Protein Disulfide Bond Formation in the Cytoplasm during Oxidative Stress

Abstract

The majority of disulfide-linked cytosolic proteins are thought to be enzymes that transiently form disulfide bonds while catalyzing oxidation-reduction (redox) processes. Recent evidence indicates that reactive oxygen species can act as signaling molecules by promoting the formation of disulfide bonds within or between select redox-sensitive proteins. However, few studies have attempted to examine global changes in disulfide bond formation following reactive oxygen species exposure. Here we isolate and identify disulfide-bonded proteins (DSBP) in a mammalian neuronal cell line (HT22) exposed to various oxidative insults by sequential nonreducing/reducing two-dimensional SDS-PAGE combined with mass spectrometry. By using this strategy, several known cytosolic DSBP, such as peroxiredoxins, thioredoxin reductase, nucleoside-diphosphate kinase, and ribonucleotide-diphosphate reductase, were identified. Unexpectedly, a large number of previously unknown DSBP were also found, including those involved in molecular chaperoning, translation, glycolysis, cytoskeletal structure, cell growth, and signal transduction. Treatment of cells with a wide range of hydrogen peroxide concentrations either promoted or inhibited disulfide bonding of select DSBP in a concentration-dependent manner. Decreasing the ratio of reduced to oxidized glutathione also promoted select disulfide bond formation within proteins from cytoplasmic extracts. In addition, an epitope-tagged version of the molecular chaperone HSP70 forms mixed disulfides with both ␤4-spectrin and adenomatous polyposis coli protein in the cytosol. Our findings indicate that disulfide bond formation within families of cytoplasmic proteins is dependent on the nature of the oxidative insult and may provide a common mechanism used to control multiple physiological processes.Oxidative stress occurs when the rate of reactive oxygen species (ROS) 1 generation exceeds the detoxification abilities of the cell, and it has been implicated in many degenerative diseases. It is frequently argued that ROS cause relatively nonspecific damage to vital cellular components such as lipids, DNA, and proteins. However, emerging evidence indicates that ROS can cause specific protein modifications that may lead to a change in the activity or function of the oxidized protein (1, 2). Several major forms of oxidative modifications can occur on amino acid residue side chains including carbonylation, nitrosylation, and oxidation of methionine to methionine sulfoxide (3). Protein sulfhydryls can be oxidized to protein disulfides and sulfenic acids as well as more highly oxidized states such as the sulfinic and sulfonic acid forms of protein cysteines (4). Under non-stressed conditions, disulfide bond formation occurs primarily in the oxidizing environment of the endoplasmic reticulum (ER) in eukaryotic cells (5). The sulfhydryl groups in the vast majority of protein cysteine residues (Cys-SH) have a pK a Ͼ8.0 and, in the reducing environment of the cytoplasm, remain protonated at physiological pH. Thus,...

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