Structural Basis of Membrane Targeting by NADPH Oxidase

描述(由申请人提供)：吞噬NADPH氧化酶(Phox)对于产生具有杀菌活性的活性氧物种至关重要。最近，非吞噬细胞NADPH氧化酶(NOx)亚型已被发现，它们能产生在信号转导中起作用的活性氧物种。这两种异构体都被脂质第二信使激活，如磷脂酰肌醇和磷脂酸，它们将氧化酶的胞液成分招募到膜上。本项目的目标是确定外周膜蛋白p47Phox、p40Phox和p47Phox同源物NOXO1识别这些激活脂类的结构基础。这项提案将一位对免疫细胞信号转导感兴趣的结构生物学家与一位在生物活性脂类对Phox调节的功能分析方面有广泛记录的合作调查者组成了团队。 目的1确定p47Phox、p40Phox和NOXO1的px结构域识别脂质的结构基础。虽然晶体结构显示了可溶性脂头基团相互作用的基础，但缺乏关于与脂类表面相互作用所需的界面的信息。我们的初步数据表明，各向同性的磷脂“双分子”是一种合适的膜模拟物，它将允许高分辨率， 磷脂双层结合蛋白的溶液状态核磁共振结构测定。 目的2验证p47Phox和NOXO1分子内相互作用通过这些蛋白调节脂质结合的假设。初步数据显示，磷脂酸结合到p47Phox的多碱区域，而NOXO1中缺乏该区域。此外，磷脂酰肌醇与全长蛋白的结合不如与分离的Px结构域结合有效。多碱区域和Px结构域似乎都与SH3结构域相互作用，这增加了脂类结合和/或其他修饰(例如多碱区域的磷酸化)改变蛋白质构象以允许与膜中的脂类和蛋白质更有效地相互作用的可能性。这些实验将提供关键的见解，以了解酶活性是如何调节的，以及吞噬和非吞噬形式的氧化酶在调节和激活方面的差异。

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.