STRUCTURE AND FUNCTION OF PLATELET ACTIVATING FACTOR ACETYLHYDROLASE TYPE-II

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 血小板活化因子乙酰水解酶（PAFAH-II）的细胞内形式通过降低作为一般抗炎清除剂的信号分子血小板活化因子（PAF）的水平而起作用，并且与哮喘、过敏反应和动脉粥样硬化有关。在生理学上，认为细胞内形式PAFAH-II在与肾和肝细胞的内小叶结合后具有功能活性。本项目的目的是阐明PAFAH-II中氧化应激反应的机制。对PAFAH-II结构和功能的分子认识将直接影响人类健康。我们将结合在一起的结构的角度来看，生物物理特性，并在体内定位和氧化调节PAFAH-II酶。我们的目的是阐明PAFAH-II的结构和界面功能之间的关系，在两个相关的方式。1)PAFAH-II在体内是N-末端肉豆蔻酰化的，并且该肉豆蔻酰基序被认为在氧化应激反应后将酶引导至细胞的内小叶。我们的目的是表征的作用，肉豆蔻酰尾膜定位使用在体外和原位方法。在大肠杆菌中的表达共表达肉豆蔻酰转移酶的大肠杆菌将允许PAFAH-II结合PC囊泡的生物物理和生物化学表征。平行地，我们将表达PAFAH-II作为与青色荧光蛋白在人肾细胞中的融合物，随后通过共聚焦显微镜定位。这将提供一个更好的了解的功能的肉豆蔻酰基序的N-末端的PAFAH-II。2)在E.大肠杆菌和人肾细胞中，我们将定义氧化应激反应背后的机制，导致PAFAH-II定位到细胞膜。初步的实验将集中在表征氧化应激的结果使用在体外系统的E。coli表达肉豆蔻酚和非肉豆蔻酰PAFAH-II。然后将在人类肾脏细胞中探索有希望的线索，以验证我们发现的生理相关性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The intracellular form of platelet activating factor acetylhydrolase (PAFAH-II) functions by reducing levels of the signaling molecule platelet activating factor (PAF) as a general anti-inflammatory scavenger and is linked to asthma, allergic reactions and atherosclerosis. Physiologically, the intracellular form PAFAH-II is believed to be functionally active after association with the inner-leaflet of kidney and liver cells. The goal of this project is to elucidate the mechanism of the oxidative stress response in PAFAH-II. The molecular understanding of structure and function of PAFAH-II will directly impact human health. We will tie together a structural perspective, biophysical characterization, and in vivo localization and oxidative regulation for the PAFAH-II enzyme. We aim to elucidate the relationship between structure and interfacial function of PAFAH-II in two related ways. 1) PAFAH-II is N-terminally myristoylated in vivo, and this myristoyl motif is believed to direct the enzyme to the inner leaflet of cells following an oxidative stress response. We aim to characterize the role of the myristoyl tail for membrane localization using both in vitro and in situ approaches. Expression in E. coli with coexpressed myristoyl transfer enzymes will allow a biophysical and biochemical characterization of PAFAH-II binding to PC vesicles. In parallel we will express PAFAH-II as a fusion with cyan fluorescent protein in human kidney cells followed by confocal microscopy localization. This will provide a better understanding of the function of the myristoyl motif on the N-terminus of PAFAH-II. 2) Following the successful expression of myristoylated PAFAH-II in E. coli and human kidney cells we will define the mechanism behind the oxidative stress response that leads to PAFAH-II localization to the cell membrane. Initial experiments will focus on characterizing the result of oxidative stress using the in vitro system of E. coli expressed myristyol and non-myristoyl PAFAH-II. Promising leads will then be explored in the human kidney cells to validate the physiological relevance of our findings.