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

描述(由申请人提供)： 这项建议描述了一项为期五年的培训计划，旨在发展基础科学的学术研究生涯。首席研究员是爱荷华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，他是解剖学和细胞生物学系的教授和主任。研究环境包括获得所需的所有设备和用品，以及由在主要研究人员感兴趣的领域拥有专门知识的教职员工组成的咨询委员会。细胞内氧化还原调节的信号通路是通过在不同的亚细胞内产生空间受控的活性氧来协调的。尽管三十多年来人们已经知道超氧化物是在原子核中局部产生的，但它在这个位置的功能和调节仍然完全不清楚。该项目的长期目标是研究核膜上产生超氧化物歧化酶是如何被激活和调节的，以及它们如何参与调控伤害反应基因的转录。我们的初步数据表明，核NADPH氧化酶亚型4(NOX4)在功能上不同于在细胞其他地方发现的NOX4复合体。我们的第一个目标是研究核NOX4复合体的亚基组成，并阐明核膜产生超氧化物的拓扑结构和通透性。在目标2中，我们假设核中NOX4的调节部分依赖于其底物NADPH的局部可获得性，并且在内毒素应激(我们知道这会提高核中NOX4活性)的条件下，核中的葡萄糖-6-磷酸脱氢酶被招募到NOX4。目的3假设核超氧化物对包括NFkB和AP-1在内的几种受氧化还原调节的转录因子的活性具有调控作用。这些转录因子受核超氧化物信号的控制将使用几种转录因子报告分析来研究。这些研究首次对核超氧化物的生理功能和调控进行了研究。

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.