Thioredoxin localization and its function in the airway

描述(由申请人提供) 这项研究提案的主要目标是确定如何 硫氧还蛋白(Trx)在细胞内的区隔作用影响其功能。 该项目的具体目标是确定TRX在以下方面的本地化 氧化应激的条件，以及这种定位如何影响基因 监管。拨款建议的目标将在四年内实现 不同的部分。首先，我们计划证明Trx优先在 氧化应激过程中的细胞核。第二，我们将操纵trx基因 以便引导它要么定位于细胞核，要么被分泌。我们 将构建含有Trx的融合质粒，其中一个Trx连接到 核定位序列或连接到分泌序列的Trx。我们 然后将比较依赖于核因子-kappaB和AP-1的启动子的差异 核Trx过表达细胞的活性与细胞的比较 过度表达分泌型Trx。这将有助于确立 Trx分区。第三，我们将确定TRX本地化是 对调节白介素8(IL-8)基因活性很重要。第四，使用 类似于为特定目的2构建的Trx表达载体，我们将 核导TRX与分泌型TRX在小鼠体内的作用比较 暴露在臭氧中。利用这个活体模型，我们计划证实这一发现 我们在体外研究中获得了。首席调查员的直接 研究目标是更好地了解涉及到的信号机制 呼吸道上皮细胞对氧化应激的反应。与此一致 目标，他目前已经收到了一个为期三年的项目的资金 探讨上调IL-8的信号机制 暴露于香烟烟雾的呼吸道上皮细胞。这是美国政府的计划 首席调查员将此职业发展奖作为优秀的 在未来五年内过渡为独立调查员的机制 好几年了。他希望这笔赠款将为他提供 需要受保护的研究时间以获得经验和 出版物需要让他成为R01资金的有竞争力的候选人。

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.