PROJECT SUMMARY The primary goal of this application is to determine the mechanisms by which pancreatic triglyceride lipase (PNLIP) variants increase the risk for chronic pancreatitis (CP) in humans. Proteotoxicity caused by misfolded mutant digestive enzymes has been recognized as a novel trypsin-independent mechanism for CP, referred to as misfolding CP. Recent studies indicate that the development of CP is often a process of discrete recurrent acute pancreatitis (RAP) driven by genetic risk factors. Restoring proteostasis in the presence of misfolded proteins is an attractive therapeutic strategy to stop RAP and prevent progression to end-stage disease in misfolding CP patients. Critical knowledge gaps remain in understanding how misfolded proteins contribute to pancreatitis onset and progression primarily stemming from the paucity of animal models that recapitulate misfolding CP in humans. We have developed a pre-clinical model of human PNLIP T221M disease. PNLIP has no known role in trypsinogen activation or trypsin inactivation. In vitro, PNLIP T221M misfolds and causes proteotoxicty by triggering unmitigated ER stress. Our preliminary data show that Pnlip T221M mice exhibit progressive pancreatic acinar atrophy, inflammatory cell infiltration, fibrosis, and apoptotic and necrotic cell death; the pathological changes occur as early as at 1 month and become severe at 3-4 month. Our working hypothesis is that proteins encoded by risk variants of PNLIP cause CP by gain-of-function proteotoxicity through protein misfolding. To test this hypothesis, first we will systematically characterize Pnlip T221 mice including CP-like phenotypes, secondary complications, and the susceptibility to pancreatic injury when being challenged with ethanol feeding. Second, we will methodically investigate early biological responses triggered by pancreatic expression of pathogenic PNLIP T221M in mice to uncover potential novel targets to halt or prevent disease onset and progression. Third, we will assess the prevalence of protein misfolding and proteotoxicity underlies the disease mechanism of PNLIP variants of uncertain significance (VUS) found in CP patients using cell cultures and another mouse model of Pnlip F300L. Completion of these studies will establish mutation-induced misfolding of digestive enzymes as a relevant disease mechanism in the pathogenesis of CP and will identify potential therapeutic targets to stop RAP and prevent CP.