Redox-mediated regulation of pattern-recognition-receptor signaling in intestinal epithelial cells

项目摘要 肠上皮细胞是介导肠上皮细胞之间的通信的关键粘膜屏障。 腔环境和免疫系统。几条证据表明稳定的活性氧 活性氧（ROS）在调节粘膜表面的细胞信号传导方面发挥着重要作用，两者都是为了响应 微生物环境和肠道炎症。稳定的ROS如过氧化氢（H2 O2）可以被 由上皮细胞通过NADPH氧化酶家族（例如NOX 1）或从其他来源产生 环境因素，如细菌。H2 O2通过氧化修饰调节细胞信号传导， 靶向蛋白质上的半胱氨酸残基，改变下游功能。然而，究竟是什么信号蛋白 和途径的影响，ROS在炎症过程中是知之甚少， 了解炎症性肠病的发病机制和 生物标志物来追踪疾病活动。 我们最近的数据表明，NOX 1产生的H2 O2可以通过水通道蛋白通道（AQP 3）转运， 并且在介导上皮环境反应中是重要的。我们的研究还表明， 上皮细胞信号传导，响应模式识别受体“危险”信号，如白细胞介素-1 β （IL-1b）也是H2 O2依赖性的。为了进一步研究蛋白质，在存在 我们对小鼠原代肠上皮细胞进行了氧化还原蛋白质组学研究。我们发现了几个 以NOX 1和AQP 3依赖性方式选择性氧化的候选蛋白，包括 Peroxiredoxin 6（PRDX 6）-已知的多功能氧化还原敏感蛋白。本R 03提案的目标1将 因此，验证PRDX 6功能对上皮模式识别受体重要的假设 发信号。Aim 2采用蛋白质组学方法，通过以下方法鉴定人类疾病中关键的氧化还原修饰蛋白质： 使用来自健康个体的类结肠研究模式识别受体反应， 炎症性肠病患者。这些研究的主要目标是确定新的氧化还原- 在炎症危险信号传导过程中肠上皮细胞中的依赖性信号传导途径及其用途 这些数据可以开发用于粘膜炎性疾病的新治疗方法。

PROJECT SUMMARY The intestinal epithelium is a critical mucosal barrier that mediates communication between the intestinal luminal environment and the immune system. Several lines of evidence suggest that stable reactive oxygen species (ROS) play an important role in regulating cell signaling at mucosal surfaces, both in response to the microbial environment and in gut inflammation. Stable ROS such as hydrogen peroxide (H2O2) can be generated by epithelial cells through the NADPH oxidase enzyme family (e.g. NOX1) or from other environmental sources such as bacteria. H2O2 modulates cellular signaling via oxidative modifications of target cysteine residues on proteins, altering downstream functions. However, exactly what signaling proteins and pathways are impacted by ROS during inflammation is poorly understood and may provide key information in understanding the pathogenesis of inflammatory bowel diseases and the development of biomarkers to track disease activity. Our recent data shows that NOX1-generated H2O2 can be transported through aquaporin channels (AQP3) and is important in mediating epithelial environmental responses. Our studies have also shown that epithelial signaling, in response to pattern-recognition-receptor ‘danger’ signals such as interleukin-1 beta (IL-1b), is also H2O2-dependent. To further investigate proteins that are selectively oxidized in the presence of IL-1b we carried out redox proteomics of mouse primary intestinal epithelial cells. We discovered several candidate proteins that are selectively oxidized in a NOX1 and AQP3-dependent manner, including Peroxiredoxin 6 (PRDX6) - a known multifunctional redox-sensitive protein. Aim 1 of this R03 proposal will therefore test the hypothesis that PRDX6 function is important for epithelial pattern-recognition-receptor signaling. Aim 2 takes a proteomics approach to identify key redox-modified proteins in human disease by investigating pattern-recognition-receptor responses using colonoids derived from healthy individuals and individuals with inflammatory bowel disease. The main goals of these studies are to identify novel redox- dependent signaling pathways in intestinal epithelial cells during inflammatory danger signaling and to use this data to develop novel therapeutic approaches for mucosal inflammatory diseases.