In Vivo Activation of PPARγ by Nitro-Fatty Acids Involves Nitroalkylation of PPARγ

FRANCISCO JOSE SCHOPFER, ALISON GROEGER, GUSTAVO BONACCI, CHENSHAN CHEN, MARSHA COLE, CARLOS BATTHYANY, PAUL BAKER, JIFENG ZHANG, TINA HALLIS, EUGENE CHEN, AND BRUCE FREEMAN

Indianapolis

Congreso; SFRBM's 15th Annual Meeting; 2008

Society for Free Radical Biology and Medicine

Nitro-fatty acids (NO2-FA) are endogenous derivatives that exert signaling actions through mechanisms that include serving as for peroxisome proliferator-activated receptor gamma (PPARg) ligands and via electrophilic reactivity. Activation of PPAR by ligand binding induces conformational changes that facilitate corepressor release and co-activator recruitment resulting in specific cellular responses. Mass spectrometric analysis of the NO2-FA treated PPARg ligand binding domain (LBD) revealed covalent adduction of Cys285 by NO2-FA. This electrophilic nitroalkylation reaction is fast, concentration-dependent and detectable by MS analysis using NO2-FA:PPARg-LBD at mol ratios as low as 1:60. When nitro-oleic acid (OA-NO2) was derivatized to an allyl ester,no PPARg nitroalkylation was detectable, indicating the relevance of the carboxylic acid for ligand activity. the identity of the NO2-FA modified tryptic peptide containing Cys285 was confirmed by comparison with nitroalkylated synthetic LBD peptide. in addition, we detected in-cell nitroalkylation of Cys285 by OA-NO2 wherein the 10- and not the 9-OA-NO2 isomer alkylated PPARg, supporting the stereospecific reaction of OA-NO2 with PPARg. Time-resolved Förster resonance energy transfer (TR-FRET) showed NO2-FA induced unique patterns of coregulator protein interactions compared with thiazolidinediones (TZDs). Importantly, TRAP220/DRIP2, a key mediator of triglyceride accumulation in adipocytes, was poorly recruited by PPARg upon NO2-FA activation. in concordance, less triglyceride accumulation occurred in adipocytes differentiated with NO2-FA versus TZDs. in summary, activation of PPARg by NO2-FA is region-specific, involves covalent reaction with Cys285 and is a potential therapeutic strategy for treating inflammation and metabolic disorders.