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Alexios Vlamis-Gardikas

Publications and source records attributed to Alexios Vlamis-Gardikas.

12 recordsLinked to original sources
PubMed2007

Characterization of the native CREB3L2 transcription factor and the FUS/CREB3L2 chimera.

CREB3L2 was first identified as the 3'-partner of FUS in a fusion gene that seems to be specific for low grade fibromyxoid sarcoma. In silico analyses suggest that the predicted CREB3L2 protein is a member of the CREB3 family of transcription factors, with its bZIP domain being highly similar to that in CREB3L1, CREB3L3, CREB3L4, CREB3, and Drosophila Bbf-2. In the present study, the authors assessed various cellular outcomes after transfection of NIH3T3 and HEK-293 cells with constructs containing full-length and truncated versions of CREB3L2 and FUS/CREB3L2. Northern blot of CREB3L2 mRNA revealed a 7.4 kbp band that contains 0.4 kbp and 5.5 kbp untranslated 5' and 3' regions, respectively. CREB3L2 constructs containing the first 120 amino acids (aa) showed the highest transcriptional activation. Much stronger transcriptional activation was consistently seen for the FUS/CREB3L2 constructs than for the corresponding CREB3L2 constructs. Transcriptional activity was achieved through the box-B element, ATF6 and CRE binding sites, as well as the GRP78 promoter. Proteins encoded by full-length CREB3L2 and FUS/CREB3L2 were localized to reticular structures of the cytoplasm, whereas the corresponding, truncated proteins lacking the transmembrane domain and the carboxy-terminal part of CREB3L2 resided within the nucleus. The results of the present study show that CREB3L2 is not only structurally, but also functionally very similar to CREB3L1. Thus, studies regarding the pathways influenced by wild-type CREB3L2 should provide valuable clues to the pathogenetic significance of the FUS/CREB3L2 chimera in low grade fibromyxoid sarcoma.

Amino Acid Sequence↗
PubMed2005

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.

Amino Acid Motifs↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2004

Stimulation of Fe-S cluster insertion into apoFNR by Escherichia coli glutaredoxins 1, 2 and 3 in vitro.

The oxygen sensor fumarate nitrate reductase regulator (FNR) of Escherichia coli contains in the active (anaerobic) state a [4Fe-4S](2+) cluster which is lost after exposure to O(2). In aerobically prepared apoFNR, or in FNR obtained by treatment of [4Fe-4S] . FNR with O(2) in vitro, intramolecular cysteine disulfides are found, including the cysteine residues which serve as ligands for the Fe-S cluster. It is shown here that the reconstitution of [4Fe-4S] . FNR from this form of aerobic apoFNR was preceded by a long lag phase when glutathione was used as the reducing agent. Addition of E. coli glutaredoxins (Grx) 1, 2 or 3 decreased the lag phase greatly and stimulated the reconstitution rate slightly (about twofold). Reconstitution of anaerobically prepared apoFNR, which has a lower cysteine disulfide content, showed only a short lag phase, which further decreased in the presence of Grx. It is concluded that in the lag phase the cysteine disulfides of apoFNR become reduced for the incorporation of the [4Fe-4S] cluster and that this reaction is stimulated by Grx. Thioredoxin (Trx) 1 showed no stimulation of FNR reconstitution in vitro. It is suggested that the function of Grx might be of significance for the insertion of FeS cluster in proteins containing disulfides.

Cysteine↗
PubMed2004

Assessment of production conditions for efficient use of Escherichia coli in high-yield heterologous recombinant selenoprotein synthesis.

The production of heterologous selenoproteins in Escherichia coli necessitates the design of a secondary structure in the mRNA forming a selenocysteine insertion sequence (SECIS) element compatible with SelB, the elongation factor for selenocysteine insertion at a predefined UGA codon. SelB competes with release factor 2 (RF2) catalyzing translational termination at UGA. Stoichiometry between mRNA, the SelB elongation factor, and RF2 is thereby important, whereas other expression conditions affecting the yield of recombinant selenoproteins have been poorly assessed. Here we expressed the rat selenoprotein thioredoxin reductase, with titrated levels of the selenoprotein mRNA under diverse growth conditions, with or without cotransformation of the accessory bacterial selA, selB, and selC genes. Titration of the selenoprotein mRNA with a pBAD promoter was performed in both TOP10 and BW27783 cells, which unexpectedly could not improve yield or specific activity compared to that achieved in our prior studies. Guided by principal component analysis, we instead discovered that the most efficient bacterial selenoprotein production conditions were obtained with the high-transcription T7lac-driven pET vector system in presence of the selA, selB, and selC genes, with induction of production at late exponential phase. About 40 mg of rat thioredoxin reductase with 50% selenocysteine content could thereby be produced per liter bacterial culture. These findings clearly illustrate the ability of E. coli to upregulate the selenocysteine incorporation machinery on demand and that this is furthermore strongly augmented in late exponential phase. This study also demonstrates that E. coli can indeed be utilized as cell factories for highly efficient production of heterologous selenoproteins such as rat thioredoxin reductase.

Arabinose↗
PubMed2003

Redox regulation of 3'-phosphoadenylylsulfate reductase from Escherichia coli by glutathione and glutaredoxins.

Inorganic sulfate (SO42-, S+VI) is reduced in vivo to sulfite (SO32-, S+IV) via phosphoadenylylsulfate (PAPS) reductase. Escherichia coli lacking glutathione reductase and glutaredoxins (gor-grxA-grxB-grxC-) barely grows on sulfate. We found that incubation of PAPS reductase with oxidized glutathione leads to enzyme inactivation with simultaneous formation of a mixed disulfide between glutathione and the active site Cys-239. A newly developed method based on thiol-specific fluorescent alkylation and gel electrophoresis showed that glutathionylated PAPS reductase is reduced by glutaredoxins via a monothiol mechanism. This glutathionylated species was also observed in poorly growing gor-grxA-grxB-grxC- cells expressing inactive glutaredoxin 2 (Grx2) C9S/C12S. However, it was absent in better growing cells expressing monothiol Grx2 C12S or wild type Grx2. Reversible glutathionylation may thus regulate the activity of PAPS reductase in vivo.

Escherichia coli↗
PubMed2003

Real-time kinetics of the interaction between the two subunits, Escherichia coli thioredoxin and gene 5 protein of phage T7 DNA polymerase.

T7 phage DNA polymerase is a tight 1:1 complex of the gene 5 protein (g5p) (80 kDa) of phage T7 and thioredoxin (12 kDa) from the Escherichia coli host. The holoenzyme is essential for the replication of the phage. We estimated the real-time kinetics and thermodynamics of the interaction of g5p with thioredoxin (wild type and mutants) using surface plasmon resonance. Thioredoxin was immobilized on a CM5 sensor chip through a six-carbon spacer (6-amino-n-hexanoic acid) using standard amine coupling. Reduced thioredoxin bound g5p but oxidized thioredoxin did not. The association and dissociation phases of the complex fit a two-exponential model with an apparent equilibrium dissociation constant (KD) of 2.2 nm for thioredoxin with 4.7 x 104.M-1.s-1 and 10.5 x 10-5.s-1 as the corresponding association (ka) and dissociation (kd) rate constants. The strong binding of g5p to thioredoxin is therefore due to fast association and very slow dissociation, a situation similar to antigen-antibody interactions. Thioredoxin mutants P34S, D26A, K57M, D26A/K57M, W31F, W31Y, K36A, K36E, and Y49F had KD values in the range of 1 to 8 nm, whereas mutant W28A had a KD of 12.5 nm. No detectable interaction was observed for mutants P40G, W31H, W31A, and C35A. The effect of temperature on KD and the changes in enthalpy (-DeltaH = 20.2 kcal.m-1) and entropy (TDeltaS =-8.4 kcal.m-1) upon formation of the complex suggested that the interaction is driven by an increase in enthalpy and opposed by a decrease in entropy.

DNA-Directed DNA Polymerase↗
PubMed2003

Characterization of the redox properties of poplar glutaredoxin.

The presence of glutaredoxins in plants is now well recognized, but their functions and natural substrates remain largely unknown. Recently, a poplar glutaredoxin has been biochemically characterized and several mutants have been engineered in order to explore its reactivity. This work focuses on some physiological functions of the enzyme. According to our findings, the poplar glutaredoxin can serve as an electron donor to the bacterial 3'-phosphoadenylylsulfate reductase as it supports both the catalysis by the enzyme in vitro and complements a methionine auxotroph strain of Escherichia coli. In addition, poplar glutaredoxin is able to reduce the Escherichia coli ribonucleotide reductase 1a (in vitro reduction of cytidine diphosphate). Although this glutaredoxin is described as an electron donor to a phloem-located peroxiredoxin, whose function is to detoxify hydroperoxides, we found that it does not directly reduce hydrogen peroxide or other alkyl hydroperoxides as described for yeast and rice glutaredoxins. However, the poplar glutaredoxin may be involved in the response to oxidative stress as its overexpression in Escherichia coli resulted in a higher resistance toward hydrogen peroxide, menadione, and tert-butyl hydroperoxide.

Amino Acid Sequence↗
PubMed2002

Protein levels of Escherichia coli thioredoxins and glutaredoxins and their relation to null mutants, growth phase, and function.

Levels of Escherichia coli thioredoxin 1 (Trx1), Trx2, glutaredoxin 1 (Grx1), Grx2, and Grx3 have been determined by novel sensitive sandwich enzyme-linked immunosorbent assay. In a wild type strain, levels of Trx1 increased from the exponential to the stationary phase of growth (1.5-fold to 3400 ng/mg), as did levels of Grx2 (from approximately 2500 to approximately 8000 ng/mg). Grx3 and Trx2 levels were quite stable during growth ( approximately 4500 and approximately 200 ng/mg, respectively). Grx1 levels decreased from approximately 600 ng/mg at the exponential phase to approximately 285 ng/mg at the stationary phase. A large elevation of Grx1 (20-30-fold), was observed in null mutants for the thioredoxin system whereas levels of the other redoxins in all combinations of examined null mutants barely exceeded a 2-3-fold increase. Measurements of thymidine incorporation in newly synthesized DNA suggested that mainly Grx1 and, to a lesser extent, Trx1 contribute to the reduction of ribonucleotides. All glutaredoxin species were elevated in catalase-deficient strains, implying an antioxidant role for the glutaredoxins. Trx1, Trx2, and Grx1 levels increased after exposure to hydrogen peroxide and decreased after exposure to mercaptoethanol. The levels of Grx2 and Grx3 behaved exactly the opposite, suggesting that the transcription factor OxyR does not regulate their expression.

Arsenates↗
PubMed2002

Expression of Escherichia coli glutaredoxin 2 is mainly regulated by ppGpp and sigmaS.

Escherichia coli glutaredoxin 2 (Grx2, encoded by grxB) differs greatly from the other two glutaredoxins in structure and catalytic properties. In a wild type strain, levels of Grx2 increased 3-fold in the stationary phase (up to 8 microg/mg). Guanosine-3',5'-tetraphoshate (ppGpp) and sigma(S), which regulate the transcription of genes in the stationary phase, dramatically affected the expression of Grx2. spoTrelA null mutants, lacking ppGpp, had very low levels of Grx2, while overproduction of full-length RelA or valine-induced starvation of isoleucine, both conditions elevating ppGpp levels, resulted in elevation of Grx2. Null mutants for the sigma(S)-specific protease ClpP, which have higher levels of sigma(S), exhibited a 3-fold Grx2 increase. sigma(S) in trans also increased the levels of Grx2. Therefore the stationary phase expression of Grx2 is determined by the sigma(S)-bound form of RNA polymerase in connection with ppGpp, while basal levels should be attributed to sigma(70)-RNA polymerase holoenzyme. Osmotic pressure and cAMP also affected the expression of Grx2, presumably via sigma(S). Furthermore, Grx2 levels were elevated in an oxyR(-) strain. In accordance with the role of Grx2 as a stationary phase protein, null mutants for grxB were shown to lyse under starvation conditions and exhibited a distorted morphology.

Bacterial Proteins↗
PubMed2001

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.

Base Sequence↗