Actin Cys374 as a nucleophilic target of alpha,beta-unsaturated aldehydes.
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.
Dalle-Donne
Isabella
I
Department of Biology, University of Milan, via Celoria 26, I-20133 Milan, Italy. quack@unimi.it
Carini
Marina
M
Vistoli
Giulio
G
Gamberoni
Luca
L
Giustarini
Daniela
D
Colombo
Roberto
R
Maffei Facino
Roberto
R
Rossi
Ranieri
R
Milzani
Aldo
A
Aldini
Giancarlo
G
D000171:Acrolein / Q000494:pharmacology; D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D010449:Peptide Mapping; D011108:Polymers / Q000737:chemistry; D050050:Protein Carbonylation / Q000187:drug effects; D011817:Rabbits
D000199:Actins; D000447:Aldehydes; D011108:Polymers; C462359:tert-4-hydroxy-2-nonenal; D000171:Acrolein; D003545:Cysteine