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Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells.

Shimizu S, Matsuoka Y, Shinohara Y, Yoneda Y, Tsujimoto Y - J. Cell Biol. (2001)

Bottom Line: Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release.When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death.Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Osaka University Graduate School of Medicine, Biomedical Research Center, Department of Medical Genetics, Osaka 565-0871, Japan.

ABSTRACT
Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

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Inhibition of VDAC activity by anti-VDAC antibodies. (A) Putative model of human VDAC1 topology. The epitopes of the three anti-VDAC antibodies (Ab#20, Ab#25, and 31HL) are shown by boxes. (B) Specificity of Ab#20 and Ab#25. Rat liver mitochondria (Mt) lysate (15 μg) and HeLa cell lysate (10 μg) were subjected to Western blotting using Ab#20 and Ab#25. (C) Inhibition of both VDAC activity and Bax-induced enhancement of VDAC activity by Ab#20 and Ab#25. 20 μl of plain liposomes or VDAC liposomes was incubated with 0.2 μg/μl of the indicated antibodies for 3 min, and then were incubated with 5 μl of [14C]sucrose (97%; 200 μCi/ml) in the presence (black bar) or absence (white bar) of rBax (0.2 μg/μl) at 25°C for the 6 min. The [14C]sucrose incorporated into the liposomes was measured as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments. (D) Lack of influence of Ab#25 on Bax channel activity. Irrelevant control protein liposomes and Bax liposomes were incubated with 0.2 μg/μl of Ab#25 or NRI for 5 min, and then incubated with 5 μl of [3H]glucose (97%; 20 Ci/mmol) at 25°C for 5 min. The [3H]glucose incorporated into the liposomes was measured as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments.
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Figure 1: Inhibition of VDAC activity by anti-VDAC antibodies. (A) Putative model of human VDAC1 topology. The epitopes of the three anti-VDAC antibodies (Ab#20, Ab#25, and 31HL) are shown by boxes. (B) Specificity of Ab#20 and Ab#25. Rat liver mitochondria (Mt) lysate (15 μg) and HeLa cell lysate (10 μg) were subjected to Western blotting using Ab#20 and Ab#25. (C) Inhibition of both VDAC activity and Bax-induced enhancement of VDAC activity by Ab#20 and Ab#25. 20 μl of plain liposomes or VDAC liposomes was incubated with 0.2 μg/μl of the indicated antibodies for 3 min, and then were incubated with 5 μl of [14C]sucrose (97%; 200 μCi/ml) in the presence (black bar) or absence (white bar) of rBax (0.2 μg/μl) at 25°C for the 6 min. The [14C]sucrose incorporated into the liposomes was measured as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments. (D) Lack of influence of Ab#25 on Bax channel activity. Irrelevant control protein liposomes and Bax liposomes were incubated with 0.2 μg/μl of Ab#25 or NRI for 5 min, and then incubated with 5 μl of [3H]glucose (97%; 20 Ci/mmol) at 25°C for 5 min. The [3H]glucose incorporated into the liposomes was measured as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments.

Mentions: Recently, we showed that Bax/Bak is able to induce cytochrome c release by opening the VDAC through direct interaction in a proteoliposome system (Shimizu et al. 1999, Shimizu et al. 2000a). However, the essential role of the VDAC in apoptotic cytochrome c release and cell death in mammalian cells was still unconfirmed. For this purpose, we produced anti-VDAC antibodies that could inhibit VDAC activity. As epitopes, we selected two oligopeptides (amino acids 104–120 and 151–165) of human VDAC1 shown in Fig. 1 A, based on previous observations that VDAC in intact bovine heart mitochondria is cleaved by trypsin at lysine residues between arginine108, which corresponds to lysine108 in human VDAC1, and arginine119 and by chymotrypsin at the tyrosine117 and tyrosine172 residues (De Pinto and Palmieri 1992), indicating that these sites are probably exposed to the cytoplasm, and also based on the proposed three-dimensional structure (De Pinto and Palmieri 1992; Song and Colombini 1996) that suggests that these oligopeptides are probably localized near the entrance of the channel whereas most of the other regions form one α-helix and 12 β-strands that cross the membrane to create a β-barrel (Fig. 1 A). Furthermore, since these residues are well conserved among three isoforms of human and mouse, the antibodies were expected to react with all isoforms of various mammals. Both antibodies raised in rabbits (Ab#20 recognizing amino acids 151–165 and Ab#25 recognizing amino acids 104–120) were affinity purified and were shown to be highly specific for human and rat VDAC (Fig. 1 B). As shown in Fig. 1 C, Ab#20 and Ab#25, but not normal rabbit IgG (NRI), efficiently inhibited VDAC activity, as assessed by [14C]sucrose uptake into VDAC liposomes. Ab#25 and Ab#20, but not NRI, also inhibited the Bax-induced enhancement of VDAC activity (Fig. 1 C). In contrast, both Ab#20 and Ab#25 showed no effect on Bax channel activity, as assessed by [3H]glucose uptake into Bax liposomes (Fig. 1 D; data not shown).


Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells.

Shimizu S, Matsuoka Y, Shinohara Y, Yoneda Y, Tsujimoto Y - J. Cell Biol. (2001)

Inhibition of VDAC activity by anti-VDAC antibodies. (A) Putative model of human VDAC1 topology. The epitopes of the three anti-VDAC antibodies (Ab#20, Ab#25, and 31HL) are shown by boxes. (B) Specificity of Ab#20 and Ab#25. Rat liver mitochondria (Mt) lysate (15 μg) and HeLa cell lysate (10 μg) were subjected to Western blotting using Ab#20 and Ab#25. (C) Inhibition of both VDAC activity and Bax-induced enhancement of VDAC activity by Ab#20 and Ab#25. 20 μl of plain liposomes or VDAC liposomes was incubated with 0.2 μg/μl of the indicated antibodies for 3 min, and then were incubated with 5 μl of [14C]sucrose (97%; 200 μCi/ml) in the presence (black bar) or absence (white bar) of rBax (0.2 μg/μl) at 25°C for the 6 min. The [14C]sucrose incorporated into the liposomes was measured as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments. (D) Lack of influence of Ab#25 on Bax channel activity. Irrelevant control protein liposomes and Bax liposomes were incubated with 0.2 μg/μl of Ab#25 or NRI for 5 min, and then incubated with 5 μl of [3H]glucose (97%; 20 Ci/mmol) at 25°C for 5 min. The [3H]glucose incorporated into the liposomes was measured as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199613&req=5

Figure 1: Inhibition of VDAC activity by anti-VDAC antibodies. (A) Putative model of human VDAC1 topology. The epitopes of the three anti-VDAC antibodies (Ab#20, Ab#25, and 31HL) are shown by boxes. (B) Specificity of Ab#20 and Ab#25. Rat liver mitochondria (Mt) lysate (15 μg) and HeLa cell lysate (10 μg) were subjected to Western blotting using Ab#20 and Ab#25. (C) Inhibition of both VDAC activity and Bax-induced enhancement of VDAC activity by Ab#20 and Ab#25. 20 μl of plain liposomes or VDAC liposomes was incubated with 0.2 μg/μl of the indicated antibodies for 3 min, and then were incubated with 5 μl of [14C]sucrose (97%; 200 μCi/ml) in the presence (black bar) or absence (white bar) of rBax (0.2 μg/μl) at 25°C for the 6 min. The [14C]sucrose incorporated into the liposomes was measured as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments. (D) Lack of influence of Ab#25 on Bax channel activity. Irrelevant control protein liposomes and Bax liposomes were incubated with 0.2 μg/μl of Ab#25 or NRI for 5 min, and then incubated with 5 μl of [3H]glucose (97%; 20 Ci/mmol) at 25°C for 5 min. The [3H]glucose incorporated into the liposomes was measured as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments.
Mentions: Recently, we showed that Bax/Bak is able to induce cytochrome c release by opening the VDAC through direct interaction in a proteoliposome system (Shimizu et al. 1999, Shimizu et al. 2000a). However, the essential role of the VDAC in apoptotic cytochrome c release and cell death in mammalian cells was still unconfirmed. For this purpose, we produced anti-VDAC antibodies that could inhibit VDAC activity. As epitopes, we selected two oligopeptides (amino acids 104–120 and 151–165) of human VDAC1 shown in Fig. 1 A, based on previous observations that VDAC in intact bovine heart mitochondria is cleaved by trypsin at lysine residues between arginine108, which corresponds to lysine108 in human VDAC1, and arginine119 and by chymotrypsin at the tyrosine117 and tyrosine172 residues (De Pinto and Palmieri 1992), indicating that these sites are probably exposed to the cytoplasm, and also based on the proposed three-dimensional structure (De Pinto and Palmieri 1992; Song and Colombini 1996) that suggests that these oligopeptides are probably localized near the entrance of the channel whereas most of the other regions form one α-helix and 12 β-strands that cross the membrane to create a β-barrel (Fig. 1 A). Furthermore, since these residues are well conserved among three isoforms of human and mouse, the antibodies were expected to react with all isoforms of various mammals. Both antibodies raised in rabbits (Ab#20 recognizing amino acids 151–165 and Ab#25 recognizing amino acids 104–120) were affinity purified and were shown to be highly specific for human and rat VDAC (Fig. 1 B). As shown in Fig. 1 C, Ab#20 and Ab#25, but not normal rabbit IgG (NRI), efficiently inhibited VDAC activity, as assessed by [14C]sucrose uptake into VDAC liposomes. Ab#25 and Ab#20, but not NRI, also inhibited the Bax-induced enhancement of VDAC activity (Fig. 1 C). In contrast, both Ab#20 and Ab#25 showed no effect on Bax channel activity, as assessed by [3H]glucose uptake into Bax liposomes (Fig. 1 D; data not shown).

Bottom Line: Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release.When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death.Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Osaka University Graduate School of Medicine, Biomedical Research Center, Department of Medical Genetics, Osaka 565-0871, Japan.

ABSTRACT
Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

Show MeSH
Related in: MedlinePlus