A Novel Role for Human Sulfiredoxin in the Reversal of Glutathionylation

Abstract

Modification of protein cysteine residues by disulfide formation with glutathione (glutathionylation) is a reversible posttranslational modification of critical importance in controlling cell signaling events following oxidative and/or nitrosative stress. Here, we show that human sulfiredoxin, a small redox protein conserved in eukaryotes, can act as a novel regulator of the redox-activated thiol switch in cells by catalyzing deglutathionylation of a number of distinct proteins in response to oxidative and/or nitrosative stress. Actin and protein tyrosine phosphatase 1B were identified in vitro as targets of sulfiredoxin 1 (Srx1)-dependent deglutathionylation and confirmed in vivo by two-dimensional gel electrophoresis analysis. In addition, we show that Srx1-dependent deglutathionylation is functionally relevant through restoration of phosphatase activity. Human sulfiredoxin contains one cysteine residue (Cys 99 ) that is conserved in all family members. Mutation of the cysteine residue inhibits deglutathionylation but did not affect its capacity to bind intracellular proteins. Furthermore, sulfiredoxin is not an acceptor molecule for the GS À moiety during the reaction process. Using two-dimensional gel electrophoresis, we identified multiple protein targets in vivo that are deglutathionylated by sulfiredoxin following oxidative and/or nitrosative stress. This novel deglutathionylation function of sulfiredoxin suggests it has a central role in redox control with potential implications in cell signaling.

The Peroxiredoxin Repair Proteins

“…HEK293 cells stably transfected with the Srx gene show a significant reduction in the levels of protein glutathionylation after treatment with PABA/NO, a potent inducer of oxidative stress via the release of nitric oxide (Findlay et al, 2006). In this study the level of glutathionylation was determined by Western blot analysis using an antibody specific for the protein-S-S-G linkage.…”

“…It will also be important to assess whether this activity has the same cofactor requirements (ATP and Mg 2+ ) as the sulfinic acid reductase activity. These initial studies suggest that Srx has therapeutic potential for a variety of diseases with aberrant protein glutathionylation Findlay et al, 2006).…”

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…However for mammalian Grx, substrate specificity towards Sglutathionylated proteins has been demonstrated [101]. It is of interest to note that sulfiredoxin also was recently demonstrated to de-glutathionylate protein targets such as actin and protein tyrosine phosphatase 1B [102]. Multiple Grx genes are known in plants and in pro-and eukaryotes; in mammalian cells, Grx1 has been described and localized in the cytoplasm.…”

Protein cysteine sulfinic acid reductase (sulfiredoxin) as a regulator of cell proliferation and drug response

“…Forward and reverse reaction kinetics frequently mimic those for protein phosphorylation and dephosphorylation. Recently, reversible reduction of protein cysteine sulfinic acid to sulfenic acid and removal of S-glutathionylation (deglutathionylation) from low pKa cysteine residues was shown to be mediated by sulfiredoxin (Srx; Biteau et al, 2003;Findlay et al, 2005Findlay et al, , 2006. Both nucleotide and amino acid sequences of Srx show high levels of conservation across species, and even phyla (Biteau et al, 2003;Basu and Koonin, 2005).…”