Limits...
Glutathionylation of beta-actin via a cysteinyl sulfenic acid intermediary.

Johansson M, Lundberg M - BMC Biochem. (2007)

Bottom Line: Glutathionylation of beta-actin by GSSG is likely to be mediated by a thiol-exchange mechanism whereas glutathionylation by GSH requires thiol oxidation. beta-actin glutathionylation by GSH was inhibited by arsenite and dimedone suggesting that the mechanism involved formation of a cysteinyl sulfenic acid residue in beta-actin.We conclude that glutathionylation of beta-actin may occur via spontaneous oxidation of a cysteinyl residue to a sulfenic acid that readily reacts with GSH to form a mixed disulfide.We also show that the reactivity and oxidation to a reactive protein thiol intermediary differ between different actin isoforms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Karolinska Institute, Department of Laboratory Medicine, Division for Clinical Microbiology, Karolinska University Hospital at Huddinge, S-141 86 Stockholm, Sweden. magnus.johansson@ki.se

ABSTRACT

Background: Cysteinyl residues in actin are glutathionylated, ie. form a mixed disulfide with glutathione, even in the absence of exogenous oxidative stress. Glutathionylation inhibits actin polymerization and reversible actin glutathionylation is a redox dependent mechanism for regulation of the cytoskeleton structure. The molecular mechanism that mediates actin glutathionylation in vivo is unclear.

Results: We have studied glutathionylation of alpha- and beta-actin in vitro using an enzyme-linked immunosorbant assay with a monoclonal anti-glutathione antibody. alpha- and beta-actin were both glutathionylated when incubated with reduced glutathione (GSH) combined with diamide as a thiol oxidant. However, beta-actin was also glutathionylated by both glutathione disulfide (GSSG) and GSH in the absence of diamide whereas alpha-actin was poorly glutathionylated by GSH or GSSG. Glutathionylation of beta-actin by GSSG is likely to be mediated by a thiol-exchange mechanism whereas glutathionylation by GSH requires thiol oxidation. beta-actin glutathionylation by GSH was inhibited by arsenite and dimedone suggesting that the mechanism involved formation of a cysteinyl sulfenic acid residue in beta-actin.

Conclusion: We conclude that glutathionylation of beta-actin may occur via spontaneous oxidation of a cysteinyl residue to a sulfenic acid that readily reacts with GSH to form a mixed disulfide. We also show that the reactivity and oxidation to a reactive protein thiol intermediary differ between different actin isoforms.

Show MeSH

Related in: MedlinePlus

Glutathionylation and deglutathionylation of β-actin. Glutathionylation of actins by different concentrations of GSH and GSSG (A). Deglutathionylation of β-actin catalyzed by Grx1 (B)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2228301&req=5

Figure 4: Glutathionylation and deglutathionylation of β-actin. Glutathionylation of actins by different concentrations of GSH and GSSG (A). Deglutathionylation of β-actin catalyzed by Grx1 (B)

Mentions: Glutathionylation of β-actin by GSH requires a thiol oxidation. However, we could not exclude that the GSH used in the experiments had undergone partial oxidation to GSSG. If GSSG was present in the samples, one possible mechanism for β-actin glutathionylation was direct disulfide exchange between GSSG and actin thiols. However, we tested the concentrations of GSH and GSSG required to induce glutathionylation of β-actin (Figure 4A). These experiments showed that a lower concentration of GSH compared to GSSG resulted in glutathionylation of the protein. We also tested the effect of adding Grx1 to the reaction after 15 min incubation with GSH (Figure 4B). Addition of Grx1 resulted in reversal of the reaction and deglutathionylation of β-actin. These experiments excluded the possibility that glutathionylation of β-actin occurred by thiol-disulfide exchange with GSSG.


Glutathionylation of beta-actin via a cysteinyl sulfenic acid intermediary.

Johansson M, Lundberg M - BMC Biochem. (2007)

Glutathionylation and deglutathionylation of β-actin. Glutathionylation of actins by different concentrations of GSH and GSSG (A). Deglutathionylation of β-actin catalyzed by Grx1 (B)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2228301&req=5

Figure 4: Glutathionylation and deglutathionylation of β-actin. Glutathionylation of actins by different concentrations of GSH and GSSG (A). Deglutathionylation of β-actin catalyzed by Grx1 (B)
Mentions: Glutathionylation of β-actin by GSH requires a thiol oxidation. However, we could not exclude that the GSH used in the experiments had undergone partial oxidation to GSSG. If GSSG was present in the samples, one possible mechanism for β-actin glutathionylation was direct disulfide exchange between GSSG and actin thiols. However, we tested the concentrations of GSH and GSSG required to induce glutathionylation of β-actin (Figure 4A). These experiments showed that a lower concentration of GSH compared to GSSG resulted in glutathionylation of the protein. We also tested the effect of adding Grx1 to the reaction after 15 min incubation with GSH (Figure 4B). Addition of Grx1 resulted in reversal of the reaction and deglutathionylation of β-actin. These experiments excluded the possibility that glutathionylation of β-actin occurred by thiol-disulfide exchange with GSSG.

Bottom Line: Glutathionylation of beta-actin by GSSG is likely to be mediated by a thiol-exchange mechanism whereas glutathionylation by GSH requires thiol oxidation. beta-actin glutathionylation by GSH was inhibited by arsenite and dimedone suggesting that the mechanism involved formation of a cysteinyl sulfenic acid residue in beta-actin.We conclude that glutathionylation of beta-actin may occur via spontaneous oxidation of a cysteinyl residue to a sulfenic acid that readily reacts with GSH to form a mixed disulfide.We also show that the reactivity and oxidation to a reactive protein thiol intermediary differ between different actin isoforms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Karolinska Institute, Department of Laboratory Medicine, Division for Clinical Microbiology, Karolinska University Hospital at Huddinge, S-141 86 Stockholm, Sweden. magnus.johansson@ki.se

ABSTRACT

Background: Cysteinyl residues in actin are glutathionylated, ie. form a mixed disulfide with glutathione, even in the absence of exogenous oxidative stress. Glutathionylation inhibits actin polymerization and reversible actin glutathionylation is a redox dependent mechanism for regulation of the cytoskeleton structure. The molecular mechanism that mediates actin glutathionylation in vivo is unclear.

Results: We have studied glutathionylation of alpha- and beta-actin in vitro using an enzyme-linked immunosorbant assay with a monoclonal anti-glutathione antibody. alpha- and beta-actin were both glutathionylated when incubated with reduced glutathione (GSH) combined with diamide as a thiol oxidant. However, beta-actin was also glutathionylated by both glutathione disulfide (GSSG) and GSH in the absence of diamide whereas alpha-actin was poorly glutathionylated by GSH or GSSG. Glutathionylation of beta-actin by GSSG is likely to be mediated by a thiol-exchange mechanism whereas glutathionylation by GSH requires thiol oxidation. beta-actin glutathionylation by GSH was inhibited by arsenite and dimedone suggesting that the mechanism involved formation of a cysteinyl sulfenic acid residue in beta-actin.

Conclusion: We conclude that glutathionylation of beta-actin may occur via spontaneous oxidation of a cysteinyl residue to a sulfenic acid that readily reacts with GSH to form a mixed disulfide. We also show that the reactivity and oxidation to a reactive protein thiol intermediary differ between different actin isoforms.

Show MeSH
Related in: MedlinePlus