Involvement of ubiquitin system on the hepatitis B virus X protein (HBx)-mediated transactivation activity

泛素系统参与乙型肝炎病毒 X 蛋白 (HBx) 介导的反式激活活性

• 批准号: 17590274
• 负责人: TOKUNAGA Fuminori
• 金额: $ 2.18万
• 依托单位: Osaka City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590274/
• 关键词: protein; enzyme; virus; transcription; 蛋白質; protein; enzyme; virus; transcription; 蛋白質

We have identified an RING-IBR-RING (RBR) family ubiquitin ligase, HOIL-1L, as a predominant intracellular isoform of HOIL-1 (heme-oxidized IRP2 ligase-1). HOIL-1L forms a high molecular mass (〜600 kDa) complex with HOIP (HOIL-1L-interacting protein) in vivo. HOIP also belongs to RBR family, and the HOIL-1L/HOIP complex exhibits ubiquitin polymerization activity in vitro. To examine the physiological function of HOIL-1L/HOIP, we first screened the effect of HOIL-1L/HOIP on transcriptional activity, since HOIL-1 has shown to bind protein kinase C and hepatitis B virus X protein, and both of them affect transcription. Then, we found by luciferase reporter assay that combined expression of HOIL-1L and HOIP, but not by single expression of respective ligase, specifically induced NF-KB activation. Indeed, co-expression of HOIL-1L/HOIP caused intranuclear translocation of p65 and p50, and enhanced phosphorylation of IKBa. Using various domain-deleted mutants, we identified that the RING domains of HOIP played a crucial role on the NF-KB activation. Notably, a dysfunctional RING-finger mutant of HOIP inhibited TNF-a-and TAK1-mediated NF-KB activation, but had no effect on NIK-mediated NF-KB activation. These results indicate that HOIL-1L/HOIP activates canonical NF-KB pathway through the ubiquitin ligase activity of HOIP. The ligase complex may have a pivotal role on the inflammation, anti-apoptosis, and immune system through the regulation of NF-KB pathway.

我们已经将戒指能力环（RBR）家族泛素连接酶HOIL-1L确定为HOIL-1的主要细胞内同工型（血红素氧化IRP2连接酶1）。 HOIL-1L在体内与Hoip（HOIL-1L相互作用蛋白）形成高分子质量（〜600 kDa）。 HOIP也属于RBR家族，HOIL-1L/HOIP复合物在体外表现出泛素聚合活性。为了检查HOIL-1L/HOIP的生理功能，我们首先筛选了HOIL-1L/HOIP对转录活性的影响，因为HOIL-1已显示蛋白激酶C和乙型肝炎B病毒X蛋白，并且两者都会影响转录。然后，我们通过荧光素酶报告基组装发现了HOIL-1L和HOIP的表达，而不是通过各个连接酶的单个表达，特别是诱导的NF-KB激活。实际上，HOIL-1L/HOIP的共表达引起了p65和p50的内部易位，并增强了IKBA的磷酸化。使用各种域删除的突变体，我们确定了HOIP的环域在NF-KB激活中起着至关重要的作用。值得注意的是，HOIP的功能失调的无名指突变体抑制了TNF-A和TAK1介导的NF-KB激活，但对NIK介导的NF-KB激活没有影响。这些结果表明，HOIL-1L/HOHIP通过HOIP的泛素连接酶活性激活规范NF-KB途径。连接酶复合物可以通过调节NF-KB途径在注射，抗凋亡和免疫系统中具有关键作用。