DJ-1 pathway impairment in alveolar type II cells in emphysema.

描述(申请人提供)：肺气肿由一种独特的肺泡壁破坏模式组成，对这种疾病的治疗有限。吸烟会导致氧化应激，是导致肺气肿的最常见原因。我们的应用旨在明确DJ-1通路损伤在本病中的作用机制。DJ-1是一种多功能蛋白质，可以保护细胞免受氧化应激的伤害。这取决于DJ-1中半胱氨酸和蛋氨酸残基的翻译后修饰。Kelch样ECH相关蛋白1(Keap1)和CR6相互作用因子1(CRIF1)也被认为是调节氧化应激的因子。DJ-1-CRIF1-Keap1相互作用诱导抗氧化防御系统对抗吸烟对肺泡上皮细胞的损伤。我们拟在体外和体外研究DJ-1对香烟烟雾致人原代肺泡上皮细胞损伤的保护作用机制。我们将使用质谱分析来确定DJ-1中半胱氨酸和蛋氨酸残基的氧化。我们将使用突变结构来鉴定可氧化半胱氨酸和蛋氨酸残基(S)对DJ-1保护香烟烟雾损伤的作用至关重要。我们将重点关注第106位半胱氨酸残基(Cys-106)，它是DJ-1内氧化应激的传感器。为了进一步确定DJ-1途径，我们将使用删除构建法分析DJ-1、CRIF1和Keap1相互作用的结构域。我们还将在我们的研究中使用从轻、中、重度肺气肿患者中分离出的肺泡II型细胞。这一独特的方法将使我们能够确定DJ-1通路损伤在肺气肿进展中的机制。我们将在体内的肺泡II型细胞中验证我们的结果。野生型小鼠将暴露在香烟中 烟雾氧化应激条件下用质谱仪测定DJ-1半胱氨酸和蛋氨酸修饰。我们还将确定腺病毒DJ-1是否能拯救DJ-1基因敲除小鼠因吸烟造成的肺泡II型细胞损伤。对肺气肿DJ-1通路损伤机制的研究可能导致开发新的药理学策略来减缓肺气肿的进展。

DESCRIPTION (provided by applicant): Emphysema consists of a unique pattern of alveolar wall destruction and there is limited therapy against this disease. Cigarette smoking induces oxidative stress and is the most common cause of pulmonary emphysema. Our application seeks to define the mechanism of DJ-1 pathway impairment in this disease. DJ-1 is a multifunctional protein that protects cells from oxidative stress. This depends on posttranslational modifications of cysteine and methionine residues within DJ-1. Kelch-like ECH-associated protein 1 (KEAP1) and CR6-interacting Factor 1 (CRIF1) have been also proposed as factors, which regulate oxidative stress. DJ-1-CRIF1-KEAP1 interaction induces antioxidant defense systems against alveolar epithelial cell injury by cigarette smoke. We propose to study the mechanism of DJ-1 cytoprotective function against human primary alveolar epithelial cell injury by cigarette smoke in vitro and ex vivo. We will determine oxidation of cysteine and methionine residues within DJ-1 using mass spectrometry analysis. We will use mutant constructs to identify, which oxidizable cysteine and methionine residue(s) are critical for DJ-1 function in protecting against injury induced by cigarette smoke. We will focus on cysteine residue at position 106 (Cys-106), which is a sensor of oxidative stress within DJ-1. To further determine the DJ-1 pathway, we will analyze DJ-1, CRIF1 and KEAP1 interacting domains using deletion constructs. We will also use in our studies alveolar type II cells isolated from patients with mild, moderate and severe emphysema. This unique approach will allow us to identify the mechanism of DJ-1 pathway impairment in emphysema progression. We will validate our results in alveolar type II cells in vivo. Wild-type mice will be exposed to cigarette smoke to determine DJ-1 cysteine and methionine modifications by mass spectrometry under oxidative stress conditions. We will also determine whether adenovirus DJ-1 will rescue of alveolar type II cell injury due to cigarette smoke in DJ-1 knockout mice. Study of the mechanism of the DJ-1 pathway impairment in emphysema may lead to the development of novel pharmacological strategies to slow the progression of this disease.