Role of the inflammasome in hepatitis C virus pathogenesis

描述（由申请人提供）：宿主脂质代谢在丙型肝炎病毒（HCV）生命周期中的作用引起了相当大的关注。宿主脂质代谢的改变是脂肪肝的原因。在理解HCV感染如何诱导固醇调节元件结合蛋白（SREBPs）的蛋白水解激活方面存在根本性差距，SREBPs是脂肪肝疾病的主要调节因子，在慢性HCV感染患者中观察到。这项研究的长期目标是确定脂质稳态改变的分子机制，这将更好地描述HCV介导的肝病的发病机制。本提案的目的是确定HCV激活SREBPs的分子机制。本申请的中心假设是HCV感染细胞中半胱天冬酶-1炎性体复合物的诱导介导SREBP的活化。这项研究的基本原理是，通过了解HCV介导的caspase-1炎性小体如何诱导SREBP的活化，可以通过新的创新方法抑制它们的活化，以阻止HCV的产生和与HCV感染相关的肝脏疾病的进展。因此，拟议的研究与NIH的使命有关，该使命涉及开发基础知识，这将可能有助于设计治疗慢性丙型肝炎感染的替代策略。 在强有力的初步数据的支持下，该提案的主要假设将在两个特定目标下进行测试：1）确定HCV激活caspase-1的机制; 2）定义SREBP蛋白水解激活的机制。在第一个目标下，肝脏特异性炎性小体复合物在caspase-1激活中的作用将使用新的双分子荧光互补（BiFC）测定，蛋白质组学/质谱，实时RT-PCR，siRNA技术，共聚焦显微镜和蛋白质-蛋白质相互作用进行研究。在第二个目标下，将使用与目标1所述类似的方法研究炎性体复合物的组分如NALP 3、ASC和半胱天冬酶-1对SCAP、Insig和COPII蛋白的调节的作用。将使用蛋白质印迹、共聚焦显微镜和基于细胞的报告基因测定研究SREBP的活化。这些研究的结果将产生新的见解改变脂质稳态，肝脏肿瘤发生，胰岛素抵抗和肥胖与慢性HCV的机制。在这些研究完成后，我们的工作预计将共同确定HCV通过与慢性炎症过程的相互作用在半胱天冬酶-1炎性体复合物的激活和SREBP的蛋白水解激活中的潜在作用。这项工作也将确定新的caspase-1炎性体复合物在人肝细胞中的诱导。这项研究意义重大，因为它有望为靶向病毒或细胞决定簇提供新的策略，以阻止HCV的产生以及与HCV感染相关的肝脏疾病的进展。此外，预计这些结果将从根本上推进HCV感染介导的肝脏炎症和脂质代谢领域。

DESCRIPTION (provided by applicant): The role of host lipid metabolism has attracted considerable attention in hepatitis C virus (HCV) life cycle. Altered host lipid metabolism is the cause of fatty-liver disease. There is a fundamental gap in understanding how HCV infection induces the proteolytic activation of sterol regulatory element binding proteins (SREBPs), the master regulator of fatty-liver disease, which is observed in patients with chronic HCV infection. The long- term goal of this research is to define the molecular mechanisms of altered lipid homeostasis that will better characterize the pathogenesis of HCV-mediated liver disease. The objective of this proposal is to identify the molecular mechanisms of activation of SREBPs by HCV. The central hypothesis of the application is that induction of the caspase-1 inflammasome complex in HCV-infected cells mediates the activation of SREBPs. The rationale for the proposed research is that, by understanding how HCV-mediated caspase-1 inflammasome induces the activation of SREBPs, their activation can be inhibited in new and innovative approaches to arrest the production of HCV and the progression of liver disease associated with HCV-infection. Thus, the proposed research is relevant to NIH mission that pertains to developing fundamental knowledge that will potentially help design the alternate strategies in the treatment of chronic hepatitis C infection. Supported by strong preliminary data, the proposal's main hypothesis will be tested under two specific aims: 1) Determine the mechanism of caspase-1 activation by HCV; 2) Define the mechanism of SREBPs proteolytic activation. Under the first aim, the role of liver-specific inflammasome complex in caspase-1 activation will be investigated using novel bimolecular fluorescence complementation (BiFC) assay, proteomics/mass spectroscopy, real-time RT-PCR, siRNA technology, confocal microscopy, and protein-protein interaction. Under the second aim, the role of the components of the inflammasome complex such as, NALP3, ASC, and caspase-1 on the regulation of SCAP, Insig, and COPII proteins will be investigated using the similar methods as described for aim 1. The activation of SREBPs will be investigated using western-blot, confocal microscopy, and cell-based reporter assays. The results from these studies will yield novel insights into mechanisms of altered lipid homeostasis, liver oncogenesis, insulin resistance, and obesity associated with chronic HCV. At the completion of these studies our work is collectively expected to establish the potential role of HCV in the activation of caspase-1 inflammasome complex and proteolytic activation of SREBPs through the interactions with chronic inflammatory processes. This work will also define the novel caspase-1 inflammasome complex induction in human hepatocytes. The proposed research is significant because it is expected to provide novel strategies for targeting the viral or cellular determinants to arrest the production of HCV as well as the progression of liver disease associated with HCV-infection. In addition, it is expected that the results will fundamentally advance the fields of liver inflammation and lipid metabolism mediated by HCV-infection.