Pathophysiology of Chronic Pancreatitis Associated with Misfolded PNLIP Risk Variants

项目总结 这项应用的主要目标是确定胰腺甘油三酯脂肪酶的机制 (PNLIP)变异会增加人类患慢性胰腺炎(CP)的风险。错误折叠引起的蛋白质毒性 突变消化酶已被认为是CP的一种新的不依赖胰酶的机制，参考 错误折叠CP。最近的研究表明，慢性胰腺炎的发展往往是一个离散的反复过程 遗传危险因素导致的急性胰腺炎(RAP)。在存在错误折叠的情况下恢复蛋白质平衡 蛋白质是一种有吸引力的治疗策略，可以阻止RAP并防止进展到终末期疾病 错误折叠的CP患者。在理解错误折叠的蛋白质如何有助于 胰腺炎的发生和发展主要源于缺乏概括的动物模型 在人类中错误折叠CP。我们已经建立了人类PNLIP T221M病的临床前模型。PNLIP 在胰酶原激活或胰酶失活中的作用尚不清楚。PNLIP T221M体外错误折叠及其原因 通过触发未减轻的内质网应激而产生的蛋白质毒性。我们的初步数据显示，Pnlip T221M小鼠表现出 进行性胰腺腺泡萎缩、炎性细胞浸润、纤维化、凋亡和坏死细胞 死亡；病变最早在1个月时出现，3-4个月时变得严重。我们的工作 假说是由PNLIP危险变异体编码的蛋白质通过功能获得性蛋白毒性引起CP 通过蛋白质的错误折叠。为了验证这一假设，我们首先将系统地描述Pnlip T221小鼠的特征 包括CP样表型、继发性并发症和胰腺损伤的易感性 用乙醇喂养来挑战。第二，我们将系统地研究早期触发的生物反应 通过在小鼠胰腺中表达致病的PNLIP T221M发现潜在的新靶点来阻止或 预防疾病的发生和发展。第三，我们将评估蛋白质错误折叠和 蛋白毒性是CP中发现的PNLIP变异体(VUS)发病机制的基础 使用细胞培养和另一种Pnlip F300L小鼠模型的患者。完成这些研究后， 建立突变诱导的消化酶错误折叠作为一种相关的疾病机制 CP的发病机制，并将确定潜在的治疗靶点，以阻止RAP和预防CP。

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.