S-nitrosothiol localization and signaling in airway epithelial cells

一氧化氮合酶（NOS）的激活可导致相关的一氧化氮疾病的形成，而不是一氧化氮自由基。其中之一是亚硝酸盐。此外，共价半胱氨酸修饰形成S亚硝基硫醇键。当这种半胱氨酸修饰信号改变蛋白质功能时，它通常被称为S亚硝基化。 这些反应越来越多地被认为代表代谢调节的细胞信号传导过程。在包括哮喘和囊性纤维化（CF）在内的一系列疾病中已经观察到气道上皮S-亚硝基化信号传导障碍。然而，S-亚硝基硫醇修饰的蛋白质的形成和定位在一般的气道上皮细胞培养中知之甚少，并且还没有在原发性假复层柱状气道上皮中进行研究。为了开始了解NOS依赖的亚硝基硫醇 为了研究与疾病相关的气道中的S-亚硝基化，我们将检验以下三个假设：1）正常人气道上皮细胞中的特定蛋白质被NOS S-亚硝基化（目的1）; 2）蛋白质S-亚硝基化发生在人气道上皮细胞中的特定亚细胞位置（目的2）;以及3）人CF气道上皮中的S-亚硝基化信号传导紊乱（目的3）。 我们选择通过诱导型和内皮型NOS（iNOS和eNOS）亚型研究S-亚硝基化，因为每种亚型在正常人气道上皮细胞中表达和活跃;并且因为CF气道上皮中INOS表达降低可能具有重要的疾病意义。选择Hsp 70/Hsp 90组织蛋白来研究iNOS和eNOS活性下游的S-亚硝基硫醇信号传导模式，因为1）其在基线处是S-亚硝基化的; 2）其S-亚硝基化随着S-亚硝基谷胱甘肽的增加而增加。 治疗; 3）其S-亚硝基化似乎对气道上皮细胞很重要 一般涉及生物学，特别涉及CF的病理生物学和治疗。我们选择主要在人气道假复层柱状上皮细胞培养物中进行研究，因为这些培养物最接近于体内人气道。我们将在小鼠模型中进行额外的体内研究。 本项目将广泛利用与本项目中其他项目的研究人员的互动，每个目标将利用该项目中的一个或多个核心。在这个项目的结论，我们预计，我们将有1）一个功能模型的机制，NOS激活导致S亚硝基化的特定蛋白质在特定的细胞位置在人类假复层柱状上皮;和2）的相关性S-亚硝基硫醇形成的疾病的CF的新疗法的发展。

Nitric oxide synthase (NOS) activation can result in formation of relevent nitrogen oxide diseases, other than nitric oxide radicals. One of these is nitrite. Additionally, covalent cysteine modifications form Snitrosothiol bonds. When this cysteine modification signals a change in protein function, it is often termed Snitrosylation. These reactions are increasingly recognized to represent metabolically regulated cell signaling processes. Disorders of ainway epithelial S-nitrosylation signaling have been observed in a range of diseases, including asthma and cystic fibrosis (CF). However, the formation and location of S-nitrosothiolmodified proteins is poorly understood in airway epithelial cell cultures in general, and has not been studied in primary, pseudostratified columnar ainway epithelium. In order to begin to understand NOS-dependent Snitrosothiol formafion in the ainways as it relates to disease, we will test the three hypotheses that 1) specific proteins in normal human airway epithelial cells are S-nitrosylated by NOS (Aim 1); 2) protein Snitrosylation occurs in specific subcellular locations in human airway epithelial cells (Aim 2); and 3) S-nitrosylation signaling is disordered in the human CF airway epithelium (Aim 3). We chose to study S-nitrosylation by the inducible and endothelial NOS (iNOS and eNOS) isoforms because each is expressed and active in normal human ainway epithelial cells; and because decreased INOS expression in the CF ainway epithelium may have important disease implications. Hsp70/Hsp90 organizing protein was chosen to study the paradigm of S-nitrosothiol signaling downstream from iNOS and eNOS activity because 1) it is S-nitrosylated at baseline; 2) its S-nitrosylation increases with Snitrosoglutathione treatment; and 3) its S-nitrosylation appears to be important to ainway epithelial cell biology in general, and to the pathobiology and treatment of CF in particular. We have chosen to perform our studies primarily in human airway pseudostratified columnar epithelial cultures because these most closely to recapitulate the human ainway in vivo. We will do additional in vivo studies in a mouse model. This project will make extensive use of interactions with investigators on the other projects in this program, and each aim will make use of one or more cores in the program. At the conclusion of this project, we anticipate that we will have 1) a functional model of mechanisms by which NOS activation leads to Snitrosylation of specific proteins in specific cellular locations in human pseudostratified columnar epithelium; and 2) the relevance of disorders of S-nitrosothiol formation to the development of new therapies for CF.