Thioredoxin localization and its function in the airway

硫氧还蛋白定位及其在气道中的功能

• 批准号: 6788738
• 负责人: Richart W Harper
• 金额: $ 12.69万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6788738
• 关键词: DNA binding protein; SDS polyacrylamide gel electrophoresis; cell nucleus; fluorescence microscopy; gel mobility shift assay; gene expression; genetically modified animals; high performance liquid chromatography; human tissue; immunocytochemistry; in situ hybridization; interleukin 8; laboratory mouse; nuclear factor kappa beta; oxidative stress; ozone; protein structure function; protein transport; respiratory epithelium; respiratory function; secretory protein; thioredoxin; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant) The primary objective of this research proposal is to identify how compartmentalization of thioredoxin (TRX) in the cell affects its function. The specific aims of this project are to identify where TRX localizes under conditions of oxidative stress, and how this localization affects gene regulation. The objectives of the grant proposal will be achieved in four distinct parts. First, we plan to show that TRX preferentially accumulates in the nucleus during oxidative stress. Second, we will manipulate the TRX gene in order to direct it to either localize in the nucleus or be secreted. We will construct TRX fusion plasmids that contain either TRX attached to a nuclear localization sequence or TRX attached to a secretory sequence. We will then compare differences in NF-kappa B- and AP-1 dependent promoter activity in cells overexpressing nuclear TRX compared with cells overexpressing secreted TRX. This will help establish the functional role of TRX compartmentalization. Third, we will establish that TRX localization is important for modulating interleukin-8 (IL-8) gene activity. Forth, using similar TRX expression plasmids as constructed for specific aim two, we will examine the effects of nucleus-directed TRX versus secreted TRX in mice exposed to ozone. Using this in vivo model, we plan to confirm the findings we obtained in our in vitro studies. The principal investigator's immediate research goals are to better understand the signaling mechanisms involved in airway epithelial cells in response to oxidative stress. Consistent with this objective, he currently has received funding for a three-year project examining the signaling mechanisms responsible for up-regulating IL-8 in airway epithelial cells exposed to cigarette smoke. It is the plan of the principle investigator to use this career development award as an excellent mechanism to transition into an independent investigator over the next five years. It is his hope that this grant funding will provide him with the protected research time needed in order to obtain the experience and publications needed to make him a competitive candidate for R01 funding.

描述（由申请人提供） 本研究计划的主要目标是确定如何 细胞内硫氧还蛋白 (TRX) 的区室化会影响其功能。 该项目的具体目标是确定 TRX 在以下位置的定位： 氧化应激的条件，以及这种定位如何影响基因 规定。 赠款提案的目标将在四个时间内实现 不同的部分。 首先，我们计划证明 TRX 优先积累在 氧化应激期间的细胞核。 其次，我们将操纵TRX基因 以引导其定位于细胞核或被分泌。 我们 将构建 TRX 融合质粒，其中包含连接到 核定位序列或连接到分泌序列的TRX。 我们 然后将比较 NF-kappa B- 和 AP-1 依赖性启动子的差异 与细胞相比，过表达核 TRX 的细胞的活性 过度表达分泌的 TRX。 这将有助于确立职能角色 TRX 分区。 第三，我们将确定TRX本地化 对于调节白细胞介素 8 (IL-8) 基因活性很重要。 四、利用 与为特定目标二构建的类似 TRX 表达质粒一样，我们将 检查小鼠中细胞核定向 TRX 与分泌 TRX 的效果 暴露于臭氧。 我们计划使用这个体内模型来确认研究结果 我们在体外研究中获得。 首席研究员的直接 研究目标是更好地理解参与的信号机制 气道上皮细胞对氧化应激的反应。 与此一致 目标，他目前已获得一个为期三年的项目的资助 检查负责上调 IL-8 的信号机制 气道上皮细胞暴露于香烟烟雾中。 这是该计划的 首席研究员将此职业发展奖作为优秀 在未来五年内过渡为独立调查员的机制 年。 他希望这笔赠款资金能为他提供 获得经验和所需的受保护的研究时间 需要发表出版物才能使他成为 R01 资助的有竞争力的候选人。