Characterization of the covalent binding of N-phenyl-N'-(2-chloroethyl)ureas to {beta}-tubulin: importance of Glu198 in microtubule stability.
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.
Unité des Biotechnologies et de Bioingénierie, Centre de Recherche, Centre Hospitalier Universitaire de Québec, Hôpital Saint-François d'Assise, 10 rue de l'Espinay, Québec, Canada. sebastien.fortin.1@ulaval.ca
Sébastien
S
Fortin
Bernadette
B
Bouchon
Christophe
C
Chambon
Jacques
J
Lacroix
Emmanuel
E
Moreau
Jean-Michel
JM
Chezal
Françoise
F
Degoul
René
R
C-Gaudreault
D000107:Acetylation; D000215:Acylation; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D008545:Melanoma / Q000188:drug therapy / Q000473:pathology; D051379:Mice; D008870:Microtubules / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D050257:Tubulin Modulators / Q000378:metabolism*; D014508:Urea / Q000031:analogs & derivatives*
D000970:Antineoplastic Agents; D014404:Tubulin; D050257:Tubulin Modulators; D014508:Urea