泛素特异性肽酶USP15对乙型肝炎病毒X蛋白水平的影响及机制

Hepatitis B virus X protein (HBx) is the most important pathogenic factor of hepatitis B virus (HBV), it participated in the progressing of hepatocellular carcinoma (HCC). Therefore, HBx protein level is crucial for its pathogenicity. Previously, by using CytoTrap yeast two-hybrid assay and GST- pull down assay, we demonstrated that ubiquitin specific peptidase 15 (USP15) may interact with HBx. USP15 is an important negative regulatory factor in ubiquitin-proteasome pathway. USP15 targets substrate proteins for proteasome mediated degradation by deubiquitylation. In the present study, interaction of USP15 with HBx and respective binding domain will be confirmed by CytoTrap yeast two-hybrid and Co-immunoprecipitation (Co-IP), then the influences of USP15 on HBx level will be evaluated by over-expression and siRNA knock-down of USP15, and the USP15 mutants or the non-related USP5 will be introduced to confirm the specificity of the effets of USP15 on HBx; finally, the ubiquitin reporter system will be established and used for the evaluation the deubiquitylation of HBx by USP15. The aim of this study is to investigate the effect of ubiquitin-proteasome pathway on HBx protein level, our study will be helpful for deep understanding of the pathogenicity of HBV.

乙型肝炎病毒X蛋白（HBx）是乙型肝炎病毒（HBV）最重要的致病因子，参与HBV相关性肝细胞癌的病程进展，HBx蛋白水平与其致病性密切相关。我们之前利用CytoTrap酵母双杂交与GST- pull down初步证实泛素特异性肽酶15（USP15）与HBx存在相互作用。USP15属于去泛素化酶，是泛素—蛋白酶体途径重要的负控因子，通过去泛素化影响底物蛋白降解。本研究先通过CytoTrap酵母双杂交及免疫共沉淀等验证HBx与USP15的相互作用并确定各自结合区域；进而探讨USP15表达水平对HBx蛋白水平的影响，再通过检测USP15活性变异体及无关USP(USP5)对HBx蛋白水平的影响，明确USP15对HBx蛋白作用的特异性；最后以泛素指示载体探讨USP15是否通过去泛素化影响HBx蛋白水平。本研究旨在阐明泛素—蛋白酶体途径对HBx蛋白水平的影响，所获结果将有助于更深入了解HBV致病机制。

乙型肝炎病毒X蛋白（HBx）在病毒的复制及HBV相关性肝细胞癌的病程进展中发挥重要作用。HBx蛋白周转快速，主要通过泛素-蛋白酶体途径降解，采用蛋白酶体抑制剂会增加细胞内HBx的蛋白水平并导致多聚泛素化HBx的积累。去泛素化酶（DUBs）可以使蛋白质去泛素化，从而增加蛋白的稳定性和/或活性。然而，目前尚无关于DUBs参与调节 HBx蛋白泛素-蛋白酶体途径降解的相关报道。本研究证明去泛素化酶USP15是HBx蛋白水平的关键调节因子，我们研究发现USP15通过与HBx第51-120位氨基酸残基之间的区域直接结合与HBx相互作用；增加USP15表达量，HBx的蛋白水平也随之增加；相反地，siRNA沉默USP15的表达，HBx的蛋白水平也随之降低。机制研究表明HBx蛋白水平及稳定性增加是由于USP15与HBx互作后使HBx蛋白去泛素化从而阻止了HBx蛋白经由泛素-蛋白酶体途径降解。并且，USP15的存在显著增强了HBx的反式激活功能。以上结果表明USP15在HBx蛋白稳定性及生物学功能方面发挥重要作用。