HBV非经典X蛋白经PCBP2介导游离铁增加及铁死亡在肝衰竭中的作用

Massive/submassive hepatocyte cell death is the main character of liver failure, however the mechanism is not fully understood. In China, the predominant cause of liver failure is hepatitis B virus (HBV) infection. The HBV DNA genome is about 3.2 kb and contains four classical open reading frames (ORFs). Recently, the 4 ORFs pattern was challenged by novel gene discovered in HBV genome. We and others found a novel ORF with 168 nucleotides upstream of the starting code of the classical X gene and termed as Pre-X. The novel ORF Pre-X has transcriptional activity and can be translated with the X gene in a frame to form non-classical HBX, termed as HBV whole-X gene (HBwx). The molecular function and involvement in diseases of the HBwx remain uncharacterized. Previously, our lab generated a substantial amount of data characterized the molecular function of HBwx. Firstly, a robust HBwx staining was found in liver tissues from patients with liver failure, which indicated that HBwx involved in liver failure. Secondly, HBwx expressing cells were more sensitive to iron induced hepatocyte cell death. Thirdly, the free iron levels were increased in HBwx expressing cells, which is an important character of ferroptosis. Moreover, LC/MS/MS and co-immunoprecipitation revealed a directed interaction between HBwx and PCBP2, which is an iron chaperone by binding iron and avoiding free iron accumulation in cytoplasm..We hypothesize that HBwx induces an increase of free iron level and ferroptosis in a PCBP2-dependent manner. Our central hypothesis will be tested by four specific aims: (1) Mapping the binding domains required to mediate interacting each other by truncated protein and co-immunoprecipitation; explorer whether HBwx may inhibit the function of iron chaperone PCBP2 by detecting binding-iron in PCBP2 after co-expression HBwx in yeast. (2) We will elucidate whether free iron accumulation and ferroptosis induced by HBwx play a role in liver failure. We also explore the mechanism of HBwx by testing free iron and ferroptosis by genetic manipulating with CRISPR/Cas9 gene editing system. (3) We will utilize genetically manipulated mice expressing HBwx in hepatocytes to explore HBwx function in liver failure mouse model and elucidate the molecular pathway by which HBwx inducing free iron accumulation and ferroptosis. (4) To confirm the HBwx-PCBP2-free iron-ferroptosis pathway by testing the molecular involved in fresh liver tissue from patients diagnosed with liver failure. This contribution will evaluate the role of ferroptosis induced by HBwx in liver failure and better our understanding of the molecular basis of liver failure.

肝细胞短期内大量死亡是肝衰竭的病理基础，其机制尚不完全清楚。HBV感染是我国肝衰竭首要病因，本课题组前期发现经典X基因上游存在前X，前X+X合称HBwx（非经典x）。并发现HBwx①在肝衰竭组织高表达、促进肝细胞死亡，提示其可能参与肝衰竭；②增加肝细胞内游离铁，这是铁死亡的特征之一；③结合铁伴侣PCBP2，后者结合并转运铁离子，避免胞浆游离铁堆积。因此，我们提出：HBwx-PCBP2-游离铁增加-铁死亡促进肝衰竭发展。本课题将利用短截蛋白技术定位HBwx/PCBP2结合部位；在酵母表达系统明确二者结合对PCBP2铁离子结合能力的影响；在细胞模型探究HBwx对游离铁、铁死亡的调控；基因敲除，以明确HBwx介导的游离铁增加和铁死亡是否依赖PCBP2；在HBwx+小鼠研究上述通路在肝衰竭中的作用；在肝衰竭患者肝组织中验证。以阐明HBV非经典X蛋白介导的铁死亡在肝衰竭中的作用，丰富肝衰竭发病机制。

肝衰竭是病毒、酒精、药物等各种因素造成的肝损害的最严重形式。尽管肝衰竭机制研究及临床救治已取得不少成果，但仍未有突破性进展。肝细胞短期内大量死亡及持续紊乱的炎症是肝衰竭的病理基础。本课题从HBV相关蛋白HBwx介导肝细胞铁死亡、自噬调节肝星状细胞表达炎症因子等方向出发和展开，探索肝衰竭发生中的具体分析机制。.研究发现：①HBwx介导游离铁增加及细胞死亡，铁死亡拮抗剂能挽救HBwx介导的细胞死亡，敲除PCBP2可阻断HBwx介导游离铁增加及细胞死亡，同时HBwx(+)肝衰竭组织中HBwx与PCBP2 定位一致，且铁死亡增加。②LPS通过活化MAPK p38抑制自噬；活化的MAPK p38诱导Ulk1磷酸化，抑制Ulk1-Atg13复合物形成，从而抑制自噬；在原代大鼠肝星状细胞中LPS通过MAPK p38/Ulk1 pathway抑制自噬，诱导的IL-1β表达。.在本期国科金支持下，课题组从HBwx这一非经典X蛋白视角阐明铁死亡在肝衰竭中的作用，同时证实肝星状细胞同样参与炎症因子分泌，丰富肝衰竭的发病机制，为肝衰竭的治疗提供可能的新思路。