@article {Fortin460, author = {S{\'e}bastien Fortin and Bernadette Bouchon and Christophe Chambon and Jacques Lacroix and Emmanuel Moreau and Jean-Michel Chezal and Fran{\c c}oise Degoul and Ren{\'e} C.-Gaudreault}, title = {Characterization of the Covalent Binding of N-Phenyl-N'-(2-chloroethyl)ureas to β-Tubulin: Importance of Glu198 in Microtubule Stability}, volume = {336}, number = {2}, pages = {460--467}, year = {2011}, doi = {10.1124/jpet.110.171082}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {N-Phenyl-N'-(2-chloroethyl)ureas (CEUs) are antimicrotubule agents interacting covalently with β-tubulin near the colchicine-binding site (C-BS). Glutamyl 198 residue in β-tubulin (Glu198), which is adjacent to the C-BS behind the two potent nucleophilic residues, Cys239 and Cys354, has been shown to covalently react with 1-(2-chloroethyl)-3-(4-iodophenyl)urea (ICEU). By use of mass spectrometry, we have now identified residues in β-tubulin that have become modified irreversibly by 1-(2-chloroethyl)-3-[3-(5-hydroxypentyl)phenyl]urea (HPCEU), 1-[4-(3-hydroxy-4-methoxystyryl)phenyl]-3-(2-chloroethyl)urea (4ZCombCEU), and N,N'-ethylenebis(iodoacetamide) (EBI). The binding of HPCEU and 4ZCombCEU to β-tubulin resulted in the acylation of Glu198, a protein modification of uncommon occurrence in living cells. Prototypical CEUs then were used as molecular probes to assess, in mouse B16F0 and human MDA-MB-231 cells, the role of Glu198 in microtubule stability. For that purpose, we studied the effect of Glu198 modification by ICEU, HPCEU, and 4ZCombCEU on the acetylation of Lys40 on α-tubulin, a key indicator of microtubule stability. We show that modification of Glu198 by prototypical CEUs correlates with a decrease in Lys40 acetylation, as observed also with other microtubule depolymerizing agents. Therefore, CEU affects the stability and the dynamics of microtubule, likewise a E198G mutation, which is unusual for xenobiotics. We demonstrate for the first time that EBI forms an intramolecular cross-link between Cys239 and Cys354 of β-tubulin in living cells. This work establishes a novel basis for the development of future chemotherapeutic agents and provides a framework for the design of molecules useful for studying the role of Asp and Glu residues in the structure/function and the biological activity of several cellular proteins under physiological conditions.}, issn = {0022-3565}, URL = {https://jpet.aspetjournals.org/content/336/2/460}, eprint = {https://jpet.aspetjournals.org/content/336/2/460.full.pdf}, journal = {Journal of Pharmacology and Experimental Therapeutics} }