Redox and Proteomic Stress Responses in Biliary Disease

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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.