Structural and functional studies of the nuclear receptor PPARgamma

描述（由申请人提供）：本研究的长期目标是揭示核受体过氧化物酶体增殖物激活受体的结构和功能关系？（PPAR?），它调节广泛的人体生理，包括脂肪细胞分化，葡萄糖稳态和炎症反应。PPAR吗?配体如罗格列酮（AvandiaTM）和吡格列酮（ActosTM）目前用于治疗人类2型糖尿病。在单核/巨噬细胞中，PPAR？能够通过抑制这些促炎基因的表达来降低细胞因子（TNFa、白细胞介素-¿和白细胞介素-6）的水平。PPAR的抗炎作用？与动脉粥样硬化的治疗高度相关。因此,PPAR吗?是治疗这类心血管疾病的有吸引力的治疗靶点。虽然许多人工合成的配体已经被开发出来，但PPAR？这仍然是一个有很大争议的问题。最近发现的内源性PPAR？硝基亚油酸（LNO2）等配体在体内丰富，可能有助于阐明PPAR的生理相关性。和NO信号在心血管疾病中的作用。

DESCRIPTION (provided by applicant): The long-term objective of this study is to reveal the structure and function relationships of the nuclear receptor peroxisome proliferator-activated receptor ? (PPAR?), which regulates a wide range of human physiology including adipocyte differentiation, glucose homeostasis, and inflammatory responses. PPAR? ligands such as rosiglitazone (AvandiaTM) and pioglitazone (ActosTM) are currently used in the treatment of type 2 diabetes in humans. In monocytes/macrophages, PPAR? is able to reduce the levels of cytokines (TNFa, interleukin-¿, and interleukin-6) by inhibiting the expression of these pro-inflammatory genes. This anti-inflammatory effect of PPAR? is highly associated with the treatment of atherosclerosis. Therefore, PPAR? is an attractive therapeutic target for the treatment of such cardiovascular diseases. While many synthetic ligands have been developed, the physiological ligands of PPAR? remain a matter of great debate. The recently identified endogenous PPAR? ligands like nitrolinoleic acid (LNO2) are abundant in vivo and may help elucidate the physiological relevance of PPAR? and NO signaling in cardiovascular diseases. As a nuclear receptor, PPAR? mediates its function through ligand binding to its C-terminal ligand-binding domain (LBD) and targeted gene promoter binding to its middle DNA-binding domain (DBD). PPAR? also contains a potent N- terminal activation domain, AF1. Until recently, structures were available only for the isolated PPAR? LBD bound to diverse synthetic ligands and to short peptide motifs of coactivators. Clearly, this limited amount of structural information is a serious deficiency considering the importance of PPAR? in diabetes and cardiovascular diseases. In this study, we propose to fill in this knowledge gap by solving the crystal structures of various PPAR? complexes. Our specific aims are focused on the crystallization and structural determination of 1) the PPAR? LBD bound to LNO2, which will provide the first view of PPAR? bound to a natural ligand; 2) the PPAR? LBD in complex with the PPAR? coactivator PGC1a; and 3) a PPAR? DBD-hinge-LBD fragment bound to RXR and a DNA of the PPAR response element (PPRE). The hypothesis for our specific aims is that the molecular interactions (protein-protein, protein-DNA, and protein-ligand) observed in those crystal structures will be crucial for understanding the biological functions of PPAR?. Following the structural determination, we will identify key structural elements by scrutinizing and analyzing the structures, and we will collaborate with Steve Kliewer (University of Texas Southwestern Medical Center), Eugene Chen (University of Michigan), and Bruce Freeman (University of Pittsburgh) on site-directed mutagenesis and cell-based transcriptional assays to validate the functional significance of the key features identified. Relevance: The structural information generated in this application will significantly enhance our understanding of the molecular mechanisms of PPAR? functions, and it should provide a rational template of drug discovery for the treatment of diabetes and heart diseases.