Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae

Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins. In Saccharomyces cerevisiae, two glutaredoxins (Grx1 and Grx2) containing a cysteine pair at the active site had been characterized as protecting yeast cells against oxidative damage. In this work, another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) that differs from the first in containing a single cysteine residue at the putative active site is described. This trait is also characteristic for a number of glutaredoxins from bacteria to humans, with which the Grx3/4/5 group has extensive homology over two regions. Mutants lacking Grx5 are partially deficient in growth in rich and minimal media and also highly sensitive to oxidative damage caused by menadione and hydrogen peroxide. A significant increase in total protein carbonyl content is constitutively observed in grx5 cells, and a number of specific proteins, including transketolase, appear to be highly oxidized in this mutant. The synthetic lethality of the grx5 and grx2 mutations on one hand and of grx5 with the grx3 grx4 combination on the other points to a complex functional relationship among yeast glutaredoxins, with Grx5 playing a specially important role in protection against oxidative stress both during ordinary growth conditions and after externally induced damage. Grx5-deficient mutants are also sensitive to osmotic stress, which indicates a relationship between the two types of stress in yeast cells.     

Nuclear monothiol glutaredoxins of Saccharomyces cerevisiae can function as mitochondrial glutaredoxins

Glutaredoxins are thiol oxidoreductases that regulate protein redox state. In Saccharomyces cerevisiae, Grx1 and Grx2 are cytosolic dithiol glutaredoxins, whereas Grx3, Grx4, and Grx5 are monothiol glutaredoxins. Grx5 locates at the mitochondrial matrix and is needed for iron/sulfur cluster biogenesis. Its absence causes phenotypes such as inactivation of iron/sulfur enzymes and sensitivity to oxidative stress. Whereas Grx5 contains a single glutaredoxin domain, in Grx3 and Grx4 a thioredoxin-like domain is fused to the glutaredoxin domain. Here we have shown that Grx3 locates at the nucleus and that the thioredoxin-like domain is required for such location. We have addressed the functional divergence among glutaredoxins by targeting Grx2/3/4 molecules to the mitochondrial matrix using the Grx5 targeting sequence. The mitochondrial forms of Grx3 and Grx4 partially rescue the defects of a grx5 null mutant. On the contrary, mitochondrially targeted Grx2 does not suppress the mutant phenotype. Both the thioredoxin-like and glutaredoxin domains are needed for the mitochondrial activity of Grx3, although none of the cysteine residues at the thioredoxin-like domain is required for rescue of the grx5 phenotypes. We have concluded that dithiol glutaredoxins are functionally divergent from monothiol ones, but the latter can interchange their biological activities when compartment barriers are surpassed.     

A single glutaredoxin or thioredoxin gene is essential for viability in the yeast Saccharomyces cerevisiae

Glutaredoxins and thioredoxins are small heat-stable oxidoreductases that have been conserved throughout evolution. The yeast Saccharomyces cerevisiae contains two gene pairs encoding cytoplasmic glutaredoxins (GRX1, GRX2) and thioredoxins (TRX1, TRX2). We report here that the quadruple trx1 trx2 grx1 grx2 mutant is inviable and that either a single glutaredoxin or a single thioredoxin (i.e. grx1 grx2 trx1, grx1 grx2 trx2, grx1 trx1 trx2, grx2 trx1 trx2) is essential for viability. Loss of both thioredoxins has been reported previously to lead to methionine auxotrophy consistent with thioredoxins being the sole reductants for 3'-phosphoadenosine 5'-phosphosulphate reductase (PAPS) in yeast. However, we present evidence for the existence of a novel yeast hydrogen donor for PAPS reductase, as strains lacking both thioredoxins assimilated sulphate under conditions that minimized the generation of reactive oxygen species (low aeration and absence of functional mitochondria). In addition, the assimilation of [35S]-sulphate was approximately 60-fold higher in the trx1 trx2 grx1 and trx1 trx2 grx2 mutants compared with the trx1 trx2 mutant. Furthermore, in contrast to the trx1 trx2 mutant, the trx1 trx2 grx2 mutant grew on minimal agar plates, and the trx1 trx2 grx1 mutant grew on minimal agar plates under anaerobic conditions. We propose a model in which the novel reductase activity normally functions in the repair of oxidant-mediated protein damage but, under conditions that minimize the generation of reactive oxygen species, it can serve as a hydrogen donor for PAPS reductase.     

Role of yeast glutaredoxins as glutathione S-transferases

The yeast Saccharomyces cerevisiae contains two glutaredoxins, encoded by GRX1 and GRX2, that are required for resistance to reactive oxygen species. We recently reported that Grx1 is active as a glutathione peroxidase and can directly reduce hydroperoxides (Collinson, E. J., Wheeler, G. L., Garrido, E. O., Avery, A. M., Avery, S. V., and Grant, C. M. (2002) J. Biol. Chem. 277, 16712-16717). We now show that Grx2 is also a general hydroperoxidase, and kinetic data indicate that both enzymes have a similar pattern of activity, which is highest with hydrogen peroxide, followed by cumene hydroperoxide and tert-butyl hydroperoxide. Furthermore, both Grx1 and Grx2 are shown be active as glutathione S-transferases (GSTs), and their activity with model substrates such as 1-chloro-2,4-dinitrobenzene is similar to their activity with hydroperoxides. Analysis of the Grx1 active site residues shows that Cys-27, but not Cys-30, is required for both the peroxidase and transferase activities, indicating that these reactions proceed via a monothiol mechanism. Deletion analysis shows that Grx1 and Grx2 have an overlapping function with yeast GSTs, encoded by GTT1 and GTT2, and are responsible for the majority of cellular GST activity. In addition, multiple mutants lacking GRX1, GRX2, GTT1, and GTT2 show increased sensitivity to stress conditions, including exposure to xenobiotics, heat, and oxidants. In summary, glutaredoxins are multifunctional enzymes with oxidoreductase, peroxidase, and GST activity, and are therefore ideally suited to detoxify the wide range of xenobiotics and oxidants that can be generated during diverse stress conditions.     

Localization and function of three monothiol glutaredoxins in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe contains two dithiol glutaredoxins (Grx1 and Grx2) and genes for three putative monothiol glutaredoxins (grx3, 4, and 5). We investigated the expression, sub-cellular localization, and functions of the three monothiol glutaredoxins. Fluorescence microscopy revealed that Grx3 is targeted to nuclear rim and endoplasmic reticulum, Grx4 primarily to the nucleus, and Grx5 to mitochondria. Null mutation of grx3 did not significantly affect growth and resistance against various oxidants, whereas grx5 mutation caused slow growth and sensitivity toward oxidants such as hydrogen peroxide, paraquat, and diamide. The grx2grx5 double mutation, deficient in all mitochondrial glutaredoxins, caused further retardation in growth and severe sensitivity toward all the oxidants tested. The grx4 mutation was not viable, suggesting a critical role of Grx4 for the physiology of S. pombe. Overproduction of Grx3 and Grx5, but not the truncated form of Grx5 without mitochondrial target sequence, severely retarded growth as Grx2 did, supporting the idea that Grx2, 3, and 5 are targeted to organellar compartments. Our results propose a distinct role for each glutaredoxin to maintain thiol redox balance, and hence the growth and stress resistance, of the fission yeast.     

Apoptosis as a mechanism for removal of mutated cells of Saccharomyces cerevisiae: The role of Grx2 under cadmium exposure

Cadmium is a strong mutagen that acts by inhibiting DNA mismatch repair, while its toxic effect seems to be related to an indirect oxidative stress that involves glutathione (GSH) mobilization. Among the roles of GSH is the protection of proteins against oxidative damage, by forming reversible mixed disulfides with cysteine residues, a process known as protein glutathionylation and catalyzed by glutaredoxins (Grx). In this current study, Saccharomyces cerevisiae cells deficient in GRX2, growing in 80 μM CdSO₄, showed high mitochondrial mutagenic rate, determined by frequency of mutants that had lost mitochondrial function (petite mutants), high tolerance and lower apoptosis induction. The mutant strain also showed decreased levels of glutathionylated-protein after cadmium exposure, which might difficult the signaling to apoptosis, leading to increased mutagenic rates. Taken together, these results suggest that Grx2 is involved with the apoptotic death induced by cadmium, a form of cellular suicide that might lead of removal of mutated cells.     

Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The Δgrx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1⁺ gene encoding a putative A-type Fe-S scaffold, in addition to mas5⁺ and hsc1⁺ genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2⁺, but not isu1⁺ and ssc1⁺, complemented the growth phenotype of Δgrx5 mutant as isa1⁺ did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.     

The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism

Trypanosoma brucei, the causative agent of African sleeping sickness, possesses two dithiol glutaredoxins (Grx1 and Grx2). Grx1 occurs in the cytosol and catalyzes protein deglutathionylations with k_cat/K_m-values of up to 2 × 10⁵ m⁻¹ s⁻¹. It accelerates the reduction of ribonucleotide reductase by trypanothione although less efficiently than the parasite tryparedoxin and has low insulin disulfide reductase activity. Despite its classical CPYC active site, Grx1 forms dimeric iron-sulfur complexes with GSH, glutathionylspermidine, or trypanothione as non-protein ligands. Thus, contrary to the generally accepted assumption, replacement of the Pro is not a prerequisite for cluster formation. T. brucei Grx2 shows an unusual CQFC active site, and orthologues occur exclusively in trypanosomatids. Grx2 is enriched in mitoplasts, and fractionated digitonin lysis resulted in a co-elution with cytochrome c, suggesting localization in the mitochondrial intermembrane space. Grx2 catalyzes the reduction of insulin disulfide but not of ribonucleotide reductase and exerts deglutathionylation activity 10-fold lower than that of Grx1. RNA interference against Grx2 caused a growth retardation of procyclic cells consistent with an essential role. Grx1 and Grx2 are constitutively expressed with cellular concentrations of about 2 μm and 200 nm, respectively, in both the mammalian bloodstream and insect procyclic forms. Trypanothione reduces the disulfide form of both proteins with apparent rate constants that are 3 orders of magnitude higher than those with glutathione. Grx1 and, less efficiently, also Grx2 catalyze the reduction of GSSG by trypanothione. Thus, the Grxs play exclusive roles in the trypanothione-based thiol redox metabolism of African trypanosomes.     

Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes

Yeast cells contain a family of three monothiol glutaredoxins: Grx3, 4, and 5. Absence of Grx5 leads to constitutive oxidative damage, exacerbating that caused by external oxidants. Phenotypic defects associated with the absence of Grx5 are suppressed by overexpression of SSQ1 and ISA2, two genes involved in the synthesis and assembly of iron/sulfur clusters into proteins. Grx5 localizes at the mitochondrial matrix, like other proteins involved in the synthesis of these clusters, and the mature form lacks the first 29 amino acids of the translation product. Absence of Grx5 causes: 1) iron accumulation in the cell, which in turn could promote oxidative damage, and 2) inactivation of enzymes requiring iron/sulfur clusters for their activity. Reduction of iron levels in grx5 null mutants does not restore the activity of iron/sulfur enzymes, and cell growth defects are not suppressed in anaerobiosis or in the presence of disulfide reductants. Hence, Grx5 forms part of the mitochondrial machinery involved in the synthesis and assembly of iron/sulfur centers.     

Structural basis for the different activities of yeast Grx1 and Grx2

Yeast glutaredoxins Grx1 and Grx2 catalyze the reduction of both inter- and intra-molecular disulfide bonds using glutathione (GSH) as the electron donor. Although sharing the same dithiolic CPYC active site and a sequence identity of 64%, they have been proved to play different roles during oxidative stress and to possess different glutathione-disulfide reductase activities. To address the structural basis of these differences, we solved the crystal structures of Grx2 in oxidized and reduced forms, at 2.10 Å and 1.50 Å, respectively. With the Grx1 structures we previously reported, comparative structural analyses revealed that Grx1 and Grx2 share a similar GSH binding site, except for a single residue substitution from Asp89 in Grx1 to Ser123 in Grx2. Site-directed mutagenesis in combination with activity assays further proved this single residue variation is critical for the different activities of yeast Grx1 and Grx2.     

Both human and soya bean ferritins highly improve the accumulation of bioavailable iron and contribute to extend the chronological life in budding yeast

Ferritin proteins have an enormous capacity to store iron in cells. In search for the best conditions to accumulate and store bioavailable iron, we made use of a double mutant null for the monothiol glutaredoxins GRX3 and GRX4. The strain grx3grx4 accumulates high iron concentrations in the cytoplasm, making the metal easily available for ferritin chelation. Here, we perform a comparative study between human (L and H) and soya bean ferritins (H1 and H2) function in the eukaryotic system Saccharomyces cerevisiae. We demonstrate that the four human and soya bean ferritin chains are successfully expressed in our model system. Upon coexpression of either both human or soya bean ferritin chains, respiratory conditions along with iron supplementation led us to obtain the maximum yields of iron stored in yeast described to date. Human and soya bean ferritin chains are functional and present equivalent properties as promoters of cell survival in iron overload conditions. The best system revealed that the four human and soya bean ferritins possess a novel function as anti-ageing proteins in conditions of iron excess. In this respect, both ferritin chains with oxidoreductase capacity (human-H and soya bean-H2) bear the highest capacity to extend life suggesting the possibility of an evolutionary conservation.     

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.     

One single in-frame AUG codon is responsible for a diversity of subcellular localizations of glutaredoxin 2 in Saccharomyces cerevisiae

Glutaredoxins belong to a family of small proteins with glutathione-dependent disulfide oxidoreductase activity involved in cellular defense against oxidative stress. The product of the yeast GRX2 gene is a protein that is localized both in the cytosol and mitochondria. To throw light onto the mechanism responsible for the dual subcellular distribution of Grx2 we analyzed mutant constructs containing different targeting information. By altering amino acid residues around the two in-frame translation initiation start sites of the GRX2 gene, we could demonstrate that the cytosolic isoform of Grx2 was synthesized from the second AUG, lacking an N-terminal extension. Translation from the first AUG resulted in a long isoform carrying a mitochondrial targeting presequence. The mitochondrial targeting properties of the presequence and the influence of the mature part of Grx2 were analyzed by the characterization of the import kinetics of specific fusion proteins. Import of the mitochondrial isoform is relatively inefficient and results in the accumulation of a substantial amount of unprocessed form in the mitochondrial outer membrane. Substitution of Met(35), the second translation start site, to Val resulted in an exclusive targeting to the mitochondrial matrix. Our results show that a plethora of Grx2 subcellular localizations could spread its antioxidant functions all over the cell, but one single A to G [corrected] mutation converts Grx2 into a typical protein of the mitochondrial matrix. The "A" denotes adenine, rather than alanine, and the "G" refers to guanine, not glycine [corrected]     

A role for yeast glutaredoxin genes in selenite-mediated oxidative stress

Since the double Δgrx1Δgrx2 mutant is hypersensitive to selenite we decided to evaluate mechanisms underlying this phenomenon and establish the roles of other components of yeast glutaredoxin system, in particular glutaredoxin 5 in the selenite resistance. We found elevation in the intracellular and mitochondrial superoxide production in the Δgrx1Δgrx2 and Δgrx5 mutants after Se(IV) treatment. The last effect was more pronounced for cells lacking the mitochondrial Grx5 protein. We also recorded selenite-induced increase in the peroxide production in all strains tested. Nonfermentable carbon sources, glycerol and ethanol, augmented selenite toxicity. Hypo- and anoxia protected against the harmful effects of Se(VI). Augmentation of the intracellular levels of two endogenous antioxidants, erythroascorbic acid and glutathione confers resistance to selenite. We recorded a strain-unspecific, selenite-mediated decrease in the level of acid-soluble thiols. Collectively, our data demonstrate that hypersensitivity to the Δgrx1Δgrx2 and Δgrx5 disruptants to selenite is mediated by altered intracellular redox equilibrium.     

Solution structure of Escherichia coli glutaredoxin-2 shows similarity to mammalian glutathione-S-transferases.

Glutaredoxin 2 (Grx2) from Escherichia coli is distinguished from other glutaredoxins by its larger size, low overall sequence identity and lack of electron donor activity with ribonucleotide reductase. However, catalysis of glutathione (GSH)-dependent general disulfide reduction by Grx2 is extremely efficient. The high-resolution solution structure of E. coli Grx2 shows a two-domain protein, with residues 1 to 72 forming a classical "thioredoxin-fold" glutaredoxin domain, connected by an 11 residue linker to the highly helical C-terminal domain, residues 84 to 215. The active site, Cys9-Pro10-Tyr11-Cys12, is buried in the interface between the two domains, but Cys9 is solvent-accessible, consistent with its role in catalysis. The structures reveal the hither to unknown fact that Grx2 is structurally similar to glutathione-S-transferases (GST), although there is no obvious sequence homology. The similarity of these structures gives important insights into the functional significance of a new class of mammalian GST-like proteins, the single-cysteine omega class, which have glutaredoxin oxidoreductase activity rather than GSH-S-transferase conjugating activity. E. coli Grx 2 is structurally and functionally a member of this new expanding family of large glutaredoxins. The primary function of Grx2 as a GST-like glutaredoxin is to catalyze reversible glutathionylation of proteins with GSH in cellular redox regulation including stress responses.     

Cloning, functional analysis, and mitochondrial localization of Trypanosoma brucei monothiol glutaredoxin-1

African trypanosomes encode three monothiol glutaredoxins (1-C-Grx1 to 3). 1-C-Grx1 has a putative CAYS active site and Cys181 as single additional cysteine. The recombinant protein forms non-covalent homodimers. As observed for other monothiol glutaredoxins, Trypanosoma brucei 1-C-Grx1 was not active in the glutaredoxin assay with hydroxyethyl disulfide and glutathione nor catalyzed the reduction of insulin disulfide. In addition, it lacked peroxidase activity and did not catalyze protein (de)glutathionylation. Upon oxidation, 1-C-Grx1 forms an intramolecular disulfide bridge and, to a minor degree, covalent dimers. Both disulfide forms are reduced by the parasite trypanothione/tryparedoxin system. 1-C-Grx1 shows mitochondrial localization. The total cellular concentration is at least 5 microm. Thus, 1-C-Grx1 is an abundant protein especially in the rudimentary organelle of the mammalian form of the parasite. Expression of 1-C-Grx1 in Grx5-deficient yeast cells with its authentic presequence targeted the protein to the mitochondria and partially restored the growth phenotype and aconitase activity of the mutant, and conferred resistance against hydroperoxides and diamide. The parasite Grx2 and 3 failed to substitute for Grx5. This is surprising because even bacterial and plant 1-Cys-glutaredoxins efficiently revert the defects, and may be due to the lack of two basic residues conserved in all but the trypanosomatid proteins.