Activation of the antioxidant enzyme 1-CYS peroxiredoxin requires glutathionylation mediated by heterodimerization with πGST

Abstract

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, can protect cells against membrane oxidation through glutathione (GSH)-dependent reduction of phospholipid hydroperoxides to corresponding alcohols. However, purified native or recombinant enzyme in vitro generally lacks GSH peroxidase (GPx) activity because of oxidation of its single conserved cysteine. Reduction of the resultant oxidized cysteine is difficult because of its protected location within the homodimer formed by the oxidized protein monomers. Partial purification of 1-cysPrx from bovine lung revealed the presence of GST in an active preparation, while purification to homogeneity yielded enzyme that inactivated with time. We show that heterodimerization of 1-cysPrx with GSH-saturated GST results in glutathionylation of the oxidized cysteine in 1-cysPrx followed by subsequent spontaneous reduction of the mixed disulfide and restoration of enzymatic activity. Maximum activation of 1-cysPrx occurred with a 1:1 molar ratio of GSH-saturated GST and a 2:1 molar ratio of GSH to 1-cysPrx. Liposome-mediated delivery of oxidized recombinant enzyme into NCI-H441 cells that lack 1-cysPrx but express GST resulted in 1-cysPrx activation, whereas activation in MCF7 cells required co-delivery of GST. Our data indicate a physiological mechanism for glutathionylation of the oxidized catalytic cysteine of 1-cysPrx by its heterodimerization with GST followed by its GSH-mediated reduction and enzyme activation. P eroxiredoxins are a superfamily of nonheme and nonselenium peroxidases that are widely distributed throughout all phyla (1-4). Of the six mammalian peroxiredoxins, five (Prx I-V) contain two conserved cysteines that participate in intramolecular disulfide͞sulfhydryl redox cycling with thioredoxin resulting in reduction of H 2 O 2 and organic hydroperoxides into corresponding alcohols (2). By contrast, 1-cys peroxiredoxin (1-cysPrx or Prx VI) † has a single conserved cysteine (5) and does not use thioredoxin as reductant (5, 6). This peroxiredoxin is expressed in all tissues but at particularly high levels in brain, eye, testes, and lung (2,7,8). Expression of 1-cysPrx protein or mRNA is decreased in a mouse that is susceptible to experimental atherosclerosis (9) and is elevated in brains of patients with Parkinsonian dementia (10), sporadic Creutzfeldt-Jacob disease (11), and Pick disease (12), in lungs from newborns (13), in malignant mesothelioma (14), in the healing edge of skin wounds (15), and in experimental cellular premature senescence (16). 1-cysPrx can reduce phospholipid and other hydroperoxides (6) and protects against cellular membrane damage (17, 18). This enzyme has phospholipase A 2 activity (19), participates in the activation of neutrophil NADPH oxidase through its interaction with p67 phox (20), and prevent methemoglobin formation in erythrocyte hemolysates (21).1-cysPrx catalysis results in the peroxide-mediated oxidation of its Cys-47 to sulfenic acid as deduced from 1-cysPrx crystallization (22). Reduction of the sul...

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

“…Importantly, mouse embryo fibroblasts from GSTp-null mice exhibited a high basal level of JNK activity, [189] and GSTp-null mice showed a phenotype of increased myeloproliferation, in association with activation of JNK and the JAK/STAT pathway [190], corroborating the functional significance of GSTp in the regulation of JNK, and potential other pathways in vivo. GSTp also is an important regulator of peroxiredoxin 6 (1-cysPrx), via a mechanism that involves heterodimerization of the oxidized catalytic cysteine of 1-cysPrx with pi GST followed by its GSH-mediated reduction and enzyme activation [191].…”

Peroxiredoxin Functions as a Peroxidase and a Regulator and Sensor of Local Peroxides

“…Major differences from other Prxs include the use of GSH instead of Trx as the physiological reductant, heterodimerization with Pi GST as part of the catalytic cycle, and the ability to hydrolyze phospholipids as a result of Ca 2ϩ -independent phospholipase A 2 (PLA 2 ) activity (35,36,(75)(76)(77). Prx VI is thus a bifunctional protein and has separate active site residues for peroxidase (Cys 47 , the C P residue) and PLA 2 (Ser 32 ) activities.…”

Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

“…Author Manuscript weight of 23,500, is of particular interest because it exhibits diverse roles in mammalian cells: it provides a defense against carcinogenesis, since it catalyzes the inactivation of known carcinogens [3]; it contributes significantly to the development of resistance to cancer chemotherapy, since GST pi levels increase in tumors and the enzyme metabolizes key anticancer drugs [4][5][6][7]; and it promotes the cellular response to oxidative stress, since GST pi has recently been reported to activate the anti-oxidant enzyme 1-Cys peroxiredoxin [8,9].…”

“…Complex production in the presence of GSH is followed by glutathionylation of 1-Cys Prx, and subsequently by the slow formation of an intermolecular disulfide between 1-Cys Prx and GST pi [9]. The disulfide is then reduced by GSH, regenerating an active 1-Cys Prx [8,9]. A plausible energy-minimized model of the complex was proposed; this 'in silico' model was constructed by docking one subunit from the crystal structure of GST pi with one subunit from the crystal structure of 1-Cys Prx using the program ZDOCKpro 1.0.…”