Effects of hepatitis C virus proteins on protein-tyrosine phosphorylation in liver cells

2006年，我们在体外培养的肝细胞（Huh-7细胞）中发现HCV非结构蛋白5A（NS 5A）与Syk有很强的相互作用，而磷脂酶C（PLC）-γ1是Syk的胞内靶点。此外，我们还对HCV NS 3和NS 4A的病理机制、新型SSPE病毒的鉴定、病毒相关的泛素连接酶的特性等进行了研究，并报道了结果。2007年，我们发现在携带HCV RNA复制子的Huh-7.5细胞和感染HCV的细胞（HCV J6/JFH-1）中也检测到了NS 5A与Syk的相互作用。缺失突变分析显示，NS 5A的N-末端部分参与与Syk的物理相互作用。体外激酶试验表明，NS 5A抑制Syk的酶活性，并且除了N-末端175个残基之外，还需要NS 5A的中心部分来抑制Syk。此外，免疫组化分析显示，内源性Syk，否则是弥漫性表达在正常肝细胞的细胞质中，被定位在细胞膜附近的补丁模式在HCV感染的肝细胞。我们的研究结果表明，NS 5A是通过抑制Syk，一种有效的肿瘤抑制剂在人类乳腺癌的肝细胞癌变的可能性。此外，我们研究了糖尿病作为HCV并发症的发病机制和HCV感染的蛋白质组分析。此外，我们研究了新的衔接蛋白3BP 2和人类遗传性疾病巨像症的作用，并发表了一篇综述文章。

The aim of this study is to investigate the effects of hepatitis C virus (HCV) proteins on protein-tyrosine phosphorylation, in particular, protein-tyrosine kinase Syk in vivo.In 2006, we identified that HCV non-structural protein 5A (NS5A) strongly interacted with Syk, and phospholipase C (PLC)-γ1 was the intracellular target of Syk in cultured liver cells (Huh-7 cells). Moreover, we investigated the pathological mechanism of HCV NS3 and NS4A, identification of novel SSPE virus, and characterization of virus-related ubiquitin ligase, and reported their results.In 2007, we found that interaction of NS5A with Syk was detected also in Huh-7.5 cells harboring an HCV RNA replicon and those infected with HCV (HCV J6/JFH-1). Deletion mutational analysis revealed that an N-terminal portion of NS5A was involved in the physical interaction with Syk. In vitro kinase assay demonstrated that NS5A inhibited enzymatic activity of Syk and that, in addition to the N-terminal 175 residues, a central portion of NS5A was required for the Syk inhibition. Moreover, immunohistochemical analysis revealed that endogenous Syk, which otherwise was expressed diffusely in the cytoplasm of normal hepatocytes, was localized near the cell membrane with a patched pattern in HCV-infected hepatocytes. Our results imply the possibility that NS5A is involved in the carcinogenesis of hepatocytes through the suppression of Syk, a potent tumor suppressor in human breast carcinoma. Furthermore, we investigated the pathogenesis of diabetes as a HCV complication and proteome analysis of HCV infection. Furthermore, we examined the role of novel adaptor protein 3BP2 and human inherited disease cherubism and published a review article.