Kanghwa Kim

Mammalian Peroxiredoxin Isoforms Can Reduce Hydrogen Peroxide Generated in Response to Growth Factors and Tumor Necrosis Factor-α

Mammalian tissues express three immunologically distinct peroxiredoxin (Prx) proteins (Prx I, II, and III), which are the products of distinct genes. With the use of recombinant proteins Prx I, II, and III, all have now been shown to possess peroxidase activity and to rely on Trx as a source of reducing equivalents for the reduction of H2O2. Prx I and II are cytosolic proteins, whereas Prx III is localized in mitochondria. Transient overexpression of Prx I or II in cultured cells showed that they were able to eliminate the intracellular H2O2 generated in response to growth factors. Moreover, the activation of nuclear factor κB (NFκB) induced by extracellularly added H2O2 or tumor necrosis factor-α was blocked by overproduction of Prx II. These results suggest that, together with glutathione peroxidase and catalase, Prx enzymes likely play an important role in eliminating peroxides generated during metabolism. In addition, Prx I and II might participate in the signaling cascades of growth factors and tumor necrosis factor-α by regulating the intracellular concentration of H2O2.

Identification of a New Type of Mammalian Peroxiredoxin That Forms an Intramolecular Disulfide as a Reaction Intermediate

Peroxidases of the peroxiredoxin (Prx) family contain a Cys residue that is preceded by a conserved sequence in the NH2-terminal region. A new type of mammalian Prx, designated PrxV, has now been identified as the result of a data base search with this conserved Cys-containing sequence. The 162-amino acid PrxV shares only ∼10% sequence identity with previously identified mammalian Prx enzymes and contains Cys residues at positions 73 and 152 in addition to that (Cys48) corresponding to the conserved Cys. Analysis of mutant human PrxV proteins in which each of these three Cys residues was individually replaced with serine suggested that the sulfhydryl group of Cys48 is the site of oxidation by peroxides and that oxidized Cys48 reacts with the sulfhydryl group of Cys152 to form an intramolecular disulfide linkage. The oxidized intermediate of PrxV is thus distinct from those of other Prx enzymes, which form either an intermolecular disulfide or a sulfenic acid intermediate. The disulfide formed by PrxV is reduced by thioredoxin but not by glutaredoxin or glutathione. Thus, PrxV mutants lacking Cys48 or Cys152 showed no detectable thioredoxin-dependent peroxidase activity, whereas mutation of Cys73 had no effect on activity. Immunoblot analysis revealed that PrxV is widely expressed in rat tissues and cultured mammalian cells and is localized intracellularly to cytosol, mitochondria, and peroxisomes. The peroxidase function of PrxV in vivo was demonstrated by the observations that transient expression of the wild-type protein, but not that of the Cys48 mutant, in NIH 3T3 cells inhibited H2O2 accumulation and activation of c-Jun NH2-terminal kinase induced by tumor necrosis factor-α.

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.

Escherichia coli Periplasmic Thiol Peroxidase Acts as Lipid Hydroperoxide Peroxidase and the Principal Antioxidative Function during Anaerobic Growth

To clarify the enzymatic property of Escherichia coli periplasmic thiol peroxidase (p20), the specific peroxidase activity toward peroxides was compared with other bacterial thiol peroxidases. p20 has the most substrate preference and peroxidase activity toward organic hydroperoxide. Furthermore, p20 exerted the most potent lipid peroxidase activity. Despite that the mutation of p20 caused the highest susceptibility toward organic hydroperoxide and heat stress, the cellular level of p20 did not respond to the exposure of oxidative stress. Expression level of p20 during anaerobic growth was sustained at the ∼50% level compared with that of the aerobic growth. Viability of aerobic p20Δ without glucose was reduced to the ∼65% level of isogenic strains, whereas viability of aerobic p20Δ with 0.5% glucose supplement was sustained. The deletion of p20 resulted in a gradual loss of the cell viability during anaerobic growth. At the stationary phase, the viability of p20Δ was down to ∼10% level of parent strains. An analysis of the protein carbonyl contents of p20Δ as a marker for cellular oxidation indicates that severe reduction of viability of anaerobic p20Δ was caused by cumulative oxidative stress. P20Δ showed hypersensitivity toward membrane-soluble organic hydroperoxides. An analysis of protein carbonyl and lipid hydroperoxide contents in the membrane of the stress-imposed p20Δ demonstrates that the severe reduction of viability was caused by cumulative oxidative stress on the membrane. Taken together, present data uncover in vivo function for p20 as a lipid hydroperoxide peroxidase and demonstrate that, as the result, p20 acts as the principal antioxidant in the anaerobic habitats.

Novel Protective Mechanism against Irreversible Hyperoxidation of Peroxiredoxin Nα-TERMINAL ACETYLATION OF HUMAN PEROXIREDOXIN II

Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (CP), cycles between thiol (CP-SH) and disulfide (–S–S–) states via a sulfenic (CP-SOH) intermediate. Hyperoxidation of the CP thiol to its sulfinic (CP-SO2H) derivative has been shown to be reversible, but its sulfonic (CP-SO3H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with CP-SO3H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that Nα-terminal acetylation (Nα-Ac) occurred exclusively on Prx II after demethionylation. Nα-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-Nα-acetylated and Nα-terminal acetylated Prx II revealed that Nα-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of Tm from 59.6 to 70.9 °C. These findings suggest that the structural maintenance of Prx II by Nα-Ac may be responsible for preventing its hyperoxidation to form CP-SO3H.

[50] Protein that prevents mercaptan-mediated protein oxidation

Publisher Summary This chapter discusses protein that prevents mercaptan-mediated protein oxidation. Several enzymes have been shown to lose catalytic activity on incubation in air with mercaptans such as dithiothreitol (DTT) and 2-mercaptoethanol and trace amounts of iron (or copper) salts. At present, proteins that are inactivated by a mercaptan-Fe 3+ -O 2 mixed-function oxidase (MFO) system include glutamine-dependent carbamoyl-phosphate synthase from Escherichia coli , 2 rhodanase from bovine liver, phosphoenolpyruvate carboxykinase from rat liver, glutamine synthetase (glutamate-ammonia ligase) from E. coli and yeast, adenylyltransferase from E. coli , pyruvate kinase and enolase from rabbit muscle, and low density lipoprotein. The mercaptan-dependent MFO inactivation is accompanied by the oxidation of unidentified amino acids as evidenced by the increase of reactive carbonyl moieties on the protein, fragmentation, and formation of higher molecular weight aggregates through radical mediated cross-linking reactions. Studies show that the autoxidation of mercaptans in the presence of iron (or copper) generates reactive oxygen species such as superoxide anion, hydrogen peroxide, and hydroxyl radical, as well as thioyl radical. Thus, Fe 3+ or Cu 2+ initiates mercaptan oxidation to produce thioyl radical as in reaction. The resulting reduced metal ion can be utilized to produce superoxide. The superoxide can undergo the dismutation reaction to form H 2 O 2 . Hydrogen peroxide in turn can react with reduced metal ion via the Fenton reaction, generating hydroxyl radical.