title: 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 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.
authors.affiliation: Cellular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Cumming
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Andon
authors.forename: Paul A
authors.initials: PA
authors.lastname: Haynes
authors.forename: Minkyu
authors.initials: M
authors.lastname: Park
authors.forename: Wolfgang H
authors.initials: WH
authors.lastname: Fischer
authors.forename: David
authors.initials: D
authors.lastname: Schubert
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000737:chemistry; D000818:Animals; D001710:Biotin / Q000737:chemistry; D002455:Cell Division; D003593:Cytoplasm / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005822:Genetic Vectors; D005978:Glutathione / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D015151:Immunoblotting; D007460:Iodoacetamide / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D009474:Neurons / Q000378:metabolism; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry; D054464:Peroxiredoxins; D011233:Precipitin Tests; D017382:Reactive Oxygen Species; D012264:Ribonucleotide Reductases / Q000737:chemistry; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry
chemical_list: D025601:Adenomatous Polyposis Coli Protein; D004220:Disulfides; D000939:Epitopes; D018840:HSP70 Heat-Shock Proteins; D017382:Reactive Oxygen Species; D001710:Biotin; D006861:Hydrogen Peroxide; D010544:Peroxidases; D054464:Peroxiredoxins; D012264:Ribonucleotide Reductases; D013880:Thioredoxin-Disulfide Reductase; D009701:Nucleoside-Diphosphate Kinase; D005978:Glutathione; D010100:Oxygen; D007460:Iodoacetamide