Thioredoxin localization and its function in the airway

描述（由申请人提供） 这项研究的主要目的是确定如何 细胞中硫氧还蛋白（TRX）的区室化影响其功能。 该项目的具体目标是确定TRX在 氧化应激的条件，以及这种定位如何影响基因 调控 拨款建议的目标将在四个方面实现 不同的部分。 首先，我们计划证明TRX优先在 氧化应激时的细胞核。 第二，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，该质粒含有连接到 核定位序列或与分泌序列连接的TRX。 我们 然后将比较NF-κ B-和AP-1依赖性启动子的差异 与细胞相比，在过表达核TRX的细胞中的活性 过表达分泌的TRX。 这将有助于确立 TRX区室化。 第三，我们将确定TRX本地化是 对于调节白细胞介素-8（IL-8）基因活性是重要的。 第四，使用 类似于为特定目的二构建的TRX表达质粒，我们将 检查核定向TRX与分泌TRX在小鼠中的作用 暴露在臭氧中。 使用这种体内模型，我们计划证实这些发现 我们在体外研究中获得的。 首席调查员会立即 研究目标是更好地了解参与的信号机制， 气道上皮细胞对氧化应激的反应。 符合本 目标，他目前已收到一个为期三年的项目的资金 检查负责上调IL-8的信号传导机制， 暴露于香烟烟雾的气道上皮细胞。 这是计划的 首席调查员使用这个职业发展奖作为一个优秀的 在未来五年内过渡为独立调查员的机制 年 他希望这笔赠款将为他提供 为了获得经验，需要保护研究时间， 出版物需要使他成为R 01资金的竞争候选人。

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.