Mediation of the antiapoptotic activity of Bcl-xL protein upon interaction with VDAC1 protein

Abstract

The mitochondrial protein, the voltage-dependent anion channel (VDAC), is implicated in the control of apoptosis, including via its interaction with the pro- and antiapoptotic proteins. We previously demonstrated the direct interaction of Bcl2 with VDAC, leading to reduced channel conductance. VDAC1-based peptides interacted with Bcl2 to prevent its antiapoptotic activity. Here, using a variety of approaches, we show the interaction of the antiapoptotic protein, Bcl-xL, with VDAC1 and reveal that this interaction mediates Bcl-xL protection against apoptosis. C-terminally truncated Bcl-xL(Δ21) interacts with purified VDAC1, as revealed by microscale thermophoresis and as reflected in the reduced channel conductivity of bilayer-reconstituted VDAC1. Overexpression of Bcl-xL prevented staurosporine-induced apoptosis in cells expressing native VDAC1 but not certain VDAC1 mutants. Having identified mutations in VDAC1 that interfere with the Bcl-xL interaction, certain peptides representing VDAC1 sequences, including the N-terminal domain, were designed and generated as recombinant and synthetic peptides. The VDAC1 N-terminal region and two internal sequences were found to bind specifically, and in a concentration- and time-dependent manner, to immobilized Bcl-xL(Δ21), as revealed by surface plasmon resonance. Moreover, expression of the recombinant peptides in cells overexpressing Bcl-xL prevented protection offered by the protein against staurosporine-induced apoptosis. These results point to Bcl-xL acting as antiapoptotic protein, promoting tumor cell survival via binding to VDAC1. These findings suggest that interfering with Bcl-xL binding to the mitochondria by VDAC1-based peptides may serve to induce apoptosis in cancer cells and to potentiate the efficacy of conventional chemotherapeutic agents.

FIGURE 1.

Bcl-xL(ΔC21) 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 before and 15 min after the addition of purified Bcl-xL(ΔC21) (10 nm). Following current recording, the cis compartment was perfused with buffer to decrease the concentration of Bcl-xL(ΔC21) by severalfold. The dashed lines indicate zero current levels. B, multichannel recordings as a function of the voltage (4 s at each voltage) are shown, as well as the average steady-state conductance of mVDAC1 before (●) and 30 min after (○) the addition of Bcl-xL(ΔC21) (10 nm) and after exchanging the solution with Bcl-xL(ΔC21)-free medium (△). Relative conductance was determined as the ratio of the conductance at a given voltage (G) to the maximal conductance (Go). C, Coomassie Brilliant Blue purified VDAC and Bcl-xL(Δ21) proteins are shown.

FIGURE 2.

Binding of VDAC to fluorescently labeled Bcl-xL(Δ21) and Bcl2(Δ23). A and B, Bcl-xL(Δ21) (A) and Bcl2(Δ23) (B) were fluorescently labeled using the NanoTemper protein-labeling kit RED. The assay was performed in PBS buffer with 0.005% Tween 20. The concentration of VDAC was varied from 2.4 nm to 20 μm, whereas the concentration of the labeled Bcl-xL was kept constant at 20 nm. After 20 min of incubation, 3–5 μl of the samples were loaded into MST-grade glass capillaries, and thermophoresis was measured using the Monolith-NT115 apparatus. ΔFNorm %, the percentage of change in normalized fluorescence.

FIGURE 3.

Bcl-xL decrease of channel conductance and protection against apoptosis observed on native VDAC1 or E65Q- but not E72Q- or E202Q-VDAC1 mutants. A, wild type, E65Q-, E72Q-, or E202Q-VDAC1 were reconstituted into a PLB, and currents in response to a voltage step from 0 to −10 mV were recorded before and 15 min after the addition of purified Bcl-xL(ΔC21) (10 nm). B, T-REx-293 cells were transiently co-transfected with plasmids pEGFP-Bcl-xL, encoding Bcl-xL, or pcDNA4/TO, encoding native, E65Q-, E72Q-, or E202Q-VDAC1 (or plasmid pcDNA4/TO, as control), respectively, and grown for 48 h in the presence of tetracycline (1 μg/ml, to induce VDAC 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.

Expression of VDAC1-based peptides in T-REx-293 cells prevents Bcl-xL-mediated protection against cell death induced by STS. A and B, T-REx-293 cells were transfected to express Bcl-xL and/or the indicated VDAC1-based peptide designed according to the original proposed topology model (17) (N terminus (N-Ter); LP1; LP2; LP3; and LP4) (A) or to the newly proposed topology model (–15) (L4–5, L14–15, L16–17, and L18–19) (B). 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).

FIGURE 5.

Selected VDAC1-based peptides interact with Bcl-xL(ΔC21) in a specific and dose-dependent manner. Interaction of purified Bcl-xL(ΔC21) with VDAC1-based synthetic peptides was revealed using real-time SPR. Bcl-xL(ΔC21) immobilized onto a GLM sensor surface was exposed to VDAC1-based peptides: A, N terminus (N-Ter) (50, 100, and 200 μm); B, LP4 (50, 100, and 200 μm); C, LP1 (50, 100, and 200 μm); D, LP2 (50, 100, and 200 μm). The peptides were run in parallel over surface strips containing immobilized Bcl-xL(ΔC21), 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. The solid and dashed arrows indicate runs with peptide-containing and peptide-free samples, respectively.

FIGURE 6.

The N-terminal domain of VDAC1 is required for its interaction with Bcl-xL(ΔC21). VDAC1 and Δ(26)VDAC1 were expressed in porin-less yeast mitochondria, purified, and reconstituted into a PLB. A, Bcl-xL reduced conductance of VDAC1, yet had no effect on the channel activity of N-terminally truncated VDAC1. Currents through bilayer-reconstituted VDAC1 or Δ(26)VDAC1 in response to a voltage step from 0 to −10 mV were recorded before and 10 min after the addition of Bcl-xL. The dashed lines indicate the zero and the maximal current levels. B, currents through the VDAC1 channel in response to a voltage step from 0 mV to voltages between −60 and +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, using VDAC1 (●) and Δ(26)VDAC1 (○) and 30 min after the addition Bcl-xL(Δ21) to Δ(26)VDAC1 (△).