Nitrated fatty acid-derived signaling.

SCHOPFER, FJ; BATTHYANY, C; BONACCI, G; BAKER, P; OBAL, G; PRITSCH, O; FREEMAN, B.A

Montevideo

Congreso; V Society for Free Radical Biology and Medicine (SFRBM-South).; 2007

Society for Free Radical Biology and Medicine-South America

Nitrated fatty acids are endogenous derivatives that exert signaling actions through different mechanisms that include serving as ligands for peroxisome proliferator-activated receptor (PPAR) activation and electrophilic reactivity. PPARs are nuclear receptors that regulate metabolism and inflammation, with endogenous ligands for this receptor presently not well defined. Activation of PPARs by ligand binding induces conformational changes that facilitate co-represor release and co-activator recruitment. Ligand-specific co-regulator protein responses induce unique patterns of receptor-dependent gene expression and clinical outcomes. PPARgamma activation by thiazolidinediones (TZDs) increase insulin sensitivity, suppress chronic inflammatory process and is one approach for treating diabetes. NO-FA are potent PPARgamma ligands that appear to induce unique differential cell responses compared with TZDs, motivating more detailed studies of PPARgamma activation by NO2-FA. Mass spectrometry analysis of PPARgamma ligand binding domain (LBD) incubated with NO2-FA revealed covalent adduction of Cys285 by electrophilic NO2-FA. This nitroalkylation is fast, concentration-dependent and detectable by MS analysis using NO2-FA at PPAR-LBD mol ratios of 1:60. When NO2-FA were derivatized to allyl esters, no nitroalkylation was detectable. The identity of the modified tryptic peptide containing Cys285 was confirmed by comparison with nitroalkylated synthetic LBD peptide. Further MS analysis also detected in vivo modification of PPARgamma by NO2-FA, with a limit of detection of 100 amol con column. Real time binding of NO2-FA to PPARgamma measured via surface Plasmon resonance showed reduced Cys285 was essential for initial docking of NO2-FA and subsequent LBD nitroalkylation, with the carboxylic acid promoting interaction with positively charged residues in the ligand binding pocket. In summary, activation of PPARgamma by NO2-FA is biologically-relevant, specific, involves covalent reaction with Cys285 and is a potential therapeutic strategy for treating inflammation and metabolic disorders.