218. Monitoring ER Stress Activation of the ATF6 Pathway Using Nanoluciferase

The endoplasmic reticulum (ER) is an invaluable component of the secretory pathway; serving as the main site for protein synthesis and modification, lipid metabolism, lipoprotein secretion, and calcium homeostasis. In response to “stress”, the ER maintains homeostasis through the unfolded protein response (UPR). The UPR is an adaptive response employed by the ER to alleviate stress caused by misfolded proteins. UPR activation is mediated by three transmembrane proteins; IRE1 (inositolrequiring protein-1), ATF6 (activating transcription factor- 6), and PERK (protein kinase RNA-like ER kinase). Here we describe the construction and characterization of a Nanoluciferase-based assay to look at the activation of the ATF6 prong of the UPR. We demonstrate the ability to simultaneously monitor ER calcium homeostasis using a novel Gaussia luciferase-based sensor. Using in vitro and in vivo assays, we demonstrate the ability to monitor ATF6 activation in human neuroblastoma cells, rat hepatoma cell line, and rat liver. With this approach we hope to gain better understanding of the contributions of ATF6 activation and ER calcium depletion in disease pathogenesis. Ongoing studies will examinegenetically or pharmacologically augmenting or inhibiting these pathways as a therapeutic approach.