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(申请人提供)：中心假说：丙型肝炎病毒(丙型肝炎病毒)E2糖蛋白是一种新型的激酶，它启动信号转导机制，调节下列通路：丙型肝炎病毒与细胞受体(CD-81、SR-B1、LDL R)和细胞紧密连接蛋白(Claudin 1和occludin)的结合是病毒最初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。网状蛋白介导的内吞作用(CME)，通过异二聚体货物适配器AP2的一个亚基-2(AP50)的位点特异性磷酸化，它是网状蛋白介导的受体内吞作用的关键调节因子，以及其他细胞替代的CME适配器蛋白，包括Numb，ARH和DAB2，它们被认为是通过假设的E2磷酸化共识位点的磷酸化来调节的。丙型肝炎病毒的E2K活性也可能通过结合/磷酸化影响其他关键的接头蛋白，包括：HIP1、2-arrestin、epsin、Calm和PD2K1。病毒颗粒的跨高尔基网络(TGN)分选和胞吐，通过异二聚体货物适配器AP1亚基的位点特异性磷酸化，AP1是TGN分选的关键调节因子，以及其他细胞TGN蛋白，包括高尔基体定位的3-Adaptin同源物和ARF结合蛋白(GGA1-3)、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通路来实现的。在初步的转染性研究和体外激酶分析中，我已经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，它与其磷酸受体Thr156上的AP50物理结合并磷酸化，Thr156是clathrin介导的内吞作用的关键步骤。此外，我们已经证明，在丙型肝炎病毒感染患者的肝脏中，E2与AP50(&lt；2)相关，并且AP50在Thr156上被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可以增加PIP2、PI3K、PDK1和Akt的表达和活性。这一信号级联促进了细胞的增殖。此外，与经典的肿瘤促进剂TGF1和EGF相比，丙型肝炎病毒E2显著刺激肝细胞DNA复制的程度更大。这项建议将研究丙型肝炎病毒与CD81、SR-B1(9)、低密度脂蛋白R(10)、细胞受体和紧密连接蛋白、claudin 1和occludin，以及磷酸化蛋白&lt；2(AP2)、Numb、ARH、DAB2、GUP/CED-6、HIP1、2-arrestin、Calm和PD2K1结合的信号转导机制，从而调节初始附着、膜融合相关事件和导致病毒进入和内吞的CME。我还将研究导致病毒胞吐的类似的TGN分选信号。这些都是由AP1(&lt；1)磷酸化(14)控制的，因此，就像内吞作用中的AP2(&lt；2)磷酸化一样，也可能受E2的调控。这项建议还将集中在E2如何诱导Akt途径并促进肝细胞增殖，从而促进肝癌的发生。 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。丙型肝炎感染宿主肝细胞的完整机制尚不清楚。首席研究员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通过首席调查员发现的新机制控制病毒的细胞进入。这些实验可以产生针对这些新途径的新疗法的合理性，并为未来的疫苗研究铺平道路。