No abstract

It is widely accepted that reactive oxygen species (ROS) promote tumorigenesis. However, the exact mechanisms are still unclear. As mice lacking the peroxidase peroxiredoxin1 (Prdx1) produce more cellular ROS and die prematurely of cancer, they offer an ideal model system to study ROS-induced tumorigenesis. Prdx1 ablation increased the susceptibility to Ras-induced breast cancer. We, therefore, investigated the role of Prdx1 in regulating oncogenic Ras effector pathways. We found Akt hyperactive in fibroblasts and mammary epithelial cells lacking Prdx1. Investigating the nature of such elevated Akt activation established a novel role for Prdx1 as a safeguard for the lipid phosphatase activity of PTEN, which is essential for its tumour suppressive function. We found binding of the peroxidase Prdx1 to PTEN essential for protecting PTEN from oxidation-induced inactivation. Along those lines, Prdx1 tumour suppression of Ras-or ErbB-2-induced transformation was mediated mainly via PTEN.

Post-translational S-glutathionylation occurs through the reversible addition of a proximal donor of glutathione to thiolate anions of cysteines in target proteins, where the modification alters molecular mass, charge, and structure/function and/or prevents degradation from sulfhydryl overoxidation or proteolysis. Catalysis of both the forward (glutathione S-transferase P) and reverse (glutaredoxin) reactions creates a functional cycle that can also regulate certain protein functional clusters, including those involved in redox-dependent cell signaling events. For translational application, S-glutathionylated serum proteins may be useful as biomarkers in individuals (who may also have polymorphic expression of glutathione S-transferase P) exposed to agents that cause oxidative or nitrosative stress.S-Glutathionylation targets cysteines in a basic environment (low pK a ) perhaps in close three-dimensional proximity to Arg, His, or Lys residues. Reports of reversible S-glutathionylation emerged as early as 1985 (1), but in concert with understanding the importance of reactive oxygen/nitrogen species (ROS/ RNS) 2 as second messengers, publications have increased substantially over the past 10 years. ROS/RNS signaling operates through a set of post-translational protein modifications that are discrete, site-specific, and reversible. Certain proteins undergo reversible chemical changes in response to altered localized redox potential. Among the most susceptible redoxsensitive targets are thiol (-SH) groups on cysteines. Signaling events are facilitated through redox-active proteins when one or more cysteines can exist as reactive thiolate anions. These cysteines are more nucleophilic and become susceptible to attack by GSH.Oxygen-based metabolism is arguably the most efficient and evolved method for producing energy from nutrients, but ROS

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...

This study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity with sn-2-linolenoyl-or sn-2-arachidonoylphosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH. Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM ؊1 min ؊1 ) were k 1 ‫؍‬ 1-3 ؋ 10 5 and k 2 ‫؍‬ 4 -11 ؋ 10 4 . In the presence of 0.36 mM GSH, apparent K m was 120 -130 M and apparent V max was 1.5-1.6 mol/min/mg of protein. Assays with H 2 O 2 and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides. Maximal enzymatic activity was at pH 7-8. Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K i 4 M. The enzyme had no GSH S-transferase activity. Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases. The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da. These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides. Thus, this enzyme may be an important component of cellular antioxidant defense systems.