Malhotra, J. 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


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.

Download full-text


Available from: Hongzhi Miao, Sep 15, 2014
36 Reads
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Journal of Molecular Endocrinology 06/2015; 55(1). DOI:10.1530/JME-15-0132 · 3.08 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Environmental Toxicology 08/2014; DOI:10.1002/tox.22039 · 3.20 Impact Factor
  • Source
    • "In mammalian cells too, glutathione stands to impact the maturation of some secreted or membrane proteins: In a study agnostic of its enzymatic activity, over-expression of wild-type cytosolic ChaC1 in mouse ganglionic eminence cells profoundly inhibited the maturation of the Notch precursor to its furin-cleaved form (Chi et al., 2012). Whereas anti-oxidants that restore impaired glutathione metabolism to normality have been shown to improve the capacity of liver cells to secrete factor VIII, a heavily disulfide bonded serum protein (Malhotra et al., 2008). Thus, conservatively interpreted, our observations lead to the conclusion that ER glutathione is not generally required to maintain protein thiol redox nor folding homeostasis in the ER of cultured mammalian cells. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER. DOI:
    eLife Sciences 07/2014; 3(3):e03421. DOI:10.7554/eLife.03421 · 9.32 Impact Factor
Show more