Hepatitis B virus (HBV) is a major etiological factor in the development of human acute and chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC).1,2 HBV is a small DNA virus belonging to the hepadnavirus family. It contains a highly conserved small open reading frame encoding HBV x protein (HBx).3 HBx is a multifunctional viral regulator that modulates protein degradation, signaling pathways, and cell responses to genotoxic stress. HBx is also known to play an important role in alternating gene expression, sensitizing cells to apoptosis, affecting cell cycle checkpoints and inducing carcinogenesis.4,5 Although modern surgery can reduce the mortality of HCC to certain degree, additional treatments such as chemotherapy and radiotherapy have resulted in no more than a 5% reduction in death rate and unpleasant side effects in patients.6 It is still a great challenge to develop novel therapeutic agents for the treatment of HBx-related diseases.
HBx could interact with a subunit of the 20S proteasome complex, PSMA7, 7 as well as the PSMC1 subunit of the 19S regulatory component which was shown by yeast two-hybrid experiments. 8 Armon et al9 originally reported a relevant ATPase activity in the 26S proteasome and its assembly. The19S particle contains six ATPases that bind to either end of the 20S proteasome. Expression of HBx in HepG2 cells causes a modest decrease in the proteasome’s chymotrypsin- and trypsin-like activities and in hydrolysis of ubiquitinated lysozyme, suggesting that HBx functions as an inhibitor of proteasome.10 The highly conserved AAA-ATPase, p97, plays an important role in ubiquitin (proteasome-dependent proteolysis). It has been implicated in preventing apoptosis, and its expression correlates with progression of solid tumors in humans.11,12
Protein bioinformatics analysis showed that a sequence in HBx is highly homologous to the “Kunitz domain,” a characteristic domain of Kunitz-type serine protease inhibitors which is essential to their inhibitory function. Mutations in these sequences completely abolish trans-activation. Indeed, HBx resembles a serine protease inhibitor or its analogue, and bring about transactivation by activating certain transcriptional factors through proteolytic cleavage alteration.13–15 In the present study, we examined whether a synthetic peptide, Chemical synthesis of a peptide from Kunitz domain (PKD), with the same sequence as Kunitz domain of HBx could suppresses the proteasome activity in HepG2 cells, and assessed whether this peptide affects the ATPase activities of proteasomes, expression of p97, cell cycle and apoptosis.
HBx sequences of hepadnaviruses used for BLAST analysis were taken from GenBank. Accession numbers were as follows: arctic ground squirrel hepatitis B virus, U29144; woodchuck hepatitis virus, M19183; woolly monkey hepatitis B virus, NC_001896; orangutan hepadnavirus, NC-002168; hepatitis B virus subtype adr, D12980; and hepatitis B virus subtype adw, M54923. Sequence alignment was performed with the software provided by Vector NTI (USA). A conserved peptide of nine residues from HBx, Phe-Val-Leu-Gly-Gly-Cys-Arg-His-Lys, was synthesized by the solid-phase method (GL Biochem Ltd., China). Purity (> 97%) was assessed by high-pressure liquid chromatography, amino acid analysis, and molecular weight determination by mass spectrometry.
Antibody used in this study was produced as a polyclonal murine antibody. The antigen is a synthesized peptide (GRLDQLIYIPLEICQRACK) which contains two short peptide sequences of p97 (637–647 and 689–696 from NP-009057). Both are located in two hydrophilic, flexible and conserved regions, and were selected for these qualities, which may encourage immunogenicity. The synthesized peptide was cross-linked to keyhole limpet hemocyanin, using a bioconjugation reagent, m-maleimidobenzoyl-N- hydroxysuccinimide ester. The synthetic peptides were dissolved in phosphate buffered saline (PBS) pH 7.5 and emulsified with Freund’s adjuvant; 50 μl of this solution was injected into each anterior muscle of Balb/c mice. Antibody was generated and affinity purified by absorption to fusion peptide immobilized on Reacti-Gel support. Specific anti-p97 antibody was eluted with 100 mmol/L glycine (pH 2.5), and dialyzed against PBS.
HepG2 cell culture
Human hepatoma HepG2 cells were maintained in RPMI 1640 medium containing 0.3 mg/ml L-glutamine, 2.0 mg/ml sodium bicarbonate, penicillin (100 IU/ml) and streptomycin (100 mg/ml) and supplemented with 10% fetal calf serum. Cells were cultured at 37°C in a humid atmosphere of 95% air and 5% CO2.
HepG2 cells were seeded in the 96-well plates (5 ×104 cells/0.2 ml per well), and pre-incubated for 1 hour with various concentrations of PKD, or incubated without PKD for 24, 48 and 72 hours, respectively. The effects of PKD on cell viability were determined by measuring the capacity of reducing enzyme activities to convert MTT to formazan crystals in viable cells as described previously.16 Cells were stained with MTT (50 mg/ml) and incubated for 4 hours at 37°C in a humidified incubator with 5% CO2. The MTT transformed crystals were dissolved in DMSO, and their absorbance was measured using a Bio-Tek spectrophotometer at 570 nm. The absorbance was proportional to the number of viable cells. All assays were done in triplicate.
HepG2 cells were grown in the 6-well plates to 80% confluence. The medium was replaced with the same fresh medium, and PKD was added for a final concentration of 10 mmol/L. After being incubated for 36 hours, the cells were harvested and lysed. Ten-μg cell lysate aliquots were analyzed using SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred onto a PVDF membrane. Nonspecific protein binding was blocked with 1% bovine serum albumin for 2 hours at room temperature. Signals were detected with anti-p97 polyclonal antibody and goat anti-mouse IgG conjugated with horseradish peroxidase; the protein bands were detected using Enhanced Chemiluminescence Reagent (ECL, Amersham, USA).
ATPase activity was measured using a colorimetric assay to determine the amount of inorganic phosphate produced from ATP hydrolysis using malachite green and ammonium molybdate.17,18 Each sample(10 μl) were mixed with 15 μl of 0.5% Nonidet P-40, 4 μl of H2O, 5 μl of 20 mmol/L ATP, and 26 μl of 2 × ATPase buffer (100 mmol/L Tris/HCl, pH7.5, 5 mmol/L Tris acetate, 10 mmol/L MgCl2, 50 μmol/L zinc acetate, 2 mmol/L DTT), then incubated at 37°C for the indicated time periods. The reaction was stopped by adding 240 μl of the malachite green/polyvinyl/ammonium molybdate reagent and 30 μl of 34% Na3-citrate/2H2O. Following a 30-minute incubation at room temperature, the absorbance of the reaction was measured at 660 nm using a spectrophotomer.
Analyses of proteasome activities
Peptidase activities of proteasomes were analyzed with the following specific substrates: N-benzyloxycarbonyl-Leu-Leu-Glu-β-naphthylamide (LLE-NA), succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (LLVY-AMC), and N-t-butyloxycarbonyl-Leu-Ser-Thr-Arg-amidomethylcou marin (LSTR-AMC).
For proteasome activity analysis, HepG2 cells were washed with cold PBS, resuspended and lysed in 25 mmol/L Tris, pH 7.5 with sonication in ice baths, 7 seconds per time for 7 times. The supernatant of the cytosolic fractions after centrifugation at 10 000 × g for 10 minutes at 4°C were used for enzymatic activity analysis. The protein concentration was determination using Bradford method. Total protein (35μg) of crude extracts and 10 mmol/L PKD were incubated for 2 hours at 37°C prior to the addition of appropriate peptide substrates. Fluorescence of fluorescent groups cleaved from proteasome peptide substrates was measured using a spectrofluorometer. Amidomethylcoumarin and β-naph thylamide were measured at 350 nm excitation with 440 nm emission, and at 335 nm excitation with 410 nm emission, respectively.
Flow cytometric analysis
HepG2 cells were cultured for 36 hours in the 6-well culture plates (2×106 cells/well in 3 ml RPMI 1640 medium) in the presence of 300 μg/ml of PKD. After aspirating the culture medium, the cells were gently washed 3 times with PBS and then harvested with 0.5 g/L trypsin and 0.2 g/L EDTA. After centrifugation 500 × g for 5 minutes, the cell pellets were resuspended. Cells were fixed in 2 ml of 70% cold ethanol at 4°C for 2 hours. The pellets were washed twice with RPMI 1640, then resuspended in 0.5 ml propidium iodide (PI)/RNase A solution. The cell mixture was incubated in the dark at room temperature for 10 minutes. Cell cycle phase and apoptosis of the stained cells was analyzed using a Becton Dickinson Flow Cytometer. Apoptotic cells were indicated by the hypodiploid peaks.
All data were presented as mean ± standard deviation (SD) and analyzed using SPSS 12.0. The data comparison was statistical analyzed using Student’s t test. Statistical significance was defined as two-tailed P <0.05.
PKD decreased the viability of HepG2 cells
MTT assay was used to determine the effect of PKD on the cell growth of HepG2 cells. As shown in Figure 1, cell viability decreased in a PKD-dose-dependent manner. While some inhibition was observed in the wells with 0 mmol/L, 5 mmol/L, and 10 mmol/L of PKD, treatment with 15 mmol/L PKD demonstrated an apparent cytotoxic effect.
PKD suppressed the expression of p97
Western blotting analysis showed that the expression of p97 decreased when cells were treated with 10 mmol/L PKD for 36 hours. The intensities of the p97 band (97 kDa) were significantly decreased in the cells with PKD treatment compared to the control cells (without treatment) (Figure 2). The result indicates that PKD inhibits the expression of p97 in HepG2 cells.
Inhibition of proteasome ATPase activity by PKD
ATPase activity of proteasomes was assayed in a time course manner for two and half hours (Figure 3). The proteasome complexes were immunoprecipitated from HepG2 cell lysates using anti-proteasome antibody and protein-G agarose beads. The cells were treated with or without 10 mmol/L PKD. The result showed that proteasome activities in the PKD-treated samples were lower than in the sample without treatment. It indicated that the treatment with PKD suppressed the ATPase activities of immunoprecipitated proteasome complexes.
PKD inhibits the chymotryptic activity of proteasomes
HepG2 cell supernatants were prepared as the source of proteasomes. The hydrolysis rates of the peptide substrates (listed in Method section) were determined in the presence or absence of 10 mmol/L PKD. The results showed that chymotryptic activity of proteasomes in HepG2 cells were significantly inhibited by PKD, while the tryptic and peptidylglutamyl peptide hydrolase activities were less inhibited (Figure 4).
PKD induces cell cycle change and apoptosis
Representative flow cytometric profiles of HepG2 cells for 36 hours, with or without PKD treatment, are shown in Figures 5A and 5B. The percentage of HepG2 cells in G0-G1, G2-M and S phases of the cell cycle, as well as apoptotic cells, are listed in Table. Our data showed that HepG2 cells accumulate largely in the G0-G1 phase after being incubated with PKD for 36 hours, while untreated control cells mainly enter into S phase. In addition, the PKD treatment significantly induces cells to proceed to apoptosis.
HBV causes a variety of fatal liver diseases including cirrhosis and hepatocellular carcinoma. The genome of HBV is compactly organized, and includes four overlapping open reading frames which encode DNA polymerase, surface antigen, core antigen, and HBx. There is a split but functional Kunitz-like serine protease inhibitor domain (61–69 and 131–142) within 154 amino acid residues of HBx.19 The function of HBx as a protease inhibitor is further supported by the identification of the proteasome as its target. Thus, these findings suggest a general role for HBx to inhibit cellular processes of protein degradation.
HBx interacts with proteasome subunits and modulates their proteolytic activities. The interaction may provide an explanation for the pleiotropic function of HBx, as the proteasome functions in diverse cellular processes including cell differentiation, cell cycle control, signal transduction, transcription activation and apoptosis.20–22 Mutagenesis studies of the first domain of HBx suggest that this domain is not important for interaction with the XAPC7 proteasome subunit. Mutagenesis analysis in the second domain indicates that this domain is responsible for XAPC7 interaction. Glycine (G136V) and histidine (H139D) mutations in the second domain eliminated this interaction.7 A partial deletion of the second domain also eliminated binding activities. A cysteine mutation (C137S) did not affect the interaction. Other conserved residues in the second domain, such as phenylalanine (aa 132), glycine (aa 135), arginine (aa 138), and lysine (aa 140), appear nonessential for the interaction between XAPC7 and HBx. This observation suggests that the interaction between the XAPC7 proteasome subunit and the second domain of HBx may play an important role in the regulation of proteasome function by HBx.23,24
There is a fully conserved nine-amino-acid sequence, FVLGGCRHK (132–140), in the second domain of HBx carboxyl terminus. We used the synthesized peptide to look for its effect on the proteasome activities. Proteasome activities of the cell samples were analyzed using spectrofluorometric assays with fluorogenic peptides. Fluorescence intensity that correlated to the peptidase activity of proteasomes was measured using a spectrofluorometer. While tryptic and peptidylglutamyl peptide hydrolase activities were slightly suppressed, the chymotryptic activity of proteasomes was strongly inhibited by PKD in HepG2 cells. Our observation agrees with a previous study that showed HBx with full-length sequence affects the peptidase activities of proteasomes.
In addition, we found that both the peptidase activity and ATPase activity of proteasomes are suppressed in cells following the treatment of PKD. ATPase activity has been detected in 26S proteasome purified from various sources such as rabbit reticulocytes, human kidney, and rat liver, or in the regulatory complex of proteasomes from bovine red cells.25–27 Our finding may suggest that when PKD blocks the peptidase activities, it alters the conformational change of the proteasome and consequently inhibits ATPase activity of the subunits in 19S particles of proteasomes.
p97 is a highly conserved 97 kDa protein with two conserved ATPase domains (AAA domains) in eukaryotes, and is one of the most abundant intracellular proteins. p97 is involved in the ubiquitin proteasome protein degradation pathway. This pathway plays an essential role in controlling the levels of various cellular proteins and regulates basic cellular processes such as cell-cycle progression, and signal transduction.28–30 RNAi and mutagenesis experiments have confirmed the essential role of p97 in the proteasome-mediated degradation of misfolded proteins.31 Functional inhibition of p97 leads to reduced proliferation and induction of apoptosis.32 In this study, the examination of p97 expression level using Western blots showed that there is a correlation between decreased expression of p97 and cell cycle changes in HepG2 cells treated with PKD.
The relation between p97 and proteasome activities may suggest these pathways are cross-regulated. The inhibition of enzymatic activity and ATPase activity of proteasomes could provide feed back to regulate the transcription, translation or degradation of p97; causing its down-regulation as we showed using Western blotting. So far, we can not rule out the possibility that PKD down regulates p97 directly.
Our results also showed that PKD is involved in apoptosis induction via blocking the activity of p97 and suppressing chymotrypsin-like activity of proteasomes. To further clarify the functions of PKD in detail, we are currently searching for target sites between PKD and various subunits of proteasomes. Taken together, this study demonstrates that a peptide derived from the Kunitz domain of HBx could be a potent proteasome inhibitor.
1. Heathcote EJ. Treatment of hepatitis B: the next five years. Clin Med 2007; 7: 472-477.
2. McMahon BJ. Epidemiology and natural history of hepatitis B. Semin Liver Dis 2005; 25 Suppl 1: 3-8.
3. Keasler VV, Hodgson AJ, Madden CR, Slagle BL. Enhancement of hepatitis B virus replication by the regulatory X protein in vitro
and in vivo.
J Virol 2007; 81: 2656-2662.
4. Murakami S. Hepatitis B virus X protein: a multifunctional viral regulator. J Gastroenterol 2001; 36: 651-660.
5. Tang H, Oishi N, Kaneko S, Murakami S. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Sci 2006; 97: 977-983.
6. Thabrew MI, Mitry RR, Morsy MA, Hughes RD. Cytotoxic effects of a decoction of Nigella sativa, Hemidesmus indicus and Smilax glabra on human hepatoma HepG2 cells. Life Sci 2005; 77: 1319-1330.
7. Huang J, Kwong J, Sun EC, Liang TJ. Proteasome
complex as a potential cellular target of hepatitis B virus X protein. J Virol 1996; 70: 5582-5591.
8. Zhang Z, Torii N, Furusaka A, Malayaman N, Hu Z, Liang TJ. Structural and functional characterization of interaction between hepatitis B virus X protein and the proteasome
complex. J Biol Chem 2000; 275: 15157-15165.
9. Armon T, Ganoth D, Hershko A. Assembly of the 26 S complex that degrades proteins ligated to ubiquitin
is accompanied by the formation of ATPase activity. J Biol Chem 1990; 265: 20723-20726.
10. Hu Z, Zhang Z, Doo E, Coux O, Goldberg AL, Liang TJ. Hepatitis B virus X protein is both a substrate and a potential inhibitor of the proteasome
complex. J Virol 1999; 73: 7231-7240.
11. Asai T, Tomita Y, Nakatsuka S, Hoshida Y, Myoui A, Yoshikawa H, et al. VCP (p97) regulates NFkappaB signaling pathway, which is important for metastasis of osteosarcoma cell line. Jpn J Cancer Res 2002; 93: 296-304.
12. Yamamoto S, Tomita Y, Hoshida Y, Iizuka N, Kidogami S, Miyata H, et al. Expression level of valosin-containing protein (p97) is associated with prognosis of esophageal carcinoma
. Clin Cancer Res 2004; 10: 5558-5565.
13. Takada S, Koike K. X protein of hepatitis B virus resembles a serine protease inhibitor. Jpn J Cancer Res 1990; 81: 1191-1194.
14. Takada S, Kido H, Fukutomi A, Mori T, Koike K. Interaction of hepatitis B virus X protein with a serine protease, tryptase TL2 as an inhibitor. Oncogene 1994; 9: 341-348.
15. Kanda T, Yokosuka O, Imazeki F, Yamada Y, Imamura T, Fukai K, et al. Hepatitis B virus X protein (HBx)-induced apoptosis
in HuH-7 cells: influence of HBV genotype and basal core promoter mutations. Scand J Gastroenterol 2004; 39: 478-485.
16. Wang QW, Lu HL, Song CC, Liu H, Xu CG. Radiosensitivity of human colon cancer cell enhanced by immunoliposomal docetaxel. World J Gastroenterol 2005; 11: 4003-4007.
17. Akiyama Y, Kihara A, Tokuda H, Ito K. FtsH (HflB) is an ATP-dependent protease selectively acting on SecY and some other membrane proteins. J Biol Chem 1996; 271: 31196-31201.
18. Yakushiji Y, Yamanaka K, Ogura T. Identification of a cysteine residue important for the ATPase activity of C. elegans fidgetin homologue. FEBS Lett 2004; 578: 191-197.
19. Chen WN, Oon CJ. Human hepatitis B virus mutants: significance of molecular changes. FEBS Lett 1999; 453: 237-242.
20. Takada S, Tsuchida N, Kobayashi M, Koike K. Disruption of the function of tumor-suppressor gene p53 by the hepatitis B virus X protein and hepatocarcinogenesis. J Cancer Res Clin Oncol 1995; 121: 593-601.
21. Miao J, Chen GG, Chun SY, Lai PP. Hepatitis B virus X protein induces apoptosis
in hepatoma cells through inhibiting Bcl-xL expression. Cancer Lett 2006; 236: 115-124.
22. Chen HY, Tang NH, Lin N, Chen ZX, Wang XZ. Hepatitis B virus X protein induces apoptosis
and cell cycle deregulation through interfering with DNA repair and checkpoint responses. Hepatol Res 2008; 38: 174-182.
23. Chen WN, Oon CJ, Goo KS. Hepatitis B virus X protein in the proteasome
of mammalian cells: defining the targeting domain. Mol Biol Rep 2001; 28: 31-34.
24. Seeger C. The hepatitis B virus X protein: the quest for a role in viral replication and pathogenesis. Hepatology 1997; 25: 496-498.
25. Hoffman L, Rechsteiner M. Nucleotidase activities of the 26 S proteasome
and its regulatory complex. J Biol Chem 1996; 271: 32538-32545.
26. Kanayama HO, Tamura T, Ugai S, Kagawa S, Tanahashi N, Yoshimura T, et al. Demonstration that a human 26S proteolytic complex consists of a proteasome
and multiple associated protein components and hydrolyzes ATP and ubiquitin
-ligated proteins by closely linked mechanisms. Eur J Biochem 1992; 206: 567-578.
27. Ugai S, Tamura T, Tanahashi N, Takai S, Komi N, Chung CH, et al. Purification and characterization of the 26S proteasome
complex catalyzing ATP-dependent breakdown of ubiquitin
-ligated proteins from rat liver. J Biochem 1993; 113: 754-768.
28. Wang Q, Song C, Li CC. Hexamerization of p97-VCP is promoted by ATP binding to the D1 domain and required for ATPase and biological activities. Biochem Biophys Res Commun 2003; 300: 253-260.
29. Song C, Wang Q, Li CC. Characterization of the aggregation-prevention activity of p97/valosin-containing protein. Biochemistry 2007; 46: 14889-14898.
30. Song C, Wang Q, Li CC. ATPase activity of p97-valosin-containing protein (VCP). D2 mediates the major enzyme activity, and D1 contributes to the heat-induced activity. J Biol Chem 2003; 278: 3648-3655.
31. Wójcik C, Yano M, DeMartino GN. RNA interference of valosin-containing protein (VCP/p97) reveals multiple cellular roles linked to ubiquitin
-dependent proteolysis. J Cell Sci 2004; 117: 281-292.
32. Yamamoto S, Tomita Y, Hoshida Y, Iizuka N, Monden M. Expression level of valosin-containing protein (p97) is correlated with progression and prognosis of non-small-cell lung carcinoma
. Ann Surg Oncol 2004; 11: 697-704.
Keywords:© 2009 Chinese Medical Association
peptides; ubiquitin; proteasome; p97 valosin-containing protein; carcinoma; hepatocellular; apoptosis