乙型肝炎病毒HBx类泛素化Neddylation修饰的功能及生物学意义

Chronic Hepatitis B virus (HBV) infection which is the main cause of hepatoma carcinoma (HCC) remains a major public health concern. The x protein of HBV (HBx) has been reported to play an important role in regulating cell proliferation, apoptosis and tumor development. However, the mechanism of how HBx protein stability being regulated is not fully understood. Our preliminary data showed that HBx can be specifically modified by Nedd8 which is a kind of ubiquitin like molecules. We also found that E3 ubiquitin ligase Hdm2 can promote HBx neddylation significantly. In order to address the significance of HBx neddylation, we propose to perform the following studies: (1) To identify the neddylation site of HBx by mass spectrometry，generate the HBx mutants and analyze whether HBx neddylation affects its stability or its activity on transcriptional regulation. (2) To examine whether Hdm2 and HBx can promote cell cycle progression synergistically by taking the approaches of flow cytometry and relevant biochemical techniques. To analyze whether Hdm2 promotes the tumor growth through HBx neddylation by using mouse model. (3) To investigate if there is a positive correlation between the protein levels of Hdm2 and HBx as well as prognostics by analyzing the clinical samples and data from HCC patients. The major goal of this project is to uncover the biological significance of HBx neddylation on regulating cell cycle and tumorigenesis.

乙型肝炎病毒（HBV）的持续性感染是肝癌发生的主要因素。 HBV编码的x蛋白（HBx）在细胞周期调控以及肝癌发生发展中发挥重要作用，但HBx蛋白稳定性的调控机制尚不明确。我们研究发现HBx能发生类泛素化Neddylation修饰，并确定Hdm2是促进HBx Neddylation修饰的E3泛素连接酶。为了探究HBx Neddylation修饰对其功能的影响及其在肝癌发生发展中的作用，我们拟开展以下研究：（1）通过质谱分析定位HBx Neddylation修饰的位点；分析HBx Neddylation修饰对其蛋白稳定性以及转录调控活性的影响；（2）通过流式细胞术等方法，分析Hdm2与HBx能否协同促进细胞周期；通过小鼠成瘤实验，研究它们是否协同促进肿瘤形成；（3）通过临床样本分析Hdm2和HBx蛋白水平的相关性以及与肝癌病人预后的关联，揭示HBx Neddylation修饰的临床意义。

乙型肝炎病毒（HBV）HBx蛋白在病毒复制和肝癌发生发展过程中起关键作用。然而，对HBx稳定性的调控机制并不为人所知。我们发现，HBx可以发生NEDD8修饰，且HDM2 E3连体酶促进HBx NEDDylation修饰，深入研究发现HBx NEDDylation修饰抑制其泛素化，因此增强了HBx的蛋白稳定性。对临床肝癌样本数据的分析表明HDM2和HBx的表达量呈正相关。通过质谱分析我们定位了 HBx 的主要 NEDDylation 位点。我们的研究表明，HBx的NEDDylation缺陷型突变体负调控对其激活下游基因转录的能力以及促进体内细胞增殖和肿瘤生长的能力。总之，我们的发现揭示了HDM2对HBx的一种新颖的翻译后修饰，它调节其稳定性、亚细胞定位和功能。这些发现表明，HDM2是HBx的重要调节分子，是HBV相关HCC的潜在诊断/治疗标志物。