Structural Basis of Membrane Targeting by NADPH Oxidase

描述（由申请方提供）：吞噬NADPH氧化酶（Phox）对于产生具有杀微生物活性的活性氧至关重要。最近，非吞噬细胞NADPH氧化酶（Nox）的异构体已被确定产生活性氧的功能，在信号转导。这两种亚型都被脂质第二信使激活，如磷脂酰肌醇和磷脂酸，它们将氧化酶的胞质组分募集到膜上。该项目的目标是确定这些激活脂质的外周膜蛋白p47 phox，p40 phox，和p47 phox同源物NOXO 1识别的结构基础。该提案由一位对免疫细胞信号传导感兴趣的结构生物学家与一位对生物活性脂质对Phox调节的功能分析有广泛记录的合作研究者组成。 目的1是确定p47 phox，p40 phox和NOXO 1的PX结构域识别脂质的结构基础。虽然晶体结构表明可溶性脂质头基相互作用的基础上，与脂质表面相互作用所需的接口的信息是缺乏的。我们的初步数据表明，各向同性的磷脂“双胞”是一种合适的膜模拟物， 磷脂双层结合蛋白质溶液状态核磁共振结构测定。 目的2检验p47 phox和NOXO 1分子内结合调节这些蛋白与脂质结合的假设。初步数据显示，磷脂酸与p47 phox的多碱基区域结合，这在NOXO 1中是缺乏的。此外，磷酸肌醇与全长蛋白质的结合不如与分离的PX结构域的结合有效。多碱基区和PX结构域似乎都与SH 3结构域相互作用，这增加了脂质结合和/或其他修饰（例如多碱基区的磷酸化）改变蛋白质构象以允许与膜中的脂质和蛋白质更有效相互作用的可能性。这些实验将提供关键的见解如何氧化酶活性的调节和调节的差异和激活的吞噬与非吞噬形式的氧化酶。

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.