Characterization of the mitochondrial association of hepatitis B virus X protein, HBx

Chronic infection with hepatitis B virus (HBV) is strongly associated with the development of hepatocellular carcinoma (HCC). HBx, a protein encoded by HBV is believed to contribute to the development of HCC. HBx was recently shown to associate with mitochondria. In this study, we mapped region(s) of HBx necessary for mitochondrial targeting and showed that a putative transmembrane region (aa 54-70) is required for mitochondrial association. In addition, amino acids in the putative alpha helical regions (aa 75-88 and aa 109-131) seem to aid in the mitochondrial targeting of this protein. We further show that the majority of HBx localizes to the outer mitochondrial membrane based on its sensitivity to trypsin and resistance to alkaline treatment. These studies suggest that the association of HBx with the outer mitochondrial membrane is its intrinsic property. These characterizations define transmembrane and alpha-helical regions of this viral protein as domains of mitochondrial targeting. These studies are further useful in the investigations concerning the physiological significance of the HBx's association with mitochondria and its impact on liver disease pathogenesis.     

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.     

Hepatitis B virus X protein induces apoptosis by enhancing translocation of Bax to mitochondria

Hepatitis B virus X protein (HBx) is essential for viral replication and plays an important role in viral pathogenesis. HBx transactivates many viral and cellular genes and participates in cellular signal transduction pathways, proliferation, and apoptosis. In the present study, we report that HBx induces apoptosis by enhancing the translocation of Bax to mitochondria, followed by inducing the loss of mitochondrial membrane potential and release of cytochrome C. In addition, Bcl-2, inhibitor of Bax, rescues the disruption of mitochondrial membrane potential and DNA fragmentation induced by serum starvation in HepG2-X cells expressing HBx. We also found that HBx binds directly to Bax and interferes with the interaction between Bax and 14-3-3epsilon to enhance the translocation of Bax to mitochondria. Taken together, our data suggest that HBx induces apoptosis by interacting with Bax and enhancing its translocation to mitochondria.     

Hepatitis B virus HBx protein localizes to mitochondria in primary rat hepatocytes and modulates mitochondrial membrane potential

Over 350 million people are chronically infected with hepatitis B virus (HBV), and a significant number of chronically infected individuals develop primary liver cancer. HBV encodes seven viral proteins, including the nonstructural X (HBx) protein. The results of studies with immortalized or transformed cells and with HBx-transgenic mice demonstrated that HBx can interact with mitochondria. However, no studies with normal hepatocytes have characterized the precise mitochondrial localization of HBx or the effect of HBx on mitochondrial physiology. We have used cultured primary rat hepatocytes as a model system to characterize the mitochondrial localization of HBx and the effect of HBx expression on mitochondrial physiology. We now show that a fraction of HBx colocalizes with density-gradient-purified mitochondria and associates with the outer mitochondrial membrane. We also demonstrate that HBx regulates mitochondrial membrane potential in hepatocytes and that this function of HBx varies depending on the status of NF-kappaB activity. In primary rat hepatocytes, HBx activation of NF-kappaB prevented mitochondrial membrane depolarization; however, when NF-kappaB activity was inhibited, HBx induced membrane depolarization through modulation of the mitochondrial permeability transition pore. Collectively, these results define potential pathways through which HBx may act in order to modulate mitochondrial physiology, thereby altering many cellular activities and ultimately contributing to the development of HBV-associated liver cancer.     

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 viral X protein overcomes inhibition of E2F1 activity by pRb on the human Rb gene promoter

Hepatitis B virus X (HBx) protein is known as an oncogenic transactivator, E2F1 as a positive regulator of the cell cycle, and pRb as a tumor suppressor. Here, we investigated the functional interactions of these proteins on the human Rb promoter. Interestingly, HBx transactivated the Rb promoter cooperatively with E2F1 in HepG2 cells but not in HeLa cells, in which the functions of p53 and pRb are inactive. Combinatorial cotransfection analyses in HepG2 cells showed that HBx overcame the inhibition of E2F1 activity by pRb but not that by p53. Domain analysis showed that aa 47-70 and aa 117-133 of HBx are important for this effect. These results suggest that HBx could inhibit the pRb tumor suppressor and increase E2F1 activity. Our data support the oncogenic potential of HBx, which may cause HBV-infected cells to grow continuously in the development of hepatocellular carcinoma.     

TFAR19促进小鼠肝线粒体膜通透性转运孔的开放

TFAR19基因 (TF 1cellapoptosisrelatedgene 19)是北京大学人类疾病基因中心从人白血病细胞株TF 1细胞中克隆到的凋亡相关新基因之一 (GenBank登记号AF0 1495 5 )。初步研究发现 ,该基因在细胞凋亡时高表达 ,并且表达产物具有抑制肿瘤细胞生长和促进凋亡作用。但是其确切的作用机制不明。线粒体膜完整性破坏所导致促凋亡因子 (如细胞色素c等因子 )的释放是细胞凋亡关键性的控制因素。线粒体膜通透性转运孔 (PTP) ,对线粒体膜完整性具有重要的调控作用。研究了重组人TFAR19蛋白在体外条件下 ,对线粒体PTP、跨膜电位 ,以及细胞色素c释放的影响。结果表明 ,TFAR19蛋白使分离的小鼠肝线粒体PTP开放、线粒体跨膜电位下降 ,以及细胞色素c释放。TFAR19对线粒体的上述作用是通过促进PTP开放起作用的。实验结果提示 ,TFAR19对线粒体凋亡信号有正反馈放大作用 ,并进一步揭示了TFAR19促进细胞凋亡的机制     

The carboxy-terminus of the hepatitis B virus X protein is necessary and sufficient for the activation of hypoxia-inducible factor-1alpha

Hepatitis B virus X protein (HBx) of the hepatitis B virus is strongly implicated in angiogenesis and metastasis during hepatocarcinogenesis. Previously, we reported that HBx enhances activity of hypoxia-inducible factor-1alpha (HIF-1alpha), a potent transactivator that induces angiogenic factors. Here, we delineate the structural region of HBx that potentiates HIF-1alpha. The carboxy-terminus of HBx increased the stability of HIF-1alpha protein, probably through inhibiting interaction with von Hippel-Lindau protein. Further, the carboxy-terminus of HBx enhanced the transactivation function of HIF-1alpha by enhancing its association with CREB binding protein (CBP). Finally, we demonstrated the physical association of HBx with the basic helix-loop-helix/PER-ARNT-SIM domain, the inhibitory domain, and the carboxy-terminal transactivation domain of HIF-1alpha in vivo.     

Participation of the mitochondrial permeability transition pore in nitric oxide-induced plant cell death

In the present study, we investigated the involvement of the mitochondrial permeability transition pore (PTP) in nitric oxide (NO)-induced plant cell death. NO donors such as sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine inhibited growth and caused death in suspension-cultured cells of Citrus sinensis. Cells treated with SNP showed chromatin condensation and fragmentation, characteristic of apoptosis. SNP caused loss of the mitochondrial membrane electrical potential, which was prevented by cyclosporin A (CsA), a specific inhibitor of PTP formation. CsA also prevented the nuclear apoptosis and subsequent Citrus cell death induced by NO. These findings indicate that mitochondrial PTP formation is involved in the signaling pathway by which NO induces apoptosis in cultured Citrus cells.     

Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential

Understanding the mechanism(s) of action of the hepatitis B virus (HBV)-encoded protein HBx is fundamental to elucidating the underlying mechanisms of chronic liver disease and hepatocellular carcinoma caused by HBV infection. In our continued attempts to identify cellular targets of HBx, we have previously reported the identification of a novel cellular protein with the aid of a yeast two-hybrid assay. This cellular gene was identified as a third member of the family of human genes that encode the voltage-dependent anion channel (HVDAC3). In the present study, physical interaction between HBx and HVDAC3 was established by standard in vitro and in vivo methods. Confocal laser microscopy of transfected cells with respective expression vectors colocalized HVDAC3 and HBx to mitochondria. This novel, heretofore unreported subcellular distribution of HBx in mitochondria implies a functional role of HBx in functions associated with mitochondria. Using a stable cationic fluorophore dye, CMXRos, we show that HBx expression in cultured human hepatoma cells leads to alteration of mitochondrial transmembrane potential. Such functional roles of HBx in affecting mitochondrial physiology have implications for HBV-induced liver injury and the development of hepatocellular carcinoma.     

Genetic Dissection of the Permeability Transition Pore

The permeability transition pore (PTP) regulates the structural re-organization of mitochondria in response to changes in cellular Ca²⁺ and is thought to be an important participant in mitochondrial responses to cell death signals. Although the proteins forming the PTP have yet to be rigorously identified, recent examination of the response of mitochondria, cells and tissues lacking putative components of the PTP have been reported. Studies on mitochondria lacking cyclophilin D (CyP-D) have proved that this protein is the target for PTP inhibition by CsA; yet they have also unequivocally demonstrated that the PTP can form and open in the absence of CyP-D. Likewise, studies in mice lacking the two adenine nucleotide translocators expressed in this species have shown that a functional PTP can form in the absence of these proteins. Thus, the inner mitochondrial membrane components of the PTP remain to be identified, and the absence of CyP-D may not preclude PTP opening in vivo – a finding that questions the conclusion that the PTP participates in cell death pathways only in response to a restricted set of challenges.     

Viroporins from RNA viruses induce caspase-dependent apoptosis

The virus-encoded viroporins are known to modify membrane permeability and play an essential role in virus budding. Here, a comparative analysis of the membrane permeabilization capacity of a number of viroporins was performed in baby hamster kidney cells. Synthesis of 6K protein from Sindbis virus, E from mouse hepatitis virus, M2 from influenza A virus, and 2B and 3A from poliovirus enhanced membrane permeability to different extents. We show that two proteins from hepatitis C virus, p7 and NS4A, also display viroporin activity to a level comparable to 6K protein. In addition to their capacity to disrupt ionic cellular homeostasis and promote bacterial cell lysis, the expressed viroporins were able to induce cell death. Degradation of internucleosomal DNA and generation of apoptotic bodies were observed upon viroporin expression. Consistently, cleavage of translation initiation factor 4GI and poly-(ADP-ribose) polymerase indicated activation of effector caspase-3. We found that poliovirus 2B localizes partially in mitochondria and induces an anomalous perinuclear distribution of these organelles. Mitochondria morphology was also altered after expression of other viroporins. Finally, detection of cytochrome c release from mitochondria suggests involvement of the mitochondrial pathway in viroporin-induced apoptosis. These findings suggest that viroporins induce caspase-dependent programmed cell death.     

The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore

The relevance of the mitochondrial permeability transition pore (PTP) in Ca2+ homeostasis and cell death has gained wide attention. Yet, despite detailed functional characterization, the structure of this channel remains elusive. Here we report on a new class of inhibitors of the PTP and on the identification of their molecular target. The most potent among the compounds prepared, Ro 68-3400, inhibited PTP with a potency comparable to that of cyclosporin A. Since Ro 68-3400 has a reactive moiety capable of covalent modification of proteins, [3H]Ro 68-3400 was used as an affinity label for the identification of its protein target. In intact mitochondria isolated from rodent brain and liver and in SH-SY5Y human neuroblastoma cells, [3H]Ro 68-3400 predominantly labeled a protein of approximately 32 kDa. This protein was identified as the isoform 1 of the voltage-dependent anion channel (VDAC). Both functional and affinity labeling experiments indicated that VDAC might correspond to the site for the PTP inhibitor ubiquinone0, whereas other known PTP modulators acted at distinct sites. While Ro 68-3400 represents a new useful tool for the study of the structure and function of VDAC and the PTP, the results obtained provide direct evidence that VDAC1 is a component of this mitochondrial pore.     

Role of Pin1 in UVA-induced cell proliferation and malignant transformation in epidermal cells

The hepatitis B virus X protein (HBx) has been implicated in the development of hepatocellular carcinoma (HCC) associated with chronic infection. As a multifunctional protein, HBx regulates numerous cellular pathways, including autophagy. Although autophagy has been shown to participate in viral DNA replication and envelopment, it remains unclear whether HBx-activated autophagy affects host cell death, which is relevant to both viral pathogenicity and the development of HCC. Here, we showed that enforced expression of HBx can inhibit starvation-induced cell death in hepatic (L02 and Chang) or hepatoma (HepG2 and BEL-7404) cell lines. Starvation-induced cell death was greatly increased in HBX-expressing cell lines treated either with the autophagy inhibitor 3-methyladenine (3-MA) or with an siRNA directed against an autophagy gene, beclin 1. In contrast, treatment of cells with the apoptosis inhibitor Z-Vad-fmk significantly reduced cell death. Our results demonstrate that HBx-mediated cell survival during starvation is dependent on autophagy. We then further investigated the mechanisms of cell death inhibition by HBx. We found that HBx inhibited the activation of caspase-3, an execution caspase, blocked the release of mitochondrial apoptogenic factors, such as cytochrome c and apoptosis-inducing factor (AIF), and inhibited the activation of caspase-9 during starvation. These results demonstrate that HBx reduces cell death through inhibition of mitochondrial apoptotic pathways. Moreover, increased cell viability was also observed in HepG2.2.15 cells that replicate HBV and in cells transfected with HBV genomic DNA. Our findings demonstrate that HBx promotes cell survival during nutrient deprivation through inhibition of apoptosis and activation of autophagy. This highlights an important potential role of autophagy in HBV-infected hepatocytes growing under nutrient-deficient conditions.     

The peripheral-type benzodiazepine receptor is involved in control of Ca2+-induced permeability transition pore opening in rat brain mitochondria

The peripheral-type benzodiazepine receptor (PBR) is an 18 kDa mitochondrial membrane protein with still elusive function in cell death. Here, we studied whether PBR is involved in Ca2+-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria (RBM). PTP opening is important in mitochondrial events leading to programmed cell death. Immunoblots revealed a single 18 kDa anti-PBR antibody-immunoreactive band in purified RBM. Adenine nucleotide transporter, a key PTP component, was found in the PBR-immunoprecipitate. In isolated intact RBM, addition of a specific anti-PBR antibody [H. Li, Z. Yao, B. Degenhardt, G. Teper, V. Papadopoulos, Cholesterol binding at the cholesterol recognition/interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 1267-1272] delayed Ca2+-induced dissipation of membrane potential (psi(m)) and diminished cyclosporine A-sensitive Ca2+ efflux, which are both indicative for the suppression of PTP opening. Moreover, anti-PBR antibody caused partial retention of Ca2+ in the mitochondrial matrix in spite of psi(m) dissipation, and reduced activation of respiratory rate at Ca2+-induced PTP opening. A release of pro-apoptotic factors, AIF and cytochrome c, from RBM was shown at threshold Ca2+ load. Anti-PBR antibody blocked the release of AIF but did not affect the cytochrome c release. Addition of ATP was able to initiate PTP closing, associated with psi(m) restoration and Ca2+ re-accumulation. At the same time mitochondrial protein phosphorylation (incorporation of 32P from [gamma-32P]ATP) occurred and anti-PBR antibody was able to inhibit phosphorylation of these proteins. The endogenous PBR ligand, protoporphyrin IX, facilitated PTP opening and phosphorylation of the mitochondrial proteins, thus, inducing effects opposite to anti-PBR antibody. This study provides evidence for PBR involvement in PTP opening, controlling the Ca2+-induced Ca2+ efflux, and AIF release from mitochondria, important stages of initiation of programmed cell death.