Redox and Proteomic Stress Responses in Biliary Disease

项目摘要 胆道系统疾病在肝移植适应症中占很大比例， 成人和儿童群体。这些疾病的发病机制可能涉及细胞对细胞因子的反应。 胆汁的有害影响，胆管的上皮细胞不断暴露于胆汁中。胆汁毒性产生 大部分来自胆盐引起的氧化损伤，其去污剂特性破坏细胞内 细胞器尤其是线粒体胆管上皮细胞，也称为胆管细胞， 除了具有强大的抗氧化防御系统之外，还存在预防胆汁毒性的策略， 在所有细胞中，以对抗正常代谢过程中产生的活性氧。 我的实验室使用斑马鱼系统的工作已经确定了胆管细胞的区域亲和性， 氧化还原应激所赋予的植物毒素biliatresone，其消费与流行性胆汁 家畜闭锁（BA）。我们最近的研究表明，热休克中区域变化的作用相当 介导的蛋白质稳态的胆管细胞暴露于胆汁酸，这些反应可以修改， cGMP信号的激活。重要的是，有令人信服的临床数据表明， 反应有助于人类BA的风险和结果，从而验证了我们采用的biliatresone模型。 本研究的目的是研究胆管细胞应激变化的机制 使用斑马鱼、小鼠和人类细胞培养模型的反应。在目标1中，我们将表征分子 斑马鱼肝内和肝外胆管细胞氧化还原应激反应的决定因素，确定 这些反应的变化是否是由胆道发育过程中出现的表观遗传因素演变而来， 使用小鼠模型将这些发现与哺乳动物胆管细胞生物学相关联。在目标2中，我们将确定 使用两种方法，在哺乳动物胆管细胞中， 方法;第一，通过下拉实验使用光活化的biliatresone类似物;第二，通过进行 针对胆甾烯松毒性所需基因的全基因组CRISPR阳性选择筛选。在目标3中，我们 探索cGMP信号和蛋白质稳态之间的联系，以及这如何影响胆管细胞对 损伤使用三种方法：首先，通过定义cGMP信号传导的下游调节因子，介导其 第二，通过将这些数据与胆汁酸毒性的综合分析相关联， 胆管细胞蛋白质组，以及它是如何修改的胆固醇和cGMP信号，第三，定义亚- cGMP信号传导活性的细胞域及其下游介质。

PROJECT SUMMARY Diseases of the biliary system comprise a significant percentage of the indications for liver transplantation in adult and pediatric populations. The pathogenesis of these disorders likely involves cellular responses to the harmful effects of bile, to which epithelial cells lining the bile ducts are continuously exposed. Bile toxicity arises largely from the oxidative damage caused by bile salts, whose detergent properties disrupt intracellular organelles, particularly mitochondria. Bile duct epithelial cells, also known as cholangiocytes, have evolved strategies for preventing bile toxicity in addition to having robust anti-oxidant defense systems, which are present in all cells to combat reactive oxygen species generated during normal metabolism. Work from my laboratory using the zebrafish system has identified regional susceptibilities of cholangiocytes to redox stress imparted by the plant toxin biliatresone, whose consumption is associated with epidemic biliary atresia (BA) in livestock. Our most recent studies indicate a comparable role for regional variantion in heat shock mediated proteostasis in cholangiocytes exposed to biliatresone, and that these responses can be modified by the activation of cGMP signaling. Importantly, there is compelling clinical data indicating the modulation of stress responses contributes to risk and outcomes in human BA, thus validating the biliatresone models we employ. The goal of this proposal is to study the mechanisms responsible for the variation in cholangiocyte stress responses using zebrafish, mouse and human cell culture models. In Aim 1, we will characterize the molecular determinants of the redox stress response in zebrafish intra-hepatic and extra-hepatic cholangiocytes, determine whether variation in these responses evolve from epigenetic factors that arise during biliary development, and correlate these findings with mammalian cholangiocyte biology using mouse models. In Aim 2, we will identify the cellular targets responsible for biliatresone-induced injury and stress in mammalian cholangiocytes using two approaches; first, via pull-down experiments using photo-activatable biliatresone analog; second, by conducting a genome-wide CRISPR positive selection screen for genes required for biliatresone toxicity. In Aim 3, we will explore links between cGMP signaling and proteostasis and how this affects susceptibility of cholangiocyte to injury using three approaches: first, by defining the downstream regulators of cGMP signaling that mediate its inhibition of biliatresone toxicity, second, by correlating these data with a comprehensive analysis of the cholangiocyte proteome, and how it is modified by biliatresone and cGMP signaling, third, defining the sub- cellular domains of cGMP signaling activity and its downstream mediators.