A rational design for hepatitis B virus X protein refolding and bioprocess development guided by second virial coefficient studies

The hepatitis B virus X (HBx) protein is well known for its role in hepatitis B virus infection that often leads to hepatocellular carcinoma. Despite the clinical importance of HBx, there is little progress in anti-HBx drug development strategies due to shortage of HBx from native sources. Consistent expression of HBx as insoluble inclusion bodies within various expression systems has largely hindered HBx manufacturing via economical biosynthesis routes. Confronted by this roadblock, this study aims to quantitatively understand HBx protein behaviour in solution that will guide the rational development of a refolding-based bioprocess for HBx production. Second virial coefficient (SVC) measurements were employed to study the effects of varying physicochemical parameters on HBx intermolecular protein interaction. The SVC results suggest that covalent HBx aggregates play a key role in protein destabilisation during refolding. The use of an SVC-optimised refolding environment yielded bioactive and soluble HBx proteins from the denatured–reduced inclusion body state. This study provides new knowledge on HBx solubility behaviour in vitro, which is important in structure–function elucidation behaviour of this hydrophobic protein. Importantly, a rational refolding-based Escherichia coli bioprocess that can deliver purified and soluble HBx at large scale is successfully developed, which opens the way for rapid preparation of soluble HBx for further clinical and characterisation studies.     

HBx-EGFP-TLM融合蛋白表达载体的构建、蛋白表达纯化及活性检测

乙型肝炎病毒X蛋白 (HBx) 具有广泛的转录激活作用,但是由于细胞类型和实验条件的差异,外源启动子介导的HBx瞬时表达水平常表现出不均一性,为其功能的研究带来困难。为了解决HBx研究过程中遇到的这些难题,利用PCR扩增获得HBx及含转导肽TLM的EGFP编码序列,经酶切后插入pGEX-4T-1原核表达载体,成功构建重组质粒pGEX-HBx-EGFP-TLM和pGEX-EGFP-TLM。重组质粒转化大肠杆菌BL21(DE3) 感受态细胞,融合蛋白经IPTG诱导表达,采用?KTATM Purifier 蛋白纯化系统纯化并进行SDS-PAGE和Western blotting分析,确定为目的蛋白。将纯化后的融合蛋白分别与AML12和SMMC-7721细胞共孵育,Western blotting和激光共聚焦显微镜检测证实TLM转导肽能够介导HBx-EGFP和EGFP进入细胞,同时进入细胞的HBx-EGFP-TLM能够发挥转录激活活性,为HBx功能的深入研究奠定了基础。     

乙型肝炎病毒X蛋白对细胞凋亡的影响存在剂量依赖性

乙型肝炎病毒(HBV)X蛋白(HBx)与HBV相关肝细胞肝癌(HCC)的发生和发展密切相关.深入研究HBx在HCC形成中的作用将为探索HBV致癌机制提供重要依据.HBx是多功能蛋白,其对细胞凋亡的影响至今仍存在分歧.许多研究表明,HBx既有促进细胞凋亡又有抑制细胞凋亡的功能,但原因不清楚.本研究中,将表达HBx的质粒短...     

乙型肝炎病毒X蛋白的优势氨基酸序列与热点突变位点的生物信息学分析

乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)全长154个氨基酸,与肝癌发生密切相关.为确定HBx的优势氨基酸序列和热点突变位点,在GenBank中下载所有HBx的氨基酸序列13950条,剔除插入突变、缺失突变和起始密码子非甲硫氨酸的序列,最后保留7126条.通过分析这7126条序列,计算出HBx每个位点的氨基酸分布情况,出现频率最高的氨基酸为该位点的优势氨基酸,其他氨基酸为突变氨基酸.154个位点的优势氨基酸组成HBx优势氨基酸序列.突变率>10.0%的热点突变位点有32个.其中第36、42、44、87、88和127位氨基酸有4种(突变率>1.0%)以上突变形式,具有较高的多态性.与肝癌密切相关的K130M/V131I双突变率为34.7%.通过7126条HBx序列与优势序列的同源性比较,随机选出其中50条序列(2条与优势序列同源性<75%,48条同源性为76%~99%),与23条参考序列及优势序列共同构建系统发生树.结果显示,HBx优势氨基酸序列属于基因型C,这与基因型C为全球主要流行型一致.本研究首次系统性分析了GenBank中HBx的优势序列,确定了32个HBx热点突变位点和6个多态性较高的位点,为基于HBx突变的基础和应用研究奠定了基础.     

肝胚瘤细胞株HepG_2中HBx蛋白对SOCS3表达的影响及其机制

目的探讨乙型肝炎病毒X蛋白(Hepatitis B virus X protein,HBx)对肝胚瘤细胞株HepG2表达信号抑制因子3(SOCS3)的影响及其机制。方法将表达HBx蛋白的重组质粒FL1-145HBx转染HepG2细胞,以不同浓度的SRC抑制剂PP2处理转染细胞,运用Western blot和RT-PCR技术分析HBx、SOCS3 mRNA和蛋白的表达情况,并以免疫细胞化学分析转染细胞中p-SRC的表达水平。结果重组质粒FL1-145HBx转染HepG2细胞后,转染细胞HBx能够表达HBx蛋白,并且细胞中SOCS3 mRNA和蛋白表达水平随着HBx蛋白的表达而显著增加(P0.05),同时p-SRC蛋白的表达增强;而SRC抑制剂PP2处理转染细胞后,随着p-SRC蛋白表达的减少SOCS3蛋白表达逐渐下降。结论在肝胚瘤细胞株HepG2中,HBx蛋白很可能通过促进SRC蛋白的磷酸化而诱导SOCS3蛋白的表达。     

COX-2 mediates hepatitis B virus X protein abrogation of p53-induced apoptosis

The oncogenic hepatitis B virus X protein (HBx) and cyclooxygenase (COX)-2 are highly co-expressed in chronic hepatitis, cirrhosis and well-differentiated hepatocellular carcinoma (HCC). Although HBx is shown to activate COX-2, the functional consequences of this interaction in hepatocarcinogenesis remain unknown. Using an engineered hepatoma cell system in which the expression of wild-type p53 can be chemically modulated, we show here that COX-2 mediates HBx actions in opposing p53. Enforced expression of HBx sequestrates p53 in the cytoplasm and significantly abolishes p53-induced apoptosis. The anti-apoptotic Mcl-1 protein is suppressed by p53 but reactivated by HBx. The abrogation of apoptosis is completely reversed by specific COX-2 inhibition, suggesting that HBx blocks p53-induced apoptosis via activation of COX-2/PGE₂ pathway. We further show that COX-2 inhibition blocks HBx reactivation of Mcl-1, linking this protein to the anti-apoptotic function of COX-2. These results demonstrate that COX-2 is an important survival factor mediating the oncogenic actions of HBx. Over-expression of HBx and COX-2 may provide a selective clonal advantage for preneoplastic or neoplastic hepatocytes and contribute to the initiation and progression of HCC.     

Interaction of the hepatitis B virus X protein (HBx) with heat shock protein 60 enhances HBx-mediated apoptosis

Understanding the function of the hepatitis B virus X protein (HBx) is fundamental to elucidating the underlying mechanisms of hepatitis and hepatocarcinogenesis caused by hepatitis B virus (HBV) infection. We identified heat shock protein 60 (Hsp60) as a novel cellular target of HBx by the combination of affinity purification and mass spectrometry. Physical interaction between HBx and Hsp60 was confirmed by standard immunoprecipitation and immunoblot methods. Analysis of HBx deletion constructs showed that amino acids 88-117 of HBx were responsible for the binding to Hsp60. Confocal laser microscopy demonstrated that HBx and Hsp60 colocalized in mitochondria. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) revealed that the introduction of Hsp60 into cells facilitated HBx-induced apoptosis. These findings suggest the importance of the molecular chaperon protein Hsp60 to the function of HBV viral proteins.     

Hepatitis B virus X protein induces size-selective membrane permeabilization through interaction with cardiolipin

Hepatitis B virus X protein (HBx) functions in a variety of cellular events during the HBV life cycle. In a previous study, we reported that the HBx protein is sufficient to induce mitochondrial membrane permeabilization; however, the exact mechanism of HBx-induced mitochondrial membrane permeabilization has been not proposed. In this study, we report that HBx specifically targets cardiolipin (CL) and induces membrane permeabilization depending on CL concentration in mitochondrial outer membrane–mimic artificial liposomes. Interestingly, HBx-induced membrane permeabilization was enhanced by liposomes containing phosphatidylethanolamine, which plays a crucial role in forming a negative curvature on the membrane. We also show that the 68-117 region of HBx, which interacts with mitochondria, is necessary for membrane permeabilization. We examined the size of the pores formed by HBx and found that HBx permeates fluorescent dyes depending on the hydrodynamic diameter with a pore size of approximately 10 nm. The results of this study suggest that CL is necessary for HBx-induced membrane permeabilization and provide important information that suggests a new strategy for anti-HBV therapy.     

Artificial chaperone-assisted refolding in a microchannel

Protein refolding using a simple dilution method in a microchannel often led to the formation of protein aggregates, which bound to the microchannel wall, resulting in low refolding yields. To inhibit aggregation and improve refolding yields, an artificial chaperone-assisted (ACA) refolding, which employed detergents and β-cyclodextrin was used. Model proteins, hen egg white lysozyme and yeast α-glucosidase, were successfully refolded in a microchannel. The microscopic observation showed that the ACA method suppressed protein aggregation and facilitated the refolding of lysozyme, whereas significant aggregation was observed when a simple dilution method was employed. The ACA method increased the lysozyme refolding yield by 40% over the simple dilution approach. Similarly, for α-glucosidase, the refolding yield using the ACA method (ca. 50%) was approximately three times compared with the simple dilution method. The ACA refolding method is a suitable approach to use in the refolding of proteins using a microfluidic system.     

Hepatitis B virus–triggered autophagy targets TNFRSF10B/death receptor 5 for degradation to limit TNFSF10/TRAIL response

Death receptors of TNFSF10/TRAIL (tumor necrosis factor superfamily member 10) contribute to immune surveillance against virus-infected or transformed cells by promoting apoptosis. Many viruses evade antiviral immunity by modulating TNFSF10 receptor signaling, leading to persistent infection. Here, we report that hepatitis B virus (HBV) X protein (HBx) restricts TNFSF10 receptor signaling via macroautophagy/autophagy-mediated degradation of TNFRSF10B/DR5, a TNFSF10 death receptor, and thus permits survival of virus-infected cells. We demonstrate that the expression of the TNFRSF10B protein is dramatically reduced both in liver tissues of chronic hepatitis B patients and in cell lines transfected with HBV or HBx. HBx-mediated downregulation of TNFRSF10B is caused by the lysosomal, but not proteasomal, degradation pathway. Immunoblotting analysis of LC3B and SQSTM1, and microscopy analysis of tandem-fluorescence-tagged LC3B revealed that HBx promotes complete autophagy. Inhibition of autophagy with a pharmacological inhibitor and LC3B knockdown revealed that HBx-induced autophagy is crucial for TNFRSF10B degradation. Immunoprecipitation and GST affinity isolation assays showed that HBx directly interacts with TNFRSF10B and recruits it to phagophores, the precursors to autophagosomes. We confirmed that autophagy activation is related to the downregulation of the TNFRSF10B protein in liver tissues of chronic hepatitis B patients. Inhibition of autophagy enhanced the susceptibility of HBx-infected hepatocytes to TNFSF10. These results identify the dual function of HBx in TNFRSF10B degradation: HBx plays a role as an autophagy receptor–like molecule, which promotes the association of TNFRSF10B with LC3B; HBx is also an autophagy inducer. Our data suggest a molecular mechanism for HBV evasion from TNFSF10-mediated antiviral immunity, which may contribute to chronic HBV infection.     

A generic hierarchical screening method for the analysis of microscale refolds using an automated robotic platform

The refolding of protein derived from inclusion bodies is often characterized by low yields of active protein. The optimization of the refolding step is achieved empirically and consequently is time-consuming slowing process development. An automated robotic platform has been used to develop a dilution refold process-screening platform upon which a hierarchical set of assays rapidly determine optimal refolding conditions at the microscale. This hierarchy allows the simplest, cheapest, and most generic high-throughput assays to first screen for a smaller subset of potentially high-yielding conditions to take forward for analysis by slower, more expensive, or protein specific assays, thus saving resources whilst maximizing information output. An absorbance assay was used to initially screen out aggregating conditions, followed by an intrinsic fluorescence assay of the soluble protein to identify the presence of native-like tertiary structure, which was then confirmed by an activity assay. Results show that fluorescence can be used in conjunction with absorbance to eliminate low-yielding conditions, leaving a significantly reduced set of conditions from which the highest yielding ones can then be identified with slower and often more costly activity or RP-HPLC assays, thus reducing bottlenecks in high-throughput analysis. The microwell-based automated process sequence with generic hierarchical assays was also used to study and minimize the effect on redox potential or misfolding, of oxygenation due to agitation, before demonstrating that the platform can be used to rapidly collect data and evaluate different refolding conditions to speed up the acquisition of process development data in a resource efficient manner.     

乙肝病毒感染对细胞基本自噬的影响

【目的】慢性乙肝病毒(Hepatitis B virus,HBV)感染在肝硬化和肝癌的发生过程中起着重要的作用,通过研究HBV感染对细胞基本自噬的影响,为HBV感染诱发肝癌以及HBV的免疫逃逸机理研究提供新的思路。【方法】本研究利用乙肝病毒表达质粒瞬时或稳定转染不同肝细胞,通过计数绿色荧光蛋白(greenfluorescent protein,GFP)聚集数目检测自噬小体形成,western blot检测LC3(microtubule-associated proteinlight chain 3,微管相关蛋白质轻链3)脂酰化和p62的降解,通过构建HBV B型和C型X蛋白(HBx)的表达质粒并瞬时转染肝癌细胞和正常肝细胞,对不同基因型X蛋白对细胞自噬的影响进行了分析。【结果】乙肝病毒感染后促进了LC3的脂酰化和p62的降解,增加了自噬小体的形成,增强了细胞的基本自噬。进一步研究发现,HBV感染增强的细胞基本自噬水平由HBx所引发,且C型HBx比B型对细胞基本自噬的增加更加显著。【结论】HBV通过HBx增强细胞的基本自噬,且不同基因型HBx对细胞基本自噬的增强程度不同,为进一步阐明HBV感染机理奠定了基础。     

CRL4在增殖的肝细胞系中上调乙型肝炎病毒抗原的分泌表达

乙型肝炎病毒（hepatitis B virus,HBV）编码的X蛋白（hepatitis B virus X protein,HBx）对HBV感染的起始和维持至关重要。HBx可能作为病毒来源的接头分子,介导Cullin-RING E3泛素连接酶4（cullin-RING ubiquitin E3 ligase 4,CRL4）复合物对染色体外DNA限制因子SMC5/6的降解。最近研究发现CRL4接头分子DNA损伤结合蛋白1（DNA damage-binding protein 1,DDB1）可不依赖与HBx的相互作用而直接上调病毒的表达和复制。本研究基于HBx基因删除（X-null）的HBV重组cccDNA（recombinant covalently closed circular DNA,rcccDNA）模型系统,在多种体外培养肝细胞系中证实上述发现。有意思的是,CRL4刺激rcccDNA^X-null转染细胞抗原分泌表达的效应能被血清饥饿实验抵消。应用尾静脉高压注射小鼠模型,同样发现CRL4并不上调rcccDNA^X-null在非增殖小鼠肝脏细胞中的表达。以上结果提示,细胞增殖特征与CRL4不依赖HBx上调病毒抗原表达的效应密切相关,有助于HBx生物学意义的准确分析和理解。     

Proteomic analysis of liver tissue from HBx‐transgenic mice at early stages of hepatocarcinogenesis

The hepatitis B virus X‐protein (HBx), a multifunctional viral regulator, participates in the viral life cycle and in the development of hepatocellular carcinoma (HCC). We previously reported a high incidence of HCC in transgenic mice expressing HBx. In this study, proteomic analysis was performed to identify proteins that may be involved in hepatocarcinogenesis and/or that could be utilized as early detection biomarkers for HCC. Proteins from the liver tissue of HBx‐transgenic mice at early stages of carcinogenesis (dysplasia and hepatocellular adenoma) were separated by 2‐DE, and quantitative changes were analyzed. A total of 22 spots displaying significant quantitative changes were identified using LC‐MS/MS. In particular, several proteins involved in glucose and fatty acid metabolism, such as mitochondrial 3‐ketoacyl‐CoA thiolase, intestinal fatty acid‐binding protein 2 and cytoplasmic malate dehydrogenase, were differentially expressed, implying that significant metabolic alterations occurred during the early stages of hepatocarcinogenesis. The results of this proteomic analysis provide insights into the mechanism of HBx‐mediated hepatocarcinogenesis. Additionally, this study identifies possible therapeutic targets for HCC diagnosis and novel drug development for treatment of the disease.     

Blocking of G1/S transition and cell death in the regenerating liver of Hepatitis B virus X protein transgenic mice

The Hepatitis B virus X (HBx) protein has been strongly implicated in the carcinogenesis of hepatocellular carcinoma (HCC). However, effects of the HBx protein on cell proliferation and cell death are controversial. This study investigates the effects of the HBx protein on liver regeneration in two independent lines of HBx transgenic mice, which developed HCC at around 14 to 16 months of age. High mortality, lower liver mass restoration, and impaired liver regeneration were found in the HBx transgenic mice post-hepatectomy. The levels of alanine aminotransferase and alpha-fetoprotein detected post-hepatectomy increased significantly in the HBx transgenic livers, indicating that they were more susceptible to damage during the regenerative process. Prolonged activation of the immediate-early genes in the HBx transgenic livers suggested that the HBx protein creates a strong effect by promoting the transition of the quiescent hepatocytes from G0 to G1 phase. However, impaired DNA synthesis and mitosis, as well as inhibited activation of G1, S, and G2/M markers, were detected. These results indicated that HBx protein exerted strong growth arrest on hepatocytes and imbalanced cell-cycle progression resulting in the abnormal cell death; this was accompanied by severe fat accumulation and impaired glycogen storage in the HBx transgenic livers. In conclusion, this study provides the first physiological evidence that HBx protein blocks G1/S transition of the hepatocyte cell-cycle progression and causes both a failure of liver functionality and cell death in the regenerating liver of the HBx transgenic mice.     

Specific inhibition of gene expression and transactivation functions of hepatitis B virus X protein and c-myc by small interfering RNAs

With a view to developing therapeutic strategies against hepatocellular carcinoma (HCC), we have recently shown that co-expression of c-myc and the X protein of hepatitis B virus (HBx) resulted in the development of HCC in the X-myc transgenic mice [Lakhtakia et al., J. Gastroenterol. Hepatol. 18 (2003) 80–91]. We now show in cell culture-based studies that small interfering RNA (siRNA) corresponding to HBx and c-myc can regulate expression and transactivation of the target genes. Expression vectors for small hairpin RNAs (shRNAs) against two different regions each of the HBx and c-myc open reading frames were constructed and their regulatory effects were investigated in COS-1 cells. A dose-dependent specific inhibition in the expression levels of HBx and c-myc was observed with individual shRNAs. Further, the recombinantly expressed shRNAs also blocked the transactivation functions of their cognate genes. Though each shRNA worked at a different efficiency, the inhibitory effects with two different shRNAs were cumulative. These results appear promising for developing a siRNA-based therapy for HCC.     

A comprehensive molecular interaction map for Hepatitis B virus and drug designing of a novel inhibitor for Hepatitis B X protein

Hepatitis B virus (HBV) infection is a leading source of liver diseases such as hepatitis, cirrhosis and hepatocellular carcinoma. In this study, we use computation methods in order to improve our understanding of the complex interactions that occur between molecules related to Hepatitis B virus (HBV). Due to the complexity of the disease and the numerous molecular players involved, we devised a method to construct a systemic network of interactions of the processes ongoing in patients affected by HBV. The network is based on high-throughput data, refined semi-automatically with carefully curated literature-based information. We find that some nodes in the network that prove to be topologically important, in particular HBx is also known to be important target protein used for the treatment of HBV. Therefore, HBx protein is the preferential choice for inhibition to stop the proteolytic processing. Hence, the 3D structure of HBx protein was downloaded from PDB. Ligands for the active site were designed using LIGBUILDER. The HBx protein's active site was explored to find out the critical interactions pattern for inhibitor binding using molecular docking methodology using AUTODOCK Vina. It should be noted that these predicted data should be validated using suitable assays for further consideration.