Structural Basis of Membrane Targeting by NADPH Oxidase

NADPH 氧化酶膜靶向的结构基础

• 批准号: 6907728
• 负责人: DAVID A HORITA
• 金额: $ 35.01万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6907728
• 关键词: NAD(P)H dehydrogenase; active sites; biological signal transduction; conformation; enzyme activity; enzyme induction /repression; enzyme mechanism; enzyme structure; free radical oxygen; inflammation; intermolecular interaction; isozymes; lipids; membrane proteins; nuclear magnetic resonance spectroscopy; phosphatidate; phosphatidylinositols; phosphorylation; protein structure function; second messengers; structural biology

DESCRIPTION (provided by applicant): Phagocytic NADPH oxidase (Phox) is critically important for generating reactive oxygen species with microbicidal activity. Recently, nonphagocytic NADPH oxidase (Nox) isoforms have been identified which generate reactive oxygen species that function in signal transduction. Both isoforms are activated by lipid second messengers such as phosphatidylinositols and phosphatidic acid which recruit cytosolic components of the oxidase to the membrane. The goal of this project is to determine the structural basis for recognition of these activating lipids by the peripheral membrane proteins p47phox, p40phox, and the p47phox homolog NOXO1. This proposal teams a structural biologist with an interest in immune cell signaling with a coinvestigator with an extensive track record of functional analysis of Phox regulation by bioactive lipids. Aim 1 is to determine the structural basis of lipid recognition by the PX domains of p47phox, p40phox, and NOXO1. While crystal structures indicate the basis for soluble lipid head group interactions, information regarding the interfaces required for interaction with a lipid surface is lacking. Our preliminary data demonstrate that isotropic, phospholipid "bicelles" are a suitable membrane mimetic that will allow high-resolution, solution-state NMR structure determination of phospholipid bilayer-bound protein. Aim 2 tests the hypothesis that intramolecular associations within p47phox and NOXO1 regulate lipid binding by these proteins. Preliminary data show that phosphatidic acid binds to the polybasic region of p47phox, which is lacking in NOXO1. In addition, binding of phosphoinositides to the full-length protein is less effective than binding to the isolated PX domain. Both the polybasic region and the PX domain appear to interact with the SH3 domains, raising the possibility that binding of lipids and/or other modifications (e.g. phosphorylation of the polybasic region) alters the conformation of the proteins to allow more effective interaction with lipids and proteins in the membrane. These experiments will provide key insights into how oxidase activity is regulated and the differences in regulation and activation of phagocytic vs. nonphagocytic forms of the oxidase.

描述（由申请人提供）：吞噬NADPH氧化酶（Phox）对于产生具有微生物活性的活性氧非常重要。最近，已经鉴定出了非晶状细胞NADPH氧化酶（NOX）同工型，从而产生在信号转导中起作用的活性氧。两种同工型都被脂质第二信使（例如磷脂酰肌醇和磷脂酸）激活，它们募集氧化酶的胞质成分到膜。该项目的目的是确定周围膜蛋白P47phox，p40phox和P47Phox同源物NOXO1识别这些激活脂质的结构基础。该提案团队由一名结构生物学家与共同评估仪对免疫细胞信号传导感兴趣，并具有广泛的生物活性脂质调节功能分析的记录。 目的1是确定p47phox，p40phox和noxo1的PX结构域的脂质识别的结构基础。尽管晶体结构表示可溶性脂质头组相互作用的基础，但缺乏有关与脂质表面相互作用所需的接口的信息。我们的初步数据表明，各向同性的磷脂“双核糖”是一种合适的膜模拟物，可以高分辨率， 溶液状态NMR结构的磷脂双层结合蛋白的测定。 AIM 2检验了以下假设：p47phox和NoxO1内分子内关联调节这些蛋白质的脂质结合。初步数据表明，磷酸酸与NoxO1缺乏的p47phox的多性区域结合。另外，磷酸肌醇与全长蛋白的结合不如与分离的PX结构域结合的有效性。多巴区域和PX结构域似乎都与SH3结构域相互作用，从而增加了脂质和/或其他修饰的结合（例如，多基质区域的磷酸化）改变了蛋白质的构象，可以使蛋白质的构象可以更有效地与膜中的脂质和蛋白质相互作用。这些实验将提供有关氧化酶活性如何受到调节以及氧化酶与非吞噬形式的调节和激活的差异的关键见解。