Hypothiocyanous acid oxidation of tubulin cysteines inhibits microtubule polymerization

Abstract

Thiol oxidation is a probable outcome of cellular oxidative stress and is linked to degenerative disease progression. In addition, protein thiol redox reactions are increasingly identified as a mechanism to regulate protein structure and function. We assessed the effect of hypothiocyanous acid on the cytoskeletal protein tubulin. Total cysteine oxidation by hypothiocyanous and hypochlorous acids was monitored by labeling tubulin with 5-iodoacetamidofluorescein and by detecting higher molecular weight inter-chain tubulin disulfides by Western blot under nonreducing conditions. Hypothiocyanous acid induced nearly stoichiometric oxidation of tubulin cysteines (1.9 mol cysteine/mol oxidant) and no methionine oxidation was observed. Because disulfide reducing agents restored all the polymerization activity that was lost due to oxidant treatment, we conclude that cysteine oxidation of tubulin inhibits microtubule polymerization. Hypothiocyanous acid oxidation of tubulin cysteines was markedly decreased in the presence of 4% glycerol, a component of the tubulin purification buffer. Due to its instability and buffer- and pH-dependent reactivity, hypothiocyanous acid studies require careful consideration of reaction conditions.

Molecular characterization and interactome analysis of Trypanosoma cruzi tryparedoxin II

“…In the case of tubulin, it has been observed that the assembly and polymerization of this protein depend on redox changes [35][36][37]. For example, in mammalian microtubule [38], cysteine oxidation to disulfides altered the ability of the proteins to promote the assembly of microtubules from tubulin.…”

The role of Nox-mediated oxidation in the regulation of cytoskeletal dynamics

“…Indeed, it has been reported that oxidation of tubulin cysteines by peroxynitrite, 2-[(1-methylpropyl)dithio]-1H-imidazole or hypothiocyanous acid inhibits microtubule polymerization [59–61]. Since cysteine residues are not part of the protein domains responsible for the dimerization and polymerization of tubulin [62], more studies are needed to determine if this is a direct effect on filament stabilization, or an effect on the binding of proteins that regulate the polymerization process; in addition, more work is needed to determine the endogenous sources of ROS that may mediate these oxidations in vivo.…”

The interplay between ROS and tubulin cytoskeleton in plants

“…57,58 Thus, it seems that apart from mechanisms that perceive cellular redox status, additional regulatory mechanisms sense the redox status of tubulin. Since the later is strongly related to the capability for MT assembly, 59,60 such mechanisms may protect tubulin against irreversible oxidation.…”