Pathophysiology of Chronic Pancreatitis Associated with Misfolded PNLIP Risk Variants

项目摘要 本申请的主要目的是确定胰甘油三酯脂肪酶 （PNLIP）变异增加了人类慢性胰腺炎（CP）的风险。错误折叠引起的蛋白毒性 突变消化酶已被认为是CP的一种新的胰蛋白酶非依赖性机制， 错误折叠的CP。近年来的研究表明，CP的发展往往是一个离散的复发过程， 急性胰腺炎（RAP）由遗传风险因素驱动。在存在错误折叠的蛋白质的情况下恢复蛋白质稳态 蛋白质是一种有吸引力的治疗策略，以阻止RAP和防止进展到终末期疾病， 错误折叠CP患者。在理解错误折叠的蛋白质如何有助于 胰腺炎的发病和进展主要源于缺乏动物模型， 在人类中错误折叠CP我们已经开发了人PNLIP T221 M疾病的临床前模型。PNLIP 在胰蛋白酶原激活或胰蛋白酶失活中没有已知的作用。PNLIP T221 M在体外的错误折叠及其原因 通过触发未减轻的ER应激而引起蛋白毒性。我们的初步数据表明，Pnlip T221 M小鼠表现出 进行性胰腺腺泡萎缩、炎性细胞浸润、纤维化、细胞凋亡和坏死 死亡;病理改变早在1个月时发生，3-4个月时加重。我们的工作 一种假说是由PNLIP风险变体编码的蛋白质通过功能获得性蛋白毒性引起CP 通过蛋白质错误折叠为了验证这一假设，首先我们将系统地表征Pnlip T221小鼠， 包括CP样表型、继发性并发症和在治疗时对胰腺损伤的易感性。 挑战乙醇喂养。第二，我们将系统地研究早期的生物反应， 通过在小鼠中胰腺表达致病性PNLIP T221 M，以揭示潜在的新靶点， 预防疾病的发生和发展。第三，我们将评估蛋白质错误折叠的普遍性， 蛋白毒性是在CP中发现的意义不确定的PNLIP变体（VUS）的疾病机制的基础 患者使用细胞培养物和另一种Pnlip F300 L小鼠模型。完成这些研究将 建立突变诱导的消化酶错误折叠作为一个相关的疾病机制， 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.