Spatial Control and Function of Nuclear Superoxide in LPS-mediated Cell Signaling

描述（由申请人提供）： 该建议描述了一个为期五年的培训计划，以发展基础科学的学术研究生涯。主要研究者是爱荷华州大学的博士后研究员。她在这个项目中的长期目标是建立一个独立的研究生涯，研究健康和疾病中氧化还原信号的细胞生物学。她的导师是约翰恩格尔哈特博士，教授和解剖学和细胞生物学系主任。研究环境包括获得所需的所有设备和用品，以及在主要研究者感兴趣的领域具有专业知识的教师咨询委员会。细胞内氧化还原调节的信号传导途径是通过空间控制的各种亚细胞区室内活性氧的产生来协调的。尽管三十多年来人们已经知道超氧化物是在细胞核中局部产生的，但其在该部位的功能和调节仍然完全未知。该项目的广泛，长期目标是研究核膜上的超氧化物生成酶如何被激活和调节，以及它们如何参与调节损伤反应基因的转录。我们的初步数据表明，核NADPH氧化酶亚型4（NOX 4）是从细胞中的其他地方发现的NOX 4复合物功能不同。我们的第一个目标将调查的亚基组成的核NOX 4复合物和阐明的拓扑结构和核膜的超氧化物生产的渗透性。在目标2中，我们假设细胞核中的NOX 4调节部分依赖于其底物NADPH的局部可用性，并且细胞核中的葡萄糖-6-磷酸脱氢酶在LPS应激条件下被募集到NOX 4（我们知道这会提高细胞核NOX 4活性）。目的3假设核超氧化物在控制包括NF κ B和AP-1在内的几种氧化还原调节转录因子的活性中起作用。这些转录因子的控制核超氧化物信号将使用几个转录因子报告分析研究。这些研究首次揭示了核超氧化物的生理功能和调控机制。 公共卫生相关性： 革兰氏阴性细菌感染释放的LPS可导致快速临床恶化和休克，通常需要在ICU环境中护理。该提案旨在了解LPS如何导致肝细胞核超氧化物自由基产生增加。这项研究将增强我们对LPS介导的损伤的理解，并可能为内毒素介导的脓毒症提供新的治疗方法。

DESCRIPTION (provided by applicant): This proposal describes a five-year training program for the development of an academic research career in basic science. The principal investigator is a postdoctoral research fellow at The University of Iowa. Her long-term goal in this project is the establishment of an independent research career studying the cell biology of redox-signaling in health and disease. Her mentor is Dr. John Engelhardt, professor and Chair of the Department of Anatomy and Cell Biology. The research environment includes access to all equipment and supplies needed as well as an advisory committee of faculty members with expertise in areas of interest to the principal investigator. Intracellular redox-regulated signaling pathways are coordinated by the spatially controlled production of reactive oxygen species within various subcellular compartments. Despite the fact that it has been known for over three decades that superoxide is produced locally in the nucleus, its function and regulation at this site remain completely unknown. The broad, long-term objective of this project is to investigate how superoxide-producing enzymes at nuclear membranes are activated and regulated, and how they may be involved in regulating the transcription of injury response genes. Our preliminary data indicate that nuclear NADPH oxidase isoform 4 (NOX4) is functionally distinct from the NOX4 complex found elsewhere in the cell. Our first aim will investigate the subunit composition of the nuclear NOX4 complex and elucidate the topology and permeability of superoxide production by nuclear membranes. In aim 2, we hypothesize that NOX4 regulation in the nucleus is in part dependent on the localized availability of its substrate NADPH and that glucose-6-phosphate dehydrogenase in the nucleus is recruited to NOX4 under conditions of LPS stress (which we know elevates nuclear NOX4 activity). Aim 3 hypothesizes that nuclear superoxide plays a role in controlling the activity of several redox-regulated transcription factors, including NFkB and AP-1. The control of these transcription factors by nuclear superoxide signals will be studied using several transcription factor reporter assays. These studies provide the first investigation of physiological functions and regulation of nuclear superoxide. PUBLIC HEALTH RELEVANCE: LPS released by gram-negative bacterial infections can cause rapid clinical deterioration and shock, often requiring care in an ICU setting. This proposal seeks to understand how LPS leads to an increase in nuclear superoxide free radical production by liver cells. The proposed research will enhance our understanding of LPS-mediated injury and may lead to new therapeutic approaches for endotoxin-mediated sepsis.