Voltage-dependent anion channel 1-based peptides interact with Bcl-2 to prevent antiapoptotic activity

Abstract

The antiapoptotic proteins of the Bcl-2 family are expressed at high levels in many types of cancer. However, the mechanism by which Bcl-2 family proteins regulate apoptosis is not fully understood. Here, we demonstrate the interaction of Bcl-2 with the outer mitochondrial membrane protein, voltage-dependent anion channel 1 (VDAC1). A direct interaction of Bcl-2 with bilayer-reconstituted purified VDAC was demonstrated, with Bcl-2 decreasing channel conductance. Expression of Bcl-2-GFP prevented apoptosis in cells expressing native but not certain VDAC1 mutants. VDAC1 sequences and amino acid residues important for interaction with Bcl-2 were defined through site-directed mutagenesis. Synthetic peptides corresponding to the VDAC1 N-terminal region and selected sequences bound specifically, in a concentration- and time-dependent manner, to immobilized Bcl-2, as revealed by the real-time surface plasmon resonance. Moreover, expression of the VDAC1-based peptides in cells over-expressing Bcl-2 prevented Bcl-2-mediated protection against staurosporine-induced apoptotic cell death. Similarly, a cell-permeable VDAC1-based synthetic peptide was also found to prevent Bcl-2-GFP-mediated protection against apoptosis. These results point to Bcl-2 as promoting tumor cell survival through binding to VDAC1, thereby inhibiting cytochrome c release and apoptotic cell death. Moreover, these findings suggest that interfering with the binding of Bcl-2 to mitochondria by VDAC1-based peptides may serve to potentiate the efficacy of conventional chemotherapeutic agents.

FIGURE 1.

Bcl-2(ΔC23) reduces bilayer-reconstituted VDAC conductance. A, VDAC was reconstituted into a PLB and current in response to a voltage step from 0 to −10 mV was recorded 2 and 25 min after the addition of purified Bcl-2(ΔC23) (1 nm). Thereafter, the voltage was switched for 2 to −60 mV, and current was recorded at −10 mV (total incubation time with Bcl-2(ΔC23) was 32 min). B, VDAC was reconstituted into a PLB, and currents through VDAC in response to a voltage step from 0 to −10 mV were recorded before and 15 min after the addition of purified Bcl-2(ΔC23) (1 nm) and after exposing the protein to high negative voltage for 2–3 min. Following current recording, the cis compartment was perfused to highly decrease the concentration of Bcl-2(ΔC23) by severalfold. The dashed lines indicate the zero current levels. C, shown are multichannel recordings as a function of the voltage (4 s at each voltage), and the average steady-state conductance of mVDAC1 before (●) and 30 min after (○) the addition of Bcl-2(ΔC23) (1 nm) and after exchanging the solution with Bcl-2(ΔC23)-free medium (▵) are presented. Relative conductance was determined as the ratio of the conductance at a given voltage (G) to the maximal conductance (Go). D, a Coomassie-stained purified VDAC and Bcl-2 proteins are shown.

FIGURE 2.

VDAC1 transmembrane topology models showing the sites of mutated amino acids, the sequences used for synthetic peptide synthesis, and the proposed domains interacting with Bcl-2. The previously proposed transmembrane topology of VDAC1 (modified from Ref. 20) (A) and a revised model based on the newly proposed structure (28–30) are shown (B). The sequences of peptides (labeled in light gray) expressed or synthesized as synthetic peptides and tested in this work are illustrated. The black circles around Glu⁶⁵, Glu⁷², and Glu²⁰² and E65Q indicate mutations in mVDAC1 used in this study and were found to eliminate Bcl-2-mediated antiapoptotic activity. The dark gray lines indicate VDAC1 sequences interacting with Bcl-2 in the peptide array experiment (Fig. 7C).

FIGURE 3.

Overexpression of Bcl-2 protects against apoptosis induced by STS in T-REx-293 cells overexpressing native mVDAC1 but not E65Q-, E72Q- or E202Q-mVDAC1 mutants. shRNA-T-Rex-293 cells were transiently cotransfected with plasmids pEGFP-Bcl-2 or pEGFP and pcDNA4/TO, respectively, encoding native, E65Q-, E72Q-, or E202Q-mVDAC1 (or pcDNA4/TO as control) and grown for 48 h in the presence of tetracycline (1 μg/ml, to induce expression) prior to exposure to STS (1.25 μm) for 4 h. Apoptosis was quantitatively analyzed by acridine orange/ethidium bromide staining. Data represent mean values ± S.E. (n = 3).

FIGURE 4.

Bcl-2(ΔC23) reduces channel conductance of bilayer-reconstituted native but not E72Q- mVDAC1. Purified recombinant, native (A) or E72Q-mVDAC1 (B) was reconstituted into a PLB in symmetrical solutions of 0.5 m NaCl, and currents through the VDAC1 channel in response to a voltage step from 0 to −10 were recorded before and 5 min after the addition Bcl-2(ΔC23) (1 nm), after applying the high voltage step (−60 mV, 2 min). The dashed lines indicate the zero and maximal current levels. C, multichannel recordings of the average steady-state conductance of mVDAC1 (●) or E72Q-mVDAC1 (○) 5 min after the addition of Bcl-2(ΔC23) (1 nm).

FIGURE 5.

Expression of VDAC1-based peptides in T-Rex-293 cells prevented Bcl-2-mediated protection against cell death induced by STS. T-REx-293 cells were transfected to express Bcl-2 and/or the indicated VDAC1-based peptide designed according to the original (AP, Trp⁶³–Asn⁷⁸; BP, Pro¹⁰⁵–Lys¹¹⁹; CP, Glu¹¹⁷–Thr¹³⁴; BP, Pro¹⁰⁵–Lys¹¹⁹) (A) or newly proposed (L4-5, Thr⁷¹–Leu⁸⁰; L14-15, Leu²⁰⁷–Arg²¹⁷; L16-17, Lys²³⁵–Gln²⁴⁸) and (L18-19, Leu²⁶²-His²⁷²) (B) VDAC1 transmembrane topology models. VDAC1-based peptide expression was induced by tetracycline (1.5 μg/ml). Apoptosis in the different cell types was induced by incubation with STS (1.25 μm, 4 h) and quantitatively analyzed by acridine orange/ethidium bromide staining. Data represent mean ± S.E. (n = 3). N-ter, N-terminal.

FIGURE 6.

Selected VDAC1-based peptides interact with Bcl-2(ΔC23) in a specific and dose-dependent manner. Interaction of purified Bcl-2(ΔC23) with VDAC1-based peptides was revealed using real-time SPR. Bcl-2 immobilized onto a GLM sensor surface was exposed to VDAC1-based peptides: A, AP (Trp⁶³–Asn⁷⁸; 50, 100, and 200 μm); B, BP (Pro¹⁰⁵–Lys¹¹⁹; 50, 100, and 200 μm); C, CP (Glu¹¹⁷–Thr¹³⁴; 50, 100, and 200 μm); D, DP (Lys¹⁹⁹–Asn²¹⁵; 50, 100, and 200 μm); and E, N-terminal domain (Met¹–Phe²⁵; 50, 100, and 200 μm). To control for nonspecific binding, the interaction of the N-terminal peptide (N-ter; F, 200 μm) with surface strips of IgG was analyzed. The peptides were run in parallel over surface strips of Bcl-2(ΔC23) and responses (resonance units, RU) as a function of peptide concentration were monitored using the ProteOn imaging system and related software tools. All experiments were carried out at 25 °C.

FIGURE 7.

The synthetic VDAC1-based peptide, DP-Antp, prevented Bcl-2-mediated protection against apoptosis and binding of Bcl-2(ΔC23) to a cellulose-bound VDAC1-based peptide array. T-REx-293 cells were transfected with pcDNA3.1 vector either empty or encoding Bcl-2. 48 h post-transfection, the cells were incubated with the synthetic peptide, DP-Antp (20 μm) for 90 min and/or STS (1.25 μm) for 3 h. Apoptotic cell death was analyzed using Annexin V-fluorescein isothiocyanate (V-FITC) and propidium iodide (PI) staining and FACS analysis, as described under “Materials and Methods.” A, representative FACS analysis data for cells treated with and without STS, and Bcl-2-expressing cells treated with STS or with both DP-Antp and STS. B, quantitative analysis of FACS experiments. Data represent mean values ± S.E. (n = 3). C, cellulose-bound peptide arrays consisting of overlapping peptides derived from VDAC1 sequence were incubated overnight with recombinant Bcl-2(ΔC23) (30 μg/ml) and blotted with anti-Bcl-2 antibodies, followed by incubation with HRP-conjugated anti-mouse IgG and detected using a chemiluminescence kit. Dark spots represent binding of Bcl-2(ΔC23) to the specific peptide corresponding to the N-terminal domain (N-Ter), and to peptide sequences overlapping with the L14-15 and L16-17 sequences (see Fig. 2). A representative blot of three similar experiments is shown.