Formation and Reversibility of BiP Protein Cysteine Oxidation Facilitate Cell Survival during and post Oxidative Stress

Abstract

Redox fluctuations within cells can be detrimental to cell function. To gain insight into how cells normally buffer against redox changes to maintain cell function, we have focused on elucidating the signaling pathways that serve to sense and respond to oxidative redox stress within the endoplasmic reticulum (ER) using yeast as a model system. Previously, we have shown that a cysteine in the molecular chaperone BiP, a Hsp70 molecular chaperone within the ER, is susceptible to oxidation by peroxide during ER-derived oxidative stress, forming a sulfenic acid (؊SOH) moiety. Here, we demonstrate that this same conserved BiP cysteine is susceptible also to glutathione modification (؊SSG). Glutathionylated BiP is detected both as a consequence of enhanced levels of cellular peroxide and also as a by-product of increased levels of oxidized glutathione (GSSG). Similar to sulfenylation, we observe glutathionylation decouples BiP ATPase and peptide binding activities, turning BiP from an ATP-dependent foldase into an ATP-independent holdase. We show glutathionylation enhances cell proliferation during oxidative stress, which we suggest relates to modified BiP's increased ability to limit polypeptide aggregation. We propose the susceptibility of BiP to modification with glutathione may serve also to prevent irreversible oxidation of BiP by peroxide.Cell homeostasis and numerous vital cell functions rely on the maintenance of a proper intracellular redox balance. Oxidative folding in the endoplasmic reticulum (ER) 2 is one intracellular system that is prone to disruption upon alterations in the redox poise. Insufficient oxidizing capacity in the ER leads to the accumulation of proteins in reduced non-native states (1, 2). Alternatively, an overly oxidizing ER results in cellular toxicity, likely caused in part by the mis-pairing of protein cysteine residues resulting from a decreased capacity to reduce nonnative disulfides (3, 4). Crucial for maintaining a continual flux of folding polypeptides through the ER are the systems that buffer against fluctuations in the ER redox environment.Recently, several proteins have been demonstrated to act as sensors of redox fluctuations in the ER. In a common theme, these proteins sense changes in the ER redox environment and respond with beneficial alterations in their enzymatic activities. The enzyme Ero1 is a major source of oxidizing equivalents in the ER, and Ero1 activity is coupled to the ER redox environment. When the ER balance shifts to overly oxidizing conditions, regulatory cysteine residues in Ero1 become oxidized to disulfides, which decreases Ero1 oxidase activity to help restore redox balance (5-7). In addition, sensors that cope with the potential for protein folding defects have emerged. These redox-dependent chaperones do not directly impact the flux of oxidizing equivalents, like Ero1, but rather are activated to help limit polypeptide aggregation during unfavorable redox conditions. Oxidation of active site cysteines in the oxidoreductase PDI has been demonstrated to...

A Conserved Cysteine within the ATPase Domain of the Endoplasmic Reticulum Chaperone BiP is Necessary for a Complete Complement of BiP Activities

“…We reported previously that addition of select Kar2 Cys63 mutant alleles allows for enhanced cell viability during cellular oxidative stress [1, 2]. These prior experiments built upon our observation that a strain containing a kar2-C63A allele was compromised for growth in the presence of exogenous or endogenous oxidants, relative to a strain containing wild-type Kar2 [1].…”

“…It has been demonstrated for both mammalian and yeast BiP that during conditions of increased ER oxidation, this cysteine becomes oxidized, which in turn augments BiP chaperone activity [1, 2, 20]. Such an alteration in BiP activity is thought to benefit cells during oxidative stress by limiting polypeptide aggregation during suboptimal redox conditions for ER protein folding.…”

“…Such an alteration in BiP activity is thought to benefit cells during oxidative stress by limiting polypeptide aggregation during suboptimal redox conditions for ER protein folding. In yeast, the conserved BiP (Kar2) cysteine undergoes modification by peroxide and/or glutathione, forming a sulfenic acid or glutathione adduct [1, 2]. In mammals (mice), the equivalent cysteine forms an intramolecular disulfide bond (with a second BiP cysteine) during overly oxidizing conditions [20].…”

“…Yet when present, the features of cysteine often afford unique function. We have shown previously that a cysteine within the ATPase domain of yeast BiP (Kar2) serves as a sensor of the endoplasmic reticulum (ER) redox environment [1, 2]. Under conditions of increased oxidant (oxidative stress) this cysteine becomes oxidized, changing Kar2 from an ATP-dependent foldase to an ATP-independent holdase.…”

Interplay between redox and protein homeostasis

“…82 Recently, it could be shown that also yeast BiP, an Hsp70 in the ER, has a cysteine which can be glutathionylated and cause enhanced chaperone holdase activity. 92 Additionally, an activating thiol-based redox switch was observed for Hsp33 of E. coli, where oxidation of Hsp33 leads to chaperone activation. 83 Taken together, the increasing number of observations that chaperones are subject to redox-regulation of their activities offers new possibilities for therapeutic intervention for neurodegenerative diseases Methods to analyze redox capacity…”