Hepatitis C: E2 Molecular Mechanisms

丙型肝炎：E2 分子机制

• 批准号: 8322071
• 负责人: MARTINA BUCK
• 金额: $ 29.09万
• 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/8322071
• 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.

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到导致病毒进入的细胞膜和随后的膜融合机制。 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），杂二聚体货物适配器AP2的亚基，杂型货物适配器AP2，网状蛋白介导的受体内吞作用的关键调节剂和其他细胞内吞噬和其他细胞替代CME适配蛋白包括在内，包括NOMB 2 E2磷酸化共识位点。 HCV E2激酶活性也可能影响其他关键衔接蛋白，包括： HIP1，2-arrestin，Epsin，Caln和PD2K1通过关联/磷酸化。 通过位点特异性磷酸化<1的位点特异性磷酸化<1杂二聚体货物适配器AP1，TGN分类和其他细胞TGN蛋白的关键调节剂，包括GOLGI-钙化，3-辅助蛋白蛋白质（Arf-anf-Coga-Copi toci offgaIns）（a） SAR和ARF。 通过宿主细胞信号转导途径（例如AKT途径）激活肝细胞增殖和肝癌发生。在试验转染研究和体外激酶测定中，我获得了令人信服的数据，这表明E2是肌动蛋白调节激酶家族（ARK/PRK激酶）的新成员，该家族（ARK/PRK激酶）与磷酸化的phosceptor thr156上的AP50相关联，并磷酸化AP50的关键一步，是隔rin介导的内层的关键。同样，我们已经表明，E2与HCV感染患者的肝脏中的AP50（<2）相关，并且在这些肝脏中，AP50在THR156上磷酸化。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增加了PIP2，PI3K，PDK1和AKT的表达和活性。该信号级联反应促进增殖。此外，HCV E2显着刺激肝细胞DNA复制，比经典肿瘤启动子TGF1和EGF更大程度。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Claudin 1和coctludin，以及磷酸化<2（AP2），ARH，ARH，ARH，DAB2，GULP/CED-6，HIP1，HIP1，HIP1，2--2--2-6，HIP1，HIP1，2--2--16，HIP1，HIP1，2--2-16，HIP1，2---调节初始附着，膜融合相关事件和CME，导致病毒入口和内吞作用。我还将研究导致病毒胞吐作用的类似TGN分选信号。这些由AP1（<1）磷酸化（14）控制，因此，在内吞作用期间的AP2（<2）磷酸化也可以受E2的控制。该建议还将集中于E2如何诱导AKT途径并增加肝细胞增殖，从而促进肝癌发生。 公共卫生相关性：丙型肝炎感染会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仍然未知。主要研究者建议研究丙型肝炎进入并退出肝细胞的机制。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来控制病毒的细胞进入。这些实验可能会产生针对这些新途径的新型治疗剂的合理性，并导致未来的疫苗研究。