HEPATOCELLULAR CARCINOMA AND HEPATITIS C: E2 MOLECULAR MECHANISMS

描述(申请人提供)：中心假设：丙型肝炎病毒(丙型肝炎病毒)E2糖蛋白是一种新的激酶，启动信号转导机制，调节以下途径：1.网状蛋白介导的内吞作用，通过网状蛋白适配器蛋白-50(AP50)的位点特异性磷酸化，网状蛋白适配器蛋白-50是网状蛋白介导的受体内吞的关键调节因子；PI3Kinase和Akt激活与肝细胞增殖和肝癌的发生 在试验性交易研究和体外激酶分析中，我获得了令人信服的数据，表明E2是肌动蛋白调节激酶家族(Ark/PRK激酶)的一个新成员，它物理上与其磷酸受体Thr156上的AP50结合并使其磷酸化，Thr156是clathrin介导的内吞作用(25，50，73)的关键步骤。此外，我们已经证明，在丙型肝炎患者的肝脏中，E2与AP50相关，并且在这些肝脏中，AP50在Thr156上被更大程度地磷酸化。 在初步研究中，我们还发现，在没有细胞外生长因子的情况下，E2能增加PIP2、PI3K、PDK1和Akt及其活性。这一信号级联促进了细胞的增殖。此外，丙型肝炎病毒E2显著刺激肝细胞DNA复制，甚至比经典的肿瘤促进剂TGF(和EGF)更大。 1.人原代肝细胞独特的丙型肝炎病毒感染系统中，丙型肝炎病毒E2与AP50相互作用与AP50磷酸化的生理学相关性。我们将分析在丙型肝炎病毒Huh-7感染系统中与AP50结合和磷酸化所必需的E2蛋白基序。 2.丙型肝炎病毒E2对丙型肝炎病毒感染的原代培养的人肝细胞和HCVHuh-7感染系统增殖的影响。 这些最近表征的丙型肝炎病毒E2机制尚未在丙型肝炎病毒感染的正常原代人肝细胞模型系统中进行探索。这将是一个非常有价值的模型来研究这些有趣的丙型肝炎病毒E2机制，可能还有其他机制，在直接从患者那里获得的具有完整生命周期的整个自然产生的丙型肝炎病毒颗粒存在的情况下。为了研究单独的E2基序在丙型肝炎病毒感染中的作用及其重要性，有必要对Huh-7型丙型肝炎病毒感染系统进行突变分析。此外，新的病毒激酶的发现和机制研究在丙型肝炎病毒、普通病毒学、网状蛋白介导的内吞作用和信号转导等领域具有广泛的意义。

DESCRIPTION (provided by applicant): Central Hypothesis: The Hepatitis C Virus (HCV) E2 glycoprotein is a novel kinase that initiates signal transduction mechanisms modulating the following pathways: 1.) Clathrin-mediated endocytosis, through a site-specific phosphorylation of the clathrin adaptor protein-50 (AP50), a key regulator of clathrin-mediated receptor endocytosis; and 2.) Hepatocyte proliferation and liver carcinogenesis through the activation of PI3 Kinase and Akt. In pilot transaction studies and in vitro kinase assays I have obtained compelling data suggesting that E2 is a novel member of the actin-regulating kinase family (Ark/Prk kinases) that associates physically with, and phosphorylates AP50 on its phospho-acceptor Thr156, a key step for clathrin-mediated endocytosis (25,50,73). Also, we have shown that E2 is associated with AP50 in livers from HCV-infected patients, and that AP50 is phosphorylated on Thr156 to a much greater extent in these livers. In preliminary studies, we have also found that E2, in the absence of extracellular growth factors, increases PIP2, PI3K, PDK1 and Akt, as well as their activities. This signaling cascade promotes proliferation. Moreover, HCV E2 markedly stimulates hepatocyte DNA replication to an even greater extent than classic tumor promoters TGF( and EGF. 1. The physiological relevance of the interaction between HCV E2 and AP50 and the phosphorylation of AP50 in a unique HCV infection system of primary human hepatocytes. We will analyze the protein motifs of E2 which are indispensable for association with and phosphorylation of AP50 in the HCV Huh-7 infection system. 2. The effects of HCV E2 on proliferation in HCV infected primary human hepatocyte cultures and in the HCV Huh-7 infection system. These recently characterized HCV E2 mechanisms have yet to be explored in an HCV-infected normal primary human hepatocyte model system. This will be an extremely valuable model to study these intriguing HCV E2 mechanisms, and possibly others, in the presence of the entire, naturally occurring HCV viral particle with a complete life cycle, obtained directly from patients. Mutational analysis in the Huh-7 HCV infection system is necessary in order to investigate the roles of the individual motifs of E2 and their importance in HCV infection. In addition, the discovery and mechanistic studies of a novel viral kinase has extensive implications in the fields of HCV, general virology, clathrin-mediated endocytosis, and signal transduction.