In the course of a high throughput screen to search for ligands of peroxisome proliferator activated receptor-gamma (PPARgamma), we identified GW9662 using a competition binding assay against the human ligand binding domain. GW9662 had nanomolar IC(50) versus PPARgamma and was 10- and 600-fold less potent in binding experiments using PPARalpha and PPARdelta, respectively. Pretreatment of all three PPARs with GW9662 resulted in the irreversible loss of ligand binding as assessed by scintillation proximity assay. Incubation of PPAR with GW9662 resulted in a change in the absorbance spectra of the receptors consistent with covalent modification. Mass spectrometric analysis of the PPARgamma ligand binding domain treated with GW9662 established Cys(285) as the site of covalent modification. This cysteine is conserved among all three PPARs. In cell-based reporter assays, GW9662 was a potent and selective antagonist of full-length PPARgamma. The functional activity of GW9662 as an antagonist of PPARgamma was confirmed in an assay of adipocyte differentiation. GW9662 showed essentially no effect on transcription when tested using both full-length PPARdelta and PPARalpha. Time-resolved fluorescence assays of ligand-modulated receptor heterodimerization, coactivator binding, and corepressor binding were consistent with the effects observed in the reporter gene assays. Control activators increased PPAR:RXR heterodimer formation and coactivator binding to both PPARgamma and PPARdelta. Corepressor binding was decreased. In the case of PPARalpha, GW9662 treatment did not significantly increase heterodimerization and coactivator binding or decrease corepressor binding. The experimental data indicate that GW9662 modification of each of the three PPARs results in different functional consequences. The selective and irreversible nature of GW9662 treatment, and the observation that activity is maintained in cell culture experiments, suggests that this compound may be a useful tool for elucidation of the role of PPARgamma in biological processes.

The 2.7 A X-ray crystal structure of the HNF4gamma ligand binding domain (LBD) revealed the presence of a fatty acid within the pocket, with the AF2 helix in a conformation characteristic of a transcriptionally active nuclear receptor. GC/MS and NMR analysis of chloroform/methanol extracts from purified HNF4alpha and HNF4gamma LBDs identified mixtures of saturated and cis-monounsaturated C14-18 fatty acids. The purified HNF4 LBDs interacted with nuclear receptor coactivators, and both HNF4 subtypes show high constitutive activity in transient transfection assays, which was reduced by mutations designed to interfere with fatty acid binding. The endogenous fatty acids did not readily exchange with radiolabeled palmitic acid, and all attempts to displace them without denaturing the protein failed. Our results suggest that the HNF4s may be transcription factors that are constitutively bound to fatty acids.

Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-l-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.