Structure and Interfacial Function of Platelet Activating Factor Acetylhydrolase

血小板激活因子乙酰水解酶的结构和界面功能

• 批准号: 7848835
• 负责人: BRIAN J BAHNSON
• 金额: $ 34.43万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-15 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7848835
• 关键词: Allergic Reaction; Anaphylaxis; Anti-Inflammatory Agents; Anti-inflammatory; Asthma; Atherosclerosis; Attention; Binding; Biochemical; Biological Assay; Cells; Cloning; Confocal Microscopy; Coupled; Cyan Fluorescent Protein; Detergents; Enzymes; Escherichia coli; Goals; Growth; Health; Hepatocyte; High Density Lipoproteins; Homeostasis; Homologous Gene; Homology Modeling; Human; In Vitro; Inflammation; Inflammatory; Insecticides; Investigation; Kidney; Kinetics; Link; Lipids; Liver; Low-Density Lipoproteins; Membrane; Membrane Proteins; Modeling; Molecular; Neurotoxins; Organophosphates; Pathway interactions; Peroxidases; Phospholipids; Physiological; Plasma; Platelet Activating Factor; Play; Proteins; Reaction; Resolution; Role; Sampling; Signaling Molecule; Site-Directed Mutagenesis; Staging; Structural Models; Structure; Surface; System; Techniques; Therapeutic; Work; aqueous; design; esterase; in vivo; inhibitor/antagonist; interest; interfacial; kidney cell; lipid metabolism; nerve agent; oxidized lipid; particle

DESCRIPTION (provided by applicant): Lipid metabolism has taken the stage front and center as a human health concern. Here much attention has been focused on the mechanisms involved in inflammation and lipid homeostasis of lipids linked to low density lipoprotein (LDL) and high density lipoprotein (HDL). Many proteins that are associated with these LDL and HDL particles have emerged as playing a critical role in these lipid pathways. The plasma form of platelet activating factor acetylhydrolase (pPAFAH) functions on the surface of LDL particles by reducing levels of the signaling molecule platelet activating factor (PAF) as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma and allergic reactions. A homologous intracellular form, referred to as PAFAH-II, is believed to have similar functions in liver and kidney cells. The phospholipid-associated pPAFAH and PAFAH-II enzymes are worthy structural targets. Physiologically, these enzymes are found associated with LDL particles or the inner leaflet of cells, and as such, are considered interfacial enzymes, which function on the lipid-aqueous interface. In addition to a role to reduce PAF levels, they have been implicated in hydrolytic activities of other pro-inflammatory agents, such as oxidized lipids. We will elucidate the relationship between structure and interfacial function for PAF AH via 4 aims: (i) The heterologous expression of and use of additives/detergents will be screened in order to obtain homogeneous forms of the pPAFAH and PAFAH-II enzymes. The quality of protein will be assessed by biophysical characterization, functional assay and protein crystal growth in order to obtain monodisperse and soluble forms of PAFAH suitable for structural and functional studies, (ii) The high-resolution crystal structure of the phospholipid- associated PAFAH enzymes will be solved. The use of detergents and amphiphilic molecules will be explored to provide higher resolution structures, as well as functionally more relevant structures, (iii) Inhibitors and substrate-mimics of PAFAH will be explored and developed via structural and kinetic characterization to elucidate in vivo physiological functions, (iv) The physiologically relevant reaction of PAFAH with organophosphate (OP) compounds will be characterized. We are interested in obtaining structural models to develop the LDL-associated pPAFAH as a practical therapeutic for people exposed to these toxic organophosphate insecticides and nerve agents.

描述（由申请人提供）：脂质代谢已成为人类健康问题的舞台方面和中心。在这里，很多关注的重点是与低密度脂蛋白（LDL）和高密度脂蛋白（HDL）相关的脂质的炎症和脂质稳态的机制。与这些LDL和HDL颗粒相关的许多蛋白质在这些脂质途径中起着至关重要的作用。血小板激活因子乙酰水合酶（PPAFAH）的血浆形式通过将信号传导分子血小板激活因子（PAF）的水平降低作为一般抗炎的清道夫而在LDL颗粒表面起作用，并与过敏性休克，哮喘和过敏反应有关。据信在肝脏和肾细胞中具有相似功能的同源细胞内形式，称为Pafah-II。磷脂相关的PPAFAH和PAFAH-II酶是值得的结构靶标。从生理上讲，这些酶与LDL颗粒或细胞内部小叶相关，因此被认为是界面酶，它们在脂质 - 水界面上起作用。除了降低PAF水平的作用外，它们还与其他促炎剂的水解活性有关，例如氧化脂质。我们将通过4个目标阐明PAF AH的结构和界面功能之间的关系：（i）将筛选添加剂/洗涤剂的异源表达和使用，以获得PPAFAH和PPAFAH-II II酶的均匀形式。蛋白质的质量将通过生物物理表征，功能测定和蛋白质晶体生长来评估，以获得适合结构和功能研究的单分散和可溶性的PAFAH，（ii）磷脂磷脂相关的Pafah酶的高分辨率晶体结构将得到解决。将探索清洁剂和两亲分子的使用，以提供更高的分辨率结构，以及在功能上更相关的结构，（iii）Pafah的抑制剂和底物模拟物，将通过结构和动力学的特性来探索和开发，以在体内的功能（（OP）中，（OP）在体液中（OP）在体内（OP），PAFAH，PAFAH，PAFAH，（OP）PAFAH，PAFAH，PAFAHS（OP）特征。我们有兴趣获得结构模型来开发与LDL相关的PPAFAH，作为暴露于这些有毒有机磷酸盐杀虫剂和神经剂的人的实际治疗方法。