Structure and Interfacial Function of Platelet Activating Factor Acetylhydrolase

描述（由申请人提供）：脂质代谢已成为人类健康问题的前沿和中心。在这里，许多注意力集中在炎症和脂质稳态的低密度脂蛋白（LDL）和高密度脂蛋白（HDL）相关的脂质的机制。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已在这些脂质途径中发挥关键作用。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）作为一般抗炎清除剂的水平而在LDL颗粒的表面上起作用，并且与过敏性休克、哮喘和过敏反应有关。被称为PAFAH-II的同源细胞内形式被认为在肝和肾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标。在生理学上，发现这些酶与LDL颗粒或细胞的内小叶相关，因此被认为是界面酶，其在脂质-水界面上起作用。除了降低PAF水平的作用外，它们还涉及其他促炎剂（如氧化脂质）的水解活性。我们将通过4个目的阐明PAF AH的结构和界面功能之间的关系：（i）将筛选添加剂/去污剂的异源表达和使用，以获得pPAFAH和PAFAH-II酶的均质形式。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，以获得适合于结构和功能研究的PAFAH的单分散和可溶形式。（ii）将解析磷脂相关PAFAH酶的高分辨率晶体结构。使用洗涤剂和两亲性分子将探索提供更高的分辨率的结构，以及功能上更相关的结构，（iii）抑制剂和底物模拟PAFAH将探索和开发通过结构和动力学表征，以阐明在体内的生理功能，（iv）生理相关的反应PAFAH与有机磷酸酯（OP）化合物的特点。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。

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.