Structure and Interfacial Function of Platelet Activating Factor Acetylhydrolase

描述（申请人提供）：脂质代谢已成为人类健康关注的前沿和中心问题。低密度脂蛋白（LDL）和高密度脂蛋白（HDL）相关的脂质在炎症和脂质稳态中的作用机制一直备受关注。许多与这些低密度脂蛋白和高密度脂蛋白颗粒相关的蛋白质在这些脂质途径中起着关键作用。血浆形式的血小板活化因子乙酰水解酶（pPAFAH）通过降低信号分子血小板活化因子（PAF）的水平在LDL颗粒表面起作用，作为一种一般的抗炎清道夫，与过敏性休克、哮喘和过敏反应有关。一种同源的细胞内形式，被称为paah - ii，被认为在肝脏和肾脏细胞中具有类似的功能。磷脂相关的pPAFAH和PAFAH-II酶是值得研究的结构靶点。生理学上，这些酶被发现与LDL颗粒或细胞内小叶有关，因此，被认为是界面酶，其作用于脂质-水界面。除了降低PAF水平外，它们还与其他促炎剂（如氧化脂质）的水解活性有关。我们将通过4个目标阐明PAFAH的结构和界面功能之间的关系：(i)筛选添加剂/洗涤剂的异源表达和使用，以获得均匀形式的pPAFAH和PAFAH-II酶。蛋白质的质量将通过生物物理特性、功能分析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形式的PAFAH。（ii）磷脂相关的PAFAH酶的高分辨率晶体结构将得到解决。将探索洗涤剂和两亲分子的使用，以提供更高分辨率的结构，以及功能上更相关的结构，（iii）将通过结构和动力学表征探索和开发PAFAH的抑制剂和底物模拟物，以阐明体内生理功能，（iv）将表征PAFAH与有机磷酸盐（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ldl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用治疗方法。

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.