STRUCTURE AND FUNCTION OF PLATELET ACTIVATING FACTOR ACETYLHYDROLASE TYPE-II

血小板活化因子乙酰水解酶II型的结构和功能

• 批准号: 7720311
• 负责人: BRIAN J BAHNSON
• 金额: $ 3.04万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720311
• 关键词: Allergic Reaction; Anti-Inflammatory Agents; Anti-inflammatory; Asthma; Atherosclerosis; Binding; Biochemical; Cell membrane; Cells; Computer Retrieval of Information on Scientific Projects Database; Confocal Microscopy; Cyan Fluorescent Protein; Enzymes; Escherichia coli; Funding; Goals; Grant; Health; Hepatocyte; Human; In Situ; In Vitro; Institution; Kidney; Link; Membrane; Molecular; Oxidative Regulation; Oxidative Stress; Physiological; Platelet Activating Factor; Research; Research Personnel; Resources; Role; Signaling Molecule; Source; Structure; System; Tail; United States National Institutes of Health; Vesicle; biological adaptation to stress; in vivo; interfacial; kidney cell; research study

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 血小板激活因子乙酰水合酶（PAFAH-II）的细胞内形式通过降低信号传导分子血小板激活因子（PAF）的水平作为一般的抗炎剂，并与哮喘，过敏反应和动脉粥样硬化有关。从生理上讲，据信，细胞内形式的pafah-ii在与肾脏和肝细胞的内叶相关后具有功能活跃。该项目的目的是阐明Pafah-II中氧化应激反应的机制。对Pafah-II的结构和功能的分子理解将直接影响人类健康。我们将对PAFAH-II酶的结构透视，生物物理表征以及体内定位和氧化调节。我们旨在以两种相关方式阐明Pafah-II的结构与界面功能之间的关系。 1）pafah-ii在体内被N末端肉豆蔻酰化，据信这种肉豆蔻酰基基序可将酶引导到氧化应激反应后将酶引导到细胞的内部小叶。我们的目的是使用体外和原位方法来表征Myristoyl尾巴在膜定位中的作用。与共表达的肉豆蔻酰转移酶在大肠杆菌中的表达将允许Pafah-II与PC囊泡结合的生物物理和生化表征。同时，我们将在人肾细胞中与青色荧光蛋白进行融合表达Pafah-II，然后将共聚焦显微镜定位。这将更好地理解Myristoyl图案对Pafah-II N末端的功能。 2）在大肠杆菌和人类肾细胞中肉豆蔻酰化的Pafah-II成功表达之后，我们将定义氧化应激反应背后的机制，从而导致Pafah-II定位到细胞膜。最初的实验将着重于使用大肠杆菌的体外系统表达氧化应激的结果。然后，将在人类肾细胞中探索有希望的铅，以验证我们发现的生理相关性。