Actin Cys374 as a nucleophilic target of α,β-unsaturated aldehydes

被引:74
作者
Dalle-Donne, Isabella
Carini, Marina
Vistoli, Giulio
Gamberoni, Luca
Giustarini, Daniela
Colombo, Roberto
Facino, Roberto Maffei
Rossi, Ranieri
Milzani, Aldo
Aldini, Giancarlo
机构
[1] Univ Milan, Dept Biol, I-20133 Milan, Italy
[2] Univ Milan, Inst Pharmaceut & Toxicol Chem Pietro Pratesi, I-20133 Milan, Italy
[3] Univ Siena, Dept Neurosci, I-53100 Siena, Italy
关键词
acrolein; 4-hydroxynonenal; actin polymerization; LC-ESI-MS/MS; cysteine; histidine; carbonyl scavenger; electrophilic lipid;
D O I
10.1016/j.freeradbiomed.2006.11.026
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have recently shown that actin can be modified by the Michael addition of 4-hydroxynonenal to Cys374. Here, we have exposed purified actin at increasing acrolein concentrations and have identified the sites of acrolein addition using LC-ESI-MS/MS. Acrolein reacted with Cys374, His87, His173, and, minimally, His40. Cys374 adduction by both 4-hydroxynonenal and acrolein negligibly affected the polymerization of aldehyde-modified (carbonylated) actin, as shown by fluorescence measurements. Differently, acrolein binding at histidine residues, when Cys374 was completely saturated, inhibited polymerization in a dose-dependent manner. Molecular modeling analyses indicated that structural distortions of the ATP-binding site, induced by four acrolein-Michael adducts, could explain the changes in the polymerization process. Aldehyde binding to Cys374 does not alter significantly actin polymerization because this residue is located in a very flexible region, whose covalent modifications do not alter the protein folding. These data demonstrate that Cys374 represents the primary target site of alpha,beta-unsaturated aldehyde addition to actin in vitro. As Cys374 is a preferential target for various oxidative/nitrosative modifications, and actin is one of the main carbonylated proteins in vivo, these findings also suggest that the highly reactive Cys374 could serve as a carbonyl scavenger of reactive alpha,beta-unsaturated aldehydes and other electrophilic lipids. (c) 2006 Elsevier Inc. All rights reserved.
引用
收藏
页码:583 / 598
页数:16
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