Redox-based Fas Signaling in Allergic Airway Disease

项目总结 过度的上皮细胞凋亡和缺乏适当的上皮修复被认为是纤维化形成的关键。 死亡受体Fas在上皮细胞凋亡中发挥重要作用，并在随后的发展中发挥作用。 肺纤维化。同样，氧化环境以及内质网(ER)的变化 应激被认为在特发性肺纤维化(IPF)的发病机制中起重要作用，提示 氧化还原和内质网紊乱、Fas、上皮细胞凋亡和纤维化之间的因果联系。在当前的 这笔赠款的资金周期我们发现，Fas作为死亡受体的效力在其 S-谷胱甘肽基化，蛋白质氧化的一种形式。S-谷胱甘肽基化代表抗氧化剂的偶联 分子谷胱甘肽转化为反应蛋白半胱氨酸(PSSG)。我们最近在肺上皮细胞中解开了这一点 并不是所有的Fas都在表面上表达，而是潜伏的Fas池没有完全加工成配体 结合形式，定位于内质网。作为对FasL刺激表面Fas的反应，蛋白质二硫键 ERp57异构酶诱导内质网内Fas的快速氧化处理。我们推测氢气 在这个过程中产生的过氧化氢(过氧化氢)负责S-谷胱甘肽基化，在一个反应中 谷胱甘肽S转移酶P(GSTP)催化。这些发现照亮了Fas的一个新维度- “配体引发内质网中潜在Fas的氧化处理”作为一种调控机制诱导细胞凋亡 调节细胞死亡的强度。内质网内触发的确切氧化事件仍不清楚，并且 内质网中产生的过氧化氢在上皮细胞凋亡中的功能作用，以及随后的发病机制 纤维化目前还不清楚。这里要解决的中心假设是ERp57催化的Fas的加工 导致内质网中过氧化氢含量增加。过氧化氢的增加反过来又需要允许GSTP催化 S-谷胱甘肽活化的Fas，增加上皮细胞的凋亡，从而导致肺纤维化。具体而言 目的#1我们将确定ERP57在氧化加工中的功能需求，随后S- 肺上皮细胞Fas的谷胱甘肽基化及由此导致的肺纤维化的发生。特定目标 #2旨在探讨GSTP在S中的功能需求--Fas的谷胱甘肽基化，以及随后的 肺纤维化的发展。在具体目标#3中，我们将评估过氧化氢的功能重要性 在Fas依赖性上皮细胞凋亡和随后的纤维化形成中产生内质网。我们将使用 补充细胞培养和小鼠转基因方法，以及对这些过程的详细分析 在IPF和NSIP患者的肺组织中，使用创新和临床相关的策略。 鉴于Fas、内质网压力和 谷胱甘肽氧化还原扰动已经独立地与纤维化的发病机制联系在一起，并且 旨在评估或靶向S谷胱甘肽基化的方法可能被证明是临床相关的，因为 诊断工具和潜在的治疗方法。

PROJECT SUMMARY Excessive epithelial apoptosis and lack of proper epithelial restitution are believed to be critical to fibrogenesis. The death receptor Fas plays a cardinal role in epithelial apoptosis, and the subsequent development of pulmonary fibrosis. Similarly, changes in the oxidative environment, as well as endoplasmic reticulum (ER) stress are believed to be important in the pathogenesis of idiopathic pulmonary fibrosis (IPF), suggesting a causal link between redox and ER perturbations, Fas, epithelial apoptosis, and fibrogenesis. During the current funding cycle of this grant we discovered that the potency of Fas as a death receptor is enhanced following its S-glutathionylation, a form of protein oxidation. S-glutathionylation represents the conjugation of the antioxidant molecule glutathione to reactive protein cysteines (PSSG). We recently unraveled that in lung epithelial cells not all Fas is expressed on the surface but that a latent pool of Fas which is not fully processed into the ligand binding form, is localized in the ER. In response to stimulation of surface Fas with FasL, the protein disulfide isomerase, ERp57, induces rapid oxidative processing of Fas within the ER. We speculate that hydrogen peroxide (H2O2) produced during this process is responsible for S-glutathionylation, in a reaction that is catalyzed by glutathione S-transferase P (GSTP). These discoveries illuminate a new dimension of Fas- induced apoptosis "ligand-triggered oxidative processing of latent Fas in the ER" as a regulatory mechanism to regulate the strength of cell death. The exact oxidative events triggered within the ER remain unknown, and the functional role of H2O2 generated within the ER for epithelial apoptosis, and subsequent pathogenesis of fibrosis are unclear. The central hypothesis to be addressed herein is that ERp57-catalyzed processing of Fas leads to increases of H2O2 content in the ER. Increases in H2O2 are in turn required to permit GSTP-catalyzed S-glutathionylation of Fas, augmenting epithelial apoptosis, thereby leading to pulmonary fibrosis. In Specific Aim #1 we will determine the functional requirement of ERp57 in oxidative processing, and subsequent S- glutathionylation of Fas in lung epithelium, and the resultant development of pulmonary fibrosis. Specific Aim #2 seeks to explore the functional requirement of GSTP in S-glutathionylation of Fas, and the subsequent development of pulmonary fibrosis. In Specific Aim #3 we will assess the functional importance of H2O2 generated in the ER in Fas-dependent epithelial apoptosis and subsequent fibrogenesis. We will use complementary cell culture and mouse transgenic approaches, coupled to detailed analysis of these processes in lung tissues from patients with IPF as well as NSIP, using innovative and clinically relevant strategies. Completion of proposed experiments is likely to exert a substantial impact given that Fas, ER stress, and glutathione redox perturbations have been independently linked to the pathogenesis of fibrosis, and methodologies aimed at assessing or targeting S-glutathionylation may prove to be clinically relevant as diagnostic tools and potential therapeutics.