Article

Malhotra, J. D.et al. Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. Proc. Natl Acad. Sci. USA105, 18525-18530

Department of Biological Chemistry, Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, MI 48109, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2008; 105(47):18525-30. DOI: 10.1073/pnas.0809677105
Source: PubMed

ABSTRACT

Protein misfolding in the endoplasmic reticulum (ER) contributes to the pathogenesis of many diseases. Although oxidative stress can disrupt protein folding, how protein misfolding and oxidative stress impact each other has not been explored. We have analyzed expression of coagulation factor VIII (FVIII), the protein deficient in hemophilia A, to elucidate the relationship between protein misfolding and oxidative stress. Newly synthesized FVIII misfolds in the ER lumen, activates the unfolded protein response (UPR), causes oxidative stress, and induces apoptosis in vitro and in vivo in mice. Strikingly, antioxidant treatment reduces UPR activation, oxidative stress, and apoptosis, and increases FVIII secretion in vitro and in vivo. The findings indicate that reactive oxygen species are a signal generated by misfolded protein in the ER that cause UPR activation and cell death. Genetic or chemical intervention to reduce reactive oxygen species improves protein folding and cell survival and may provide an avenue to treat and/or prevent diseases of protein misfolding.

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Available from: Hongzhi Miao, Sep 15, 2014
    • "This H 2 O 2 is required for the re-oxidation of the reduced PDI protein by ER-localised glutathione peroxidases (GPx7 and GPx8) and potentially also by peroxiredoxin 4 (Prdx4). On the other hand, the activation of the ER stress pathway, which may lead to apoptotic cell death, has also been associated to the fulminant ROS generation in the ER (Malhotra et al. 2008, Bhandary et al. 2012, Cao & Kaufman 2014). "
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    ABSTRACT: Oxidative folding of nascent proteins in the endoplasmic reticulum (ER), catalysed by one or more members of the protein disulfide isomerase (PDI) family, and the sulfhydryl oxidase ER oxidoreductin 1 (ERO-1) is accompanied by generation of hydrogen peroxide (H2O2). Because of the high rate of insulin biosynthesis and the low expression of H2O2-inactivating enzymes in pancreatic beta cells, it has been proposed that the luminal H2O2 concentration might be very high. As the role of this H2O2 in ER stress and proinsulin processing is still unsolved, an ER-targeted and luminal-active catalase variant, ER-Catalase N244, was expressed in insulin-secreting INS-1E cells. In these cells the influence of ER-specific H2O2 removal on cytokine-mediated cytotoxicity and ER stress, insulin gene expression, insulin content and secretion was analysed. The expression of ER-Catalase N244 reduced the toxicity of exogenously added H2O2 significantly with a threefold increase of the EC50 value for H2O2. However, the expression of cytokine-induced ER stress genes and viability after incubation with beta cell toxic cytokines (IL-1β alone or together with TNF-α +IFN-γ) was not affected by ER-Catalase N244. In control and ER-Catalase N244 expressing cells insulin secretion and proinsulin content was identical, while removal of luminal H2O2 reduced insulin gene expression and insulin content in ER-Catalase N244 expressing cells. These data show that ER-Catalase N244 reduced H2O2 toxicity but did not provide protection against pro-inflammatory cytokine-mediated toxicity and ER stress. Insulin secretion was not affected by decreasing H2O2 in the ER in spite of a reduced insulin transcription and processing.
    No preview · Article · Jun 2015 · Journal of Molecular Endocrinology
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    • "On one hand it seems that luminal H 2 O 2 is reutilized through ER-specific peroxidases such as glutathione peroxidases 7 and 8 (GPx7 and GPx8) and probably also peroxiredoxin 4 (PRDXIV) to maintain or even booster oxidative protein folding [3] [5]. In contrast to these findings an excessive ER-derived H 2 O 2 production due to overwhelming protein processing is associated with the activation of ER-stress pathways leading to apoptotic cell death [6] [7] [8]. These mechanisms have been considered to be of great importance for the development of nonalcoholic steatohepatitis or type 2 diabetes mellitus [9] [10]. "
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    ABSTRACT: Disulfide bond formation during protein folding of nascent proteins is associated with the generation of H2O2 in the endoplasmic reticulum (ER). Approaches to quantify H2O2 directly within the ER failed due to the oxidative environment in the ER lumen and ER-specific catalase expression to detoxify high H2O2 concentrations resulted in an inactive protein due to N-glycosylation. Therefore, the N-glycosylation motives at asparagine-244 and -439 of the human catalase protein were deleted by side-directed mutagenesis. The ER-targeted expression of these variants revealed that only the deletion of the N-glycosylation motive at asparagine-244 (N244) was associated with the maintenance of full enzymatic activity in the ER. Expression of catalase N244 in the ER (ER-Catalase N244) was ER-specific and protected the cells significantly against exogenously added H2O2. With the expression of ER-Catalase N244, a highly effective H2O2 inactivation within the ER was achieved for the first time. Catalase has a high H2O2 inactivation capacity without the need of reducing cofactors, which might interfere with the ER redox homeostasis, and is not involved in protein folding. With these characteristics ER-Catalase N244 is an ideal tool to explore the impact of ER-generated H2O2 for the generation of disulfide bonds or to study the induction of ER-stress pathways through protein folding overload and accumulation of H2O2. Copyright © 2014. Published by Elsevier Inc.
    Full-text · Article · Dec 2014 · Free Radical Biology and Medicine
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    • "differentiated PC12 cells (Kusunoki et al., 2008) and rat testicular sertoli cells (Vermes et al., 1995) exposed to nonylphenol . Additionally, our results (data not shown) and other studies indicated that 2,4-DCP could induce overproduction of ROS (Bukowska et al., 2007; Bors et al., 2011), which might be another inducer to trigger ER stress (Malhotra et al., 2008). Overall, the results of this study demonstrated firstly that 2,4-DCP could induce ER stress contributing to CPs cytotoxicity. "
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    ABSTRACT: 2,4-Dichlorophenol (2,4-DCP) has been widely used to produce herbicides and pharmaceutical intermediates, which exhibits various toxic effects including apoptosis. However, the mechanisms underlying 2,4-DCP-induced apoptosis, especially mediated by endoplasmic reticulum (ER) stress, are still unknown. In the present study, the mouse embryonic fibroblasts (MEFs) were used as an in vitro model system to figure out whether 2,4-DCP could induce ER stress, and further to elucidate the role of ER stress in 2,4-DCP-induced apoptosis. The results showed that 2,4-DCP dramatically caused the decrease of cell viability, the increase of apoptotic cells, the collapse of mitochondrial membrane potential (MMP) and the activation of caspase-3, suggesting that 2,4-DCP did induce apoptosis. Meanwhile, 2,4-DCP acted similarly as ER stress agonist tunicamycin (Tu) to activate all three branches (IRE1α, ATF6 and eIF2α) of ER stress. Furthermore, repression of ER stress or inhibition of eIF2α dephosphorylation significantly alleviated 2,4-DCP-induced apoptosis. Taking these results together, the present study firstly showed that 2,4-DCP induced ER stress-mediated apoptosis via eIF2α dephosphorylation in mammalian cells. These findings will provide new insights into the mechanisms underlying apoptosis after chlorophenols exposure. © 2014 Wiley Periodicals, Inc. Environ Toxicol, 2014.
    Full-text · Article · Aug 2014 · Environmental Toxicology
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