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      Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress

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          Abstract

          Oxidative protein folding in the endoplasmic reticulum (ER) has emerged as a potentially significant source of cellular reactive oxygen species (ROS). Recent studies suggest that levels of ROS generated as a byproduct of oxidative folding rival those produced by mitochondrial respiration. Mechanisms that protect cells against oxidant accumulation within the ER have begun to be elucidated yet many questions still remain regarding how cells prevent oxidant-induced damage from ER folding events. Here we report a new role for a central well-characterized player in ER homeostasis as a direct sensor of ER redox imbalance. Specifically we show that a conserved cysteine in the lumenal chaperone BiP is susceptible to oxidation by peroxide, and we demonstrate that oxidation of this conserved cysteine disrupts BiP's ATPase cycle. We propose that alteration of BiP activity upon oxidation helps cells cope with disruption to oxidative folding within the ER during oxidative stress.

          DOI: http://dx.doi.org/10.7554/eLife.03496.001

          eLife digest

          The endoplasmic reticulum is the cellular compartment where approximately one third of the cell's proteins are made. Inside, chaperone molecules bind to newly made protein chains and help them to fold into the three-dimensional structure required for the protein to work correctly. A chaperone called Ero1 helps to facilitate this folding process by catalyzing a reaction that forms strong chemical bonds, which help stabilize the final protein structures. However, this help from Ero1 comes at a cost: forming a stabilizing bond this way also produces a peroxide molecule as a byproduct.

          Peroxide is a ‘reactive oxygen species’: a chemical that can oxidize and damage proteins and DNA, which can potentially kill the cell. Three other enzymes in the endoplasmic reticulum can convert peroxide into water, to protect the cells from reactive oxygen species build-up. However, not all cells that use Ero1 have these other enzymes, suggesting that other pathways must exist to manage reactive oxygen species.

          Wang et al. took advantage of yeast cells containing a hyperactive mutant version of the Ero1 enzyme to look for alternative detoxifying mechanisms that occur when the cell is stressed by an excess of reactive oxygen species. In these cells, Wang et al. observed that the high levels of reactive oxygen species caused part of a chaperone molecule called BiP to oxidize. This modification of BiP acts like a switch that the reactive oxygen species flip on. When activated by the reactive oxygen species, BiP enhances its activity as a folding molecular chaperone, keeping proteins apart. This is thought to allow BiP to minimize the protein misfolding that may otherwise occur in the wake of the damage caused by the building levels of peroxide. Wang et al. created a mutant BiP chaperone that mimics the oxidized form, and found that it also protects cells from the damage inflicted by the excess of reactive oxygen species.

          Wang et al. propose that the BiP chaperone may be an important sensor of reactive oxygen species that changes its activity when these harmful chemicals are present and helps to protect the cell from damage. The success in mimicking the protective effects of oxidized BiP with a mutant BiP suggest that in the future one may be able to design small molecule drugs that bind to BiP to produce the activity of the modified form.

          DOI: http://dx.doi.org/10.7554/eLife.03496.002

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          Most cited references46

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          Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae.

          Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain. Copyright 1999 John Wiley & Sons, Ltd.
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            Protein folding in the cell.

            In the cell, as in vitro, the final conformation of a protein is determined by its amino-acid sequence. But whereas some isolated proteins can be denatured and refolded in vitro in the absence of other macromolecular cellular components, folding and assembly of polypeptides in vivo involves other proteins, many of which belong to families that have been highly conserved during evolution.
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              The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins.

              Two glucose-regulated proteins, GRP78 and GRP94, are major constituents of the endoplasmic reticulum (ER) of mammalian cells. These proteins are synthesized constitutively in detectable amounts under normal growth conditions; they can also be induced under a variety of conditions of stress including glucose starvation and treatment with drugs that inhibit cellular glycosylation, with calcium ionophores or with amino-acid analogues. Unlike the closely-related heat shock protein (HSP) family, the GRPs are not induced significantly by high temperature. Recently, GRP78 has been identified as the immunoglobulin heavy chain binding protein (BiP) (ref. 5 and Y.K. et al., in preparation) which binds transiently to a variety of nascent, wild-type secretory and transmembrane proteins and permanently to malfolded proteins that accumulate within the ER. We have tested the hypothesis that the presence of malfolded proteins may be the primary signal for induction of GRPs by expressing wild-type and mutant forms of influenza virus haemagglutinin (HA) in simian cells. Only malfolded HAs, whose transport from the ER is blocked, induced the synthesis of GRPs 78 and 94. Additional evidence is presented that malfolding per se, rather than abnormal glycosylation, is the proximal inducer of this family of stress proteins.
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                Author and article information

                Contributors
                Role: Reviewing editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                22 July 2014
                2014
                : 3
                : e03496
                Affiliations
                [1 ]Department of Molecular Medicine, Cornell University , Ithaca, United States
                [2 ]Department of Biology, Massachusetts Institute of Technology , Cambridge, United States
                University of Cambridge , United Kingdom
                University of Cambridge , United Kingdom
                Author notes
                [* ]For correspondence: css224@ 123456cornell.edu
                Article
                03496
                10.7554/eLife.03496
                4132286
                25053742
                14ff9a4f-5cc0-4b17-a2b6-956970fc98a1
                Copyright © 2014, Wang et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 28 May 2014
                : 20 July 2014
                Funding
                Funded by: National Institute of General Medical Sciences FundRef identification ID: http://dx.doi.org/10.13039/100000057
                Award ID: GM105858
                Award Recipient :
                Funded by: Cornell University
                Award ID: Startup funds
                Award Recipient :
                Funded by: National Science Foundation FundRef identification ID: http://dx.doi.org/10.13039/100000001
                Award ID: NSF GRF
                Award Recipient :
                Funded by: National Institute of General Medical Sciences FundRef identification ID: http://dx.doi.org/10.13039/100000057
                Award ID: GM46941
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Biochemistry
                Cell Biology
                Custom metadata
                0.7
                Direct modification by endogenous peroxide of a conserved cysteine in the molecular chaperone BiP decouples its ATPase and peptide-binding activities, allowing for enhanced polypeptide holdase activity during oxidative stress.

                Life sciences
                bip,endoplasmic reticulum,ero1,oxidative folding,ros,s. cerevisiae
                Life sciences
                bip, endoplasmic reticulum, ero1, oxidative folding, ros, s. cerevisiae

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