Spectroscopic analyses of the binding kinetics of 15d-PGJ2 to the PPARgamma ligand-binding domain by multi-wavelength global fitting.

PPARgamma (peroxisome proliferator-activated receptor gamma) is a nuclear receptor that is activated by natural lipid metabolites, including 15d-PGJ2 (15-deoxy-Delta(12,14)-prostaglandin J2). We previously reported that several oxidized lipid metabolites covalently bind to PPARgamma through a Michael-addition to activate transcription. To separate the ligand-entering (dock) and covalent-binding (lock) steps in PPARgamma activation, we investigated the binding kinetics of 15d-PGJ2 to the PPARgamma LBD (ligand-binding domain) by stopped-flow spectroscopy. We analysed the spectral changes of 15d-PGJ2 by multi-wavelength global fitting based on a two-step chemical reaction model, in which an intermediate state represents the 15d-PGJ2-PPARgamma complex without covalent binding. The extracted spectrum of the intermediate state in wild-type PPARgamma was quite similar to the observed spectrum of 15d-PGJ2 in the C285S mutant, which cannot be activated by 15d-PGJ2, indicating that the complex remains in the inactive, intermediate state in the mutant. Thus 'lock' rather than 'dock' is one of the critical steps in PPARgamma activation by 15d-PGJ2.

Alpha,beta-unsaturated ketone is a core moiety of natural ligands for covalent binding to peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptor gamma (PPARgamma) functions in various biological processes, including macrophage and adipocyte differentiation. Several natural lipid metabolites have been shown to activate PPARgamma. Here, we report that some PPARgamma ligands, including 15-deoxy-Delta12,14-prostaglandin J2, covalently bind to a cysteine residue in the PPARgamma ligand binding pocket through a Michael addition reaction by an alpha,beta-unsaturated ketone. Using rhodamine-maleimide as well as mass spectroscopy, we showed that the binding of these ligands is covalent and irreversible. Consistently, mutation at the cysteine residue abolished abilities of these ligands to activate PPARgamma, but not of BRL49653, a non-covalent synthetic agonist, indicating that covalent binding of the alpha,beta-unsaturated ketone in the natural ligands was required for their transcriptional activities. Screening of lipid metabolites containing the alpha,beta-unsaturated ketone revealed that several other oxidized metabolites of hydroxyeicosatetraenoic acid, hydroxyeicosadecaenoic acid, and prostaglandins can also function as novel covalent ligands for PPARgamma. We propose that PPARgamma senses oxidation of fatty acids by recognizing such an alpha,beta-unsaturated ketone as a common moiety.