Interactions of Bax and tBid with Lipid Monolayers

The release of cytochrome c from mitochondria to the cytosol is a crucial step of apoptosis that involves interactions of Bax and tBid proteins with the mitochondrial membrane. We investigated Bax and tBid interactions with (i) phosphatidylcholine (PC) monolayer as the main component of the outer leaflet of the outer membrane, (ii) with phosphatidylethanolamine (PE) and phosphatidylserine (PS) that are present in the inner leaflet and (iii) with a mixed PC/PE/Cardiolipin (CL) monolayer of the contact sites between the outer and inner membranes. These interactions were studied by measuring the increase of the lipidic monolayer surface pressure induced by the proteins. Our measurements suggest that tBid interacts strongly with the POPC/DOPE/CL, whereas Bax interaction with this monolayer is about 12 times weaker. Both tBid and Bax interact moderately half as strongly with negatively charged DOPS and non-lamellar DOPE monolayers. TBid also slightly interacts with DOPC. Our results suggest that tBid but not Bax interacts with the PC-containing outer membrane. Subsequent insertion of these proteins may occur at the PC/PE/CL sites of contact between the outer and inner membranes. It was also shown that Bax and tBid being mixed in solution inhibit their insertion into POPC/DOPE/CL monolayer. The known 3-D structures of Bax and Bid allowed us to propose a structural interpretation of these experimental results.     

Activation of Bax by joint action of tBid and mitochondrial outer membrane: Monte Carlo simulations

The mitochondrial pathway of apoptosis proceeds when molecules, such as cytochrome c, sequestered between the outer and inner mitochondrial membranes are released to the cytosol by mitochondrial outer membrane (MOM) permeabilization. Bax, a member of the Bcl-2 protein family, plays a pivotal role in mitochondrion-mediated apoptosis. In response to apoptotic stimuli, Bax integrates into the MOM, where it mediates the release of cytochrome c from the intermembrane space into the cytosol, leading to caspase activation and cell death. The pro-death action of Bax is regulated by interactions with both other prosurvival proteins, such as tBid, and the MOM, but the exact mechanisms remain largely unclear. Here, the mechanisms of integration of Bax into a model membrane mimicking the MOM were studied by Monte Carlo simulations preceded by a computer prediction of the docking of tBid with Bax. A novel model of Bax activation by tBid was predicted by the simulations. In this model, tBid binds to Bax at an interaction site formed by Bax helices α1, α2, α3 and α5 leading, due to interaction of the positively charged N-terminal fragment of tBid with anionic lipid headgroups, to Bax reorientation such that a hydrogen-bonded pair of residues, Asp98 and Ser184, is brought into close proximity with negatively charged lipid headgroups. The interaction with these headgroups destabilizes the hydrogen bond which results in the release of helix α9 from the Bax-binding groove, its insertion into the membrane, followed by insertion into the membrane of the α5–α6 helical hairpin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Investigating interaction of ligands with voltage dependent anion channel through noise analyses

Ion channel-protein complexes inserted in the membrane act as molecular gates for transport across the membrane. The opening and closing of these gates can be controlled by one or more variables like ligands (small molecules, proteins, etc.), transmembrane voltage, and the concentration gradient of a chemical across the membrane. We have shown how current noise profile of voltage dependent anion channel can be used to monitor change in the gating of the channel after its modulation by various ligands. This is being proposed as a novel method to probe the interaction of ion channels with ligands.     

Purification and properties of the voltage-dependent anion channel of the outer mitochondrial membrane

The methods for the purification of functionally active mitochondrial porin or voltage-dependent anion channel of the outer mitochondrial membrane are critically evaluated. Two rapid and efficient methods are now available. Both make use of a hydroxyapatite/celite column as a single chromatographic step. However, in one method with long polar head-group detergents, porin passes through the column, whereas in the other method, with shorter polar headgroup detergents, porin is first bound to the column and then eluted by the addition of salts. On the basis of these results, a model for the arrangement of porin in the detergent-protein micelles is proposed.     

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.     

tBid Undergoes Multiple Conformational Changes at the Membrane Required for Bax Activation

Bid is a Bcl-2 family protein that promotes apoptosis by activating Bax and eliciting mitochondrial outer membrane permeabilization (MOMP). Full-length Bid is cleaved in response to apoptotic stimuli into two fragments, p7 and tBid (p15), that are held together by strong hydrophobic interactions until the complex binds to membranes. The detailed mechanism(s) of fragment separation including tBid binding to membranes and release of the p7 fragment to the cytoplasm remain unclear. Using liposomes or isolated mitochondria with fluorescently labeled proteins at physiological concentrations as in vitro models, we report that the two components of the complex quickly separate upon interaction with a membrane. Once tBid binds to the membrane, it undergoes slow structural rearrangements that result in an equilibrium between two major tBid conformations on the membrane. The conformational change of tBid is a prerequisite for interaction with Bax and is, therefore, a novel step that can be modulated to promote or inhibit MOMP. Using automated high-throughput image analysis in cells, we show that down-regulation of Mtch2 causes a significant delay between tBid and Bax relocalization in cells. We propose that by promoting insertion of tBid via a conformational change at the mitochondrial outer membrane, Mtch2 accelerates tBid-mediated Bax activation and MOMP. Thus the interaction of Mtch2 and tBid is a potential target for therapeutic control of Bid initiated cell death.     

tBid forms a pore in the liposome membrane

We investigated the ability of tBid (truncated form of Bid) to bind and permeabilize the liposomes (large unilamellar vesicles, LUVs) and release fluorescent marker molecules (fluorescein-isothiocyanate-conjugated dextrans, FITC-dextrans) of various molecular diameters (FD-20, FD-70, FD-250S) from LUVs. Obtained data showed that tBid was more efficient in promoting leakage of FITC-dextrans from LUVs composed of cardiolipin and dioleoylphosphatidylcholine (DOPC) than LUVs made of dioleoylphosphatidic acid or dioleoylphosphatidylglycerol and DOPC. The leakage efficiency was reduced with increasing amount of dioleoylphosphatidylethanolamine or dielaidoylphosphatidylethanolamine. Phospholipid monolayer assay and fluorescence quenching measurements revealed that tBid inserted deeply into the hydrophobic acyl chain of acidic phospholipids. Taking into account the tBid three-dimensional structure, we propose that tBid could penetrate into the hydrophobic core of membrane, resulting in the leakage of entrapped content from LUVs via a pore-forming mechanism.     

Fluoxetine (Prozac) interaction with the mitochondrial voltage-dependent anion channel and protection against apoptotic cell death

Fluoxetine (Prozac) is a potent antidepressant compound inhibiting serotonin reuptake, but also Na+, K+ and Ca2+ channels and reported to both trigger and prevent apoptosis. Recently, fluoxetine was found to increase the voltage sensitivity of the mitochondrial voltage-dependent anion channel (VDAC). VDAC which functions in transporting metabolites across the mitochondria also plays a crucial role in apoptosis. Here, we demonstrate that fluoxetine interacted with VDAC and decreased its conductance. Fluoxetine inhibited the opening of the mitochondrial permeability transition pore, the release of cytochrome c, and protected against staurosporine-induced apoptotic cell death. These findings may explain some of the reported fluoxetine side effects.     

Phosphorylation of rat brain mitochondrial voltage-dependent anion as a potential tool to control leakage of cytochrome c

Apoptosis is a controlled form of cell death that participates in development, elimination of damaged cells and maintenance of cell homeostasis. Also, it plays a role in neurodegenerative disorders like Alzheimer's disease. Recently, mitochondria have emerged as being pivotal in controlling apoptosis. They house a number of apoptogenic molecules, such as cytochrome c, which are released into the cytoplasm at the onset of apoptosis. When rat brain mitochondrial voltage-dependent anion channel (VDAC), an outer mitochondrial membrane protein, interacts with Bcl-2 family proteins Bax and tBid, its pore size increases, leading to the release of cytochrome c and other apoptogenic molecules into the cytosol and causing cell death. Regulation of this tBid- and Bax-induced increase in pore size of VDAC is a significant step to control cell death induced by cytochrome c. In this work, we have shown, through bilayer electrophysiological experiments, that the increase in VDAC conductance as a result of its interaction with Bax and tBid is reduced because of the action of cyclic AMP-dependent protein kinase A (PKA) in the presence of ATP. This indicates that the increase in the pore size of VDAC after its interaction with Bax and tBid is controlled via phosphorylation of this channel by PKA. This, we believe, could be a mechanism of controlling cytochrome c-mediated cell death in living cells.     

Interaction of mitochondrial voltage-dependent anion channel from rat brain with plasminogen protein leads to partial closure of the channel

Voltage-dependent anion channel (VDAC) is reported to be the receptor for plasminogen kringle5. In this paper, the interaction of VDAC from rat brain mitochondria with plasminogen protein has been investigated through bilayer electrophysiological studies. We report for the first time that interaction of plasminogen with VDAC leads to partial closure of the channel on a lipid bilayer. This could be a mechanism of modulation of VDAC gating in a cellular system.     

Bax interacts with the voltage-dependent anion channel and mediates ethanol-induced apoptosis in rat hepatocytes

Acute ethanol exposure induces oxidative stress and apoptosis in primary rat hepatocytes. Previous data indicate that the mitochondrial permeability transition (MPT) is essential for ethanol-induced apoptosis. However, the mechanism by which ethanol induces the MPT remains unclear. In this study, we investigated the role of Bax, a proapoptotic Bcl-2 family protein, in acute ethanol-induced hepatocyte apoptosis. We found that Bax translocates from the cytosol to mitochondria before mitochondrial cytochrome c release. Bax translocation was oxidative stress dependent. Mitochondrial Bax formed a protein complex with the mitochondrial voltage-dependent anion channel (VDAC). Prevention of Bax-VDAC interactions by a microinjection of anti-VDAC antibody effectively prevented hepatocyte apoptosis by ethanol. In conclusion, these data suggest that Bax translocation from the cytosol to mitochondria leads to the subsequent formation of a Bax-VDAC complex that plays a crucial role in acute ethanol-induced hepatocyte apoptosis.     

Transmembrane arrangement of mitochondrial porin or voltage-dependent anion channel (VDAC)

Porin or voltage-dependent anion-selective channel (VDAC) is the main protein responsible for the high permeability of the outer mitochondrial membrane. The mitochondrial porin is mainly composed of sided -strands, in analogy with bacterial porin, whose structure has been resolved at 1.8 Å resolution. In mitochondrial porins the N-terminal region forms an amphipathic -helix, a structure conserved in organisms very distant from the evolutionary point of view. This part of the protein is exposed to the water phase, as demonstrated by ELISA assays. Various extramembranous loops have been identified by specific proteolytic cleavages. These overall, combined results were used to draw a model of the transmembrane arrangement of mammalian porin. This model is compared to other mitochondrial and bacterial porin models.     

Identification of Bax-voltage-dependent anion channel 1 complexes in digitonin-solubilized cerebellar granule neurons

Mitochondrial outer membrane Bax oligomers are critical for cytochrome c release, but the role of resident mitochondrial proteins in this process remains unclear. Membrane-associated Bax has primarily been studied using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) as the solubilizing agent, as it does not induce conformational artifacts, although recent evidence indicates it may have other artifactual effects. The objective of this study was to investigate digitonin as an alternative detergent to assess Bax oligomeric state, and possible interaction with voltage-dependent anion channel (VDAC)1 in cerebellar granule neurons. VDAC1 co-immunoprecipitated with Bax in digitonin extracts from healthy and apoptotic neurons. Two-dimensional blue native-SDS-PAGE revealed five Bax and VDAC1 oligomers having similar masses from 120 to 500 kDa. The levels of two VDAC1 oligomers in Bax 1D1 immunodepleted extracts negatively correlated with levels of co-precipitated VDAC1, indicating the co-precipitated VDAC1 was derived from these oligomers. Immunodepletion with the 6A7 antibody modestly reduced the levels of Bax oligomers from apoptotic but not healthy neurons. A sixth 170 kDa oligomer containing exclusively 6A7 Bax and no VDAC1 was identified after apoptosis induction. CHAPS failed to solubilize VDAC1, and additionally yielded no distinct oligomers. We conclude that digitonin is a potentially useful detergent preserving Bax-VDAC1 interactions that may be disrupted with CHAPS.     

The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore

The relevance of the mitochondrial permeability transition pore (PTP) in Ca2+ homeostasis and cell death has gained wide attention. Yet, despite detailed functional characterization, the structure of this channel remains elusive. Here we report on a new class of inhibitors of the PTP and on the identification of their molecular target. The most potent among the compounds prepared, Ro 68-3400, inhibited PTP with a potency comparable to that of cyclosporin A. Since Ro 68-3400 has a reactive moiety capable of covalent modification of proteins, [3H]Ro 68-3400 was used as an affinity label for the identification of its protein target. In intact mitochondria isolated from rodent brain and liver and in SH-SY5Y human neuroblastoma cells, [3H]Ro 68-3400 predominantly labeled a protein of approximately 32 kDa. This protein was identified as the isoform 1 of the voltage-dependent anion channel (VDAC). Both functional and affinity labeling experiments indicated that VDAC might correspond to the site for the PTP inhibitor ubiquinone0, whereas other known PTP modulators acted at distinct sites. While Ro 68-3400 represents a new useful tool for the study of the structure and function of VDAC and the PTP, the results obtained provide direct evidence that VDAC1 is a component of this mitochondrial pore.     

Susceptibility of Hep3B cells in different phases of cell cycle to tBid

tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC.     

Direct evidence for membrane pore formation by the apoptotic protein Bax

Direct imaging of the interaction of the apoptotic protein, Bax, with membrane bilayers shows the presence of toroidal-shaped pores using atomic force microscopy. These pores are sufficiently large to allow passage of proteins from the intermitochondrial space. Both the perturbation of the membrane and the amount of protein bound to the bilayer are increased in the presence of calcium. The results from the imaging are consistent with leakage studies from liposomes of the same composition. The work shows that Bax by itself can form pores in membrane bilayers.     

Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c

The Bcl-2 family of proteins, consisting of anti-apoptotic and pro-apoptotic members, regulates cell death by controlling mitochondrial membrane permeability that is crucial for apoptotic signal transduction. We have recently shown that some of these proteins, such as Bcl-x(L), Bax, and Bak, directly modulate the mitochondrial voltage-dependent anion channel (VDAC) and thus regulate apoptogenic cytochrome c release and potential loss. To elucidate the molecular mechanisms of VDAC regulation by Bcl-2 family proteins, an electrophysiological study was carried out. It was found that VDAC and pro-apoptotic Bax created a large pore, with conductance levels 4- and 10-fold greater than those of the VDAC and Bax channels, respectively. Although the VDAC and Bax channels both show ion selectivity and voltage-dependent modulation of their activity, the VDAC-Bax channel had neither of their properties. Anti-apoptotic Bcl-x(L) and its BH4 oligopeptide completely closed the VDAC, in contrast to the Bax. Cytochrome c passed through a single VDAC-Bax channel but not through the VDAC or Bax channel in a planar lipid bilayer. These data provide direct evidence that VDAC forms a novel large pore together with Bax.     

A soluble mitochondrial protein increases the voltage dependence of the mitochondrial channel,VDAC

A soluble protein isolated from mitochondria has been found to modulate the voltage-dependent properties of the mitochondrial outer membrane channel, VDAC. This protein, called the VDAC modulator, was first found inNeurospora crassa and then discovered in species from other eukaryotic kingdoms. The modulator-containing fraction (at a crude protein concentration of 20 µg/ml) increases the voltage dependence of VDAC channels over 2–3-fold. At higher protein concentrations (50–100 µg/ml), some channels seem to remain in a closed state or be blocked while others display the higher voltage dependence and are able to close at low membrane potentials. By increasing the steepness of the voltage-dependent properties of VDAC channels, this modulator may serve as an amplifierin vivo to increase the sensitivity of the channels in response to changes in the cell's microenvironment, and consequently, regulate the metabolic flux across the outer mitochondrial membrane by controlling the gating of VDAC channels.     

Phosphorylation by Nek1 regulates opening and closing of voltage dependent anion channel 1

VDAC1 is a key component of the mitochondrial permeability transition pore. To initiate apoptosis and certain other forms of cell death, mitochondria become permeable such that cytochrome c and other pre-apoptotic molecules resident inside the mitochondria enter the cytosol and activate apoptotic cascades. We have shown recently that VDAC1 interacts directly with never-in-mitosis A related kinase 1 (Nek1), and that Nek1 phosphorylates VDAC1 on Ser193 to prevent excessive cell death after injury. How this phosphorylation regulates the activity of VDAC1, however, has not yet been reported. Here, we use atomic force microscopy (AFM) and cytochrome c conductance studies to examine the configuration of VDAC1 before and after phosphorylation by Nek1. Wild-type VDAC1 assumes an open configuration, but closes and prevents cytochrome c efflux when phosphorylated by Nek1. A VDAC1-Ser193Ala mutant, which cannot be phosphorylated by Nek1 under identical conditions, remains open and constitutively allows cytochrome c efflux. Conversely, a VDAC1-Ser193Glu mutant, which mimics constitutive phosphorylation by Nek1, remains closed by AFM and prevents cytochrome c leakage in the same liposome assays. Our data provide a mechanism to explain how Nek1 regulates cell death by affecting the opening and closing of VDAC1.