Molecular mechanism of PIN1-mediated regulation of the nuclear receptor PPARy

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Project Summary/Abstract Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand sensitive nuclear receptor and master regulator of adipogenesis. There exist FDA approved drugs that bind the ligand binding domain (LBD) of PPARγ to induce a well characterized conformational change, which alters the activity of this transcription factor. However, the functionally critical N-terminal intrinsically disordered AF1 domain remains poorly understood from a structural standpoint due to the limited number of biophysical methods that can provide atomic level detail of binding interactions and AF1 conformational states. Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1) is a known protein binding partner of PPARγ that exerts its catalytic activity specifically at pSer/pThr-Pro motifs, facilitating the cis/trans isomerization of proline bonds along the peptide backbone. Preliminary data from our lab and others suggest PIN1 binds the AF1 region of PPARγ with a much greater affinity than the LBD, an observation which presents the opportunity to better understand the role of AF1 function in PPARγ biology. This binding interaction suggests a cascade of post translational modifications (PTMs), including kinase-mediated phosphorylation and PIN1 enzyme-catalyzed proline cist/trans isomerization of the AF1 region, may be responsible for tuning the transcriptional activity of PPARγ and maintaining activation of adipogenic gene programs. In this project I will use nuclear magnetic resonance (NMR) spectroscopy to monitor PTM (phosphorylation and isomerization) induced changes in AF1 conformational states and correlate this data with cellular function associated with PPARγ-mediated gene programs that influence adipogenesis and insulin sensitization. Aim 1 of this project outlines the use of NMR methods to monitor and describe the phosphorylation- dependent and isomerization-dependent conformational changes the AF1 domain undergoes. These NMR studies will be complemented by kinetic and structural insight gleaned from biolayer interferometry (BLI) experiments to measure binding affinity of AF1 peptides to PIN1, X-ray crystallography studies of PIN1 in complex with AF1 peptides, and cross-linking mass spectrometry (XL-MS) using full length PPARγ and PIN1 to identify intermolecular protein contacts which may not be exclusive to the AF1 domain. To correlate the structural and molecular mechanisms described by Aim 1 with functional effects observed in cells, Aim 2 of this project uses structure-guided mutagenesis, transcriptional reporter assays, and knockdown experiments in pre- adipocytes to understand the role of PIN1 as a mediator of PPARγ function during adipogenesis. Gene expression analysis and assays that measure cellular endpoints associated with PPARγ function will further assess the roles of these PTMs within the AF1 domain in promoting adipogenic function.