Hyun Ae Woo

Characterization of Mammalian Sulfiredoxin and Its Reactivation of Hyperoxidized Peroxiredoxin through Reduction of Cysteine Sulfinic Acid in the Active Site to Cysteine

Peroxiredoxins (Prxs) are a family of peroxidases that reduce hydroperoxides. The cysteine residue in the active site of certain eukaryotic Prx enzymes undergoes reversible oxidation to sulfinic acid (Cys-SO2H) during catalysis, and sulfiredoxin (Srx) has been identified as responsible for reversal of the resulting enzyme inactivation in yeast. We have now characterized mammalian orthologs of yeast Srx with an assay based on monitoring of the reduction of sulfinic Prx by immunoblot analysis with antibodies specific for the sulfinic state. Sulfinic reduction by mammalian Srx was found to be a slow process (kcat = 0.18/min) that requires ATP hydrolysis. ATP could be efficiently replaced by GTP, dATP, or dGTP but not by CTP, UTP, dCTP, or dTTP. Both glutathione and thioredoxin are potential physiological electron donors for the Srx reaction, given that their Km values (1.8 mm and 1.2 μm, respectively) are in the range of their intracellular concentrations, and the Vmax values obtained with the two reductants were similar. Although its pKa is relatively low (∼7.3), the active site cysteine of Srx remained reduced even when the active site cysteine of most Prx molecules became oxidized. Finally, depletion of human Srx by RNA interference suggested that Srx is largely responsible for reduction of the Cys-SO2H of Prx in A549 human cells.

Multiple Functions of Peroxiredoxins: Peroxidases, Sensors and Regulators of the Intracellular Messenger H2O2, and Protein Chaperones

Peroxiredoxins (Prxs) are a family of peroxidases that reduce peroxides, with a conserved cysteine residue (the peroxidatic Cys) serving as the site of oxidation by peroxides. Peroxides oxidize the peroxidatic Cys-SH to Cys-SOH, which then reacts with another cysteine residue (typically the resolving Cys [C(R)]) to form a disulfide that is subsequently reduced by an appropriate electron donor. On the basis of the location or absence of the C(R), Prxs are classified into 2-Cys, atypical 2-Cys, and 1-Cys Prx subfamilies. In addition to their peroxidase activity, members of the 2-Cys Prx subfamily appear to serve as peroxide sensors for other proteins and as molecular chaperones. During catalysis, the peroxidatic Cys-SOH of 2-Cys Prxs is occasionally further oxidized to Cys-SO(2)H before disulfide formation, resulting in inactivation of peroxidase activity. This hyperoxidation, which is reversed by the ATP-dependent enzyme sulfiredoxin, modulates the sensor and chaperone functions of 2-Cys Prxs. The peroxidase activity of 2-Cys Prxs is extensively regulated via tyrosine and threonine phosphorylation, which allows modulation of the local concentration of the intracellular messenger H(2)O(2). Finally, 2-Cys Prxs interact with a variety of proteins, with such interaction having been shown to modulate the function of the binding partners in a reciprocal manner.

Reversible Oxidation of the Active Site Cysteine of Peroxiredoxins to Cysteine Sulfinic Acid IMMUNOBLOT DETECTION WITH ANTIBODIES SPECIFIC FOR THE HYPEROXIDIZED CYSTEINE-CONTAINING SEQUENCE

We previously suggested that oxidation of the active site cysteine of peroxiredoxin (Prx) I or Prx II to cysteine sulfinic acid in H2O2-treated cells is reversible (Woo, H. A., Chae, H. Z., Hwang, S. C., Yang, K.-S., Kang, S. W., Kim, K., and Rhee, S. G. (2003) Science 300, 653–656). In contrast, it was recently proposed that sulfinylation of Prx II, but not that of Prx I or Prx III, is reversible (Chevallet, M., Wagner, E., Luche, S., van Dorssealaer, A., Leize-Wagner, E., and Rabilloud, T. (2003) J. Biol. Chem. 278, 37146–37153). The detection of sulfinylated proteins in both of these previous studies relied on complex proteomics analysis. We now describe a simple immunoblot assay for the detection of sulfinylated Prx enzymes that is based on antibodies produced in response to a sulfonylated peptide modeled on the conserved active site sequence. These antibodies recognized both sulfinic and sulfonic forms of Prx equally well and allowed the detection of sulfinylated Prx enzymes in H2O2-treated cells with high sensitivity and specificity. With the use of these antibodies, we demonstrated that not only the cytosolic enzymes Prx I and Prx II but also the mitochondrial enzyme Prx III undergo reversible sulfinylation. The generation of antibodies specific for sulfonylated peptides should provide insight into protein function similar to that achieved with antibodies to peptides containing phosphoserine or phosphothreonine.

Irreversible Oxidation of the Active-site Cysteine of Peroxiredoxin to Cysteine Sulfonic Acid for Enhanced Molecular Chaperone Activity

The thiol (–SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (–SO2H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (–SO3H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H2O2 concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO3H) by mass spectrometry. Tsa1p-SO3H was not an autoxidation product of Tsa1p-SO2H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO3H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO3H multimer lost its peroxidase activity, it gained ∼4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO3H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO3H is a marker of cumulative oxidative stress in cells.

Peroxiredoxin III and sulfiredoxin together protect mice from pyrazole-induced oxidative liver injury.

AIMS To define the mechanisms underlying pyrazole-induced oxidative stress and the protective role of peroxiredoxins (Prxs) and sulfiredoxin (Srx) against such stress. RESULTS Pyrazole increased Srx expression in the liver of mice in a nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent manner and induced Srx translocation from the cytosol to the endoplasmic reticulum (ER) and mitochondria. Pyrazole also induced the expression of CYP2E1, a primary reactive oxygen species (ROS) source for ethanol-induced liver injury, in ER and mitochondria. However, increased CYP2E1 levels only partially accounted for the pyrazole-mediated induction of Srx, prompting the investigation of CYP2E1-independent ROS generation downstream of pyrazole. Indeed, pyrazole increased ER stress, which is known to elevate mitochondrial ROS. In addition, pyrazole up-regulated CYP2E1 to a greater extent in mitochondria than in ER. Accordingly, among Prxs I to IV, PrxIII, which is localized to mitochondria, was preferentially hyperoxidized in the liver of pyrazole-treated mice. Pyrazole-induced oxidative damage to the liver was greater in PrxIII(-/-) mice than in wild-type mice. Such damage was also increased in Srx(-/-) mice treated with pyrazole, underscoring the role of Srx as the guardian of PrxIII. INNOVATION The roles of Prxs, Srx, and ER stress have not been previously studied in relation to pyrazole toxicity. CONCLUSION The concerted action of PrxIII and Srx is important for protection against pyrazole-induced oxidative stress arising from the convergent induction of CYP2E1-derived and ER stress-derived ROS in mitochondria.