1906 Research Article The VDAC1 N-terminus is essential both for 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‡ Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected])

Accepted 26 February 2009 Journal of Cell Science 122, 1906-1916 Published by The Company of Biologists 2009 doi:10.1242/jcs.040188

Summary The release of mitochondrial-intermembrane-space pro- apoptosis, induced by various stimuli. Employing a variety of apoptotic proteins, such as cytochrome c, is a key step in experimental approaches, we show that and Bcl2 initiating apoptosis. Our study addresses two major questions confer protection against apoptosis through interaction with the in apoptosis: how are mitochondrial pro-apoptotic proteins VDAC1 N-terminal region. We also demonstrate that apoptosis released and how is this process regulated? Accumulating induction is associated with VDAC oligomerization. These evidence indicates that the voltage-dependent anion channel results show VDAC1 to be a component of the apoptosis (VDAC) plays a central role in mitochondria-mediated machinery and offer new insight into the mechanism of apoptosis. Here, we demonstrate that the N-terminal domain cytochrome c release and how anti-apoptotic proteins regulate of VDAC1 controls the release of cytochrome c, apoptosis and apoptosis and promote tumor cell survival. the regulation of apoptosis by anti-apoptotic proteins such as hexokinase and Bcl2. Cells expressing N-terminal truncated VDAC1 do not release cytochrome c and are resistant to Key words: Apoptosis, Bcl2, hexokinase, mitochondria, VDAC1

Introduction 2007; Lemasters et al., 1999; Shoshan-Barmatz et al., 2006; Programmed cell death – apoptosis – is a genetically predetermined Tsujimoto and Shimizu, 2002; Zamzami and Kroemer, 2003). mechanism elicited by several molecular pathways (Kroemer et al., Indeed, a variety of apoptotic stimuli were shown to trigger 2007). Defects in the regulation of apoptosis are often associated apoptosis by modulation of VDAC and implicate VDAC1 as a

Journal of Cell Science with disease, such as neuronal degenerative diseases, tumorigenesis, component of the apoptosis machinery (Shoshan-Barmatz et al., autoimmune disorders, and viral infections (Reed, 2002; Thompson, 2008; Shoshan-Barmatz et al., 2006; Tsujimoto and Shimizu, 2002; 1995). Mitochondria are potent integrators and coordinators of Zheng et al., 2004). By contrast, VDAC proteins have been reported apoptosis by releasing pro-apoptotic agents and/or disrupting to be dispensable for Ca2+- and oxidative stress-induced permeability cellular energy . Following an apoptotic stimulus, transition pore (PTP) opening (Baines et al., 2007). Nonetheless, various proteins that normally reside in the intermembrane space recent studies have elucidated that VDAC1 is an indispensable of the mitochondria, including cytochrome c and apoptosis-inducing protein for PTP opening and induction of apoptosis (Tajeddine et factor (AIF), are released to initiate the activation of pro-caspases, al., 2008; Yuan et al., 2008). It was found that reducing VDAC1 the protease mediators of cell death (Green and Reed, 1998; levels by siRNA attenuates endostatin-induced apoptosis in Halestrap et al., 2000). It remains unclear, however, how these endothelial cells, and that endostatin-induced PTP opening is apoptotic initiators cross the outer mitochondrial membrane (OMM) accompanied by upregulation of VDAC1 expression (Yuan et al., and are released into the cytosol. Some models predict that release 2008). Similarly, siRNA-mediated depletion of VDAC1 strongly is facilitated by swelling of the mitochondrial matrix and subsequent reduced cisplatin-induced cytochrome c release and apoptotic cell rupture of the OMM, whereas other models predict the formation death (Tajeddine et al., 2008). These recent reports and previous of protein-conducting channels that are large enough to allow the accumulated evidence strongly suggest that VDAC serves a key passage of cytochrome c and other proteins into the cytosol, without function in apoptosis (Crompton, 1999; Halestrap et al., 2000; compromising the integrity of the OMM (Halestrap et al., 2000; Kroemer et al., 2007; Lemasters et al., 1999; Shoshan-Barmatz et Shoshan-Barmatz et al., 2008). The finding that cytochrome c can al., 2008; Shoshan-Barmatz et al., 2006; Tsujimoto and Shimizu, leak from intact mitochondria (Doran and Halestrap, 2000; Martinou 2002; Zamzami and Kroemer, 2003). VDAC is the major OMM et al., 2000; Shoshan-Barmatz et al., 2006) supports the latter transporter and plays an important role in the controlled passage of models. Release of cytochrome c is regulated by different proteins, adenine nucleotides, Ca2+ and other metabolites into and out of some of which interact with the OMM protein, VDAC (voltage- mitochondria, thereby assuming an important role in energy dependent anion channel), also known as mitochondrial . production through the control of metabolite traffic (Colombini, VDAC is thus believed to participate in the release of cytochrome 2004; Lemasters and Holmuhamedov, 2006). Recently, the three- c and to interact with anti- and pro-apoptotic proteins, and is dimensional (3D) structures of recombinant human and murine considered a probable candidate for such an OMM pore-forming VDAC1 were determined by NMR spectroscopy and X-ray protein (Crompton, 1999; Halestrap et al., 2000; Kroemer et al., crystallography (Bayrhuber et al., 2008; Hiller et al., 2008; Ujwal (Δ26)VDAC1-mediated apoptotic resistance 1907

et al., 2008). VDAC1was shown to adopt a β-barrel architecture comprising 19 β-strands and an α-helix located within the pore. The α-helix of the N-terminal segment is oriented against the interior wall, causing a partial narrowing at the center of the pore. As such, this helix is ideally positioned to regulate the conductance of ions and metabolites passing through the VDAC pore. These structural and other studies (De Pinto et al., 2007) further indicate that the helical region of the VDAC N-terminal domain forms a distorted α-helix that does not span the whole sequence but only part of it. Although these recently determined VDAC1 structures suggest that the hydrophilic N-terminus of the protein is nestled within the pore (Bayrhuber et al., 2008; Hiller et al., 2008; Ujwal et al., 2008), other approaches point to the N-terminal α-helix as being exposed to the cytoplasm (De Pinto et al., 2003), to cross the membrane (Colombini, 2004) or to lie on the membrane surface (Reymann et al., 1995). Further evidence suggesting that the N-terminal region of VDAC1 in fact constitutes a mobile component of the protein comes from the observations that the N-terminal α-helix exhibits motion during voltage gating (Mannella, 1997), that anti-VDAC antibodies raised against the N-terminal region of the protein interact with membranal VDAC (Abu-Hamad et al., 2006; Junankar et al., 1995; Shoshan-Barmatz et al., 2004) and that mobility of the N- α terminal -helix may modulate the accessibility of apoptosis- Fig. 1. N-terminally truncated VDAC1 restores growth in T-REx293 cells regulating proteins of the Bcl2 family (i.e. Bax and Bcl-xL) to their silenced for hVDAC1 expression by shRNA-hVDAC1. (A) T-REx293 cells binding sites on VDAC1 (Shi et al., 2003). (᭹) silenced for hVDAC1 expression by shRNA-hVDAC (᭺) showed ᭢ In the present study, the role of the VDAC1 N-terminal domain inhibited cell growth, which was restored by expression of either native ( ) or Δ(26)mVDAC1 (᭞) under the control of 1 μg/ml tetracycline. Cells were in regulating VDAC1 activity in cell life and apoptotic cell death stained with trypan blue and counted under a microscope. (B) Oxygen was investigated. Using Δ(26)mVDAC1, an N-terminal truncated consumption was determined polarographically using a Clark oxygen form of murine VDAC1 expressed in human (h)VDAC1-shRNA electrode, as described in Materials and Methods. Oxygen utilization of T- cells expressing low levels of endogenous hVDAC1, the N-terminal REx293, hVDAC1-shRNA T-REx293 cells expressing stable native or Δ ϫ 6 region of VDAC1 was shown to be required for the release of (26)mVDAC1 (2 10 ) cells was measured in the absence (black bars) and presence (grey bars) of rotenone (5 μM) for up to 10 minutes each. Rotenone cytochrome c and apoptotic cell death. Furthermore, the VDAC1 was added directly into the respiration chamber and measurement of oxygen N-terminal α-helix was shown to mediate the interaction of VDAC1 consumption was continued. The data represent the means from two different with the anti-apoptotic, pro-survival factors, hexokinase (HK)-I, experiments. (C) A representative immunoblot analysis of VDAC expression level in the various cell types used in experiment B, using anti-VDAC Journal of Cell Science HK-II and Bcl2. The findings of this study thus indicate that VDAC1 antibodies. (D) hVDAC1-shRNA-T-REx293 expressing GFP, mVDAC1-GFP is a critical component in mitochondria-mediated apoptosis and its or Δ(26)mVDAC1-GFP were visualized using confocal microscopy. Scale bar: regulation and point to the N-terminal domain of VDAC1 as being 10 μm. (E) Immunoblot analysis of GFP, mVDAC1-GFP or Δ(26)mVDAC1- a critical component in the regulation of cytochrome c release and GFP expression in T-REx293 cells using anti-GFP antibodies. apoptosis.

Results The N-terminal region of VDAC1 is not required for cell growth Δ(26)mVDAC1 can substitute for native VDAC1 in supporting the and localization to mitochondria metabolite exchange between the mitochondria and the cytosol in The N-terminal domain of VDAC1 has been the object of cells expressing low levels of VDAC1 (Abu-Hamad et al., 2006). considerable interest and was proposed to possess functionally Expressing N-truncated VDAC1 in porin-less yeast restored their relevant properties (Colombini et al., 1987; De Pinto et al., 2007; growth to levels of porin-less yeast expressing native VDAC (data Peng et al., 1992; Stanley et al., 1995; Ujwal et al., 2008; Yehezkel not shown). et al., 2007). VDAC provides the major pathway for nucleotides The oxygen consumption rates of control T-REx cells, shRNA- and other metabolites moving across the OMM. To verify that the hVDAC1 cells with a low VDAC1 expression level, and cells stably N-terminal region of VDAC1 is necessary for such transport expressing native or N-terminal-truncated mVDAC1 were measured activity, N-terminally truncated murine VDAC1 [Δ(26)mVDAC1] as an index of in situ mitochondrial metabolism (Fig. 1B). The was expressed in human T-REx293 cells in which endogenous oxygen consumption rate of shRNA-VDAC1 cells was about 67% VDAC1 expression had been suppressed by ~85% using a single that of control cells, suggesting that VDAC1 is required for normal hVDAC1-shRNA (Abu-Hamad et al., 2006). Such cells proliferated mitochondrial respiration. When such cells were transfected to slowly, compared with normal cells (Fig. 1A), owing to low levels express either native or Δ(26)mVDAC1, the respiration rate was of ATP and reduced ATP synthesis capacity, both of which could increased to almost the level obtained by control cells. Rotenone be restored upon expression of mVDAC1 under the control of inhibited oxygen consumption in all cell types (Fig. 1B). The tetracycline (Abu-Hamad et al., 2006). Δ(26)mVDAC1 expression expression levels of VDAC, as analyzed by immunoblotting using in hVDAC1-silenced cells (shRNA-hVDAC1) also restored cell polyclonal anti-VDAC antibodies, clearly show that the level of growth (Fig. 1A), suggesting that the 26 N-terminal residues are VDAC expression in VDAC1-shRNA cells was dramatically not essential for VDAC1 function in cell growth and that reduced by over 90%. That value could be restored to control levels 1908 Journal of Cell Science 122 (11)

Fig. 2. Channel properties of bilayer-reconstituted Δ(26)mVDAC1. Δ(26)mVDAC1 functions as a channel but shows no voltage-dependence conductance. mVDAC1 and Δ(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 (᭹) and Δ(26)mVDAC1 (ᮀ). (B) HK had no effect on the channel activity of N-terminal truncated VDAC1. Currents through bilayer-reconstituted mVDAC1 or Δ(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.

by transfecting the cells with plasmids encoding mVDAC1 or Δ(26)mVDAC1 assumed a stable highly conductive state, as would Δ(26)mVDAC1 (Fig. 1C). These results suggest that be expected for a voltage-insensitive channel (Fig. 2A), HK-I had Δ(26)mVDAC1 is as active as native mVDAC1. no effect on the channel activity of Δ(26)VDAC1. These results We then demonstrated, by confocal microscopy, that mVDAC1- indicate that the VDAC1 N-terminal region is required for HK-I GFP and Δ(26)mVDAC1-GFP expressed in T-REx293 cells showed modification of channel conductance. similarly distributed punctuated fluorescence, which was confined to the mitochondria (Fig. 1D). The expression of Δ(26)mVDAC1- N-terminally truncated mVDAC1-expressing cells do not GFP in these cells was also shown by immunoblot analysis (Fig. release cytochrome c and do not undergo apoptosis 1E). The results indicate that both native and truncated mVDAC1 It has been previously demonstrated that overexpression of human, were localized to the mitochondria, suggesting that the 26 N- murine, yeast or rice VDAC1, VDAC1-GFP or mutated VDAC1 terminal residues do not act as a targeting sequence. induces apoptotic cell death (Abu-Hamad et al., 2006; Bae et al., 2003; Zaid et al., 2005). Accordingly, upon overexpression of Channel activity of Δ(26)mVDAC1 and its modulation by mVDAC1 in T-REx293 cells silenced for hVDAC expression by hexokinase hVDAC1-shRNA (using 2.5 μg/ml tetracycline), growth continued Δ Journal of Cell Science We further demonstrated that (26)mVDAC1 functions as a for 3 days before cells starting to die, with all cells having died by channel-forming protein. Δ(26)mVDAC1 or mVDAC1 were the fifth day (Fig. 3A). By contrast, cells expressing Δ(26)mVDAC1 expressed in the mitochondria of porin-less yeast, purified and continued to grow for >8 days, with only 5-8% of the cells reconstituted into a planar (PLB), where VDAC channel succumbing to apoptosis, as reflected by enhanced nuclear activity was analyzed. Single-channel recording gave a maximal fragmentation, visualized by Acridine Orange and ethidium bromide conductance of 4 nS (at 1 M NaCl) for both the native and the N- staining (Fig. 3B). The overexpression of native and N-terminal terminally truncated proteins. However, whereas bilayer truncated mVDAC1 in cells stably expressing these proteins is reconstituted-mVDAC1 showed a typical voltage-dependence shown in Fig. 3C. conductance, with the highest values being obtained at In the light of the findings that cells overexpressing N-terminal- transmembrane potentials between –20 and +20 mV and decreasing truncated VDAC1 had lost their ability to undergo mitochondria- at both high negative and positive potentials (Fig. 2A), mediated apoptosis (Fig. 3), we tested for the involvement of the Δ(26)mVDAC1 showed no voltage dependence and exhibited high N-terminal region of VDAC1 in apoptosis, as induced by various conductance at all the tested voltages (Fig. 2A) in agreement with stimuli. hVDAC1-shRNA-T-REx293 cells expressing mVDAC1 or previous results (De Pinto et al., 2008; Koppel et al., 1998; Popp Δ(26)mVDAC1 were challenged with various apoptosis-inducing et al., 1996). Since there is no significant membrane potential across agents, all of which affect the mitochondria but produce their effect the OMM, the loss of voltage sensitivity of Δ(26)mVDAC1 may by a different mechanisms (Fig. 4). Staurosporine (STS), curcumin, not have physiological significance, as reflected in the finding that As2O3 and cisplatin, all induced death in cells expressing native its ability to support cell growth was similar to that of the intact mVDAC1 but not in those expressing Δ(26)mVDAC1. By contrast, protein (Fig. 1A). tumor necrosis factor alpha (TNFα), an agent that activates apoptosis In our previous studies (Abu-Hamad et al., 2008; Azoulay-Zohar via a mitochondria-independent pathway (Schmitz et al., 2000), et al., 2004), we demonstrated that hexokinase (HK) interacts with induced a similar extent of cell death in native mVDAC1- and Δ(26)- VDAC to decrease it channel activity. The interaction of the VDAC1 mVDAC1-expressing cells (Fig. 4A). N-terminal region with purified rat brain HK-I was studied using Similar results were obtained by flow cytometric analysis, using bilayer-reconstituted mVDAC1 or Δ(26)mVDAC1. Addition of annexin V-FITC and propidium iodide (PI; Fig. 4B,C). The results HK-I to bilayer-reconstituted native mVDAC1 induced the channel, showed that induction of mVDAC1 overexpression by a high fluctuating between the fully open and a sub-conducting state, to concentration of tetracycline (2.5 μg/ml) resulted in an increase in adopt a stable, long-lived low-conducting state (Fig. 2B). Although cell death of approximately three- to fourfold, but reduced to 1.4- (Δ26)VDAC1-mediated apoptotic resistance 1909

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 (᭹) or Δ(26)mVDAC1 (᭺) 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 Fig. 4. Cells expressing Δ(26)mVDAC1 are resistant to apoptotic induction. degraded nuclei (stained with Acridine Orange). Arrowhead indicates late Cells overexpressing native but not N-terminal-truncated VDAC1 undergo apoptotic state (stained with Acridine Orange and ethidium bromide). Scale mitochondria-mediated apoptosis. (A) T-REx293 cells silenced for hVDAC1 bars: 15 μm. (C) Western blot analysis of VDAC levels in control cells and expression and expressing mVDAC1 (black bars) or Δ(26)mVDAC1 (grey cells transfected to express mVDAC1 or Δ(26)mVDAC1 (15 μg) using bars), as induced by tetracycline (1 μg/ml), were treated with curcumin polyclonal anti-VDAC antibodies. For loading controls, actin levels in the (200 μM, 48 hours), As O (60 μM, 48 hours), STS (1.25 μM, 5 hours), samples (15 μg) were compared using anti-actin antibodies. 2 3 cisplatin (50 μM, 30 hours) or TNFα (25 ng/ml, 7 hours). In addition, overexpression (as induced by tetracycline, 2.5 μg/ml) of mVDAC1 but not of Δ(26)mVDAC1 led to apoptosis. Statistical analysis of apoptosis in the fold or less (Fig. 4B,C) when Δ(26)mVDAC1 was overexpressed. different treatments was performed by ANOVA and t-tests; P<0.01. Data are = Exposure of cells expressing mVDAC to As2O3 resulted in 47.5% means ± s.e.m. (n 4). (B) FACS analysis of apoptotic cell death, as induced by overexpression of mVDAC1 or Δ(26)mVDAC1 or by As2O3 was carried out Journal of Cell Science annexin V-FITC-positive and PI-positive cells (i.e. late apoptotic using annexin V-FITC and propidium iodide (PI), as described in Materials cells), as compared with the 22.5% value obtained with cells and Methods. A representative of three similar FACS analyses of unstained expressing Δ(26)mVDAC1 (Fig. 4C). These results indicate that in and double stained cells with annexin V-FITC and PI for each treatment is cells expressing Δ(26)mVDAC1, apoptosis is inhibited. shown. (C) Quantitative analysis of apoptosis in the FACS experiments shown To test whether the apoptotic resistance of Δ(26)mVDAC1- in B for cells expressing mVDAC1 (hatched bars) or Δ(26)mVDAC1 (white expressing cells is due to an inability of the cells to release bars). The same experiments were analyzed by Acridine Orange and ethidium Δ bromide staining for cells expressing mVDAC1 (black bars) or cytochrome c, cells expressing native mVDAC1 or (26)mVDAC1 Δ(26)mVDAC1 (grey bars). Data shown in C are the mean ± s.e.m. of three were exposed to As2O3, and the localization of cytochrome c, before independent experiments. and after exposure to the apoptosis inducers, was analyzed by immunocytochemistry (Fig. 5A). Although in cells expressing native mVDAC1 the punctuate mitochondrial localization of cytochrome VDAC, and apoptosis was induced by various stimuli. As with cells c (i.e. colocalized with MitoTracker, a dye that specifically labels possessing low level of VDAC1 (Fig. 4), all cell types showed mitochondria in living cells) changed upon exposure to apoptosis insensitivity to apoptosis induction by reagents that act through stimuli to a diffused cytoplasmic location (Fig. 5B), no such change activation of cytochrome c release, but not by TNFα, when in cytochrome c distribution was observed in cells expressing expressing Δ(26)mVDAC1 (Fig. 6). These results show that Δ(26)mVDAC1 (Fig. 5C). Cytochrome c release into the cytosol although the cells express endogenous hVDAC1, the presence of was also analyzed by western blotting using anti-cytochrome c the N-terminal-truncated mVDAC1 mutant, completely prevented monoclonal antibodies (Fig. 5D). STS or cisplatin induced the induction of apoptosis. cytochrome c release in cells expressing native mVDAC1 but not those expressing Δ(26)mVDAC1. These findings clearly show that A synthetic peptide corresponding to the VDAC1 N-terminal the N-terminal region of VDAC1 is essential for cytochrome c region interacts with HK and Bcl2 and, when expressed in release. cells, prevents their anti-apoptotic effects HK-I has been shown to interact with VDAC and protect against The Δ(26)mVDAC1 mutant has a dominant-negative effect apoptotic cell death as induced by STS or VDAC1 overexpression To test whether the resistance to apoptosis conferred by N-terminal- (Abu-Hamad et al., 2008; Arzoine et al., 2009; Azoulay-Zohar et truncated mVDAC1 has a dominant negative effect, Δ(26)mVDAC1 al., 2004; Zaid et al., 2005). As shown in Fig. 2B, purified HK-I was expressed in three different cell lines expressing endogenous did not modify the channel conductance of bilayer-reconstituted 1910 Journal of Cell Science 122 (11)

Fig. 5. Cytochrome c release as induced by As2O3, cisplatin or staurosporine in cells expressing native but not N-terminal truncated VDAC1. Cells overexpressing Δ(26)mVDAC1 are resistant to mitochondria-mediated apoptosis. T-REx293 cells (A), shRNA-hVDAC1-T- REx293 cells stably expressing native (B) or Δ(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 Mito- Tracker 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 Δ(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 Δ(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.

Δ Journal of Cell Science (26)mVDAC1. Thus, to further assess the interaction of HK-I and terminal peptide was diffuse throughout the cytosol (Fig. 8A). Bcl2 with the VDAC1 N-terminal region, HK-I (Fig. 7A) and Similar results were obtained when peptide A (AP), but not peptide recombinant Bcl2(Δ23) (Fig. 7B) or, as a control, rabbit IgG, were B (BP) was expressed (Fig. 8A). These results indicate that the N- coupled to surface plasmon resonance (SPR) biosensor chips. terminal peptide can detach or prevent HK-I binding to the Increasing concentrations of a synthetic peptide corresponding to mitochondria. the N-terminal region of VDAC1 were injected onto the sensor Next, we tested whether expression of the VDAC1 N-terminal 26- chips, and binding to purified immobilized HK-I (Fig. 7C), amino-acid peptide in cells overexpressing Bcl2-GFP would interfere Bcl2(Δ23) (Fig. 7D) or IgG was monitored. The N-terminal peptide with Bcl2- or HK-mediated protection against apoptosis, as induced bound to immobilized HK-I or Bcl2(Δ23) in a concentration- and by STS. Upon exposure of control cells to STS for 5 hours, about time-dependent manner, rapidly associating with and dissociating 50% of the cells underwent apoptosis (Fig. 8B). By contrast, cells from the immobilized proteins. The binding of the VDAC1 N- transformed to overexpress Bcl2-GFP (Fig. 8D) had complete terminal peptide to HK-I and Bcl2(Δ23) was specific, since no signal protection against STS-induced apoptosis (Fig. 8B). However, in cells was obtained with the IgG-immobilized chip. Moreover, a different overexpressing both Bcl2-GFP and the VDAC1 N-terminal peptide, VDAC1-based peptide (designated BP), in a loop configuration (18 Bcl2-GFP did not confer protection against STS-induced cell death amino acids, E157 to T174), did not interact with either HK-I (Fig. (Fig. 8B). Since an inducible plasmid for VDAC1 N-terminal peptide 7A) or Bcl2(Δ23) (Fig. 7B). The results thus demonstrate the direct expression was used, inhibition of the anti-apoptotic effect of Bcl2- and specific interaction of VDAC1-N-terminal peptide with HK-I GFP by the peptide was observed only when its expression was and Bcl2. induced by tetracycline (Fig. 8B). As with Bcl2, overexpression of The interaction of the VDAC1-N-terminal peptide with HK-I was HK-I or HK-II (Fig. 8C,E) also conferred protection against cell death further demonstrated at the cellular level by expressing the HK- induced by STS. This protection was prevented upon expression of I–GFP fusion protein alone or together with the N-terminal peptide the VDAC1 N-terminal peptide (Fig. 8C). (1-26 amino acids) or other VDAC1-based peptides (i.e. peptides A, 63W-N76, and B, 157E-T174) and then visualizing HK-I–GFP VDAC oligomerization is enhanced by STS and by cellular distribution (Fig. 8A). Confocal fluorescence microscopy overexpression of native and Δ(26)mVDAC1 showed that in transformed control cells, HK-I–GFP fluorescence In a previous study, we employed chemical cross-linking with the was punctuated, as expected for a mitochondria distribution. membrane-permeable cross-linker, EGS, to show that VDAC However, HK-I–GFP fluorescence in cells expressing the N- assumes an oligomeric form in isolated mitochondria (Zalk et al., (Δ26)VDAC1-mediated apoptotic resistance 1911

oligomeric states were obtained upon cross-linking with EGS. Using monoclonal anti-VDAC1 antibodies that specifically interact with the N-terminal region of VDAC1, no VDAC cross-linked products were detected in cells expressing Δ(26)mVDAC1 (Fig. 9D). These results suggest that the N-terminal region of VDAC1 is not required for VDAC1 oligomerization but is essential for apoptosis induction involving a mechanism proposed below.

Discussion We have shown that cells expressing a N-terminal deletion mutant Fig. 6. Cells expressing both endogenous VDAC and N-truncated mVDAC1 of VDAC1 are resistant to mitochondria-mediated apoptosis. While are resistant to apoptosis induction, a dominant negative effect. Expression of the channel still localizes to mitochondria and functions normally, N-terminal-truncated mVDAC1 in different cell lines expressing endogenous hVDAC1, prevented apoptosis induction. (A-C) HEK293, HeLa and MCF-7 releases of cytochrome c and apoptotic cell death were inhibited. cells were transfected to express mVDAC1 (black bars) or Δ(26)mVDAC1 Moreover, the anti-apoptotic proteins HK and Bcl2 were shown to (grey bars) and treated with STS (1.25 μM, 5 hours), cisplatin (50 μM, 30 confer protection against apoptosis through interaction with the hours) or TNFα (25 ng/ml, 7 hours). Apoptotic cell death was analyzed by VDAC1 N-terminal region. Finally, we demonstrated that apoptosis Acridine Orange and ethidium bromide staining, as in Fig. 3. Statistical analysis of apoptosis as a result of the different treatments was performed by induction is associated with VDAC oligomerizaion. In this ANOVA and t-tests; P<0.01. Data are means ± s.e.m. (n=4). Discussion section, we consider these findings in light of a model we propose for VDAC1 and its N-terminal function in apoptosis induction and regulation. 2005). Fig. 9 shows that upon apoptosis induction by STS, the dynamic equilibrium between monomeric and oligomeric forms of N-terminal VDAC1 is essential for cytochrome c release and VDAC was shifted towards the formation of dimers, trimers, apoptosis tetramers and multimers. VDAC oligomerization was, moreover, Lying in the OMM, VDAC appears to be a convergence point for dramatically increased (up to sixfold) in both HEK293 and B-16 a variety of cellular survival and death signals. Here, we have shown cells upon exposure to STS (Fig. 9A,B). that Δ(26)mVDAC1 functions as a channel and, like native VDAC1 overexpression not only encouraged apoptotic cell mVDAC1, could restore the growth and oxygen consumption of death (Figs 3 and 4), but also led to enhanced VDAC cells silenced for hVDAC1 expression by hVDAC1-shRNA, oligomerization. Upon mVDAC1 overexpression, VDAC oligomers suggesting that the N-terminal region of VDAC1 is not essential (dimers to hexamers and multimers) became apparent even in the for VDAC1 transport activity and, hence, for mitochondrial energy absence of chemical cross-linking (Fig. 9D). The appearance of production. trimers, tetramers (and possibly hexamers and multimers) upon However, the N-terminal α-helix of VDAC1 is required for overexpression of mVDAC1 was, nonetheless, apparent upon cytochrome c release and apoptosis when induced by various stimuli chemical cross-linking with EGS (Fig. 9C). Similar results were (Figs 3-6). Overexpression of human, murine, yeast or rice VDAC1, Δ Journal of Cell Science obtained upon expression of (26)mVDAC1, as visualized using VDAC1-GFP and mutated VDAC1 was shown to induce apoptotic polyclonal anti-VDAC antibodies (Fig. 9C). The Δ(26)mVDAC1 cell death, regardless of the cellular host (Abu-Hamad et al., 2006; dimers (as expected, with a lower molecular mass) and higher Bae et al., 2003; Godbole et al., 2003; Muller et al., 2000; Zaid et

Fig. 7. VDAC1 N-terminal peptide binding to HK-I and Bcl2(Δ23). Synthetic peptide corresponding to the VDAC1 N- terminal region interacts with HK and Bcl2. Interaction of purified HK-I and Bcl2(Δ23) 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(Δ23) (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(Δ23) (D) used in these experiments. 1912 Journal of Cell Science 122 (11)

(Figs 3 and 4). Moreover, induction of apoptosis by STS, curumin, cisplatin and As2O3, each relying on different pathways (Duvoix et al., 2005; Schmitz et al., 2000; Zaid et al., 2005; Zhang et al., 2005), all ultimately led to cytochrome c release (Fig. 5) and cell death (Figs 3, 4 and 6) in cells expressing native but not Δ(26)mVDAC1. Thus, by expressing N-terminal-truncated VDAC1, we have constructed an apoptosis-resistant cell line, namely, a line for which a highly active apoptosis stimulus did not induce cytochrome c release or cell death. Recently, it has been shown that overexpression of hVDAC1 in COS cells increased the number of cells with depolarized mitochondria, a phenomenon associated with apoptosis, and that this effect could not be obtained upon overexpression of (Δ19)hVDAC1 (De Pinto et al., 2007). These findings are consistent with our results showing that the VDAC1 N-terminal region is necessary for mitochondria-mediated apoptosis.

The VDAC1 N-terminal region interacts with HK and Bcl2 to prevent their anti-apoptotic effects HK-I and HK-II are highly expressed in cancer cells, with over 70% of the protein being bound to mitochondria (Arora et al., 1990; Pedersen et al., 2002), or more precisely, to VDAC (Abu-Hamad et al., 2008; Arzoine et al., 2009; Azoulay-Zohar et al., 2004; Pastorino et al., 2002). Similarly, Bcl2 is overexpressed in more than half of all human cancers, with its broad expression in tumors being linked to a conferring of resistance to chemotherapy-induced apoptosis (Ding et al., 2000). Both HK (Abu-Hamad et al., 2008; Arzoine et al., 2009; Azoulay-Zohar et al., 2004; Pastorino et al., 2002) and members of the Bcl2 family (Pastorino et al., 2002; Shi et al., 2003; Shimizu et al., 1999; Tsujimoto and Shimizu, 2002) are though to act via interaction with VDAC. Recently, those VDAC1 amino acid residues important for HK- I binding and conferring the anti-apoptotic effects of the protein were identified (Abu-Hamad et al., 2008). Clearly, the results

Journal of Cell Science presented here indicate that the N-terminal region of VDAC1 is essential for the HK-mediated reduction in channel conductance (Fig. 2B) and for HK-I and HK-II protection against apoptosis (Fig. Fig. 8. Expression of the VDAC1-N-terminal peptide in T-REx293 cells 8C). Both HK-I and Bcl2 interact with a synthetic peptide prevents HK-I-, HK-II- and Bcl2-mediated protection against STS-induced cell death. Expression of VDAC1 N-terminal peptide inhibited the anti- corresponding to the VDAC1 N-terminus, as revealed using SPR apoptotic effect of Bcl2, HK-I and HK-II. (A) T-REx293 cells or shRNA- technology (Fig. 7A,B). Moreover, expression of this N-terminal hVDAC1–T-REx293 cells stably expressing native or Δ(26)mVDAC1 under peptide in cells overexpressing HK-I, HK-II or Bcl2 prevented their the control of 1 μg/ml tetracycline were transfected with HK-I–GFP or co- protective effects against apoptosis, as induced by STS (Fig. 8). transfected with HK-I–GFP and pcDNA4TO-AP, pcDNA4TO-BP or Our findings suggest that the expressed peptide competes with pcDNA4TO-NP. After 40 hours cells were visualized using confocal microscopy. (B) T-REx293 cells and cells expressing Bcl2-GFP and/or the N- VDAC1 for binding to Bcl2, HK-I and HK-II and thus interferes terminal 26-amino-acid peptide (NP) under the control of tetracycline were with their binding to VDAC1, an interaction that prevents apoptosis. exposed for 5 hours to 1.25 μM STS, and apoptotic cell death was followed. It has been shown that the N-terminal region of VDAC1 modulates (C) An experiment similar to that in B was carried out with T-REx293 cells the accessibility of VDAC1 to Bax and Bcl-xL (Shi et al., 2003). 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 Our observations, therefore, suggest that the anti-apoptotic activity Orange and ethidium bromide staining, as in Fig. 2. Data are means ± s.e.m. of HK and Bcl2 is mediated through interactions with the VDAC1 (n=2-4). (D,E) Western blot analysis of Bcl2-GFP (D) or HK-I or HK-II (E) N-terminal region, pointing to the N-terminal region of VDAC1 as levels in control cells and cells transfected to overexpress either of these participating in the regulation of apoptosis by anti-apoptotic μ proteins and/or NP. Aliquots (50 g) were analyzed for HK-I and HK-II levels, proteins. This novel mechanism is presented in Fig. 10. 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. The mobile VDAC1 N-terminal α-helix participates in the formation of a cytochrome c release pore: a proposed model Numerous studies have focused on the importance of the N-terminal al., 2005), suggesting that VDAC1 is a conserved mitochondrial α-helical segment in channel function. These studies have yielded element of the death machinery. In accordance with previous results, a wide range of predictions as to the functional disposition of this overexpression of mVDAC dramatically triggers cell death. By domain, ranging from it forming a segment of the channel wall to contrast, overexpression of Δ(26)mVDAC1, although distributed acting as the voltage sensor (Colombini et al., 1996; Koppel et al., to the mitochondria (Fig. 1B), triggered no such apoptotic cell death 1998), to regulating the conductance of ions and metabolites (Δ26)VDAC1-mediated apoptotic resistance 1913

Fig. 9. Apoptosis induction by STS or VDAC1 overexpression induces VDAC oligomerization of native and Δ(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 Δ(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 Δ(26)mVDAC1 dimers, respectively.

Journal of Cell Science passing through the VDAC1 pore, based on its position in the pore apoptosis is demonstrated here, with the demonstration that the (Bayrhuber et al., 2008; Ujwal et al., 2008). In addition, supramolecular assembly of VDAC in cultured cells is highly conformational instability of the N-terminal part of hVDAC1 was enhanced upon apoptosis induction (Fig. 9). Apoptosis induction suggested, with the domain being predicted to switch between by STS or by overexpression of mVDAC1 resulted in several-fold different conformations, during which hydrogen bonds within the increase in VDAC oligomerization. Similarly, the apoptosis- N-terminus and the strands of the barrel are transiently formed and inducing effect of As2O3 was attributed to an induction of VDAC broken (Bayrhuber et al., 2008; Ujwal et al., 2008). Therefore, the homodimerization that was prevented by overexpression of the anti- N-terminal domain may adopt different conformations, depending apoptotic protein, Bcl-XL (Yu et al., 2007; Zheng et al., 2004). on various factors, such as protein-protein interactions, ion However, since (Δ26)mVDAC1 is able to undergo oligomerization interactions and lipid environment. Re-arrangement and (Fig. 9) but not to induce apoptosis (Figs 3, 4 and 6), it appears coordination of the N-terminal helix is suggested here to be that the N-terminal region is not required for VDAC oligomerization involved in VDAC1 function in apoptosis. but is important for cytochrome c release and subsequent apoptosis. The precise mechanisms regulating cytochrome c release, a key Together, these observations can be explained by the following initial step in the apoptotic process, remain unknown. Similarly, model (Fig. 10). Once formed, an oligomeric VDAC1 the molecular architecture of the cytochrome-c-conducting channel intermolecular pore can serve as a conduit for a protein. Oligomers has yet to be determined. We hypothesize that a protein-conducting of VDAC1 β-barrels would, however, form a hydrophobic channel channel is formed within a VDAC1 homo-oligomer or a hetero- that would not allow charged proteins, such as cytochrome c to oligomer containing VDAC1 and pro-apoptotic proteins (Shoshan- pass. However, if the amphipathic N-terminal region (containing Barmatz et al., 2008; Shoshan-Barmatz et al., 2006; Zalk et al., three positive and two negative residues, but with an overall charge 2005; Zheng et al., 2004). Indeed, further support for the supra- of zero) of each VDAC1 molecule is moved to line the external molecular organization of VDAC, comprising monomers to surface of the β-barrel, a hydrophilic pore would form (Fig. 10). tetramers, hexamers and higher oligomers, has come from recent In such a scenario, the now hydrophilic pore, lined by several α- studies applying atomic force microscopy (Goncalves et al., 2007; helix N-terminal regions, would permit protein transport across the Hoogenboom et al., 2007) and NMR (Bayrhuber et al., 2008; Malia OMM (Fig. 10B). In this regard, it should be noted that a recent and Wagner, 2007). It was also demonstrated that VDAC study suggested that the N-terminus of VDAC in aqueous medium oligomerization is encouraged in the presence of cytochrome c (Zalk is uncoiled, requiring a hydrophobic and negatively charged et al., 2005). The function of this VDAC oligomerization in environment to gain its α-helical structure (De Pinto et al., 2007). 1914 Journal of Cell Science 122 (11)

mechanisms of the cancer cells, thereby promoting apoptosis and increasing sensitivity to chemotherapy.

Materials and Methods Materials Staurosporine (STS), poly-D-lysine (PDL) and propidium iodide were purchased from Sigma (St Louis, MO). Mito-Tracker red dye CMXPos was from Molecular Probes. Annexin V-FITC kit was purchased from Beender MedSystem. Monoclonal anti-VDAC antibodies raised to the N-terminal region of VDAC1 came from Calbiochem-Novobiochem (Nottingham, UK), and rabbit polyclonal anti-VDAC antibodies, prepared against residues 150-250 of human (h)VDAC1, were from Abcam. Monoclonal anti-cytochrome c antibodies for immunoblots (1:2000; cat. no. 556433) and for immunostaining (1:800; cat. no. 556432) were obtained from BD Biosciences Pharmingen. Monoclonal anti-GFP antibodies were obtained from Santa Cruz Biotechnology. Metefectene was purchased from Biotex (Munich, Germany). RPMI 1640 and DMEM growth media and the supplements, fetal calf serum, L-glutamine and penicillin-streptomycin were purchased from Biological Industries (Beit Haemek, Israel). Blasticidin and zeocin were purchased from InvivoGen (San Diego, CA). Puromycin was purchased from ICN Biomedicals Fig. 10. Model for VDAC1 N-terminal region-mediated cytochrome c release. (Eschwege, Germany). (A) Side-view across a membranal VDAC1 with the amphipathic α-helix N- terminal region in various locations: cytoplasmically exposed (De Pinto et al., Plasmids and site-directed mutagenesis 2003), membrane-spanning (Colombini, 2004), lying on the membrane surface Vectors expressing N-terminally truncated murine (m)VDAC1 or the N-terminal 26- (Reymann et al., 1995) and positioned in the pore (Bayrhuber et al., 2008; amino-acid peptide were generated by PCR and cloned into the BamHI-NotI or Hiller et al., 2008; Ujwal et al., 2008). (B) Upon an apoptotic signal, VDAC BamHI-EcoRI sites of the tetracycline-inducible pcDNA4/TO vector (Invitrogen), oligomerization is enhanced and the amphipathic α-helix N-terminal region of using mVDAC1 cDNA as template and the following primers: 5Ј-CGGGATCCAT- each VDAC molecule flips inside the hydrophobic pore formed by the β- GATAAAACTTGATTTGAAAACG-3Ј (F) and 5Ј-GCGGCCGCTTATGCTTGAA - Ј Δ Ј barrels, making the pore hydrophilic and capable of conducting cytochrome c ATTCCAGTCC-3 (R) for ( 26)mVDAC1 and 5 -GCC AGATCTATGGCTGTG - CCACCCACGTA-3Ј (F) and 5Ј-CGAATTCTCATAAGCCAAATCCATAGCCCTT- release. (C) Interaction of anti-apoptotic proteins (HK, Bcl2) with the N- Ј terminal region of VDAC1 prevents its translocation and thus the formation of 3 (R) for the 26 amino acid peptide. The pcDNA3–HK-II and pcDNA3–HK-I plasmids were kindly provided by J. E. the hydrophilic pore, so inhibiting cytochrome c release. Wilson (Michigan State University). HK-I–GFP was generated as described previously(Abu-Hamad et al., 2008). For construction of C-terminally truncated Bcl2, Bcl2-GFP cDNA was sub-cloned from plasmid pcDNA3 using the primers: 5Ј-CTCCATGGCGATGGCGCACGC - With N-terminally truncated VDAC1, such a hydrophilic protein- TGGGAGAACG-3Ј (F) containing a NcoI restriction site and 5Ј-CGGAATTC- conducting pore could not be formed, and hence, cytochrome c TTACAGAGACAGCCAGGAGAAATC-3Ј (R), containing a stop codon and an EcoRI restriction site. The generated product, encoding Bcl2(Δ23), was then cloned would not be released (Fig. 5). The finding that the VDAC1 N- into a pHISparallel vector for expression in E. coli BL21 cells. terminal α-helix specifically interacts with cytochrome c (Mannella et al., 1987) supports the proposed model. Cell culture MCF7 cells are a human breast carcinoma cell line, HEK293 cells are a transformed The ability of a synthetic peptide, corresponding to the N-terminal primary human embryonal kidney cell line, T-REx293 cells are tetracycline repressor-

Journal of Cell Science region of VDAC1, to bind both HK-I and Bcl2 (Fig. 7), to detach expressing HEK293 cells and HeLa cells are a cervical cancer-derived cell line. These mitochondrial-bound HK-I–GFP (Fig. 8A) and to prevent Bcl2-, and hVDAC1-shRNA T-REx293 cells were grown in Dulbecco’s modified Eagle’s medium (Biological Industries), supplemented with 10% fetal calf serum, 2 mM L- HK-I- and HK-II-mediated protection against apoptosis (Fig. 8B,C), glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin and nonessential amino suggests that the function of the α-helix N-terminal portion of acids (all from Biological Industries) and maintained in a humidified atmosphere, at VDAC1 in apoptosis can be modulated by anti-apoptotic proteins. 37°C with 5% CO2. hVDAC1-shRNA T-REx293 cells stably expressing the pSUPERretro vector encoding shRNA-targeting hVDAC1 were transfected with According to our proposal, the anti-apoptotic activities of HK-I, plasmids pcDNA4/TO-mVDAC1 or pcDNA4/TO-Δ(26)mVDAC1 and grown as HK-II and Bcl2 result from their interaction with the mobile N- described previously (Abu-Hamad et al., 2006). T-REx293 cells were transiently co- terminal region of VDAC1 and preventing its reorientation into the transfected with plasmids pEGFP-Bcl2, pcDNA3.1–HK-I or pcDNA3.1–HK-II and pcDNA/4TO encoding the N-terminal 26 amino acids and grown for 48 hours with cytochrome c-releasing pore (Fig. 10C). tetracycline (1 μg/ml), prior to a 5 hour exposure to STS (1.25 μM). Overexpression Taken together, the requirement for the VDAC1 N-terminal of mVDAC1- or Δ(26)mVDAC1-pcDNA4/TO was induced by tetracycline (2.5 region for cytochrome c release and apoptosis induction, as well μg/ml) for 96-100 hours. Apoptosis was analyzed by Acridine Orange and ethidium bromide staining (Zaid et al., 2005) or by FACS. as its interaction with anti-apoptotic proteins, point to the VDAC1 N-terminal region as being a key element in regulating apoptosis Oxygen consumption and the target for anti-apoptotic proteins (Fig. 10). Oxygen utilization was measured polarographically with a thermostatically controlled (37°C) Clark oxygen electrode (Strathkelvin 782 Oxygen System). T-REx293, Finally, mitochondria-mediated apoptosis plays a crucial role in hVDAC1-shRNA T-REx293 cells expressing stable native or Δ(26)mVDAC1 the pathophysiology of many diseases, including heart attack and (2ϫ106) were washed, suspended in a serum-free DMEM medium [1000 mg/l stroke, neurodegenerative disorders, such as Parkinson’s disease and glucose pyridoxine, HCl and NaHCO3, without phenol red (Sigma D-5921)], supplemented with L-glutamine (4 mM) and L-pyruvate (1 mM), and then added Alzheimer’s disease, as well as mitochondrial encephalomyopathies to a 1 ml water-jacketed chamber. Cells respiration was determined in absence and and cancer (Mattson, 2000; Thompson, 1995). Although some presence of rotenone (5 μM, added directly into the respiration chamber) for up to diseases, such as cancer, are caused by suppression of apoptosis, 10 minutes. other diseases, such as Alzheimer’s disease, are characterized by Detachment of mitochondria-bound HK-I–GFP by VDAC1-based increased apoptosis. The VDAC1 N-terminal region offers a peptides tempting target for the development of therapeutic agents designed Cells (5ϫ104) were grown on PDL-coated coverslips in a 24-well dish and transfected to inhibit apoptosis. In cancer therapeutics, targeting VDAC1 N- with plasmid pEGFP–HK-I alone or with DNA encoding the N-terminal peptide (NP), peptide A (amino acids 63W-78N, AP, previously named LP1) and peptide B (157E- terminal peptide to tumor cells overexpressing anti-apoptotic 174T, BP, previously named LP3) that were generated and cloned into the tetracycline- proteins, such as Bcl2 and HK-I, would minimize the self-defense inducible pcDNA4/TO vector (Invitrogen), as described previously (Arzione et al., (Δ26)VDAC1-mediated apoptotic resistance 1915

2009). Peptides A and B were flanked by a tryptophan zipper motif, i.e. the SWTWE References amino acid sequence, at the N-terminus of the peptide, and the KWTWK sequence Abu-Hamad, S., Sivan, S. and Shoshan-Barmatz, V. (2006). The expression level of the at the C-terminus, to form a loop (Arzione et al., 2009). voltage-dependent anion channel controls life and death of the cell. Proc. Natl. Acad. Cells were fixed for 15 minutes with 4% paraformaldehyde and rinsed with PBS Sci. USA 103, 5787-5792. for 30 minutes prior to imaging by confocal microscopy (using an Olympus 1X81 Abu-Hamad, S., Zaid, H., Israelson, A., Nahon, E. and Shoshan-Barmatz, V. (2008). Hexokinase-I protection against apoptotic cell death is mediated via interaction with the microscope). voltage-dependent anion channel-1: mapping the site of binding. J. Biol. Chem. 283, 13482-13490. Cytochrome c release Arora, K. K., Fanciulli, M. and Pedersen, P. L. (1990). Glucose phosphorylation in Immunoblot analysis tumor cells. Cloning, sequencing, and overexpression in active form of a full-length For immunoblot analysis, cells expressing native or Δ(26)mVDAC1 were grown for cDNA encoding a mitochondrial bindable form of hexokinase. J. Biol. Chem. 265, 6481- 72 hours, exposed to STS, cisplatine or As2O3 and analyzed for cytochrome c release 6488. (Abu-Hamad et al., 2008). Aliquots of the cytosolic fraction were separated on Tris- Arzoine, L., Zilberberg, N., Ben-Romano, R. and Shoshan-Barmatz, V. (2009). Voltage- Tricine gels (13% polyacrylamide) and immunoprobed using anti-cytochrome c dependent anion channel-1-based peptides interact with hexokinase to prevent its anti- apoptotic activity. J. Biol. Chem. 284, 3946-3955. antibodies. To rule out mitochondrial contamination, the cytosolic fraction was Azoulay-Zohar, H., Israelson, A., Abu-Hamad, S. and Shoshan-Barmatz, V. (2004). immunoprobed for VDAC using anti-VDAC antibodies, and then with HRP- In self-defence: hexokinase promotes voltage-dependent anion channel closure and conjugated anti-mouse IgG as a secondary antibody (1:10,000). An enhanced prevents mitochondria-mediated apoptotic cell death. Biochem. J. 377, 347-355. chemiluminiscent substrate (Pierece Chemical, Rockford, IL) was used for detection Bae, J. H., Park, J. W. and Kwon, T. K. (2003). Ruthenium red, inhibitor of mitochondrial of HRP. Ca2+ uniporter, inhibits curcumin-induced apoptosis via the prevention of intracellular Ca2+ depletion and cytochrome c release. Biochem. Biophys. Res. Commun. 303, 1073- Immunostaining 1079. For immunostaining, cells (5ϫ104) were grown on poly-D-lysine (PDL)-coated Baines, C. P., Kaiser, R. A., Sheiko, T., Craigen, W. J. and Molkentin, J. D. (2007). Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death. coverslip in a six-well plate. After 48 hours, cells were treated with As2O3 (60 μM, 24 hours). Then, the cells were treated with MitoTracker red dye (25 nM) for 15 Nat. Cell Biol. 9, 550-555. Bayrhuber, M., Meins, T., Habeck, M., Becker, S., Giller, K., Villinger, S., Vonrhein, minutes in an incubator (37°C, 5% CO2), washed three times with PBS, fixed for 15 C., Griesinger, C., Zweckstetter, M. and Zeth, K. (2008). Structure of the minutes with 4.0% paraformaldehyde and rinsed three times with PBS. Cells were human voltage-dependent anion channel. Proc. Natl. Acad. Sci. USA 105, 15370- treated with methanol for 15 minutes (–20°C), then incubated for 1 hour with blocking 15375. solution (5% normal goat serum (NGS) and 0.2% Triton X-100), followed by Colombini, M. (2004). VDAC: the channel at the interface between mitochondria and the incubation with anti-cytochrome c antibodies (cat. no. 556432, 1:800, diluted in cytosol. Mol. Cell Biochem. 256-257, 107-115. blocking solution) for 1 hour. After washing with PBS, cells were incubated for 1 Colombini, M., Yeung, C. L., Tung, J. and Konig, T. (1987). The mitochondrial outer hour with Alexa-Fluor-488-conjugated antibodies (1:500, diluted in blocking solution) , VDAC, is regulated by a synthetic polyanion. Biochim. Biophys. and washed with PBS. Coverslips were mounted on glass slides with mounting Acta 905, 279-286. Colombini, M., Blachly-Dyson, E. and Forte, M. (1996). VDAC, a channel in the outer medium and sealed with nail polish. Cells were visualized by confocal microscopy mitochondrial membrane. Ion Channels 4, 169-202. (Olympus 1X81). Crompton, M. (1999). The mitochondrial permeability transition pore and its role in cell death. Biochem. J. 341, 233-249. Flow cytometry De Pinto, V., Messina, A., Accardi, R., Aiello, R., Guarino, F., Tomasello, M. F., Apoptotic cells were estimated using a commercial annexin V-FITC and PI detection Tommasino, M., Tasco, G., Casadio, R., Benz, R. et al. (2003). New functions of an kit or following PI uptake and FACS analysis (FACScan, Beckton-Dickinson, San old protein: the eukaryotic porin or voltage dependent anion selective channel (VDAC). Jose, CA) and ModFIT-lt2.0 software. Ital. J. Biochem. 52, 17-24. De Pinto, V., Tomasello, F., Messina, A., Guarino, F., Benz, R., La Mendola, D., Magri, Protein purification A., Milardi, D. and Pappalardo, G. (2007). Determination of the Conformation of the Δ human VDAC1 N-terminal peptide, a protein moiety essential for the functional properties mVDAC1 and (26)mVDAC1 were expressed in the Saccharomyces cerevisiae of the pore. Chembiochem 8, 744-756. por1-mutant strain M22-2 (Zaid et al., 2005) and purified from mitochondria De Pinto, V., Reina, S., Guarino, F. and Messina, A. (2008). Structure of the voltage

Journal of Cell Science (Shoshan-Barmatz and Gincel, 2003). HK-I was purified from rat brain mitochondria dependent anion channel: state of the art. J. Bioenerg. Biomembr. 40, 139-147. (Azoulay-Zohar et al., 2004). Bcl2(Δ23) was expressed in E. coli BL21, induced Ding, Z., Yang, X., Pater, A. and Tang, S. C. (2000). Resistance to apoptosis is correlated with isopropyl β-D-1-thiogalactopyranoside. Cells were grown for 30-60 minutes with the reduced caspase-3 activation and enhanced expression of antiapoptotic proteins at 20°C, sonicated, and the soluble fraction (5-10% of the total) was purified by Ni- in human cervical multidrug-resistant cells. Biochem. Biophys. Res. Commun. 270, 415- NTA chromatography. 420. Doran, E. and Halestrap, A. P. (2000). Cytochrome c release from isolated rat liver mitochondria can occur independently of outer-membrane rupture: possible role of contact Cross-linking experiments sites. Biochem. J. 348, 343-350. For cross-linking, T-REx293, HEK293 or B-16 cells (2-3 mg/ml) were incubated Duvoix, A., Blasius, R., Delhalle, S., Schnekenburger, M., Morceau, F., Henry, E., with different concentrations of EGS [ethylene glycol bis(succinimidylsuccinate)] in Dicato, M. and Diederich, M. (2005). Chemopreventive and therapeutic effects of PBS, pH 8.3 (15 minutes, 30°C). Samples (50 μg) were subjected to SDS-PAGE and curcumin. Cancer Lett. 223, 181-190. immunoblotting using anti-VDAC antibodies. Gincel, D., Zaid, H. and Shoshan-Barmatz, V. (2001). Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in Single-channel recording and analysis mitochondrial function. Biochem. J. 358, 147-155. Godbole, A., Varghese, J., Sarin, A. and Mathew, M. K. (2003). VDAC is a conserved Reconstitution of purified native mVDAC1 and Δ(26)mVDAC1 into a planar lipid element of death pathways in plant and animal systems. Biochim. Biophys. Acta 1642, bilayer (PLB), channel recording, and analysis were carried out as described 87-96. previously (Gincel et al., 2001). Goncalves, R. P., Buzhynskyy, N., Prima, V., Sturgis, J. N. and Scheuring, S. (2007). Supramolecular assembly of VDAC in native mitochondrial outer membranes. J. Mol. Real-time surface plasmon resonance Biol. 369, 413-418. Purified rat brain HK-I, recombinant Bcl2 and rabbit IgG were immobilized on a Green, D. R. and Reed, J. C. (1998). Mitochondria and apoptosis. Science 281, 1309- CM5 sensor surface according to the manufacturer’s instructions. N-terminal VDAC1 1312. peptide was diluted in running buffer [150 mM NaCl, 0.005% Tween 20, 4% (v/v) Halestrap, A. P., Doran, E., Gillespie, J. P. and O’Toole, A. (2000). Mitochondria and cell death. Biochem. Soc. Trans. 28, 170-177. DMSO, PBS, pH 7.4] and injected onto the sensor chip at varying concentrations μ Hiller, S., Garces, R. G., Malia, T. J., Orekhov, V. Y., Colombini, M. and Wagner, G. (flow rate of 40 l/minute, 25°C). Experiments were carried out using the SPR (2008). Solution structure of the integral human membrane protein VDAC-1 in detergent ProteOn-XPR36 system (Bio-Rad). Peptides were synthesized by the oligonucleotide micelles. Science 321, 1206-1210. and peptide synthesis unit, Weizmann Institute, Israel. Hoogenboom, B. W., Suda, K., Engel, A. and Fotiadis, D. (2007). The supramolecular assemblies of voltage-dependent anion channels in the native membrane. J. Mol. Biol. This research was supported, in part, by grants from the Israel Science 370, 246-255. Junankar, P. R., Dulhunty, A. F., Curtis, S. M., Pace, S. M. and Thinnes, F. P. (1995). Foundation, the Israel Cancer Association and by the Johnson and Porin-type 1 proteins in sarcoplasmic reticulum and plasmalemma of striated muscle Johnson Corporate Office of Science and Technology. The authors thank fibres. J. Muscle Res. Cell Motil. 16, 595-610. Bio-Rad, Israel, for providing their ProteOn instrument and Zafrir Koppel, D. A., Kinnally, K. W., Masters, P., Forte, M., Blachly-Dyson, E. and Mannella, C. A. (1998). Bacterial expression and characterization of the mitochondrial Bravman for helping with the SPR experiments and Dorit Ben-Shahar outer membrane channel. Effects of n-terminal modifications. J. Biol. Chem. 273, 13794- for helping with oxygen consumption experiments. 13800. 1916 Journal of Cell Science 122 (11)

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