Hepatitis C: E2 Molecular Mechanisms

丙型肝炎：E2 分子机制

• 批准号: 7887884
• 负责人: MARTINA BUCK
• 金额: $ 35.4万
• 依托单位: VETERANS MEDICAL RESEARCH FDN/SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7887884
• 关键词: Actins; Adaptor Signaling Protein; Affect; Arrestins; Binding; Binding Proteins; Biological Assay; Biological Models; CD81 gene; Cell membrane; Cellular Membrane; Cessation of life; Clathrin; Co-Immunoprecipitations; Coat Protein Complex I; Complex; Consensus; DNA; DNA Replication Induction; DNA biosynthesis; Data; EGF gene; Endocytosis; Event; Exocytosis; Family; Future; Golgi Apparatus; Growth Factor; Hepatitis C; Hepatitis C virus; Hepatocarcinogenesis; Hepatocyte; Homologous Gene; Human; In Vitro; Individual; Infection; Laser Scanning Confocal Microscopy; Lead; Length; Liver; Low-Density Lipoproteins; MAP Kinase Gene; Mediating; Medical; Membrane Fusion; Molecular; Mutate; Mutation; Mutation Analysis; Nonstructural Protein; PDPK1 gene; Pathway interactions; Patients; Phosphatidylinositol 4,5-Diphosphate; Phosphorylation; Phosphotransferases; Physiological; Point Mutation; Principal Investigator; Proteins; RNA; Recombinant Proteins; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Sorting - Cell Movement; Stimulus; System; Tight Junctions; Transcription Factor AP-1; Transfection; Tumor Promoters; Vaccines; Viral; Viral Proteins; Virus; Virus Activation; Virus Receptors; arrestin 2; claudin-1 protein; env Gene Products; epsin; extracellular; hepatitis C virus envelope 2 protein; hepatoma cell; liver cell proliferation; member; mutant; new therapeutic target; novel; occludin; particle; public health relevance; receptor; receptor mediated endocytosis; research study; trans-Golgi Network; viral RNA

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: HCV association with cellular receptors (CD-81, SR-B1, LDL R) and cellular tight junction proteins (Claudin 1, and occludin) that are indispensible for initial viral binding to the cellular membrane and subsequent membrane fusion mechanisms that lead to viral entry. Clathrin-mediated endocytosis (CME), through site-specific phosphorylations of <2(AP50), a subunit of the heterodimeric cargo adaptor AP2, a key regulator of clathrin-mediated receptor endocytosis and other cellular alternative CME adaptor proteins including NUMB, ARH, and Dab2 that are known to be regulated through phosphorylation at the postulated E2 phosphorylation consensus site. HCV E2 kinase activity may also affect other critical adaptor proteins including; HIP1, 2-arrestin, epsin, CALM, and PD2K1 by association/phosphorylation. Trans Golgi network (TGN) sorting and exocytosis of the viral particle, through site-specific phosphorylations of <1 a subunit of the heterodimeric cargo adaptor AP1, a key regulator of TGN sorting and other cellular TGN proteins including the Golgi-localized, 3-adaptin homologue and ARF- binding proteins (GGA1-3), COPI, COPII, Sar and ARF. Hepatocyte proliferation and liver carcinogenesis through the activation of host cellular signal transduction pathways, such as the Akt pathway. In pilot transfection 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. Also, we have shown that E2 is associated with AP50 (<2) 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 the expression and activities of PIP2, PI3K, PDK1 and Akt. This signaling cascade promotes proliferation. Moreover, HCV E2 markedly stimulates hepatocyte DNA replication to an even greater extent than classic tumor promoters TGF1 and EGF. This proposal will study the signal transduction mechanisms by which HCV associates with CD81, SR- B1(9), LDL R(10), cellular receptors and tight junction proteins, claudin 1 and occludin, and phosphorylates <2 (AP2), NUMB, ARH, Dab2, GULP/CED-6 , HIP1, 2-arrestin, CALM, and PD2K1 thereby modulating initial attachment, membrane-fusion associated events and CME resulting in viral entry and endocytosis. I will also investigate the analogous TGN sorting signals that lead to viral exocytosis. These are controlled by AP1 (<1) phosphorylation (14) and so like the AP2 (<2) phosphorylation during endocytosis may also be governed by E2. This proposal will also focus on how E2 induces the Akt pathway and increases hepatocyte proliferation, thereby facilitating liver carcinogenesis. PUBLIC HEALTH RELEVANCE: Hepatitis C infection can cause serious medical problems and death. The complete mechanisms by which Hepatitis C infects the host liver cell remain unknown. The Principal Investigator proposes to study the mechanisms by which hepatitis C enters and exits the liver cell. Preliminary results suggest that the envelope protein 2 on the outside of the virus controls the cellular entry of the virus through novel mechanisms discovered by the Principal Investigator. These experiments could generate the rational for novel therapeutics targeting these new pathways and lead the way to future vaccine studies.

描述（由申请人提供）：中心假设：丙型肝炎病毒（HCV） E2糖蛋白是一种启动信号转导机制的新型激酶，调节以下途径：HCV与细胞受体（CD-81, SR-B1, LDL - R）和细胞紧密连接蛋白（Claudin 1和occludin）的关联，它们是初始病毒与细胞膜结合和随后导致病毒进入的膜融合机制所必需的。网格蛋白介导的内吞作用（CME），通过<2（AP50）的位点特异性磷酸化，AP50是异二聚体货物接头AP2的一个亚基，是网格蛋白介导的受体内吞作用和其他细胞替代CME接头蛋白的关键调节剂，包括NUMB， ARH和Dab2，已知通过在假设的E2磷酸化共识位点磷酸化来调节。HCV E2激酶活性也可能影响其他关键的接头蛋白，包括；HIP1, 2-骤停素，epsin， CALM和PD2K1的关联/磷酸化。反式高尔基网络（Trans Golgi network， TGN）的分选和病毒颗粒的胞外分泌，通过异二聚体载物接头AP1 <1 a亚基的位点特异性磷酸化，AP1是TGN分选和其他细胞TGN蛋白的关键调控因子，包括高尔基定位、3-适应蛋白同源物和ARF结合蛋白（GGA1-3）、COPI、COPII、Sar和ARF。肝细胞增殖和肝癌发生通过激活宿主细胞信号转导通路，如Akt通路。在中试转染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）的新成员，它与磷酸化受体Thr156上的AP50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。此外，我们已经证明E2与hcv感染患者肝脏中的AP50相关（<2），并且这些肝脏中AP50在Thr156上磷酸化的程度要大得多。在前期研究中，我们还发现E2在缺乏细胞外生长因子的情况下，可以增加PIP2、PI3K、PDK1和Akt的表达和活性。这种信号级联促进了细胞增殖。此外，与经典的肿瘤启动子TGF1和EGF相比，HCV E2显著刺激肝细胞DNA复制的程度更大。该提案将研究HCV与CD81， SR- B1(9), LDL R(10)，细胞受体和紧密连接蛋白，claudin 1和occludin结合的信号转导机制，并磷酸化<2 (AP2)， NUMB， ARH, Dab2, GULP/CED-6, HIP1, 2-阻滞蛋白，CALM和PD2K1，从而调节初始附着，膜融合相关事件和CME导致病毒进入和内吞作用。我还将研究导致病毒胞吐的类似TGN分选信号。这些是由AP1（<1）磷酸化控制的（14），因此像AP2（<2）磷酸化一样，内吞作用也可能由E2控制。本研究还将关注E2如何诱导Akt通路，增加肝细胞增殖，从而促进肝癌的发生。