Structure and Interfacial Function of Platelet Activating Factor Acetylhydrolase

描述(申请者提供)：脂类代谢已成为人类健康关注的焦点。低密度脂蛋白(LDL)和高密度脂蛋白(高密度脂蛋白)相关的脂类在炎症和脂平衡中的作用机制引起了人们的极大关注。与这些低密度脂蛋白和高密度脂蛋白颗粒相关的许多蛋白质已经出现，在这些脂质途径中发挥着关键作用。血浆形式的血小板激活因子乙酰水解酶(PPAFAH)通过降低信号分子血小板激活因子(PAF)作为一般抗炎清除剂的水平在低密度脂蛋白颗粒表面发挥作用，与过敏性休克、哮喘和过敏反应有关。一种被称为PAFAH-II的同源细胞内形式被认为在肝脏和肾脏细胞中具有相似的功能。磷脂相关的pPAFAH和PAFAH-II酶是有价值的结构靶点。生理上，这些酶被发现与低密度脂蛋白颗粒或细胞内叶相关，因此，被认为是界面酶，在脂质-水界面上发挥作用。除了降低PAF水平的作用外，它们还参与了其他促炎剂的水解性活动，如氧化脂质。我们将通过四个目标来阐明PAF AH的结构和界面功能之间的关系：(I)筛选不同的添加剂/洗涤剂的表达和使用，以获得均一形式的pPAFAH和PAFAH-II酶。将通过生物物理表征、功能测定和蛋白质晶体生长来评估蛋白质的质量，以获得适合于结构和功能研究的单分散和可溶形式的PAFAH。(Ii)将解决与磷脂相关的PAFAH酶的高分辨晶体结构。将探索洗涤剂和两亲性分子的使用，以提供更高分辨率的结构以及更相关的结构，(Iii)将通过结构和动力学表征来探索和开发多环芳烃的抑制剂和底物模拟物，以阐明体内的生理功能，(Iv)将表征多环芳烃与有机磷(OP)化合物的生理相关反应。我们有兴趣获得结构模型，以开发与低密度脂蛋白相关的pPAFAH，作为暴露于这些有毒有机磷杀虫剂和神经毒剂的人的实际治疗方法。

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.