title: Evidence for thiol/disulfide exchange reactions between tubulin and glyceraldehyde-3-phosphate dehydrogenase.
abstract: While thiol redox reactions are a common mechanism to regulate protein structure and function, protein disulfide bond formation is a marker of oxidative stress that has been linked to neurodegeneration. Both tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contain multiple cysteines that have been identified as targets for oxidation to disulfides, S-nitrosation and S-glutathionylation. We show that GAPDH is one of three prominent brain microtubule-associated proteins (MAPs), in addition to MAP-2 and tau, with reactive cysteines. We detected a threefold to fourfold increase in tubulin cysteine oxidation by hydrogen peroxide in the presence of rabbit muscle GAPDH by 5-iodoacetamidofluorescein labeling and by Western blot detection of higher molecular weight inter-chain tubulin disulfides. In thiol/disulfide exchange experiments, tubulin restored ∼50% of oxidized GAPDH cysteines and the equilibrium favored reduced GAPDH. Further, we report that oxidized GAPDH is repaired by the thioredoxin reductase system (TRS). Restoration of GAPDH activity after reduction by both tubulin and the TRS was time-dependent suggesting conformational changes near the active site cysteine149. The addition of brain MAPs to oxidized tubulin reduced tubulin disulfides and labeling of MAP-2 and of GAPDH decreased. Because the extent of tubulin repair of oxidized GAPDH was dependent on buffer strength, we conclude that electrostatics influence thiol/disulfide exchange between the two proteins. The novel interactions presented herein may protect GAPDH from inhibition under oxidative stress conditions.
authors.affiliation: Department of Chemistry, The College of William and Mary, Williamsburg, Virginia.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Landino
authors.forename: Tara D
authors.initials: TD
authors.lastname: Hagedorn
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Kennett
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004220:Disulfides; D008869:Microtubule-Associated Proteins; D013438:Sulfhydryl Compounds; D014404:Tubulin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
keywords: GAPDH; cysteine oxidation; microtubule-associated proteins; thiol/disulfide exchange; thioredoxin; thioredoxin reductase; tubulin