Glutathione, a major low molecular weight thiol in mammalian cells, has long been considered to be a major redox buffer in maintaining a reduced state of protein thiols in cells (1
). Protein glutathionylation, greatly elevated under oxidative stress, is considered to be a protective mechanism to prevent protein thiols from being hyperoxidized to their irreversible derivatives, since glutathionylated proteins are readily deglutathionylated in the presence of deglutathionylating enzymes. In recent years, this redox-sensitive, reversible protein covalent modification has emerged as a major regulatory mechanism in cellular regulation and in cell signaling mediated by redox signals known to be generated in response to ligation of various cell surface receptors (3
). In vitro
studies revealed that glutathionylation leads either to inactivation or activation of dozens of enzymes (6
). However, to date, only a few studies have revealed the glutathionylation-dependent changes in enzyme specific activity or protein cytoskeletal function in a physiological context (7
). They include studies of the protein tyrosine phosphatase 1B (5
), ras (11
), mitochondrial complex II (12
), and actin (13
). Nevertheless, given the fact that glutathione is present in cells in the millimolar range and enzymic activity of a given protein can be altered by its glutathionylation, reversible protein glutathionylation has emerged as a major cellular regulatory mechanism.
Peroxiredoxins (Prxs), a family of peroxidases, known to catalyze the removal of H2
and organic hydroperoxides, have the ability to reversibly assemble into a doughnut-shaped homodecamer or even higher-order oligomeric structures (15
). Prx I to Prx IV belong to the 2-Cys Prx enzymes, which reduce H2
through the use of reducing equivalents provided by thioredoxin (Trx). The peroxidative cysteine, Cys52 for Prx I, is located in the NH2
-terminal region. It is selectively oxidized by H2
to the cysteine-sulfenic derivative that in turn reacts with the thiol moiety of Cys173(for Prx I) in the COOH-terminal region of a different subunit to form an intersubunit disulfide. The disulfide is then reduced by Trx. During catalysis, the thiol group of the peroxidative cysteine is occasionally overoxidized to sulfinic acid, resulting in the inactivation of its peroxidase activity (16
). Recently, it was reported that the formation of high molecular weight complexes is favored when the peroxidatic cysteine is in an overoxidized form due to oxidative stress or heat shock stress (17
). The structural change from low molecular weight oligomers to high molecular weight complexes accompanies a functional change from peroxidase to molecular chaperone. The chaperone activity can protect a protein substrate from thermally induced aggregation, resembling the function of heat shock proteins that can also form well-ordered oligomers (17
In addition, the peroxidase activity of the Prx I and Prx II isoforms is known to be regulated by phosphorylation. On one hand, Prx I is phosphorylated by cyclin-dependent kinase Cdk1 at Thr 90 and causes a greater than 80% decrease in its peroxidase activity (18
). On the other hand, Prx II is known to bind Cdk5/p35, which in turn phosphorylates its Thr89 in cells treated with 1-methyl-4-phenylpyridinium ion (MPP+
). Like Prx I, this Thr phosphorylation also diminishes Prx II peroxidase activity. These observations can be attributed to the introduction of a negative charge by phosphorylation at the Thr residue. Moreover, Jang et al.
showed that the mutation of Thr90 to Asp to mimic the phosphorylation status induced a significant increase in chaperone activity and an elevation of the population of high molecular weight oligomers (20
Based on previous studies, the regulation and switching of the dual functions of Prxs appear to be mediated by posttranslational modifications, such as sulfinylation and phosphorylation. Recently, we showed that Prx I can be glutathionylated at three of its four cysteine residues (21
). In this study, we reveal for the first time that glutathionylation can alter the activity a dual-function enzyme, Prx I, mediated through its ability to regulate the oligomeric status of the protein.