S-nitrosothiol localization and signaling in airway epithelial cells

S-亚硝基硫醇在气道上皮细胞中的定位和信号传导

• 批准号: 8375026
• 负责人: Benjamin Gaston
• 金额: $ 39.03万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8375026
• 关键词: Address; Affect; Air; Asthma; Biology; Cell Culture Techniques; Cell Signaling Process; Cellular biology; Cilia; Columnar Epithelium; Cysteine; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Development; Disease; Dissociation; Endoplasmic Reticulum; Epithelial; Epithelial Cells; Epithelium; Esters; Functional disorder; Heat-Shock Proteins 90; Human; Humulus; In Vitro; Liquid substance; Location; Lung diseases; Maps; Metabolism; Modeling; Modification; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Nitrogen Oxides; Post-Translational Protein Processing; Process; Protein Isoforms; Protein S; Proteins; Reaction; Research Personnel; S-Nitrosoglutathione; S-Nitrosothiols; Signal Pathway; Signal Transduction; Stress; Sulfhydryl Compounds; Technology; Testing; Ubiquitination; Up-Regulation; airway epithelium; base; cystic fibrosis airway epithelia; cystic fibrosis mouse; cystic fibrosis patients; experience; genetic regulatory protein; human NOS2A protein; human NOS3 protein; in vivo; mouse model; programs; protein function; respiratory; trafficking

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.

一氧化氮合酶（NOS）激活可能导致形成相关的氮氧化物疾病，而不是一氧化氮自由基。其中之一是亚硝酸盐。另外，共价半胱氨酸修饰形成了索硫醇键。当这种半胱氨酸的修饰信号标志着蛋白质功能的变化时，通常称为sn硝化。 这些反应越来越多地识别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AINWay上皮S-硝基化信号的疾病。然而，在气道上皮细胞培养物中，S-硝基硫醇化蛋白的形成和位置尚不清楚，并且在原代，假柱状柱状AINWay上皮中尚未研究。为了开始理解NOS依赖性的Snitrosothiol 与疾病有关的Ainways中的形式，我们将检验三个假设，即1）正常人类气道上皮细胞中的特定蛋白质通过NOS进行S-硝基化（AIM 1）； 2）蛋白质的sn依蛋白发生发生在人气道上皮细胞的特定亚细胞位置（AIM 2）； 3）S-亚硝基化信号在人CF气道上皮中无序（AIM 3）。 我们选择通过诱导和内皮NOS（iNOS和eNOS）同工型研究S-硝基化，因为每个人都在正常的人类Ainway上皮细胞中表达并活跃。并且因为CF AINWAY上皮中的iNOS表达降低可能具有重要的疾病意义。选择HSP70/HSP90组织蛋白来研究Inos和eNOS活性下游的S-硝基硫醇信号传导的范式，因为1）它在基线时已被S-硝基基化； 2）其S-亚硝基化随着Snitrosoglutathione的增加而增加 治疗; 3）其S-亚硝基化似乎对Ainway上皮细胞很重要 一般而言，生物学，特别是CF的病理生物学和治疗。我们选择主要在人类气道伪造的柱状上皮培养物中进行研究，因为这些培养物最紧密地概括了体内人类Ainway。我们将在小鼠模型中进行其他体内研究。 该项目将广泛使用该计划中其他项目的研究人员的互动，每个目标都将利用该计划中的一个或多个核心。在该项目的结论中，我们预计我们将有1）一个机制的功能模型，通过该模型，NOS激活会导致人类假性分层柱状上皮的特定细胞位置中特定蛋白质的sn依蛋白； 2）S-硝基硫醇形成疾病与开发新疗法的相关性。