Covalent binding of 15-deoxy-delta12,14-prostaglandin J2 to PPARγ

Since 15-deoxy-delta12,14-prostaglandin J2 (15dPGJ2) has been identified as an endogenous ligand of PPARγ thus inducing adipogenesis, it has been reported to play active parts in numerous cellular regulatory mechanisms. As 15dPGJ2 has been shown to covalently bind several peptides and proteins, we investigated whether it also covalently binds PPARγ. We first observed that after incubation of 15dPGJ2 with recombinant PPARγ, the quantity of free 15dPGJ2 measured was always lower than the initial amount. We then measured the ability of the labeled agonist rosiglitazone to displace the complex PPARγ2/15dPGJ2 obtained after pre-incubation. We observed that the binding of rosiglitazone was dependent on the initial concentration of 15dPGJ2. Finally using MALDI-TOF mass spectrometry analysis, after trypsinolysis of an incubate of the PPARγ2 ligand binding domain (GST-LBD) with 15dPGJ2, we found a fragment (m/z =1314.699) corresponding to the addition of 15dPGJ2 (m/z =316.203) to the GST-LBD peptide (m/z =998.481). All these observations demonstrate the existence of a covalent binding of 15dPGJ2 to PPARγ, which opens up new perspectives to study the molecular basis for selective activities of PPARs. [Copyright &y& Elsevier]