Article Figures & Data
Figures
- Fig. 1.
Effects of proteasome inhibitors on HBX expression. (A) HBX expression. HBX tagged with a Flag epitope was expressed by an adenovirus vector via infection or by a CMV-driven expression plasmid via transient transfection. AdHBX0 and plasmid pCEP4 were used as controls; 108 PFU of virus or 15 μg of the plasmid was used per 10-cm-diameter dish of cells. At 24 h after infection or 42 h after transfection, cells were treated with various inhibitors (20 μM each) for 6 h and then lysed and subjected to immunoprecipitation with antibody M2. The immunoprecipitates were subjected to SDS-PAGE (15% gel) and immunoblotted with antibody M2. (B) HBX degradation. Three 10-cm-diameter dishes of HepG2 cells were transiently transfected with pCEPHBXFlag (15 μg per dish); 18 h later, the cells were evenly distributed among 10 60-mm-diameter dishes. Cells transfected with plasmid pCEP4 were used as a control. After another 24 h, cells were incubated with methionine-free medium for 30 min, pulse-labeled with [35S]methionine (100 μCi/ml) for 20 min, and then chased with medium containing excessive unlabeled methionine (50 μg/ml). Cells were lysed at the end of the labeling and various times after chase. One set of cells was exposed to MG132 (20 μM) starting 1 h before the pulse-labeling and continuing during the chase. The cell lysates were immunoprecipitated with antibody M2. The control represents time zero lysate of pCEPHBX-transfected cells incubated with mouse immunoglobulin G. The immunoprecipitates were electrophoresed on an SDS–15% polyacrylamide gel. The gel was dried, and the levels of HBX expression were analyzed with a PhosphorImager (B). The values of HBX signals at time zero were arbitrarily set at 100, and the signal intensities of other time points were adjusted accordingly. The half-lives (T1/2) are indicated in panel C.
- Fig. 2.
Ubiquitin conjugation to HBX. HepG2 cells were plated in 10-cm-diameter dishes and transiently transfected with control plasmid pCEP4 (lane 1), pCMVUb (15 μg; lane 2), pCEPHBXFlag (5 μg; lane 3), or pCMVUb (15 μg) and pCEPHBXFlag (5 μg) (lanes 4 and 5), respectively. The control plasmid pCEP4 was used to equalize the total amount of transfected plasmid at 20 μg; 42 h after transfection, the cells were treated with or without MG132 (20 μM) as indicated for 6 h. The cells were then lysed and denatured in the presence of 1% SDS and 1% β-mercaptoethanol followed by boiling for 5 min. The denatured lysate was then diluted 1:3 with standard lysis buffer and subjected to immunoprecipitation with anti-Flag antibody M2. The immunoprecipitates were divided equally into three aliquots, analyzed by SDS-PAGE (15% gel), and Western blotted with antibodies against ubiquitin (A), the HA epitope (12CA5) (B), and the Flag epitope (M2) (C).
- Fig. 3.
Interaction of HBX and proteasome complex in vivo. (A) Coimmunoprecipitation of HBX and proteasome in the presence of proteasome inhibitors. HepG2 cells were plated in 10-cm-diameter dishes and infected with AdHBX or AdHBX0 (108 PFU per dish); 24 h after infection, cells were treated with or without proteasome inhibitor MG132 or lactacystin (20 μM) for 6 h. The cells were then lysed and subjected to immunoprecipitation with antibody M2. The immunoprecipitates were divided equally into four aliquots, subjected to SDS-PAGE on a 15% gel, and probed with antibodies against three different proteasome subunits as indicated and antibody M2. Partially purified proteasomes from uninfected HepG2 cells was used as positive controls for immunoblotting of various proteasome subunits. (B) Colocalization of HBX and proteasome by sucrose gradient centrifugation. Partially purified proteasome from HBX-expressing cell lysate as described above was subjected to sucrose gradient centrifugation as described in Materials and Methods; 0.5-ml fractions were collected from the top (fractions 1 to 10) and used for Western immunoblotting with antibodies against two 20S proteasome subunits (PSMA1 and PSMA7) and two 19S subunits (PSMC1 and PSMC3) and immunoprecipitation followed by Western blotting with antibody M2.
- Fig. 4.
Effects of proteasome inhibitors on HBX transactivation. (A and B) Effects on complex promoter. HepG2 cells were cotransfected with RSV-Luc and pCDHBX or pCD1 as a control at a ratio of 1 to 5 with a total DNA of 0.6 μg per well in a six-well plate; 20 h after transfection, the cells were treated with various inhibitors at a concentration of 20 μM (A) or at different concentrations (B); 12 h later, the cells were lysed and luciferase (Luc) activities were measured. Data presented are means ± standard deviations in triplicates, and the results are representative of three separate experiments. (C) Effect on simple promoter. HepG2 cells were cotransfected with reporter plasmids AP1-CAT, AP2-CAT, and SP1-Luc as indicated and pCDHBX (░) or pCD1 (control; ■) at a ratio of 1 to 5 with a total DNA of 0.6 μg per well in a six-well plate. At 20 h after transfection, the cells were treated for 12 h in the presence of 20 μM MG132 or 30 μM lactacystin. The cells were lysed, and luciferase or CAT activities were measured. Data presented are means ± standard deviations in triplicates, and the results are representative of three separate experiments.
- Fig. 5.
Reversibility of inhibition of HBX transactivation by proteasome inhibitors. HepG2 cells were cotransfected with RSV-Luc and pCDHBX or pCD1 as a control (CTRL) at a ratio of 1 to 5 with a total DNA of 0.6 μg per well in a six-well plate; 20 h after transfection, the cells were treated for 6 h with 10 μM MG132, a reversible inhibitor of the proteasome, or 20 μM lactacystin, an irreversible proteasome inhibitor. At the end of 6 h, one set of cells treated with or without inhibitors was harvested for luciferase determination. The second set of cells was incubated for another 6 h; the third set of cells was washed with PBS and exposed to fresh medium without inhibitors for another 6 h. At the end of 6 h, both sets of cells were harvested for luciferase determination. Data presented are the means ± standard deviations in triplicates, and the results are representative of three separate experiments.
- Fig. 6.
HBX transactivation is dose dependent and does not exhibit a squelching phenomenon. (A) HepG2 cells were grown in a six-well plate and cotransfected with 0.1 μg of RSV-Luc and increasing amounts (0.02, 0.05, 0.1, 0.5, 0.8, and 1 μg) of pCDHBXFlag. pCD1 was added to make the total transfected DNA 1.1 μg per well. The cells were lysed 48 h later, and luciferase activities were measured. (B) HepG2 cells were cotransfected with RSV-Luc and three different amounts of pCDHBXFlag as indicated; 20 h after transfection, the cells were treated with proteasome inhibitor MG132 at concentrations of 10 and 20 μM for the transactivation assay (top) or 20 μM for measurement of HBX levels (bottom). The cells were lysed and assayed for either luciferase activities or HBX levels by Western blot analysis using antibody M2. The results are shown as fold induction, calculated by dividing the luciferase activity of HBX transfected cells by that of control. Data presented are means ± standard deviations in triplicates, and the results are representative of three separate experiments.
- Fig. 7.
Inhibition of proteasome activities by HBX. HepG2 cells were transiently transfected with pHookHBX or control plasmid pHookLacZ0. Transfected cells were isolated by pHook magnetic beads as described in Materials and Methods. The proteasomes were partially purified by differential centrifugation from the isolated cells. The proteasome contents of the preparations were shown to be similar by immunoblotting with anti-PSMA1 antibody. (A) Peptidase activities. The proteasome preparations (3 μg) were incubated with the three fluorogenic peptide substrates specific for each active site (LLVY for chymotrysin-like activity, LRR for trypsin-like activity, and LLE for post-acidic activity) with or without MG132 (1 μM). The resulting fluorescence reflecting the peptidase activity of proteasomes was measured with a spectrofluorometer. The fluorescence signal from the proteasome of pHookLacZ0-transfected cells without MG132 treatment was standardized as 100% activity, and all the other fluorescence activities were calculated as percent activities. (B) Degradation of 125I-ubiquitin-lysozyme. The partially purified proteasome (3 μg) was incubated with125I-ubiquitin-lysozyme with or without MG132 at 37°C for 20 min and then precipitated with 10% TCA. The TCA-soluble fraction reflecting the degraded 125I-ubiquitin-lysozyme was measured with a γ counter. The soluble counts per minute from the pHookLacZ-transfected cells was standardized as 100% activity, and all other counts were calculated as percent activity. As controls, the partially purified proteasomes from pHookLacZ-transfected cells (A) and from both HBX- and LacZ-transfected cells (B) were treated with 1 μM MG132 to confirm the proteasome’s involvement in these reactions.
- Fig. 8.
HBX inhibits the peptidase activities of both 20S and 26S proteasome complexes. HepG2 cells were plated in 10-cm-diameter dishes and infected with recombinant AdHBX or AdHBX0(108 PFU per dish); 24 h after infection, proteasomes were partially purified as described above and subjected to activity gel analysis (A) or sucrose gradient centrifugation (B) followed by analysis of peptidase activities of each fraction (0.25 ml). In panel A, purified yeast proteasomes treated with or without 0.02% SDS were analyzed on an activity gel (left). The activity gel of proteasome preparation from HBX-expressing or control cells is shown in the middle, and Western immunoblot analysis using anti-PSMA1 antibody is shown on the right. The activity gel and sucrose gradient centrifugation are described in Materials and Methods. The locations of 20S and 26S bands in the sucrose gradient are indicated. Signal intensities of the 20S and 26S bands on the activity gel and Western blot were quantitated by using the ImageQuant software, and the value of the 26S band of the control (AdHBX0) in each gel is arbitrarily set at 100. The data are shown below the gel as means ± standard deviations from three separate experiments.
Tables
- Table 1.
Potency of inhibitors in blocking HBX transactivation correlates with potency against pure 20S proteasomes
Inhibitor Ki (μM) for inhibition of: HBX function in vivoa LLVY hydrolysisb aLLM None >1 aLLN 40–50 0.14 MG115 10–25 0.021 MG132 2.5–10 0.011 a Half-maximal concentrations for inhibition of HBX transactivation are derived from Fig. 4B.
b Ki’s for the chymotrypsin-like activity are given in reference38.
