB (grxB(-)) was constructed and combined with other mutations. Null mutants for grxB or all three glutaredoxin genes were viable in rich and minimal media with little changes in their growth properties. Expression of leaderless alkaline phosphatase showed that Grx1 and Grx2 (but not Grx3) contributed in the reduction of cytosolic protein disulfides. Moreover, Grx1 could catalyze disulfide formation in the oxidizing cytosol of combined null mutants for glutathione reductase and thioredoxin 1. grxB(-) cells were more sensitive to hydrogen peroxide and other oxidants and showed increased carbonylation of intracellular proteins, particularly in the stationary phase. Significant up-regulation of catalase activity was observed in null mutants for thioredoxin 1 and the three glutaredoxins, whereas up-regulation of glutaredoxin activity was observed in catalase-deficient strains with additional defects in the thioredoxin pathway. The expression of catalases is thus interconnected with the thioredoxin/glutaredoxin pathways in the antioxidant response.     

Redox-dependent mechanisms of regulation of breast epithelial cell proliferation

Activation of free radical oxidation in various types of cells, including breast epithelial cells, can lead to damage to macromolecules, particularly proteins involved in regulation of proliferation and programmed cell death. The glutathione, glutaredoxin and thioredoxin systems play an essential role in maintaining intracellular redox homeostasis. In this regard, modulation of the redox status of cells by means of a blocker and a protector of SH-groups of proteins can be used as a model for studying the role of redox proteins and glutathione in regulation of cell proliferation during the development of various pathological processes. In this study the state of thioredoxin, glutaredoxin, glutathione systems and their role in the regulation of HBL-100 breast epithelial cell proliferation during modulation of the redox status by using N-ethylmaleimide (NEM) and 1,4-dithioerythritol (DTE) have been investigated. The modulation of the redox status of the breast epithelial cells by the blocker (NEM) and the protector (DTE) of thiol groups of proteins and peptides influenced the functional activity of glutathione-dependent enzymes, glutaredoxin, thioredoxin, and thioredoxin reductase by changing concentrations of GSH and GSSG. Modulation of the redox status of HBL-100 cells was accompanied by an increase in the number of cells in the S phase of the cell cycle and a decrease of cells in G₀/G₁ and G₂/M phases as compared with the intact cell culture. The proposed method for evaluating the proliferative activity of cells during modulation of their redox state can be used in the development of new therapeutic approaches for treatment of diseases accompanied by the development of oxidative stress.     

Structural Analysis of Glutaredoxin Domain of Mus musculus Thioredoxin Glutathione Reductase

Thioredoxin glutathione reductase (TGR) is a member of the mammalian thioredoxin reductase family that has a monothiol glutaredoxin (Grx) domain attached to the thioredoxin reductase module. Here, we report a structure of the Grx domain of mouse TGR, determined through high resolution NMR spectroscopy to the final backbone RMSD value of 0.48±0.10 Å. The structure represents a sandwich-like molecule composed of a four stranded β-sheet flanked by five α–helixes, with the CxxS active motif located on the catalytic loop. We structurally characterized the glutathione-binding site in the protein and describe sequence and structural relationships of the domain with glutaredoxins. The structure illuminates a key functional center that evolved in mammalian TGRs to act in thiol-disulfide reactions. Our study allows us to hypothesize that Cys105 might be functionally relevant for TGR catalysis. In addition, the data suggest that the N-terminus of Grx acts as a possible regulatory signal also protecting the protein active site from unwanted interactions in cellular cytosol.     

In Silica Analysis of Thioredoxins and Glutaredoxins

Thioredoxins (TRXs) and glutaredoxins (GRXs) are ubiquitous small redox proteins belonging to the thioredoxin (TRX) superfamily. They regulate several cellular functions via mediating a dithiolldisulphide exchange in target proteins. Thioredoxins have been classified into several subgroups based on their structural homologs. In an attempt to identify thioredoxin proteins which have not been characterized, an EST database survey of Lycopersicon esculentum, Glycine max, Helianthus annus, Secale cereale, Solanum tuberosum, Apis mellifera ligustica, Oncorhynchus mykiss, Salmo salar, and whole genome survey for Drosophila melanogaster, Rattus norvegicus and Caenorhabditis briggsae was performed. Several glutaredoxin and glutaredoxin-like proteins from Ricinus communis, Vercinia fordii, Lycopersicon esculentum, Tilia platyphyllos, Populus tremuloides, Triticum aestivum and Oryza sativa were also characterized. The deduced amino acid sequences were aligned and phylogenetic trees were constructed to determine the consensus sequences and for establishing inter-relationships amongst the new and established thioredoxin and glutaredoxins. Based on the alignments, proteins were designated to their respective classes and subcellular localization predictions were used to predict their possible site of actions. In silico analysis has identified several new thioredoxins, glutaredoxins and related proteins and provided insight into their evolutionary relationships.