Hepatitis C: E2 Molecular Mechanisms

丙型肝炎：E2 分子机制

• 批准号: 8071218
• 负责人: MARTINA BUCK
• 金额: $ 29.69万
• 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/8071218
• 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糖蛋白是一种新型激酶，可启动调节以下途径的信号转导机制：HCV与细胞受体的结合（CD-81、SR-B1、LDL R）和细胞紧密连接蛋白封闭蛋白（Claudin 1和occludin）是初始病毒与细胞膜结合以及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α 2（AP 50）的位点特异性磷酸化，α 2（AP 50）是异源二聚体货物衔接子AP 2的亚基，是网格蛋白介导的受体内吞作用的关键调节剂，其他细胞替代CME衔接蛋白包括NUMB、ARH和Dab 2，已知其通过在假定的E2磷酸化共有位点处的磷酸化来调节。HCV E2激酶活性也可能通过缔合/磷酸化影响其他关键衔接蛋白，包括HIP 1、2-抑制蛋白、胰蛋白酶、CALM和PD 2K 1。 通过异源二聚体货物衔接子AP 1的α亚基的位点特异性磷酸化，跨高尔基体网络（TGN）分选和病毒颗粒的胞吐，所述异源二聚体货物衔接子AP 1是TGN分选和其它细胞TGN蛋白的关键调节子，所述其它细胞TGN蛋白包括高尔基体定位的3-衔接蛋白同源物和ARF结合蛋白（GGA 1 -3）、COPI、COPII、Sar和ARF。 肝细胞增殖和肝癌的发生是通过激活宿主细胞信号转导通路，如Akt通路。在初步转染研究和体外激酶测定中，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）的一个新成员，与其磷酸化受体Thr 156上的AP 50发生物理关联和磷酸化，这是网格蛋白介导的内吞作用的关键步骤。此外，我们已经表明，E2与HCV感染患者肝脏中的AP 50（<2）相关，并且在这些肝脏中，AP 50在Thr 156上的磷酸化程度要高得多。在初步研究中，我们还发现，E2，在细胞外生长因子的情况下，增加PIP 2，PI 3 K，PDK 1和Akt的表达和活性。这种信号级联促进增殖。此外，HCV E2显着刺激肝细胞DNA复制的程度甚至超过经典的肿瘤促进剂TGF 1和EGF。该提案将研究HCV与CD 81、SR-B1（9）、LDL R（10）、细胞受体和紧密连接蛋白、claudin 1和occludin以及磷酸化<2（AP 2）、NUMB、ARH、Dab 2、GULP/CED-6、HIP 1、2-arrestin、CALM和PD 2K 1相关的信号转导机制，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作用的类似TGN分选信号。这些由AP 1（<1）磷酸化控制（14），因此与内吞作用期间的AP 2（<2）磷酸化一样，也可能由E2控制。该提案还将关注E2如何诱导Akt通路并增加肝细胞增殖，从而促进肝癌发生。 公共卫生相关性：丙型肝炎感染可导致严重的医疗问题和死亡。丙型肝炎病毒感染宿主肝细胞的完整机制仍然未知。主要研究者建议研究丙型肝炎进入和离开肝细胞的机制。初步结果表明，病毒外部的包膜蛋白2通过主要研究者发现的新机制控制病毒进入细胞。这些实验可以为靶向这些新途径的新疗法提供理论依据，并为未来的疫苗研究铺平道路。