Structural and functional studies of the nuclear receptor PPARgamma

核受体 PPARgamma 的结构和功能研究

• 批准号: 7479184
• 负责人: H Eric XU
• 金额: $ 45.5万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7479184
• 关键词: 9-deoxy-delta-9-prostaglandin D2; Achievement; Acids; Adverse effects; Anti-Inflammatory Agents; Anti-inflammatory; Atherosclerosis; Binding; Biochemical; Biological Assay; Biological Process; Blood Vessels; C-terminal; Cardiovascular Diseases; Cells; Clinical; Complex; Consensus; Crystallization; DNA; DNA Binding; DNA Binding Domain; DNA-Protein Interaction; Diabetes Mellitus; Elements; Fatty Acids; Gene Targeting; Genes; Glass; Heart Diseases; Hormones; Human; Inflammatory; Inflammatory Response; Interleukin-6; Interleukins; Knowledge; Ligand Binding; Ligand Binding Domain; Ligands; Linoleic Acids; Malignant Neoplasms; Mediating; Medical center; Medicine; Michigan; Molecular; Mutation; N-terminal; Nitrates; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Receptors; PPAR gamma; Peptides; Peroxisome Proliferator-Activated Receptors; Pharmaceutical Preparations; Physiological; Physiology; Pioglitazone; Proteins; RXR; Range; Research; Research Personnel; Response Elements; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Structure; Structure-Activity Relationship; Testing; Texas; Therapeutic Uses; Transcriptional Activation; Universities; adipocyte differentiation; base; blood glucose regulation; clinical application; cofactor; cytokine; design; diabetic; dimer; drug discovery; in vivo; insight; intermolecular interaction; macrophage; monocyte; next generation; nitrate; programs; promoter; receptor binding; response; rosiglitazone; therapeutic target

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.

描述(申请人提供)：这项研究的长期目标是揭示核受体过氧化物体增殖物激活受体的结构和功能关系？(PPAR？)，它调节广泛的人类生理，包括脂肪细胞分化、葡萄糖动态平衡和炎症反应。PPAR？罗格列酮(AvandiaTM)和吡格列酮(ActosTM)等配体目前用于治疗人类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、白介素β和白介素6)的水平。PPAR的这种抗炎作用？与动脉粥样硬化的治疗密切相关。因此，PPAR？是治疗此类心血管疾病的一个有吸引力的治疗靶点。虽然已经开发出了许多合成配体，但PPAR？仍然是一个极具争议性的问题。最近发现的内源性PPAR？亚硝基亚油酸(LNO2)等配体在体内含量丰富，可能有助于阐明PPAR？心血管疾病中的NO信号。 作为核受体，PPAR？通过与其C末端配体结合域(LBD)的配体结合和其中间DNA结合域(DBD)的靶基因启动子结合来介导其功能。PPAR？还含有一个有效的N-末端激活结构域AF1。直到最近，结构只适用于孤立的PPAR？LBD与不同的合成配体结合，并与辅活化子的短肽基序结合。显然，考虑到PPAR的重要性，这种有限的结构信息是一个严重的缺陷？糖尿病和心血管疾病。在本研究中，我们建议通过求解各种PPAR？的晶体结构来填补这一知识空白。复合体。我们的具体目标是：1)PPAR？LBD绑定到LNO2，哪一项将提供PPAR的第一个视图？与天然配体结合；2)PPAR？LBD与PPAR的复合体？辅活化子PGC1a；3)PPAR？与RXR结合的DBD-Hinger-LBD片段和PPAR反应元件(PPRE)的DNA。我们特定目的的假设是，在这些晶体结构中观察到的分子相互作用(蛋白质-蛋白质、蛋白质-DNA和蛋白质-配体)将对理解PPAR？的生物学功能至关重要。 在结构确定之后，我们将通过仔细检查和分析结构来确定关键结构元素，我们将与德克萨斯大学西南医学中心的Steve Kliewer、密歇根大学的Eugene Chen和匹兹堡大学的Bruce Freeman合作进行定点突变和基于细胞的转录分析，以验证已确定的关键功能的功能意义。相关性：在这一应用中产生的结构信息将显著增强我们对PPAR分子机制的理解？它应该为糖尿病和心脏病的治疗提供一个合理的药物发现模板。