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 beta4-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.
Highlights
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
H2O2 and Diamide Induce Protein Mixed Disulfides with Glutathione—We examined the effect of a variety of oxidative conditions on protein disulfide bond formation using the murine hippocampal cell line HT22
During oxidative stress the levels of GSH decrease, GSSG levels increase, and mixed disulfides form between GSH and redox-sensitive proteins, all of which are theorized to promote protein disulfide bond formation [29]
Summary
Oxidative stress occurs when the rate of reactive oxygen species (ROS) generation exceeds the detoxification abilities of the cell, and it has been implicated in many degenerative diseases. 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 pKa Ͼ8.0 and, in the reducing environment of the cytoplasm, remain protonated at physiological pH. Many redox-sensitive proteins form transient disulfide bonds while catalyzing the reduction of thiol groups [6]. The reduced form of thioredoxin (TRX) binds to substrate proteins containing a disulfide bond, and a dithioldisulfide exchange reaction occurs in which the active site cysteine residues of TRX are oxidized, whereas the cysteine residues in the substrate protein are reduced [8]. This paper is available on line at http://www.jbc.org
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