Redox and Proteomic Stress Responses in Biliary Disease

胆道疾病中的氧化还原和蛋白质组应激反应

• 批准号: 10636916
• 负责人: MICHAEL A PACK
• 金额: $ 45.14万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-15 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10636916
• 关键词: Adult; Affect; Antioxidants; Award; Bile Duct Diseases; Bile Duct Epithelium; Bile fluid; Biliary; Biliary Atresia; Biology; Body Fluids; CRISPR screen; Caustics; Cell Culture Techniques; Cell Line; Cell physiology; Cells; Characteristics; Child; Childhood; Client; Clinical Data; Clustered Regularly Interspaced Short Palindromic Repeats; Congenital atresia of extrahepatic bile duct; Consumption; Cyclic GMP; DNA-Directed DNA Polymerase; Data Correlations; Detergents; Development; Disease; Disease Outbreaks; Duct (organ) structure; Ductal Epithelial Cell; Epidemic; Epigenetic Process; Epithelial Cells; Exposure to; Extrahepatic; Genes; Glutathione; Glutathione Disulfide; Glutathione Metabolism Pathway; Goals; Heat-Shock Response; Heterogeneity; Human; In Vitro; Incidence; Injury; Knowledge; Laboratories; Link; Liver Failure; Livestock; Mass Spectrum Analysis; Mediating; Mediator; Metabolism; Mitochondria; Modeling; Molecular; Molecular Chaperones; Mus; Mutate; Mutation; Organelles; Outcome; Oxidation-Reduction; Oxidative Stress; Pathogenesis; Physiological; Plants; Play; Population; Predisposition; Property; Proteome; Proteomics; Publishing; Reactive Oxygen Species; Reduced Glutathione; Regional Anatomy; Research; Risk; Risk Factors; Role; Signal Transduction; Stress; System; Toxic effect; Toxin; Variant; Vertebrates; Work; Zebrafish; analog; bile duct; bile salts; biliary tract; biological adaptation to stress; cellular targeting; cholangiocyte; combat; disorder risk; experimental study; genome-wide; human disease; in vivo; intrahepatic; liver transplantation; macromolecule; mouse model; multicatalytic endopeptidase complex; novel; novel therapeutics; oxidative damage; prevent; proteostasis; proteotoxicity; regional difference; response

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.

项目摘要 胆道系统的疾病占肝移植适应症的很大一部分 成人和小儿种群。这些疾病的发病机理可能涉及对细胞的反应 胆汁的有害作用，胆管内壁的上皮细胞不断暴露。胆汁毒性出现 主要来自胆汁盐造成的氧化损伤，其洗涤剂特性破坏了细胞内 细胞器，特别是线粒体。胆管上皮细胞，也称为胆管细胞，已经进化 除了具有强大的抗氧化防御系统外，还可以防止胆汁毒性的策略 在所有细胞中，应对正常代谢过程中产生的活性氧。 使用斑马鱼系统从我的实验室进行的工作已确定胆管细胞的区域敏感性 植物毒素胆汁酮赋予的氧化还原应激，其消耗与流行病有关 牲畜的闭锁（BA）。我们最近的研究表明，在热休克中，区域变异的作用可比 暴露于胆汁酮的胆管细胞中介导的蛋白抑制剂，这些反应可以通过 CGMP信号的激活。重要的是，有令人信服的临床数据表明压力调节 响应有助于人类BA的风险和结果，从而验证了我们采用的胆汁酮模型。 该提案的目的是研究负责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AIM 1中，我们将表征分子 斑马鱼内和肝外胆管细胞中氧化还原应激反应的决定因素，确定 这些反应的变化是否来自胆道发育过程中出现的表观遗传因素以及 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AIM 2中，我们将确定 使用两个哺乳动物胆管细胞中胆汁酮诱导损伤和应激的细胞靶标的细胞靶标 方法；首先，通过使用可相连的胆道类似物进行下拉实验；第二，通过进行 全基因组CRISPR的阳性选择屏幕，用于胆红素毒性所需的基因。在AIM 3中，我们将 探索CGMP信号传导与蛋白质的联系，这如何影响胆管细胞的敏感性 使用三种方法受伤：首先，通过定义介导其介导的CGMP信号的下游调节剂 抑制胆红素毒性，其次是通过将这些数据与对 胆管细胞蛋白质组，以及如何通过胆汁酮和CGMP信号（第三）修饰它 CGMP信号活性及其下游介体的细胞结构域。