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First published online May 20, 2009
doi: 10.1242/10.1242/jcs.040188

Journal of Cell Science 122, 1906-1916 (2009)
Published by The Company of Biologists 2009
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The VDAC1 N-terminus is essential both for apoptosis and the protective effect of anti-apoptotic proteins

Salah Abu-Hamad*, Nir Arbel*, Doron Calo, Laetitia Arzoine, Adrian Israelson, Nurit Keinan, Ronit Ben-Romano, Orr Friedman and Varda Shoshan-Barmatz{ddagger}

Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel


Figure 1
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  		Fig. 1. N-terminally truncated VDAC1 restores growth in T-REx293 cells silenced for hVDAC1 expression by shRNA-hVDAC1. (A) T-REx293 cells (bullet) silenced for hVDAC1 expression by shRNA-hVDAC ({circ}) showed inhibited cell growth, which was restored by expression of either native ({blacktriangledown}) or {Delta}(26)mVDAC1 ({triangledown}) under the control of 1 µg/ml tetracycline. Cells were stained with trypan blue and counted under a microscope. (B) Oxygen consumption was determined polarographically using a Clark oxygen electrode, as described in Materials and Methods. Oxygen utilization of T-REx293, hVDAC1-shRNA T-REx293 cells expressing stable native or {Delta}(26)mVDAC1 (2x106) cells was measured in the absence (black bars) and presence (grey bars) of rotenone (5 µM) for up to 10 minutes each. Rotenone was added directly into the respiration chamber and measurement of oxygen consumption was continued. The data represent the means from two different experiments. (C) A representative immunoblot analysis of VDAC expression level in the various cell types used in experiment B, using anti-VDAC antibodies. (D) hVDAC1-shRNA-T-REx293 expressing GFP, mVDAC1-GFP or {Delta}(26)mVDAC1-GFP were visualized using confocal microscopy. Scale bar: 10 µm. (E) Immunoblot analysis of GFP, mVDAC1-GFP or {Delta}(26)mVDAC1-GFP expression in T-REx293 cells using anti-GFP antibodies.

 

Figure 2
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  		Fig. 2. Channel properties of bilayer-reconstituted {Delta}(26)mVDAC1. {Delta}(26)mVDAC1 functions as a channel but shows no voltage-dependence conductance. mVDAC1 and {Delta}(26)mVDAC1 were expressed in porin-less yeast mitochondria, purified, and then reconstituted into a PLB. (A) Currents through the VDAC1 channel in response to a voltage step from 0 mV to voltages between –60 to +60 mV were recorded. Relative conductance was determined as the ratio of conductance at a given voltage (G) to the maximal conductance (Go). A representative of four similar experiments is shown, mVDAC1 (bullet) and {Delta}(26)mVDAC1 ({square}). (B) HK had no effect on the channel activity of N-terminal truncated VDAC1. Currents through bilayer-reconstituted mVDAC1 or {Delta}(26)mVDAC1 in response to a voltage step from 0 to –40 mV were recorded before and 10 minutes after the addition of HK-I (28 mIU/ml). The dashed lines indicate the zero and the maximal current levels.

 

Figure 3
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  		Fig. 3. Overexpression of native but not N-truncated mVDAC1 triggers cell death. (A) In T-REx293 cells silenced for hVDAC1 expression, overexpression of native mVDAC1 (bullet) or {Delta}(26)mVDAC1 ({circ}) was induced by 2.5 µg/ml tetracycline. A representative of three similar experiments is shown. (B) Apoptotic cell death visualized by Acridine Orange and ethidium bromide staining. Arrow indicates cells in an early apoptotic state, reflected by degraded nuclei (stained with Acridine Orange). Arrowhead indicates late apoptotic state (stained with Acridine Orange and ethidium bromide). Scale bars: 15 µm. (C) Western blot analysis of VDAC levels in control cells and cells transfected to express mVDAC1 or {Delta}(26)mVDAC1 (15 µg) using polyclonal anti-VDAC antibodies. For loading controls, actin levels in the samples (15 µg) were compared using anti-actin antibodies.

 

Figure 4
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  		Fig. 4. Cells expressing {Delta}(26)mVDAC1 are resistant to apoptotic induction. Cells overexpressing native but not N-terminal-truncated VDAC1 undergo mitochondria-mediated apoptosis. (A) T-REx293 cells silenced for hVDAC1 expression and expressing mVDAC1 (black bars) or {Delta}(26)mVDAC1 (grey bars), as induced by tetracycline (1 µg/ml), were treated with curcumin (200 µM, 48 hours), As2O3 (60 µM, 48 hours), STS (1.25 µM, 5 hours), cisplatin (50 µM, 30 hours) or TNF{alpha} (25 ng/ml, 7 hours). In addition, overexpression (as induced by tetracycline, 2.5 µg/ml) of mVDAC1 but not of {Delta}(26)mVDAC1 led to apoptosis. Statistical analysis of apoptosis in the different treatments was performed by ANOVA and t-tests; P<0.01. Data are means ± s.e.m. (n=4). (B) FACS analysis of apoptotic cell death, as induced by overexpression of mVDAC1 or {Delta}(26)mVDAC1 or by As2O3 was carried out using annexin V-FITC and propidium iodide (PI), as described in Materials and Methods. A representative of three similar FACS analyses of unstained and double stained cells with annexin V-FITC and PI for each treatment is shown. (C) Quantitative analysis of apoptosis in the FACS experiments shown in B for cells expressing mVDAC1 (hatched bars) or {Delta}(26)mVDAC1 (white bars). The same experiments were analyzed by Acridine Orange and ethidium bromide staining for cells expressing mVDAC1 (black bars) or {Delta}(26)mVDAC1 (grey bars). Data shown in C are the mean ± s.e.m. of three independent experiments.

 

Figure 5
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  		Fig. 5. Cytochrome c release as induced by As2O3, cisplatin or staurosporine in cells expressing native but not N-terminal truncated VDAC1. Cells overexpressing {Delta}(26)mVDAC1 are resistant to mitochondria-mediated apoptosis. T-REx293 cells (A), shRNA-hVDAC1-T-REx293 cells stably expressing native (B) or {Delta}(26)mVDAC1 (C) under the control of 1 µg/ml tetracycline were grown on poly-D-lysine (PDL)-coated coverslips. After 48 hours, cells were treated with As2O3 (60 µM, 24 hours). Then, cells were treated with MitoTracker and anti-cytochrome c antibodies as described in Materials and Methods and visualized using confocal microscope (Olympus 1X81). Scale bar: 10 µm. (D) Immunoblot analysis of cytochrome c released to the cytosol in T-REx293 cells silenced for hVDAC1 expression and expressing mVDAC1 or {Delta}(26)mVDAC1, as triggered by STS (1.25 µM, 5 hours) or by cisplatin (40 µM, 24 hours). Actin levels in cells confirmed that equal amounts of cells were used. (E) Western blot analysis of VDAC levels in control cells and cells transfected to express mVDAC1 or {Delta}(26)mVDAC1 (20 µg) using polyclonal anti-VDAC antibodies. For loading controls, actin levels in the samples (20 µg) were compared using anti-actin antibodies.

 

Figure 6
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  		Fig. 6. Cells expressing both endogenous VDAC and N-truncated mVDAC1 are resistant to apoptosis induction, a dominant negative effect. Expression of N-terminal-truncated mVDAC1 in different cell lines expressing endogenous hVDAC1, prevented apoptosis induction. (A-C) HEK293, HeLa and MCF-7 cells were transfected to express mVDAC1 (black bars) or {Delta}(26)mVDAC1 (grey bars) and treated with STS (1.25 µM, 5 hours), cisplatin (50 µM, 30 hours) or TNF{alpha} (25 ng/ml, 7 hours). Apoptotic cell death was analyzed by Acridine Orange and ethidium bromide staining, as in Fig. 3. Statistical analysis of apoptosis as a result of the different treatments was performed by ANOVA and t-tests; P<0.01. Data are means ± s.e.m. (n=4).

 

Figure 7
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  		Fig. 7. VDAC1 N-terminal peptide binding to HK-I and Bcl2({Delta}23). Synthetic peptide corresponding to the VDAC1 N-terminal region interacts with HK and Bcl2. Interaction of purified HK-I and Bcl2({Delta}23) with VDAC1-based peptides was revealed using real-time surface plasmon resonance. Different concentrations (40, 100, 200 µM) of the VDAC1 N-terminal or VDAC1 inter-membranal loop (BP) peptides were run in parallel over surface-strips of HK-I (A) or Bcl2({Delta}23) (B) and analyzed using ProteOn software. As a control for non-specific binding, the interaction of the peptide (200 µM) with IgG was analyzed. Responses (resonance units, RU), as a function of peptide concentration, were monitored using the ProteOn imaging system and related SW-tools. (C,D) Coomassie Blue staining of purified HK-I (C) and Bcl2({Delta}23) (D) used in these experiments.

 

Figure 8
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  		Fig. 8. Expression of the VDAC1-N-terminal peptide in T-REx293 cells prevents HK-I-, HK-II- and Bcl2-mediated protection against STS-induced cell death. Expression of VDAC1 N-terminal peptide inhibited the antiapoptotic effect of Bcl2, HK-I and HK-II. (A) T-REx293 cells or shRNA-hVDAC1–T-REx293 cells stably expressing native or {Delta}(26)mVDAC1 under the control of 1 µg/ml tetracycline were transfected with HK-I–GFP or co-transfected with HK-I–GFP and pcDNA4TO-AP, pcDNA4TO-BP or pcDNA4TO-NP. After 40 hours cells were visualized using confocal microscopy. (B) T-REx293 cells and cells expressing Bcl2-GFP and/or the N-terminal 26-amino-acid peptide (NP) under the control of tetracycline were exposed for 5 hours to 1.25 µM STS, and apoptotic cell death was followed. (C) An experiment similar to that in B was carried out with T-REx293 cells that had been transformed to express HK-I, HK-II and/or NP. Apoptosis in the different cell types was analyzed quantitatively with the aid of Acridine Orange and ethidium bromide staining, as in Fig. 2. Data are means ± s.e.m. (n=2-4). (D,E) Western blot analysis of Bcl2-GFP (D) or HK-I or HK-II (E) levels in control cells and cells transfected to overexpress either of these proteins and/or NP. Aliquots (50 µg) were analyzed for HK-I and HK-II levels, using polyclonal anti-HK-I and anti-HK-II antibodies, respectively, and for Bcl2-GFP, using anti-GFP antibodies. For control loading, actin levels in the samples were compared, using anti-actin antibodies.

 

Figure 9
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  		Fig. 9. Apoptosis induction by STS or VDAC1 overexpression induces VDAC oligomerization of native and {Delta}(26)mVDAC1. STS-induced VDAC oligomerization revealed by EGS cross-linking of HEK293 (A) or B-16 (B) cells before and after 1.5 or 3 hours of incubation with STS (1.25 µM). Cells were washed with PBS and incubated with the indicated concentration of EGS at 30°C for 15 minutes, followed by SDS-PAGE (2.6–13% gel gradient or 9% acrylamide for A and B, respectively), and western blotted using monoclonal anti-VDAC. Anti-actin antibodies were used for the loading control. Cross-linking of isolated mitochondria (Mito) is also shown. In C and D, mVDAC1 and {Delta}(26)mVDAC1 were overexpressed in T-REx293 cells under the control of tetracycline (2.5 µg/ml). The cells were exposed to EGS (125 or 250 µM) and analyzed for VDAC-containing cross-linked products using polyclonal (C) or monoclonal (directed to the N-terminal) anti-VDAC antibodies (D). A single and double black asterisk indicate native endogenous VDAC and {Delta}(26)mVDAC1 dimers, respectively.

 

Figure 10
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  		Fig. 10. Model for VDAC1 N-terminal region-mediated cytochrome c release. (A) Side-view across a membranal VDAC1 with the amphipathic {alpha}-helix N-terminal region in various locations: cytoplasmically exposed (De Pinto et al., 2003Go), membrane-spanning (Colombini, 2004Go), lying on the membrane surface (Reymann et al., 1995Go) and positioned in the pore (Bayrhuber et al., 2008Go; Hiller et al., 2008Go; Ujwal et al., 2008Go). (B) Upon an apoptotic signal, VDAC oligomerization is enhanced and the amphipathic {alpha}-helix N-terminal region of each VDAC molecule flips inside the hydrophobic pore formed by the β-barrels, making the pore hydrophilic and capable of conducting cytochrome c release. (C) Interaction of anti-apoptotic proteins (HK, Bcl2) with the N-terminal region of VDAC1 prevents its translocation and thus the formation of the hydrophilic pore, so inhibiting cytochrome c release.

 

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© The Company of Biologists Ltd 2009