HEPATOCELLULAR CARCINOMA AND HEPATITIS C: E2 MOLECULAR MECHANISMS

描述（由申请人提供）：中心假设：丙型肝炎病毒（HCV）E2糖蛋白是一种新型激酶，可启动调节以下途径的信号转导机制：1.）网格蛋白介导的内吞作用，通过网格蛋白衔接蛋白-50（AP 50）的位点特异性磷酸化，网格蛋白衔接蛋白-50（AP 50）是网格蛋白介导的受体内吞作用的关键调节剂;和2.）PI 3激酶和Akt激活对肝细胞增殖和肝癌发生的影响 在试验性交易研究和体外激酶试验中，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（方舟/Prk激酶）的新成员，与其磷酸受体Thr 156上的AP 50物理相关并使其磷酸化，这是网格蛋白介导的内吞作用的关键步骤（25，50，73）。此外，我们已经表明，E2与HCV感染患者肝脏中的AP 50相关，并且在这些肝脏中，AP 50在Thr 156上磷酸化的程度要大得多。 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增加PIP 2，PI 3 K，PDK 1和Akt，以及它们的活性。这种信号级联促进增殖。此外，HCV E2显着刺激肝细胞DNA复制的程度甚至大于经典的肿瘤促进剂TGF β和EGF。 1. HCV E2和AP 50之间相互作用的生理相关性以及原代人肝细胞独特HCV感染系统中AP 50的磷酸化。我们将分析E2的蛋白基序，这些基序对于HCV Huh-7感染系统中AP 50的缔合和磷酸化是必不可少的。 2. HCV E2对HCV感染的原代人肝细胞培养物和HCV Huh-7感染系统中增殖的影响。 这些最近的特点HCV E2机制尚未在HCV感染的正常人原代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，研究这些有趣的HCV E2机制，可能还有其他机制，在整个自然发生的HCV病毒颗粒的存在下，具有完整的生命周期，直接从患者中获得。Huh-7 HCV感染系统中的突变分析是必要的，以研究E2的各个基序的作用及其在HCV感染中的重要性。此外，一种新型病毒激酶的发现和机制研究在HCV、普通病毒学、网格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.