Hepatitis C: E2 Molecular Mechanisms

丙型肝炎：E2 分子机制

基本信息

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
项目类别：
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

项目摘要

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（CD-81、SR-B1、LDL R）的关联和细胞紧密连接蛋白（Claudin 1 和 Occludin），它们对于最初病毒与细胞膜的结合以及随后导致病毒进入的膜融合机制是必不可少的。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，<2(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的关键调节剂和其他细胞替代 CME 接头蛋白，包括 NUMB、ARH、已知通过假定的 E2 磷酸化共有位点的磷酸化来调节 Dab2 和 Dab2。 HCV E2 激酶活性也可能影响其他关键的接头蛋白，包括： HIP1、2-arrestin、epsin、CALM 和 PD2K1 通过关联/磷酸化。跨高尔基网络 (TGN) 分选和病毒颗粒的胞吐作用，通过异二聚体货物接头 AP1 亚基的位点特异性磷酸化，AP1 是 TGN 分选和其他细胞 TGN 蛋白（包括高尔基定位的 3-）的关键调节因子。适应素同源物和 ARF 结合蛋白 (GGA1-3)、COPI、COPII、Sar 和 ARF。通过激活宿主细胞信号转导途径（例如 Akt 途径）来促进肝细胞增殖和肝癌发生。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（Ark/Prk 激酶）的新成员，与磷酸受体 Thr156（一种磷酸化受体 Thr156）物理结合并磷酸化 AP50。网格蛋白介导的内吞作用的关键步骤。此外，我们还发现，在 HCV 感染患者的肝脏中，E2 与 AP50 (<2) 相关，并且 AP50 在这些肝脏中的 Thr156 上磷酸化程度要高得多。在初步研究中，我们还发现，在缺乏细胞外生长因子的情况下，E2会增加PIP2、PI3K、PDK1和Akt的表达和活性。这种信号级联促进增殖。此外，HCV E2 比经典肿瘤促进剂 TGF1 和 EGF 更能显着刺激肝细胞 DNA 复制。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 以及磷酸盐 <2 (AP2)、NUMB、 ARH、Dab2、GULP/CED-6、HIP1、2-arrestin、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作用的类似 TGN 分选信号。这些受 AP1 (<1) 磷酸化 (14) 控制，因此内吞作用期间的 AP2 (<2) 磷酸化也可能受 E2 控制。该提案还将重点关注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。丙型肝炎感染宿主肝细胞的完整机制仍不清楚。首席研究员提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通过首席研究员发现的新机制控制病毒进入细胞。这些实验可以为针对这些新途径的新疗法提供理论基础，并为未来的疫苗研究指明方向。