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.

Two Tau binding sites on tubulin revealed by thiol-disulfide exchanges

“…Wang et al . showed the presence of a disulfide bond between the α-C347 of α-tubulin and the cysteine of a synthetic stathmin-like peptide (Ncap) that prevents MTs formation 41 and more recently, a thiol-disulfide exchange reaction between tubulin and GAPDH has been demonstrated 42 . Our result reveals for the first time the ability of Tau to perform a thiol/disulfide exchange with tubulin/MTs.…”

Redox regulation in tumor cell epithelial–mesenchymal transition: molecular basis and therapeutic strategy

“… 40 In the presence of H 2 O 2 or HOCl, tubulin can form tetramers by oxidative modification. 41 In addition, treatment with paraquat, a ROS inducer, results in cytoskeletal injury by diminishing tubulin and microtubules. 42 These studies indicate that oxidative stress monitors cytoskeleton remodeling via the redox regulation of both actin and tubulin.…”

Interplay between oxidant species and energy metabolism

“…Finally, GAPDH Cys149 and other nucleophilic Cys and His residues can be modified by nitroalkenes, inactivating the enzyme [58] . Some reports show that besides from its known role as an intermediary metabolism enzyme, GAPDH can participate in cell signaling, DNA repair, transcriptional regulation of genes, membrane fusion, tubulin bundling, and apoptotic signaling [182] , [183] , [184] . Importantly, nitroalkylation of GAPDH promotes its migration to cell membranes and might be responsible for changes in the subcellular distribution and function of GAPDH in certain situations [58] .…”