Lack of an Efficient Endoplasmic Reticulum-localized Recycling System Protects Peroxiredoxin IV from Hyperoxidation

Typical 2-Cys peroxiredoxins are required to remove hydrogen peroxide from several different cellular compartments. Their activity can be regulated by hyperoxidation and consequent inactivation of the active-site peroxidatic cysteine. Here we developed a simple assay to quantify the hyperoxidation of peroxiredoxins. Hyperoxidation of peroxiredoxins can only occur efficiently in the presence of a recycling system, usually involving thioredoxin and thioredoxin reductase. We demonstrate that there is a marked difference in the sensitivity of the endoplasmic reticulum-localized peroxiredoxin to hyperoxidation compared with either the cytosolic or mitochondrial enzymes. Each enzyme is equally sensitive to hyperoxidation in the presence of a robust recycling system. Our results demonstrate that peroxiredoxin IV recycling in the endoplasmic reticulum is much less efficient than in the cytosol or mitochondria, leading to the protection of peroxiredoxin IV from hyperoxidation.     

Nrf2 Activates Augmenter of Liver Regeneration (ALR) via Antioxidant Response Element and Links Oxidative Stress to Liver Regeneration

Liver regeneration can be impaired by permanent oxidative stress and activation of nuclear factor erythroid 2–related factor 2 (Nrf2), known to regulate the cellular antioxidant response, and has been shown to improve the process of liver regeneration. A variety of factors regulate hepatic tissue regeneration, among them augmenter of liver regeneration (ALR), attained great attention as being survival factors for the liver with proproliferative and antiapoptotic properties. Here we determined the Nrf2/antioxidant response element (ARE) regulated expression of ALR and show ALR as a target gene of Nrf2 in vitro and in vivo. The ALR promoter comprises an ARE binding site and, therefore, ALR expression can be induced by ARE-activator tertiary butylhydroquinone (tBHQ) in hepatoma cells and primary human hepatocytes (PHH). Promoter activity and expression of ALR were enhanced after cotransfection of Nrf2 compared with control and dominant negative mutant of Nrf2. Performing partial hepatectomy in livers from Nrf2^+/+ mice compared with Nrf2^−/− knock-out (KO) mice, we found increased expression of ALR in addition to known antioxidant ARE-regulated genes. Furthermore, we observed increased ALR expression in hepatitis B virus (HBV) compared with hepatitis C virus (HCV) positive hepatoma cells and PHH. Recently, it was demonstrated that HBV infection activates Nrf2 and, now, we add results showing increased ALR expression in liver samples from patients infected with HBV. ALR is regulated by Nrf2, acts as a liver regeneration and antioxidative protein and, therefore, links oxidative stress to hepatic regeneration to ensure survival of damaged cells.     

Serum oxidative stress-induced repression of Nrf2 and GSH depletion: a mechanism potentially involved in endothelial dysfunction of young smokers

Background

Although oxidative stress plays a major role in endothelial dysfunction (ED), the role of glutathione (GSH), of nuclear erythroid-related factor 2 (Nrf2) and of related antioxidant genes (ARE) are yet unknown. In this study we combined an in vivo with an in vitro model to assess whether cigarette smoking affects flow-mediated vasodilation (FMD), GSH concentrations and the Nrf2/ARE pathway in human umbilical vein endothelial cells (HUVECs).

Methods and Results

52 healthy subjects (26 non-smokers and 26 heavy smokers) were enrolled in this study. In smokers we demonstrated increased oxidative stress, i.e., reduced concentrations of GSH and increased concentrations of oxidation products of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) in serum and in peripheral blood mononuclear cells (PBMC), used as in vivo surrogates of endothelial cells. Moreover we showed impairment of FMD in smokers and a positive correlation with the concentration of GSH in PBMC of all subjects. In HUVECs exposed to smokers'' serum but not to non-smokers'' serum we found that oxidative stress increased, whereas nitric oxide and GSH concentrations decreased; interestingly the expression of Nrf2, of heme oxygenase-1 (HO-1) and of glutamate-cysteine ligase catalytic (GCLC) subunit, the rate-limiting step of synthesis of GSH, was decreased. To test the hypothesis that the increased oxidative stress in smokers may have a causal role in the repression of Nrf2/ARE pathway, we exposed HUVECs to increasing concentrations of oxPAPC and found that at the highest concentration (similar to that found in smokers'' serum) the expression of Nrf2/ARE pathway was reduced. The knockdown of Nrf2 was associated to a significant reduction of HO-1 and GCLC expression induced by oxPAPC in ECs.

Conclusions

In young smokers with ED a novel further consequence of increased oxidative stress is a repression of Nrf2/ARE pathway leading to GSH depletion.     

Gualou Guizhi Granule Protects Against Oxidative Injury by Activating Nrf2/ARE Pathway in Rats and PC12 Cells

Stroke involves numerous pathophysiological processes and oxidative stress is considered as a main cellular event in its pathogenesis. The nuclear factor erythroid-2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway plays a key role in inducing phase II detoxifying enzymes and antioxidant proteins and is now considered as a interesting therapeutic target for the treatment of stroke. The objective of this study is to investigate the protective effect of Gualou Guizhi granule (GLGZG) against oxidative stress and explore the protective mechanism of the Nrf2/ARE pathway. In vivo, administration of GLGZG in a rat model of focal cerebral ischemia significantly suppressed oxidative injury by increasing the activity of superoxide dismutase and glutathione level and decreasing reactive oxygen species and malondialdehyde levels. Western blot analysis showed that GLGZG induced nuclear translocation of Nrf2, and combined with real-time PCR results, which indicated that GLGZG up-regulated the Nrf2/ARE pathway. In addition, in cultured PC12 cells, GLGZG protected against H₂O₂ induced oxidative injury and activated the Nrf2/ARE pathway. All the results demonstrated that GLGZG in the management of cerebral ischemia and H₂O₂ induced oxidative injury may be associated with activation of Nrf2/ARE signaling pathway.     

Nrf2及天然产物导向的Nrf2激活剂研究进展

氧化应激能够破坏细胞内氧化还原平衡,造成系统和组织损伤,最终引起一系列疾病的产生.转录因子E2相关因子2(Nrf2),受Kelch样环氧氯丙烷相关蛋白1(Keap1)蛋白的调控,是细胞氧化应激反应中的关键因子,在氧化应激条件下,Nrf2从Keap1中分离,然后进入细胞核与抗氧化反应元件(ARE)结合,增加了Ⅱ相解毒酶的...     

氧化和化学应激的防御性转导通路——Nrf2/ARE

Nrf2/ARE是近年新发现的机体抵抗内外界氧化和化学等刺激的防御性转导通路．生理条件下,NF-E2相关因子2(Nrf2,NF-E2-related factor 2)在细胞质中与Keap1结合处于非活性、易降解的状态.在内外界自由基和化学物质刺激时,Keap1的构象改变或者Nrf2直接被磷酸化,导致Nrf2与Keap1解离而活化．活化的Nrf2进入细胞核,与抗氧化反应元件(ARE)结合,启动ARE下游的Ⅱ相解毒酶、抗氧化蛋白、蛋白酶体/分子伴侣等基因转录和表达以抵抗内外界的有害刺激．MAPK、PI3K/AKT、PKC等信号通路分子广泛参与了Nrf2的活化和核转位过程,但是具体何种通路被激动、何种通路发挥主导作用,取决于刺激物种类、刺激方式和细胞类型．本文就Nrf2分子结构、Nrf2活化机制、Nrf2/ARE调控的下游基因、与Nrf2相关的信号通路分子以及其在肿瘤、炎症、衰老等应用领域的最新进展进行综述．     

Involvement of Nrf2 and Keap1 in the activation of antioxidant responsive element (ARE) by chemopreventive agent peptides from soft-shelled turtle

The antioxidant response element (ARE) is a cis-acting enhancer sequence located in the region containing genes related to antioxidant and detoxification. Under oxidative stress, the induction of nuclear factor-E2-related factor 2 (Nrf2)/ARE is considered as a fundamental process involved in defending reactive oxygen species (ROS) and providing protection against toxic xenobiotics. In this study, we obtained seven antioxidant peptides from soft-shelled turtle and concluded that Glu-Asp-Tyr-Gly-Ala (EDYGA) is the most potent ARE-luciferase inducer. To gain fundamental insights into the role of EDYGA in oxidative stress, we evaluated the effects of EDYGA on the Nrf2/Keap1 system in HepG₂ cells. The results revealed that EDYGA modulated the Nrf2/ARE pathway by enhancing Nrf2 level through the stabilization of Nrf2, which was accomplished by a decrease in the level of Keap1. These actions eventually led to an increase in nuclear Nrf2 accumulation and ARE-binding activity. Moreover, silencing Nrf2 markedly reduced ARE-driven activity induced by EDYGA. Docking results proved that glutamate residues of peptide EDYGA directly bind to Arg 415 of Kelch domain receptor pocke. The results were helpful in understanding the antioxidant activity of peptides from soft-shelled turtle which have potential to be used in foods and drugs as functional ingredients.     

Evidence for the formation of a covalent thiosulfinate intermediate with peroxiredoxin in the catalytic mechanism of sulfiredoxin

The typical 2-Cys peroxiredoxins are thiol-peroxidases involved in the physiology of hydrogen peroxide not only as a toxic but also as a signaling molecule. Coordination of these functions depends on the sulfinylation of the catalytic Cys, a modification reversed by ATP-dependent sulfiredoxin, which specifically reduces the sulfinic acid group of overoxidized 2-Cys peroxiredoxins into a sulfenic acid. Sulfiredoxin was originally proposed to operate by covalent catalysis, with formation of a peroxiredoxin-sulfiredoxin intermediate linked by a thiosulfinate bond between the catalytic Cys of both partners, a hypothesis rejected by a study of the human enzyme. To settle the argument, we investigated the catalytic mechanism of Saccharomyces cerevisiae sulfiredoxin, by the characterization of the nature and kinetics of formation of the protein species formed between sulfiredoxin and its substrate in the presence of ATP, using mutants of the non-essential Cys residues of both proteins. We observed the formation of a dithiothreitol-reducible peroxiredoxin-sulfiredoxin species using SDS-PAGE and Western blot analysis, and its mass was shown to correspond to a thiosulfinate complex by high resolution mass spectrometry coupled to liquid chromatography. We next measured indirectly and directly a rate constant of formation of the thiosulfinate species of approximately 2 min(-1), for both wild-type and mutant sulfiredoxins, at least equal to the steady-state rate constant of the reaction, with a stoichiometry of 1:1 relative to peroxiredoxin. Taken altogether, our results strongly argue in favor of the formation of a covalent thiosulfinate peroxiredoxin-sulfiredoxin species as an intermediate on the catalytic pathway.