Substoichiometric inhibition of microtubule formation by acetaldehyde-tubulin adducts

Abstract

We have shown previously that acetaldehyde forms stable covalent adducts with tubulin, resulting in impaired microtubule formation. The present study explored the mechanism responsible for impaired microtubule formation caused by the substoichiometric stable binding of acetaldehyde to tubulin. The free tubulin dimer was much more reactive with acetaldehyde than microtubules, binding more than twice as much aldehyde. The dimer also formed nearly twice as many stable adducts on its α-chain as on its β-chain, whereas microtubules exhibited an equal distribution of adducts between the two subunits. These data confirm that the α-chain of free tubulin, but not microtubules, has an accessible highly reactive lysine (HRL) residue that is a preferential target of acetaldehyde binding. Adduct formation with the HRL residue also correlated with impaired tubulin polymerization, and only 0.08 moles of acetaldehyde bound per mole of HRL was required for complete inhibition; however, adducts with other lysine residues (bulk adducts) did not affect assembly. Adducts to microtubule-associated proteins (MAPs) also impaired the assembly of tubulin, but were much less effective than HRL adducts. In a copolymerization assay, HRL-adducted tubulin, in addition to being itself assembly incompetent, also interfered with polymerization of normal (unadducted) tubulin. Bulk adducts did not alter assembly and were incorporated normally into the growing polymer. When tubulin was cleaved by the proteolytic enzyme, subtilisin, microtubule formation could readily take place in the absence of MAPs. In this polymerization system, HRL adducts, but not bulk adducts, still markedly inhibited assembly. When low concentrations of acetaldehyde (50 μM) were used to generate HRL adducts, an adduct on only 1 out of 20 tubulin molecules was sufficient to totally block polymerization. These findings indicate that substoichiometric amounts of acetaldehyde bound to HRL of tubulin can markedly inhibit microtubule formation via direct interference of dimer-dimer interactions, and further suggest that low concentrations of acetaldehyde could generate sufficient amounts of HRL adducts in cellular systems to alter microtubule formation and function.