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.
Footnotes
This work was supported by the Canadian Institutes of Health Research [Grants MOP-79334, MOP-89707, CGD-83623 (to S.F.)], the Institut National de la Santé et de la Recherche Médicale, the Université d'Auvergne, and the FRONTENAC program from le Fond Québécois de la Recherche sur la Nature et les Technologies (FQRNT) (to S.F.).
This work will be published as part of a PhD thesis: Fortin S, Modélisation moléculaire, synthèse chimique, évaluation de l'activité antiproliférative et détermination du mécanisme d'action de nouveaux dérivés d'arylchloroéthylurées hybrides, July 2010, l'Université Laval, Québec, QC, Canada.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.171082.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
-
ABBREVIATIONS:
- CEU
- N-phenyl-N′-(2-chloroethyl)urea
- tBCEU
- [14C, urea]-1-(4-tert-butylphenyl)-3-(2-chloroethyl)urea
- MALDI-TOF
- matrix-assisted laser desorption ionization/time of flight
- ICEU
- 1-(2-chloroethyl)-3-(4-iodophenyl)urea
- HPCEU
- 1-(2-chloroethyl)-3-[3-(5-hydroxypentyl)phenyl]urea
- 4ZCombCEU
- 1-[4-(3-hydroxy-4-methoxystyryl)phenyl]-3-(2-chloroethyl)urea
- EBI
- N,N′-ethylenebis(iodoacetamide)
- T138067
- 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene
- PAGE
- polyacrylamide gel electrophoresis
- DMSO
- dimethyl sulfoxide
- C-BS
- colchicine-binding site
- ESI-MS/MS
- electrospray ionization-tandem mass spectrometry
- TBST
- Tris-buffered saline with Tween 20
- IPG
- immobilized pH gradient
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
- RPR112378
- ottelione A.
- Received June 4, 2010.
- Accepted October 25, 2010.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|