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Cited by 168Cell death mechanisms and regulationMitochondrial Function and PathologyCardiac Ischemia and Reperfusion

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Voltage-dependent Anion Channel 1-based Peptides Interact with Bcl-2 to Prevent Antiapoptotic Activity

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Arbel, Nir, Shoshan-Barmatz, Varda·2010·Journal of Biological Chemistry
DOI 10.1074/jbc.m109.082990PMC PMC2825399
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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.

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Figures

FIGURE1 Bcl-2( 𝚫 C23)reducesbilayer-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( 𝚫 C23) (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( 𝚫 C23) 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( 𝚫 C23) (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 Bcl2( 𝚫 C23) 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 ( F ) and 30 min after ( E ) the addition of Bcl-2( 𝚫 C23) (1 nM) and after exchanging the solution with Bcl-2( 𝚫 C23)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.
FIGURE1 Bcl-2( 𝚫 C23)reducesbilayer-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( 𝚫 C23) (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( 𝚫 C23) 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( 𝚫 C23) (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 Bcl2( 𝚫 C23) 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 ( F ) and 30 min after ( E ) the addition of Bcl-2( 𝚫 C23) (1 nM) and after exchanging the solution with Bcl-2( 𝚫 C23)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.CC-BY · From the PDF
Image 2
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FIGURE 3 Overexpression of Bcl-2 protects against apoptosis induced by STS in T-REx-293 cells overexpressing native mVDAC1 but not E65Q-, E72Q-orE202Q-mVDAC1mutants. 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 𝛍 g/ml, to induce expression) prior to exposure to STS (1.25 𝛍 M) for 4 h. Apoptosis was quantitatively analyzed by acridine orange/ethidium bromide staining. Data represent mean values ± S.E. ( n = 3).
FIGURE 3 Overexpression of Bcl-2 protects against apoptosis induced by STS in T-REx-293 cells overexpressing native mVDAC1 but not E65Q-, E72Q-orE202Q-mVDAC1mutants. 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 𝛍 g/ml, to induce expression) prior to exposure to STS (1.25 𝛍 M) for 4 h. Apoptosis was quantitatively analyzed by acridine orange/ethidium bromide staining. Data represent mean values ± S.E. ( n = 3).CC-BY · From the PDF
Image 4
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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, Trp 63 -Asn 78 ; BP, Pro 105 -Lys 119 ; CP, Glu 117 Thr 134 ; BP, Pro 105 -Lys 119 ) ( A ) or newly proposed (L4-5, Thr 71 -Leu 80 ; L14-15, Leu 207 -Arg 217 ; L16-17, Lys 235 Gln 248 ) and (L18-19, Leu 262 -His 272 ) ( B ) VDAC1transmembranetopologymodels.VDAC1-basedpeptideexpression was induced by tetracycline (1.5 𝛍 g/ml). Apoptosis in the different cell types was induced by incubation with STS (1.25 𝛍 M, 4 h) and quantitatively analyzed by acridine orange/ethidium bromide staining. Data represent mean ± S.E. ( n = 3). N-ter , N-terminal.
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, Trp 63 -Asn 78 ; BP, Pro 105 -Lys 119 ; CP, Glu 117 Thr 134 ; BP, Pro 105 -Lys 119 ) ( A ) or newly proposed (L4-5, Thr 71 -Leu 80 ; L14-15, Leu 207 -Arg 217 ; L16-17, Lys 235 Gln 248 ) and (L18-19, Leu 262 -His 272 ) ( B ) VDAC1transmembranetopologymodels.VDAC1-basedpeptideexpression was induced by tetracycline (1.5 𝛍 g/ml). Apoptosis in the different cell types was induced by incubation with STS (1.25 𝛍 M, 4 h) and quantitatively analyzed by acridine orange/ethidium bromide staining. Data represent mean ± S.E. ( n = 3). N-ter , N-terminal.CC-BY · From the PDF
Image 6
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FIGURE 6 Selected VDAC1-based peptides interact with Bcl-2( 𝚫 C23 ) in a specific and dose-dependent manner. Interaction of purified Bcl-2( 𝚫 C23) 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 (Trp 63 -Asn 78 ; 50, 100, and 200 𝛍 M); B , BP (Pro 105 -Lys 119 ; 50, 100, and 200 𝛍 M); C , CP (Glu 117 -Thr 134 ; 50, 100, and 200 𝛍 M); D , DP (Lys 199 -Asn 215 ; 50, 100, and 200 𝛍 M); and E , N-terminal domain (Met 1 -Phe 25 ; 50, 100, and 200 𝛍 M). To control for nonspecific binding, the interaction of the N-terminal peptide ( N-ter ; F , 200 𝛍 M) with surface strips of IgG was analyzed. The peptides were run in parallel over surface strips of Bcl-2( 𝚫 C23) 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 6 Selected VDAC1-based peptides interact with Bcl-2( 𝚫 C23 ) in a specific and dose-dependent manner. Interaction of purified Bcl-2( 𝚫 C23) 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 (Trp 63 -Asn 78 ; 50, 100, and 200 𝛍 M); B , BP (Pro 105 -Lys 119 ; 50, 100, and 200 𝛍 M); C , CP (Glu 117 -Thr 134 ; 50, 100, and 200 𝛍 M); D , DP (Lys 199 -Asn 215 ; 50, 100, and 200 𝛍 M); and E , N-terminal domain (Met 1 -Phe 25 ; 50, 100, and 200 𝛍 M). To control for nonspecific binding, the interaction of the N-terminal peptide ( N-ter ; F , 200 𝛍 M) with surface strips of IgG was analyzed. The peptides were run in parallel over surface strips of Bcl-2( 𝚫 C23) 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.CC-BY · From the PDF
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Image 9
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Research works printed but not linked: 4

  1. Krajewski, 1993, Cancer Res., 53, 4701
  2. De Pinto, 2003, Ital. J. Biochem., 52, 17
  3. Dias, 2002, Mol. Cancer Ther., 1, 347
  4. Leone, 2003, Cancer Res., 63, 8118

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Metadata

Authors
Arbel, N., & Shoshan-Barmatz, V.
Affiliations
Ben-Gurion University of the Negev (IL)
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2010-02
Volume
285
Issue
9
Pages
6053-6062
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m109.082990
PMC
PMC2825399
OpenAlex
W1998225262
Cited by
168
References
68
Citations by year
2026: 5 · 2025: 8 · 2024: 8 · 2023: 5 · 2022: 8 · 2021: 11 · 2020: 14 · 2019: 8 · 2018: 18 · 2017: 18 · 2016: 11 · 2015: 9 · 2014: 15 · 2013: 9 · 2012: 11
FWCI
2.82
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
Israel Science Foundation
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Cardiac Ischemia and Reperfusion
Keywords
VDAC1, Bcl-2, Bcl-2 family proteins, apoptosis, anti-apoptotic effects, mitochondrial outer membrane, cytochrome c release, staurosporine-induced apoptosis, tumor cell survival, chemosensitization, channel conductance
MeSH terms
Animals, Cell Line, Electric Conductivity, Electrophysiology, Humans, Mitochondria, Peptide Fragments, Protein Binding, Sheep, Mutagenesis, Site-Directed, Apoptosis, Proto-Oncogene Proteins c-bcl-2, Surface Plasmon Resonance, Voltage-Dependent Anion Channel 1

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