Cloning, expression and characterization of human thioltransferase (glutaredoxin) in <i>E. coli</i>

Chrestensen, Carol A.; Eckman, Christopher B.; Starke, David W.; Mieyal, John J.

doi:10.1016/0014-5793(95)01066-n

Cited by 41 publications

(39 citation statements)

References 18 publications

(27 reference statements)

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“…Previous studies have shown that GRX contains two redox-active cysteine residues, Thr-Cys 22 -Pro-Tyr-Cys 25 -Arg in an active catalytic center (42). However, unlike TRX, GRX is highly selective for glutathione-containing disulfides, and its catalytic cycle involves a covalent glutathionyl-enzyme disulfide intermediate (GRX-SSG) rather than an intramolecular disulfide at its active site (31).…”

Section: Discussionmentioning

confidence: 99%

Role of Glutaredoxin in Metabolic Oxidative Stress

Song

Rhee

Suntharalingam

et al. 2002

Journal of Biological Chemistry

235

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Section: Discussionmentioning

confidence: 99%

Role of Glutaredoxin in Metabolic Oxidative Stress

Song

Rhee

Suntharalingam

et al. 2002

Journal of Biological Chemistry

235

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“…Glutaredoxins catalyze the cleavage of mixed disulfides in the presence of low concentrations of GSH and may therefore protect cells by reducing any mixed disulfides formed during oxidative stress (Chrestensen et al, 1995). In addition, in vitro experiments indicate that glutaredoxins can reactivate a number of oxidized enzymes by reducing the mixed disulfides formed as a result of thiol oxidation (Terada et al, 1992;Terada, 1994;Yoshitake et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

The YeastSaccharomyces cerevisiaeContains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

Luikenhuis

Perrone

Dawes

et al. 1998

MBoC

218

217

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Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designated GRX1 and GRX2, which share 40 -52% identity and 61-76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae. Strains deleted for both GRX1 and GRX2 were viable but lacked heat-stable oxidoreductase activity using ␤-hydroxyethylene disulfide as a substrate. Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo. Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 and GRX2. In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide. Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains. The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth. Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins. INTRODUCTIONGlutaredoxin from Escherichia coli was first discovered as a small, heat-stable protein required for the glutathione-dependent synthesis of deoxyribonucleotides catalyzed by ribonucleotide reductase (Holmgren, 1976). Glutaredoxin 1 is a 9-kDa protein that acts as a reduced glutathione (GSH)-dependent disulfide oxidoreductase by virtue of the two cysteine residues in its active site (Holmgren and Aslund, 1995). Later studies in mutants that lacked both glutaredoxin and thioredoxin revealed that E. coli actually contains three glutaredoxins (Grx1-3), with glutaredoxin 3 also able to function in ribonucleotide synthesis (Aslund et al., 1994). In contrast, glutaredoxin 2 was proposed to be the first member of a novel class of glutaredoxins that lack activity as hydrogen donors for ribonucleotide reductase (Vlamis-Gardikas et al., 1997). Glutaredoxins have subsequently been identified and isolated from various eukaryotes, including human, bovine, pig, yeast, and rice (Minakuchi et al., 1994;Holmgren and Aslund, 1995). The structure of these proteins has been highly conserved throughout evolution, particularly in the region of the active site (Wells et al., 1993;Holmgren and Aslund, 1995). However, despite extensive structural analysis, little is known regarding the biochemical function of these eukaryotic glutaredoxins in vivo.There appears to be considerable functional overlap between the glutaredoxin and thioredoxin systems. Similar to glutaredoxin, thioredoxin is a small, heatstable pro...

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“…Thioredoxin reductase (bovine and Escherichia coli) was purchased from Sigma and American Diagonstica (Greenwich, CT Enzymes-Recombinant human thioltransferases (wild type and mutant) were expressed and purified from E. coli as described previously (5,27). The thioltransferase enzymes and bovine GSSG reductase were diluted in PGN buffer (100 mM sodium/potassium phosphate, pH 7.5, 0.5 mM GSH, and 0.2 mM NADPH) for inhibition experiments.…”

Section: Methodsmentioning

confidence: 99%

“…Transfection-The hygromycin-resistant, constitutively active expression vectors pRep4 and pRep10 were modified by insertion of thioltransferase cDNA (27) into the multicloning region (identical but reversed in the two plasmids) between the BamHI and HindIII sites to construct sense (pRep 10) and antisense (pRep 4) plasmids. Transfection was accomplished with Lipofectamine using manufacturer's protocols, and cells were photographed at 6 weeks.…”

Section: Methodsmentioning

confidence: 99%

Acute Cadmium Exposure Inactivates Thioltransferase (Glutaredoxin), Inhibits Intracellular Reduction of Protein-glutathionyl-mixed Disulfides, and Initiates Apoptosis

Chrestensen¹,

Starke

Mieyal

2000

Journal of Biological Chemistry

Self Cite

297

248

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Oxidative stress broadly impacts cells, initiating regulatory pathways as well as apoptosis and necrosis. A key molecular event is protein S-glutathionylation, and thioltransferase (glutaredoxin) is a specific and efficient catalyst of protein-SSG reduction. In this study 30-min exposure of H9 and Jurkat cells to cadmium inhibited intracellular protein-SSG reduction, and this correlated with inhibition of the thioltransferase system, consistent with thioltransferase being the primary intracellular catalyst of deglutathionylation. The thioredoxin system contributed very little to total deglutathionylase activity. Thioltransferase and GSSG reductase in situ displayed similar dose-response curves (50% inhibition near 10 M cadmium in extracellular buffer). Acute cadmium exposure also initiated apoptosis, with H9 cells being more sensitive than Jurkat. Moreover, transfection with antisense thioltransferase cDNA was incompatible with cell survival. Collectively, these data suggest that thioltransferase has a vital role in sulfhydryl homeostasis and cell survival. In separate experiments, cadmium inhibited the isolated component enzymes of the thioltransferase and thioredoxin systems, consistent with the vicinal dithiol nature of their active sites: thioltransferase (IC 50 Ϸ 1 M), GSSG reductase (IC 50 Ϸ 1 M), thioredoxin (IC 50 Ϸ 8 M), thioredoxin reductase (IC 50 Ϸ 0.2 M). Disruption of the vicinal dithiol on thioltransferase (via oxidation to C22-SS-C25; or C25S mutation) protected against cadmium, consistent with a dithiol chelation mechanism of inactivation.

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Cloning, expression and characterization of human thioltransferase (glutaredoxin) in E. coli

Cited by 41 publications

References 18 publications

Role of Glutaredoxin in Metabolic Oxidative Stress

Role of Glutaredoxin in Metabolic Oxidative Stress

The YeastSaccharomyces cerevisiaeContains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

Acute Cadmium Exposure Inactivates Thioltransferase (Glutaredoxin), Inhibits Intracellular Reduction of Protein-glutathionyl-mixed Disulfides, and Initiates Apoptosis

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