Mechanism of ER Protein Misfolding-Induced Mitochondrial Dysfunction

项目总结 在西方世界，非酒精性脂肪性肝病(NAFLD)的患病率几乎为30%， 预计在未来十年将会上升。NAFLD的特征是内质中的蛋白质错误折叠 网状结构(ER)激活未折叠蛋白反应(UPR)。长期的内质网应激条件进一步导致 氧化应激，蛋白质聚集，细胞器损伤，细胞生物能量崩溃，最终 细胞死亡。最近的研究表明，内质网中的蛋白质错误折叠导致肝细胞衰竭 与NAFLD、非酒精性脂肪性肝炎(NASH)和肝细胞癌(HCC)相关。 通过对肝细胞功能的详细描述，我们的初步结果引导我们提出内质网 蛋白质错误折叠导致线粒体功能障碍，从而导致进一步的蛋白质错误折叠，最终导致 肝细胞衰竭。尽管先前的研究表明，肝细胞内质网中的蛋白质错误折叠 与代谢综合征和非酒精性脂肪肝有关，但从来没有仔细描述过 ER蛋白错误折叠导致灾难性的细胞事件，导致氧化损伤、纤维化和细胞 死亡。我们已经证明，凝血因子(FVIII)在内质网中的错误折叠破坏了内质网的复合体1。 电子传输链。令人惊讶的是，我们证明了线粒体靶向治疗 抗氧化剂纠正线粒体功能缺陷，并促进细胞内内质网FVIII折叠 文化。这一史无前例的发现表明，ER蛋白错误折叠之间存在未被认可的联系 和有缺陷的线粒体生物能量学。在拟议的研究中，我们将使用两个不同的模型来 在体内诱导肝细胞内质网应激；(I)高果糖饮食的过量营养，或(Ii)FVIII的表达 为了阐明ER蛋白错误折叠是如何扰乱线粒体生物能量学和动力学的。目前， 正在进行的临床研究正在使用FVIII病毒传递到血友病A患者的肝细胞。 此外，我们将应用代谢组学和RNA-Seq的无偏见技术，使用新的基因 小鼠模型阐明蛋白质错误折叠如何导致钙离子从内质网泄漏并进入内质网 线粒体基质破坏线粒体生物能量学和/或动力学。有证据支持ER 蛋白质错误折叠和线粒体功能缺陷存在于所有退行性疾病中。此外, 广泛的研究结果表明，内质网中的蛋白质错误折叠在许多情况下与肝功能衰竭有关 常见的急性情况，包括病毒感染、乙醇中毒、对乙酰氨基酚中毒和缺血 再灌注损伤。我们对未知领域的基本小说探索无疑将产生 ER蛋白错误折叠对线粒体电子传递影响的新假说 链条。这些研究将提供对内质网和线粒体功能如何的基本机制洞察 相互调节，并导致治疗NAFLD和其他蛋白质错误折叠疾病的新疗法。

PROJECT SUMMARY The prevalence of non-alcoholic fatty liver disease (NAFLD) is almost 30% in the western world and is expected to rise in the next decade. NAFLD is characterized by protein misfolding in the endoplasmic reticulum (ER) activating the unfolded protein response (UPR). Prolonged ER stress conditions further lead to oxidative stress, protein aggregation, organelle damage, cellular bioenergetic collapse, and eventually cell death. Recent studies indicate that protein misfolding in the ER contributes to hepatocyte failure associated with NAFLD, non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). Through detailed characterization of hepatocyte function, our preliminary results lead us to propose that ER protein misfolding causes mitochondrial dysfunction that leads to further protein misfolding culminating in hepatocyte failure. Although previous studies demonstrated that protein misfolding in the ER of hepatocytes is associated with metabolic syndrome and NAFLD, there has never been a careful characterization of how ER protein misfolding causes catastrophic cellular events leading to oxidative damage, fibrosis and cell death. We have shown that misfolding of coagulation factor VIII (FVIII) in the ER disrupts complex 1 of the electron transport chain. Amazingly, we demonstrated that treatment with a mitochondrial-targeted antioxidant corrects he defective mitochondrial function and also improves FVIII folding in the ER in cell culture. This unprecedented finding indicates an unappreciated association between ER protein misfolding and defective mitochondrial bioenergetics. In the proposed studies, we will use two separate models to induce ER stress in vivo in hepatocytes; (i) excess nutrition of a high fructose diet, or (ii) expression of FVIII to elucidate how ER protein misfolding disrupts mitochondrial bioenergetics and dynamics. Presently, ongoing clinical studies are using viral delivery of FVIII to hepatocytes in hemophilia A patients. Furthermore, we will apply non-biased technologies of metabolomics and RNA-Seq using novel genetic murine models to elucidate how protein misfolding causes Ca2+ leak from the ER and entry into the mitochondrial matrix to disrupt mitochondrial bioenergetics and/or dynamics. Evidence supports that ER protein misfolding and defective mitochondrial function exist in all degenerative diseases. In addition, extensive findings demonstrate that protein misfolding in the ER is associated with liver failure in a number of common acute conditions including viral infection, ethanol toxicity, acetaminophen toxicity and ischemia reperfusion injury. Our fundamental novel exploration into unchartered territory will undoubtedly generate new hypotheses concerning the impact of ER protein misfolding on the mitochondrial electron transport chain. The studies will provide fundamental mechanistic insight into how ER and mitochondrial functions are reciprocally regulated and lead to novel therapies for NAFLD and other diseases of protein misfolding.