Multi-domain CGFS-type glutaredoxin Grx4 regulates iron homeostasis via direct interaction with a repressor Fep1 in fission yeast

The fission yeast Schizosaccharomyces pombe contains two CGFS-type monothiol glutaredoxins, Grx4 and Grx5, which are localized primarily in the nucleus and mitochondria, respectively. We observed involvement of Grx4 in regulating iron-responsive gene expression, which is modulated by a repressor Fep1. Lack of Grx4 caused defects not only in growth but also in the expression of both iron-uptake and iron-utilizing genes regardless of iron availability. In order to unravel how Grx4 is involved in Fep1-mediated regulation, interaction between them was investigated. Co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) revealed that Grx4 physically interacts with Fep1 in vivo. BiFC revealed localized nuclear dots produced by interaction of Grx4 with Fep1. Mutation of cysteine-172 in the CGFS motif to serine (C172S) produced effects similarly observed under Grx4 depletion, such as the loss of iron-dependent gene regulation and the absence of nuclear dots in BiFC analysis. These results suggest that the ability of Grx4 to bind iron, most likely Fe-S cofactor, could be critical in interacting with and modulating the activity of Fep1.     

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.     

Structure-function analysis of yeast Grx5 monothiol glutaredoxin defines essential amino acids for the function of the protein

Grx5 defines a family of yeast monothiol glutaredoxins that also includes Grx3 and Grx4. All three proteins display significant sequence homology with proteins found from bacteria to humans. Grx5 is involved in iron/sulfur cluster assembly at the mitochondria, but the function of Grx3 and Grx4 is unknown. Three-dimensional modeling based on known dithiol glutaredoxin structures predicted a thioredoxin fold structure for Grx5. Positionally conserved amino acids in this glutaredoxin family were replaced in Grx5, and the effect on the biological function of the protein has been tested. For all changes studied, there was a correlation between the effects on several different phenotypes: sensitivity to oxidants, constitutive protein oxidation, ability for respiratory growth, auxotrophy for a number of amino acids, and iron accumulation. Cys(60) and Gly(61) are essential for Grx5 function, whereas other single or double substitutions in the same region had no phenotypic effects. Gly(115) and Gly(116) could be important for the formation of a glutathione cleft on the Grx5 surface, in contrast to adjacent Cys(117). Substitution of Phe(50) alters the beta-sheet in the thioredoxin fold structure and inhibits Grx5 function. None of the substitutions tested affect the structure at a significant enough level to reduce protein stability.     

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.     

An arsenate-activated glutaredoxin from the arsenic hyperaccumulator fern Pteris vittata L. regulates intracellular arsenite

To elucidate the mechanisms of arsenic resistance in the arsenic hyperaccumulator fern Pteris vittata L., a cDNA for a glutaredoxin (Grx) Pv5-6 was isolated from a frond expression cDNA library based on the ability of the cDNA to increase arsenic resistance in Escherichia coli. The deduced amino acid sequence of Pv5-6 showed high homology with an Arabidopsis chloroplastic Grx and contained two CXXS putative catalytic motifs. Purified recombinant Pv5-6 exhibited glutaredoxin activity that was increased 1.6-fold by 10 mm arsenate. Site-specific mutation of Cys(67) to Ala(67) resulted in the loss of both GRX activity and arsenic resistance. PvGrx5 was expressed in E. coli mutants in which the arsenic resistance genes of the ars operon were deleted (strain AW3110), a deletion of the gene for the ArsC arsenate reductase (strain WC3110), and a strain in which the ars operon was deleted and the gene for the GlpF aquaglyceroporin was disrupted (strain OSBR1). Expression of PvGrx5 increased arsenic tolerance in strains AW3110 and WC3110, but not in OSBR1, suggesting that PvGrx5 had a role in cellular arsenic resistance independent of the ars operon genes but dependent on GlpF. AW3110 cells expressing PvGrx5 had significantly lower levels of arsenite when compared with vector controls when cultured in medium containing 2.5 mm arsenate. Our results are consistent with PvGrx5 having a role in regulating intracellular arsenite levels, by either directly or indirectly modulating the aquaglyceroporin. To our knowledge, PvGrx5 is the first plant Grx implicated in arsenic metabolism.     

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.     

The Glutaredoxin Family in Oxygenic Photosynthetic Organisms

Glutaredoxins (GRXs) are small redox proteins of the thioredoxin (TRX) superfamily. Compared to TRXs, much less information on the GRX family is available, especially in photosynthetic organisms since GRXs have been mainly studied in E. coli, yeast and mammal cells. The analysis of the TRX family in oxygenic photosynthetic organisms revealed an unsuspected multiplicity of TRXs but it is not known if the same situation holds for GRXs. Despite the availability of genome sequences from different oxygenic photosynthetic organisms, the number of GRXs and the different groups present in these organisms are still undescribed. This paper presents a comparative analysis of the GRX families present in Arabidopsis, Chlamydomonas and Synechocystis which were found to contain 30, 6 and 3 GRX genes, respectively. The putative subcellular localization of each GRX and its relative expression level, based on EST data, have been investigated. This analysis reveals the presence of three major classes of GRXs, the CPYC type, the CGFS type and a previously undescribed type, called the CC type that appears specific to higher plants. These data are discussed in view of recent results suggesting a complex cross-regulation between the TRX and GRX systems.     

The thioredoxin superfamily in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The thioredoxin (TRX) superfamily includes redox proteins such as thioredoxins, glutaredoxins (GRXs) and protein disulfide isomerases (PDI). These proteins share a common structural motif named the thioredoxin fold. They are involved in disulfide oxido-reduction and/or isomerization. The sequencing of the Arabidopsisgenome revealed an unsuspected multiplicity of TRX and GRX genes compared to other organisms. The availability of full Chlamydomonasgenome sequence offers the opportunity to determine whether this multiplicity is specific to higher plant species or common to all photosynthetic eukaryotes. We have previously shown that the multiplicity is more limited in Chlamydomonas for TRX and GRX families. We extend here our analysis to the PDI family. This paper presents a comparative analysis of the TRX, GRX and PDI families present in Arabidopsis,Chlamydomonas and Synechocystis. The putative subcellular localization of each protein and its relative expression level, based on EST data, have been investigated. This analysis provides a large overview of the redox regulatory systems present in Chlamydomonas. The data are discussed in view of recent results suggesting a complex cross-talk between the TRX, GRX and PDI redox regulatory networks.