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

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.     

Key regions of VDAC1 functioning in apoptosis induction and regulation by hexokinase

The voltage-dependent anion channel (VDAC), located in the mitochondrial outer membrane, functions as gatekeeper for the entry and exit of mitochondrial metabolites, and thus controls cross-talk between mitochondria and the cytosol. VDAC also serves as a site for the docking of cytosolic proteins, such as hexokinase, and is recognized as a key protein in mitochondria-mediated apoptosis. The role of VDAC in apoptosis has emerged from various studies showing its involvement in cytochrome c release and apoptotic cell death as well as its interaction with proteins regulating apoptosis, including the mitochondria-bound isoforms of hexokinase (HK-I, HK-II). Recently, the functional HK-VDAC association has shifted from being considered in a predominantly metabolic light to the recognition of its major impact on the regulation of apoptotic responsiveness of the cell. Here, we demonstrate that the HK-VDAC1 interaction can be disrupted by mutating VDAC1 and by VDAC1-based peptides, consequently leading to diminished HK anti-apoptotic activity, suggesting that disruption of HK binding to VDAC1 can decrease tumor cell survival. Indeed, understanding structure-function relationships of VDAC is critical for deciphering how this channel can perform such a variety of differing functions, all important for cell life and death. By expressing VDAC1 mutants and VDAC1-based peptides, we have identified VDAC1 amino acid residues and domains important for interaction with HK and protection against apoptosis. These include negatively- and positively-charged residues, some of which are located within β-strands of the protein. The N-terminal region of VDAC1 binds HK-I and prevents HK-mediated protection against apoptosis induced by STS, while expression of a VDAC N-terminal peptide detaches HK-I-GFP from mitochondria. These findings indicate that the interaction of HK with VDAC1 involves charged residues in several β-strands and in the N-terminal domain. Displacing HK, serving as the ‘guardian of the mitochondrion’, from its binding site on VDAC1 may thus be exploited as an approach to cancer therapy.     

NMR structural investigation of the mitochondrial outer membrane protein VDAC and its interaction with antiapoptotic Bcl-xL

Bcl-2 family proteins are essential regulators of cell death and exert their primary pro- or antiapoptotic roles at the mitochondrial outer membrane. Previously, pro- and antiapoptotic Bcl-2 proteins have been shown to interact with the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. VDAC is a 283-residue integral membrane protein that forms an aqueous pore in the outer mitochondrial membrane, through which metabolites and other small molecules pass between the cytosol and intermembrane space. The essential life-sustaining function of VDAC in metabolite trafficking is believed to be regulated by proteins of the Bcl-2 family. The protective role of antiapoptotic Bcl-xL may be through its interaction with VDAC. Here, VDAC has been expressed, purified, and refolded into a functional form amenable to NMR studies. Various biophysical experiments indicate that micelle-bound VDAC is in intermediate exchange between monomer and trimer. Using NMR spectroscopy, gel filtration, and chemical cross-linking, we obtained direct evidence for binding of Bcl-xL to VDAC in a detergent micelle system. The VDAC-interacting region of Bcl-xL was characterized by NMR with chemical shift perturbation and transferred cross-saturation. The interaction region was mapped to a putative helical hairpin motif of Bcl-xL that was found to insert into detergent micelles. Our results suggest that Bcl-xL can bind to one or two VDAC molecules forming heterodimers and heterotrimers. Our characterization of the VDAC/Bcl-xL complex offers initial structural insight into the role of antiapoptotic Bcl-xL in regulating apoptotic events in the mitochondrial outer membrane.     

The Association of Receptor of Activated Protein Kinase C 1(RACK1) with Infectious Bursal Disease Virus Viral Protein VP5 and Voltage-dependent Anion Channel 2 (VDAC2) Inhibits Apoptosis and Enhances Viral Replication

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Our previous report indicates that IBDV VP5 induces apoptosis via interaction with voltage-dependent anion channel 2 (VDAC2). However, the underlying molecular mechanism is still unclear. We report here that receptor of activated protein kinase C 1 (RACK1) interacts with both VDAC2 and VP5 and that they could form a complex. We found that overexpression of RACK1 inhibited IBDV-induced apoptosis in DF-1 cells and that knockdown of RACK1 by small interfering RNA induced apoptosis associated with activation of caspases 9 and 3 and suppressed IBDV growth. These results indicate that RACK1 plays an antiapoptotic role during IBDV infection via interaction with VDAC2 and VP5, suggesting that VP5 sequesters RACK1 and VDAC2 in the apoptosis-inducing process.     

VDAC1-based peptides: novel pro-apoptotic agents and potential therapeutics for B-cell chronic lymphocytic leukemia

The voltage-dependent anion channel 1 (VDAC1), localized in the outer mitochondrial membrane, mediates metabolic cross-talk between the mitochondrion and the cytoplasm and thus serves a fundamental role in cell energy metabolism. VDAC1 also plays a key role in mitochondria-mediated apoptosis, interacting with anti-apoptotic proteins. Resistance of cancer cells to apoptosis involves quenching the mitochondrial apoptotic pathway by over-expression of anti-apoptotic/pro-survival hexokinase (HK) and Bcl-2 family proteins, proteins that mediate their anti-apoptotic activities via interaction with VDAC1. Using specifically designed VDAC1-based cell-penetrating peptides, we targeted these anti-apoptotic proteins to prevent their pro-survival/anti-apoptotic activities. Anti-apoptotic proteins are expressed at high levels in B-cell chronic lymphocytic leukemia (CLL), an incurable disease requiring innovative new approaches to improve therapeutic outcome. CLL is characterized by a clonal accumulation of mature neoplastic B cells that are resistant to apoptosis. Specifically, we demonstrate that the VDAC1-based peptides (Antp-LP4 and N-Terminal-Antp) selectively kill peripheral blood mononuclear cells (PBMCs) obtained from CLL patients, yet spare those obtained from healthy donors. The cell death induction competence of the peptides was well correlated with the amount of double positive CD19/CD5 cancerous CLL PBMCs, further illustrating their selectivity toward cancer cells. Moreover, these VDAC1-based peptides induced apoptosis by activating the mitochondria-mediated pathway, reflected in membrane blebbing, condensation of nuclei, DNA fragmentation, release of mitochondrial cytochrome c, loss of mitochondrial membrane potential, decreased cellular ATP levels and detachment of HK, all leading to apoptotic cell death. Thus, the mode of action of the peptides involves decreasing energy production and inducing apoptosis. Over 27 versions of cell-penetrating VDAC1-based peptides were designed and screened to identify the most stable, short and apoptosis-inducing peptides toward CLL-derived lymphocytes. In this manner, three optimized peptides suitable for in vivo studies were identified. This study thus reveals the potential of VDAC1-based peptides as an innovative and effective anti-CLL therapy.     

Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.     

Structure-based analysis of VDAC1: N-terminus location, translocation, channel gating and association with anti-apoptotic proteins

Structural studies place the VDAC1 (voltage-dependent anion channel 1) N-terminal region within the channel pore. Biochemical and functional studies, however, reveal that the N-terminal domain is cytoplasmically exposed. In the present study, the location and translocation of the VDAC1 N-terminal domain, and its role in voltage-gating and as a target for anti-apoptotic proteins, were addressed. Site-directed mutagenesis and cysteine residue substitution, together with a thiol-specific cross-linker, served to show that the VDAC1 N-terminal region exists in a dynamic equilibrium, located within the pore or exposed outside the β-barrel. Using a single cysteine-residue-bearing VDAC1, we demonstrate that the N-terminal region lies inside the pore. However, the same region can be exposed outside the pore, where it dimerizes with the N-terminal domain of a second VDAC1 molecule. When the N-terminal region α-helix structure was perturbed, intra-molecular cross-linking was abolished and dimerization was enhanced. This mutant also displays reduced voltage-gating and reduced binding to hexokinase, but not to the anti-apoptotic proteins Bcl-2 and Bcl-xL. Replacing glycine residues in the N-terminal domain GRS (glycine-rich sequence) yielded less intra-molecular cross-linked product but more dimerization, suggesting that GRS provides the flexibility needed for N-terminal translocation from the internal pore to the channel face. N-terminal mobility may thus contribute to channel gating and interaction with anti-apoptotic proteins.     

Apoptosis is regulated by the VDAC1 N-terminal region and by VDAC oligomerization: release of cytochrome c,AIF and Smac/Diablo

Mitochondria, central to basic life functions due to their generation of cellular energy, also serve as the venue for cellular decisions leading to apoptosis. A key protein in mitochondria-mediated apoptosis is the voltage-dependent anion channel (VDAC), which also mediates the exchange of metabolites and energy between the cytosol and the mitochondria. In this study, the functions played by the N-terminal region of VDAC1 and by VDAC1 oligomerization in the release of cytochrome c, Smac/Diablo and apoptosis-inducing factor (AIF) and subsequent apoptosis were addressed. We demonstrate that cells undergoing apoptosis induced by STS or cisplatin and expressing N-terminally truncated VDAC1 do not release cytochrome c, Smac/Diablo or AIF. Ruthenium red (RuR), AzRu, DIDS and hexokinase-I (HK-I), all known to interact with VDAC, inhibited the release of cytochrome c, Smac/Diablo and AIF, while RuR-mediated inhibition was not observed in cells expressing RuR-insensitive E72Q-VDAC1. These findings suggest that VDAC1 is involved in the release of not only cytochrome c but also of Smac/Diablo and AIF. We also demonstrate that apoptosis induction is associated with VDAC oligomerization, as revealed by chemical cross-linking and monitoring in living cells using Bioluminescence Resonance Energy Transfer. Apoptosis induction by STS, H₂O₂ or selenite augmented the formation of VDAC oligomers several fold. The results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the functions of VDAC1 oligomerization in apoptosis and of the VDAC1 N-terminal domain in the release of apoptogenic proteins as well as into regulation of VDAC by anti-apoptotic proteins, such as HK and Bcl2.     

Critical role for antiapoptotic Bcl-xL and Mcl-1 in human macrophage survival and cellular IAP1/2 (cIAP1/2) in resistance to HIV-Vpr-induced apoptosis

Macrophages are resistant to HIV cytopathic effects, which contributes to viral persistence and reservoir formation. HIV viral protein R (Vpr) is a potent apoptosis-inducing agent for primary monocytes. Because the biologically active Vpr is found in serum and cerebrospinal fluid of HIV-infected patients, we investigated the apoptotic effect of Vpr on monocyte-derived macrophages and phorbol 12-myristate 13-acetate-activated THP1 macrophages. Our results show that primary monocytes and THP1 cells develop resistance to Vpr-induced apoptosis following differentiation into macrophages. To determine the effect of Vpr on the expression of antiapoptotic proteins, we show that in contrast to the undifferentiated cells, Vpr did not down-regulate the expression of antiapoptotic inhibitors of apoptosis (IAPs) and Bcl2 family members in macrophages, suggesting their involvement in resistance to Vpr-induced apoptosis. However, knocking down Bcl-xL and Mcl-1 proteins induced spontaneous apoptosis with no impact on susceptibility to Vpr-induced apoptosis. In contrast, down-regulation of cellular IAP1 (cIAP1) and cIAP2 by using siRNAs and SMAC (second mitochondria-derived activator of caspases) mimetic sensitized macrophages to Vpr-induced apoptosis. Overall, our results suggest that resistance to Vpr-induced apoptosis is specifically mediated by cIAP1/2 genes independent of Bcl-xL and Mcl-1, which play a key role in maintaining cell viability. Moreover, IAP modulation may be a potential strategy to eliminate HIV persistence in macrophages.     

One-step on-column affinity refolding purification and functional analysis of recombinant human VDAC1

The outer mitochondrial membrane porin, voltage-dependent anion-selective channel (VDAC), is believed to play an important role in mediating mitochondria-dependent apoptosis. However, detailed structure-function studies of VDAC have been hindered by the difficulties to obtain a soluble, correctly folded, and fully active form of the recombinant VDAC and its mutant variants due to its transmembrane nature. Here we report a high-throughput one-step chromatographic procedure in purification of recombinant human VDAC1 (rhVDAC1) protein overexpressed in bacteria. The improved methodology could generate a large quantity of rhVDAC1 with correct folding in terms of the secondary structure, with full biological activities in mediating cytochrome c release and in interaction with Bcl-X(L). The method will significantly benefit genetic, biochemical, and structural studies of this critical channel protein.     

Structure-based analysis of VDAC1 protein: defining oligomer contact sites

The outer mitochondrial membrane protein, the voltage-dependent anion channel (VDAC), is increasingly implicated in the control of apoptosis. Oligomeric assembly of VDAC1 was shown to be coupled to apoptosis induction, with oligomerization increasing substantially upon apoptosis induction and inhibited by apoptosis blockers. In this study, structure- and computation-based selection of the predicated VDAC1 dimerization site, in combination with site-directed mutagenesis, cysteine replacement, and chemical cross-linking, were employed to identify contact sites between VDAC1 molecules in dimers and higher oligomers. The predicted weakly stable β-strands were experimentally found to represent the interfaces between VDAC1 monomers composing the oligomer. Replacing hydrophobic amino acids with charged residues in β-strands 1, 2, and 19 interfered with VDAC1 oligomerization. The proximity of β-strands 1, 2, and 19 within the VDAC1 dimer and the existence of other association sites involving β-strand 16 were confirmed when a cysteine was introduced at defined positions in cysteineless VDAC1 mutants, together with the use of cysteine-specific cross-linker bis(maleimido)ethane. Moreover, the results suggest that VDAC1 also exists as a dimer that upon apoptosis induction undergoes conformational changes and that its oligomerization proceeds through a series of interactions involving two distinct interfaces. Dissection of VDAC1 dimerization/oligomerization as presented here provides structural insight into the oligomeric status of cellular VDAC1 under physiological and apoptotic conditions.     

The BH4 Domain of Anti-apoptotic Bcl-XL,but Not That of the Related Bcl-2, Limits the Voltage-dependent Anion Channel 1 (VDAC1)-mediated Transfer of Pro-apoptotic Ca2+ Signals to Mitochondria

Excessive Ca²⁺ fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca²⁺-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP₃Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca²⁺-dependent apoptosis by binding and inhibiting IP₃Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP₃Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca²⁺ uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1^−/− cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca²⁺ uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca²⁺ release in the cytosol or to prevent apoptosis, as done instead by an IP₃R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca²⁺ uptake into the mitochondria.     

Yes-associated Protein (YAP) Promotes Cell Survival by Inhibiting Proapoptotic Dendrin Signaling

Kidney podocytes are highly specialized terminally differentiated cells that form the final barrier to urinary protein loss. Podocytes are a target for injury by metabolic, autoimmune, hereditary, inflammatory, and other stressors. Persistence of podocyte injury leads to podocyte death and loss, which results in progressive kidney damage and ultimately kidney failure. Dendrin is a dual compartment protein with proapoptotic signaling properties. Nuclear relocation of dendrin in response to glomerular injury promotes podocyte apoptosis. Here we show that Yes-associated protein (YAP), a downstream target of Hippo kinases and an inhibitor of apoptosis, is expressed in the nucleus of podocytes. The WW domains of YAP mediate the interaction with the PPXY motifs of dendrin. This interaction is functionally relevant because YAP binding to dendrin reduces dendrin-dependent, staurosporine-induced apoptosis in co-transfected HEK293 cells. Moreover gene silencing of YAP in podocytes increases adriamycin-induced podocyte apoptosis. It also increases staurosporine-induced caspase-3/7 activity, which is rescued by dendrin depletion in YAP knockdown cells. Our findings elucidate YAP binding to dendrin as a prosurvival mechanism. The antiapoptotic signaling properties of YAP in podocytes could hold significance in the quest for targeted therapeutics aimed at preventing podocyte loss.     

A novel inhibitory mechanism of mitochondrion-dependent apoptosis by a herpesviral protein

Upon viral infection, cells undergo apoptosis as a defense against viral replication. Viruses, in turn, have evolved elaborate mechanisms to subvert apoptotic processes. Here, we report that a novel viral mitochondrial anti-apoptotic protein (vMAP) of murine gamma-herpesvirus 68 (gammaHV-68) interacts with Bcl-2 and voltage-dependent anion channel 1 (VDAC1) in a genetically separable manner. The N-terminal region of vMAP interacted with Bcl-2, and this interaction markedly increased not only Bcl-2 recruitment to mitochondria but also its avidity for BH3-only pro-apoptotic proteins, thereby suppressing Bax mitochondrial translocation and activation. In addition, the central and C-terminal hydrophobic regions of vMAP interacted with VDAC1. Consequently, these interactions resulted in the effective inhibition of cytochrome c release, leading to the comprehensive inhibition of mitochondrion-mediated apoptosis. Finally, vMAP gene was required for efficient gammaHV-68 lytic replication in normal cells, but not in mitochondrial apoptosis-deficient cells. These results demonstrate that gammaHV-68 vMAP independently targets two important regulators of mitochondrial apoptosis-mediated intracellular innate immunity, allowing efficient viral lytic replication.     

Underphosphorylated BAD interacts with diverse antiapoptotic Bcl-2 family proteins to regulate apoptosis

Survival factors activate kinases which, in turn, phosphorylate the proapoptotic Bcl-xl/Bcl-2-associated death promoter homolog (BAD) protein at key serine residues. Phosphorylated BAD interacts with 14-3-3 proteins, and overexpression of 14-3-3 attenuates BAD-mediated apoptosis. Although BAD is known to interact with Bcl-2, Bcl-w, and Bcl-xL, the exact relationship between BAD and anti- or proapoptotic Bcl-2 proteins has not been analyzed systematically. Using the yeast two-hybrid protein interaction assay, we found that BAD interacted negligibly with proapoptotic Bcl-2 proteins. Even though wild type BAD only interacted with selected numbers of antiapoptotic proteins, underphosphorylated mutant BAD interacted with all antiapoptotic Bcl-2 proteins tested (Bcl-2, Bcl-w, Bcl-xL, Bfl-1/A1, Mcl-1, Ced-9, and BHRF-1). Using nonphosphorylated recombinant BAD expressed in bacteria, direct interactions between BAD and diverse antiapoptotic Bcl-2 members were also observed. Furthermore, apoptosis induced by BAD was blocked by coexpression with Bcl-2, Bcl-w, and Bfl-1. Comparison of BAD orthologs from zebrafish to human indicated the conservation of a 14-3-3 binding site and the BH3 domain during evolution. Thus, highly conserved BAD interacts with diverse antiapoptotic Bcl-2 members to regulate apoptosis.     

Retinoid x receptor agonists increase bcl2a1 expression and decrease apoptosis of naive T lymphocytes

Vitamin A affects many aspects of T lymphocyte development and function. The vitamin A metabolites all-trans- and 9-cis-retinoic acid regulate gene expression by binding to the retinoic acid receptor (RAR), while 9-cis-retinoic acid also binds to the retinoid X receptor (RXR). Naive DO11.10 T lymphocytes expressed mRNA and protein for RAR-alpha, RXR-alpha, and RXR-beta. DNA microarray analysis was used to identify RXR-responsive genes in naive DO11.10 T lymphocytes treated with the RXR agonist AGN194204. A total of 128 genes was differentially expressed, including 16 (15%) involved in cell growth or apoptosis. Among these was Bcl2a1, an antiapoptotic Bcl2 family member. Quantitative real-time PCR analysis confirmed this finding and demonstrated that Bcl2a1 mRNA expression was significantly greater in nonapoptotic than in apoptotic T lymphocytes. The RXR agonist 9-cis-retinoic acid also increased Bcl2a1 expression, although all-trans-retinoic acid and ligands for other RXR partner receptors did not. Treatment with AGN194204 and 9-cis-retinoic acid significantly decreased apoptosis measured by annexin V staining but did not affect expression of Bcl2 and Bcl-xL. Bcl2a1 promoter activity was examined using a luciferase promoter construct. Both AGN194204 and 9-cis-retinoic acid significantly increased luciferase activity. In summary, these data demonstrate that RXR agonists increase Bcl2a1 promoter activity and increase expression of Bcl2a1 in naive T lymphocytes but do not affect Bcl2 and Bcl-xL expression in naive T lymphocytes. Thus, this effect on Bcl2a1 expression may account for the decreased apoptosis seen in naive T lymphocytes treated with RXR agonists.     

Disruption of the VDAC2–Bak interaction by Bcl-xS mediates efficient induction of apoptosis in melanoma cells

The proapoptotic B-cell lymphoma (Bcl)-2 protein Bcl-x_S encloses the Bcl-2 homology (BH) domains BH3 and BH4 and triggers apoptosis via the multidomain protein Bak, however, the mechanism remained elusive. For investigating Bcl-x_S efficacy and pathways, an adenoviral vector was constructed with its cDNA under tetracycline-off control. Bcl-x_S overexpression resulted in efficient apoptosis induction and caspase activation in melanoma cells. Indicative of mitochondrial apoptosis pathways, Bcl-x_S translocated to the mitochondria, disrupted the mitochondrial membrane potential and induced release of cytochrome c, apoptosis-inducing factor and second mitochondria-derived activator of caspases. In melanoma cells, Bcl-x_S resulted in significant Bak activation, and Bak knockdown as well as Bcl-x_L overexpression abrogated Bcl-x_S-induced apoptosis, whereas Mcl-1 (myeloid cell leukemia-1) knockdown resulted in a sensitization. With regard to the particular role of voltage-dependent anion channel 2 (VDAC2) for inhibition of Bak, we identified here a notable interaction between Bcl-x_S and VDAC2 in melanoma cells, which was proven in reciprocal coimmunoprecipitation analyses. On the other hand, Bcl-x_S showed no direct interaction with Bak, and its binding to VDAC2 appeared as also independent of Bak expression. Suggesting a new proapoptotic mechanism, Bcl-x_S overexpression resulted in disruption of the VDAC2–Bak interaction leading to release of Bak. Further supporting this pathway, overexpression of VDAC2 strongly decreased apoptosis by Bcl-x_S. New proapoptotic pathways are of principle interest for overcoming apoptosis deficiency of melanoma cells.