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 α,β-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 α,β-unsaturated aldehydes and other electrophilic lipids.
|Titolo:||Actin Cys374 as a nucleophilic target of α,β-unsaturated aldehydes|
|Citazione:||DALLE DONNE, I., Carini, M., Vistoli, G., Gamberoni, L., Giustarini, D., Colombo, R., et al. (2007). Actin Cys374 as a nucleophilic target of α,β-unsaturated aldehydes. FREE RADICAL BIOLOGY & MEDICINE, 42(5), 583-598.|
|Appare nelle tipologie:||1.1 Articolo in rivista|