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Thioredoxins function as deglutathionylase enzymes in the yeast Saccharomyces cerevisiae.

Greetham D, Vickerstaff J, Shenton D, Perrone GG, Dawes IW, Grant CM - BMC Biochem. (2010)

Bottom Line: Glutaredoxins and thioredoxins are key oxidoreductases which have been implicated in regulating glutathionylation/deglutathionylation in diverse organisms.We have confirmed that thioredoxins, but not glutaredoxins, catalyse deglutathionylation of model glutathionylated substrates using purified thioredoxin and glutaredoxin proteins.Furthermore, we show that the deglutathionylase activity of thioredoxins is required to reduce the high levels of glutathionylation in stationary phase cells, which occurs as cells exit stationary phase and resume vegetative growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: The University of Manchester, Faculty of Life Sciences, Manchester M13 9PT, UK.

ABSTRACT

Background: Protein-SH groups are amongst the most easily oxidized residues in proteins, but irreversible oxidation can be prevented by protein glutathionylation, in which protein-SH groups form mixed disulphides with glutathione. Glutaredoxins and thioredoxins are key oxidoreductases which have been implicated in regulating glutathionylation/deglutathionylation in diverse organisms. Glutaredoxins have been proposed to be the predominant deglutathionylase enzymes in many plant and mammalian species, whereas, thioredoxins have generally been thought to be relatively inefficient in deglutathionylation.

Results: We show here that the levels of glutathionylated proteins in yeast are regulated in parallel with the growth cycle, and are maximal during stationary phase growth. This increase in glutathionylation is not a response to increased reactive oxygen species generated from the shift to respiratory metabolism, but appears to be a general response to starvation conditions. Our data indicate that glutathionylation levels are constitutively high in all growth phases in thioredoxin mutants and are unaffected in glutaredoxin mutants. We have confirmed that thioredoxins, but not glutaredoxins, catalyse deglutathionylation of model glutathionylated substrates using purified thioredoxin and glutaredoxin proteins. Furthermore, we show that the deglutathionylase activity of thioredoxins is required to reduce the high levels of glutathionylation in stationary phase cells, which occurs as cells exit stationary phase and resume vegetative growth.

Conclusions: There is increasing evidence that the thioredoxin and glutathione redox systems have overlapping functions and these present data indicate that the thioredoxin system plays a key role in regulating the modification of proteins by the glutathione system.

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Overexpression of the thioredoxin system reduces glutathionylation. The wild-type strain containing pRS426 (v), thioredoxin system (Trx1 and Trr1) or glutaredoxin system (Grx1 and Glr1) was grown to exponential phase in minimal SD media. Reduced (GSH), oxidized (GSSG) and protein bound (GSSP) glutathione levels were determined. Values shown are the means of at least three independent determinations and are given in nmol/ml/A600.
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Figure 5: Overexpression of the thioredoxin system reduces glutathionylation. The wild-type strain containing pRS426 (v), thioredoxin system (Trx1 and Trr1) or glutaredoxin system (Grx1 and Glr1) was grown to exponential phase in minimal SD media. Reduced (GSH), oxidized (GSSG) and protein bound (GSSP) glutathione levels were determined. Values shown are the means of at least three independent determinations and are given in nmol/ml/A600.

Mentions: In a further effort to define the role of glutaredoxins and thioredoxins in protein glutathionylation, we examined whether their overexpression alters GSSP levels (Fig. 5). Multi-copy vectors were used to overexpress the thioredoxin (TRX1, TRR1) or glutaredoxin (GRX1, GLR1) systems. We have previously used these vectors to confirm that overexpression of GRX1 and TRX1 confers resistance to hydrogen peroxide [17]. Overexpression of the thioredoxin system did not significantly affect the levels of oxidized (GSSG) or reduced (GSH) glutathione (Fig. 5). In agreement with the idea that thioredoxins maintain low exponential phase GSSP levels, overexpression of the thioredoxin system was found to decreases GSSP levels by approximately 50%. In contrast, overexpression of the glutaredoxin system resulted in an approximate 66% increases in GSSP levels, confirming that glutaredoxins do not act as general deglutathionylases in vivo. GSH levels were unaffected, whereas, GSSG levels were significantly lowered by the glutaredoxin system presumably due to the activity of Glr1. These data further confirm that there does not appear to be any strong correlation between cellular GSSP and GSSG levels.


Thioredoxins function as deglutathionylase enzymes in the yeast Saccharomyces cerevisiae.

Greetham D, Vickerstaff J, Shenton D, Perrone GG, Dawes IW, Grant CM - BMC Biochem. (2010)

Overexpression of the thioredoxin system reduces glutathionylation. The wild-type strain containing pRS426 (v), thioredoxin system (Trx1 and Trr1) or glutaredoxin system (Grx1 and Glr1) was grown to exponential phase in minimal SD media. Reduced (GSH), oxidized (GSSG) and protein bound (GSSP) glutathione levels were determined. Values shown are the means of at least three independent determinations and are given in nmol/ml/A600.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2836980&req=5

Figure 5: Overexpression of the thioredoxin system reduces glutathionylation. The wild-type strain containing pRS426 (v), thioredoxin system (Trx1 and Trr1) or glutaredoxin system (Grx1 and Glr1) was grown to exponential phase in minimal SD media. Reduced (GSH), oxidized (GSSG) and protein bound (GSSP) glutathione levels were determined. Values shown are the means of at least three independent determinations and are given in nmol/ml/A600.
Mentions: In a further effort to define the role of glutaredoxins and thioredoxins in protein glutathionylation, we examined whether their overexpression alters GSSP levels (Fig. 5). Multi-copy vectors were used to overexpress the thioredoxin (TRX1, TRR1) or glutaredoxin (GRX1, GLR1) systems. We have previously used these vectors to confirm that overexpression of GRX1 and TRX1 confers resistance to hydrogen peroxide [17]. Overexpression of the thioredoxin system did not significantly affect the levels of oxidized (GSSG) or reduced (GSH) glutathione (Fig. 5). In agreement with the idea that thioredoxins maintain low exponential phase GSSP levels, overexpression of the thioredoxin system was found to decreases GSSP levels by approximately 50%. In contrast, overexpression of the glutaredoxin system resulted in an approximate 66% increases in GSSP levels, confirming that glutaredoxins do not act as general deglutathionylases in vivo. GSH levels were unaffected, whereas, GSSG levels were significantly lowered by the glutaredoxin system presumably due to the activity of Glr1. These data further confirm that there does not appear to be any strong correlation between cellular GSSP and GSSG levels.

Bottom Line: Glutaredoxins and thioredoxins are key oxidoreductases which have been implicated in regulating glutathionylation/deglutathionylation in diverse organisms.We have confirmed that thioredoxins, but not glutaredoxins, catalyse deglutathionylation of model glutathionylated substrates using purified thioredoxin and glutaredoxin proteins.Furthermore, we show that the deglutathionylase activity of thioredoxins is required to reduce the high levels of glutathionylation in stationary phase cells, which occurs as cells exit stationary phase and resume vegetative growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: The University of Manchester, Faculty of Life Sciences, Manchester M13 9PT, UK.

ABSTRACT

Background: Protein-SH groups are amongst the most easily oxidized residues in proteins, but irreversible oxidation can be prevented by protein glutathionylation, in which protein-SH groups form mixed disulphides with glutathione. Glutaredoxins and thioredoxins are key oxidoreductases which have been implicated in regulating glutathionylation/deglutathionylation in diverse organisms. Glutaredoxins have been proposed to be the predominant deglutathionylase enzymes in many plant and mammalian species, whereas, thioredoxins have generally been thought to be relatively inefficient in deglutathionylation.

Results: We show here that the levels of glutathionylated proteins in yeast are regulated in parallel with the growth cycle, and are maximal during stationary phase growth. This increase in glutathionylation is not a response to increased reactive oxygen species generated from the shift to respiratory metabolism, but appears to be a general response to starvation conditions. Our data indicate that glutathionylation levels are constitutively high in all growth phases in thioredoxin mutants and are unaffected in glutaredoxin mutants. We have confirmed that thioredoxins, but not glutaredoxins, catalyse deglutathionylation of model glutathionylated substrates using purified thioredoxin and glutaredoxin proteins. Furthermore, we show that the deglutathionylase activity of thioredoxins is required to reduce the high levels of glutathionylation in stationary phase cells, which occurs as cells exit stationary phase and resume vegetative growth.

Conclusions: There is increasing evidence that the thioredoxin and glutathione redox systems have overlapping functions and these present data indicate that the thioredoxin system plays a key role in regulating the modification of proteins by the glutathione system.

Show MeSH
Related in: MedlinePlus