The Proapoptotic Protein tBid Forms Both Superficially Bound and Membrane-Inserted Oligomers

Bid is a proapopotic activator protein of the Bcl-2 family that plays a pivotal role in controlling mitochondrial outer membrane permeabilization during apoptosis. Here, we characterized the interaction of fluorescently labeled truncated Bid (tBid) with a mitochondria-like supported lipid bilayer at the single-molecule level. The proteins observed at the membrane exhibited a very wide range of mobility. Confocal images of the membrane displayed both diffraction-limited Gaussian spots and horizontal streaks, corresponding to immobile and mobile tBid species, respectively. We observed 1), fast-diffusing proteins corresponding to a loosely, probably electrostatically bound state; 2), slowly diffusing proteins, likely corresponding to a superficially inserted state; and 3), fully immobilized proteins, suggesting a fully inserted state. The stoichiometry of these proteins was determined by normalizing their fluorescence intensity by the brightness of a tBid monomer, measured separately using fluorescence fluctuation techniques. Strikingly, the immobile species were found to be mainly tetramers and higher, whereas the mobile species had on average a significantly lower stoichiometry. Taken together, these results show that as soluble Bid progresses toward a membrane-inserted state, it undergoes an oligomerization process similar to that observed for Bax.     

Multiple partners can kiss-and-run: Bax transfers between multiple membranes and permeabilizes those primed by tBid

During apoptosis Bid and Bax are sufficient for mitochondrial outer membrane permeabilization, releasing pro-apoptotic proteins such as cytochrome c and Smac/Diablo into the cytoplasm. In most cells, both Bid and Bax are cytoplasmic but bind to mitochondrial outer membranes to exert pro-apoptotic functions. Binding to membranes is regulated by cleavage of Bid to truncated Bid (tBid), by conformation changes in tBid and Bax, and by interactions with other proteins. At least at the peripherally bound stage, binding is reversible. Therefore, regulation of apoptosis is closely linked with the interactions of tBid and Bax with mitochondria. Here we use fluorescence techniques and cell-free systems containing mitochondria or liposomes that faithfully mimic tBid/Bax-dependent membrane permeabilization to study the dynamic interactions of the proteins with membranes. We confirm that the binding of both proteins to the membrane is reversible by quantifying the binding affinity of proteins for the membrane. For Bax, both peripherally bound (inactive) and oligomerized (active) proteins migrate between membranes but much slower than and independent of tBid. When re-localized to a new membrane, Bax inserts into and permeabilizes it only if primed by an activator. In the case of tBid, the process of transfer is synergetic with Bax in the sense that tBid ‘runs'' faster if it has been ‘kissed'' by Bax. Furthermore, Mtch2 accelerates the re-localization of tBid at the mitochondria. In contrast, binding to Bcl-XL dramatically impedes tBid re-localization by lowering the off-rate threefold. Our results suggest that the transfer of activated tBid and Bax to different mitochondria is governed by dynamic equilibria and potentially contributes more than previously anticipated to the dissemination of the permeabilization signal within the cell.     

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.     

Real time single cell analysis of Bid cleavage and Bid translocation during caspase-dependent and neuronal caspase-independent apoptosis

Bcl-2 homology domain (BH) 3-only proteins couple stress signals to evolutionarily conserved mitochondrial apoptotic pathways. Caspase 8-mediated cleavage of the BH3-only protein Bid into a truncated protein (tBid) and subsequent translocation of tBid to mitochondria has been implicated in death receptor signaling. We utilized a recombinant fluorescence resonance energy transfer (FRET) Bid probe to determine the kinetics of Bid cleavage and tBid translocation during death receptor-induced apoptosis in caspase 3-deficient MCF-7 cells. Cells treated with tumor necrosis factor-alpha (200 ng/ml) showed a rapid cleavage of the Bid-FRET probe occurring 75.4 +/- 12.6 min after onset of the tumor necrosis factor-alpha exposure. Cleavage of the Bid-FRET probe coincided with a translocation of tBid to the mitochondria and a collapse of the mitochondrial membrane potential (DeltaPsim). We next investigated the role of Bid cleavage in a model of caspase-independent, glutamate-induced excitotoxic apoptosis. Rat cerebellar granule neurons were transfected with the Bid-FRET probe and exposed to glutamate for 5 min. In contrast to death receptor-induced apoptosis, neurons showed a translocation of full-length Bid to the mitochondria. This translocation occurred 5.6 +/- 1.7 h after the termination of the glutamate exposure and was also paralleled with a collapse of the DeltaPsim. Proteolytic cleavage of the FRET probe also occurred, however, only 25.2 +/- 3.5 min after its translocation to the mitochondria. Subfractionation experiments confirmed a translocation of full-length Bid from the cytosolic to the mitochondrial fraction during excitotoxic apoptosis. Our data demonstrate that both tBid and full-length Bid have the capacity to translocate to mitochondria during apoptosis.     

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.     

Permeabilization of the mitochondrial outer membrane by Bax/truncated Bid (tBid) proteins as sensitized by cardiolipin hydroperoxide translocation: mechanistic implications for the intrinsic pathway of oxidative apoptosis

Cytochrome c (cyt c) release upon oxidation of cardiolipin (CL) in the mitochondrial inner membrane (IM) under oxidative stress occurs early in the intrinsic apoptotic pathway. We postulated that CL oxidation mobilizes not only cyt c but also CL itself in the form of hydroperoxide (CLOOH) species. Relatively hydrophilic CLOOHs could assist in apoptotic signaling by translocating to the outer membrane (OM), thus promoting recruitment of the pro-apoptotic proteins truncated Bid (tBid) and Bax for generation of cyt c-traversable pores. Initial testing of these possibilities showed that CLOOH-containing liposomes were permeabilized more readily by tBid plus Ca(2+) than CL-containing counterparts. Moreover, CLOOH translocated more rapidly from IM-mimetic to OM-mimetic liposomes than CL and permitted more extensive OM permeabilization. We found that tBid bound more avidly to CLOOH-containing membranes than to CL counterparts, and binding increased with increasing CLOOH content. Permeabilization of CLOOH-containing liposomes in the presence of tBid could be triggered by monomeric Bax, consistent with tBid/Bax cooperation in pore formation. Using CL-null mitochondria from a yeast mutant, we found that tBid binding and cyt c release were dramatically enhanced by transfer acquisition of CLOOH. Additionally, we observed a pre-apoptotic IM-to-OM transfer of oxidized CL in cardiomyocytes treated with the Complex III blocker, antimycin A. These findings provide new mechanistic insights into the role of CL oxidation in the intrinsic pathway of oxidative apoptosis.     

The mitochondrial TOM complex is required for tBid/Bax-induced cytochrome c release

Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.     

Bcl-2 family member Bfl-1/A1 sequesters truncated bid to inhibit is collaboration with pro-apoptotic Bak or Bax

Following caspase-8 mediated cleavage, a carboxyl-terminal fragment of the BH3 domain-only Bcl-2 family member Bid transmits the apoptotic signal from death receptors to mitochondria. In a screen for possible regulators of Bid, we defined Bfl-1/A1 as a potent Bid interacting protein. Bfl-1 is an anti-apoptotic Bcl-2 family member, whose preferential expression in hematopoietic cells and endothelium is controlled by inflammatory stimuli. Its mechanism of action is unknown. We find that Bfl-1 associates with both full-length Bid and truncated (t)Bid, via the Bid BH3 domain. Cellular expression of Bfl-1 confers protection against CD95- and Trail receptor-induced cytochrome c release. In vitro assays, using purified mitochondria and recombinant proteins, demonstrate that Bfl-1 binds full-length Bid, but does not interfere with its processing by caspase-8, or with its mitochondrial association. Confocal microscopy supports that Bfl-1, which at least in part constitutively localizes to mitochondria, does not impede tBid translocation. However, Bfl-1 remains tightly and selectively bound to tBid and blocks collaboration between tBid and Bax or Bak in the plane of the mitochondrial membrane, thereby preventing mitochondrial apoptotic activation. Lack of demonstrable interaction between Bfl-1 and Bak or Bax in the mitochondrial membrane suggests that Bfl-1 generally prevents the formation of a pro-apoptotic complex by sequestering BH3 domain-only proteins.     

Monte Carlo simulations of tBid association with the mitochondrial outer membrane

Bid, a BH3-only pro-apoptopic member of the BCL-2 protein family, regulates cell death at the level of mitochondrial cytochrome c efflux. Bid consists of 8 α-helices (H1–H8, respectively) and is soluble cytosolic protein in its native state. Proteolysis of the N-terminus (encompassing H1 and H2) of Bid by caspase 8 in apoptosis yields activated “tBid” (truncated Bid), which translocates to the mitochondria and induces the efflux of cytochrome c. The release of cytochrome c from mitochondria to the cytosol constitutes a critical control point in apoptosis that is regulated by interaction of tBid protein with mitochondrial membrane. tBid displays structural homology to channel-forming bacterial toxins, such as colicins or transmembrane domain of diphtheria toxin. By analogy, it has been hypothesized that tBid would unfold and insert into the lipid bilayer of the mitochondria outer membrane (MOM) upon membrane association. However, it has been shown recently that unlike colicins and the transmembrane domain of diphtheria toxin, tBid binds to the lipid bilayer maintaining α-helical conformation of its helices without adopting a transmembrane orientation by them. Here, the mechanism of the association of tBid with the model membrane mimicking the mitochondrial membrane is studied by Monte Carlo simulations, taking into account the underlying energetics. A novel two-stage hierarchical simulation protocol combining coarse-grained discretization of conformational space with subsequent refinements was applied which was able to generate the protein conformation and its location in the membrane using modest computational resources. The simulations show that starting from NMR-established conformation in the solution, the protein associates with the membrane without adopting the transmembrane orientation. The configuration (conformation and location) of tBid providing the lowest free energy for the system protein/membrane/solvent has been obtained. The simulations reveal that tBid upon association with the membrane undergoes significant conformational changes primarily due to rotations within the loops between helices H4 and H5, H6 and H7, H7 and H8. It is established that in the membrane-bound state of tBid-monomer helices H3 and H5 have the locations exposed to the solution, helices H6 and H8 are partly buried and helices H4 and H7 are buried into the membrane at shallow depth. The average orientation of tBid bound to the membrane in the most stable configuration reported here is in satisfactory agreement with the evaluations obtained by indirect experimental means. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Membrane-insertion fragments of Bcl-xL, Bax, and Bid

Apoptosis regulators of the Bcl-2 family associate with intracellular membranes from mitochondria and the endoplasmic reticulum, where they perform their function. The activity of these proteins is related to the release of apoptogenic factors, sequestered in the mitochondria, to the cytoplasm, probably through the formation of ion and/or protein transport channels. Most of these proteins contain a C-terminal putative transmembrane (TM) fragment and a pair of hydrophobic alpha helices (alpha5-alpha6) similar to the membrane insertion fragments of the ion-channel domain of diphtheria toxin and colicins. Here, we report on the membrane-insertion properties of different segments from antiapoptotic Bcl-x(L) and proapoptotic Bax and Bid, that correspond to defined alpha helices in the structure of their soluble forms. According to prediction methods, there are only two putative TM fragments in Bcl-x(L) and Bax (the C-terminal alpha helix and alpha-helix 5) and one in activated tBid (alpha-helix 6). The rest of their sequence, including the second helix of the pore-forming domain, displays only weak hydrophobic peaks, which are below the prediction threshold. Subsequent analysis by glycosylation mapping of single alpha-helix segments in a model chimeric system confirms the above predictions and allows finding an extra TM fragment made of helix alpha1 of Bax. Surprisingly, the amphipathic helices alpha6 of Bcl-x(L) and Bax and alpha7 of Bid do insert in membranes only as part of the alpha5-alpha6 (Bcl-x(L) and Bax) or alpha6-alpha7 (Bid) hairpins but not when assayed individually. This behavior suggests a synergistic insertion and folding of the two helices of the hairpin that could be due to charge complementarity and additional stability provided by turn-inducing residues present at the interhelical region. Although these data come from chimeric systems, they show direct potentiality for acquiring a membrane inserted state. Thus, the above fragments should be considered for the definition of plausible models of the active, membrane-bound species of Bcl-2 proteins.     

Dynamic interaction of cBid with detergents, liposomes and mitochondria

The BH3-only protein Bid plays a key role in the induction of mitochondrial apoptosis, but its mechanism of action is still not completely understood. Here we studied the two main activation events of Bid: Caspase-8 cleavage and interaction with the membrane bilayer. We found a striking reversible behaviour of the dissociation-association events between the Bid fragments p15 and p7. Caspase-8 cleavage does not induce per se separation of the two Bid fragments, which remain in a stable complex resembling the full length Bid. Detergents trigger a complete dissociation, which can be fully reversed by detergent removal in a range of protein concentrations from 100 μM down to 500 nM. Incubation of cBid with cardiolipin-containing liposomes leads to partial dissociation of the complex. Only p15 (tBid) fragments are found at the membrane, while p7 shows no tendency to interact with the bilayer, but complete removal of p7 strongly increases the propensity of tBid to become membrane-associated. Despite the striking structural similarities of inactive Bid and Bax, Bid does not form oligomers and reacts differently in the presence of detergents and membranes, highlighting clear differences in the modes of action of the two proteins. The partial dissociation of cBid triggered by the membrane is suggested to depend on the strong and specific interaction between p15 and p7. The reversible disassembly and re-assembly of the cBid molecules at the membrane was as well proven by EPR using spin labeled cBid in the presence of isolated mitochondria. The observed dynamic dissociation of the two Bid fragments could allow the assistance to the pore-forming Bax to occur repeatedly and may explain the proposed "hit-and-run" mode of action of Bid at the bilayer.     

Structural Insights of tBid,the Caspase-8-activated Bid,and Its BH3 Domain

The Bcl-2 family proteins regulate mitochondria-mediated apoptosis through intricate molecular mechanisms. One of the pro-apoptotic proteins, tBid, can induce apoptosis by promoting Bax activation, Bax homo-oligomerization, and mitochondrial outer membrane permeabilization. Association of tBid on the mitochondrial outer membrane is key to its biological function. Therefore knowing the conformation of tBid on the membrane will be the first step toward understanding its crucial role in triggering apoptosis. Here, we present NMR characterization of the structure and dynamics of human tBid in 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] micelles. Our data showed that tBid is monomeric with six well defined α-helices in the micelles. Compared with the full-length Bid structure, a longer flexible loop between tBid helix α₄ and α₅ was observed. Helices in tBid do not pack into a compact-fold but form an extended structure with a C-shape configuration in the micelles. All six tBid helices were shown to interact with LPPG micelles, with helix α₆ and α₇ being more embedded. Of note, the BH₃-containing helix α₃, which was previously believed to be exposed above the membrane surface, is also membrane associated, suggesting an “on the membrane” binding mode for tBid interaction with Bax. Our data provided structural details on the membrane-associated state of tBid and the functional implications of its membrane-associated BH₃ domain.     

Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak

Recently, we discovered that Humanin (HN), a small endogenous peptide of 24 amino acids, binds to and inhibits the proapoptotic protein Bax. We show here that HN also interacts with the BH3-only Bcl-2/Bax family protein, Bid, as well as a truncated form of Bid (tBid) associated with protease-mediated activation of this proapoptotic protein. Synthetic HN peptide binds purified Bid and tBid in vitro and blocks tBid-induced release of cytochrome c and SMAC from isolated mitochondria, whereas mutant peptides that fail to bind Bid or tBid lack this activity. Moreover, HN peptide also retained protective activity on bax-/-mitochondria, indicating that HN can block tBid-induced release of apoptogenic proteins from these organelles in a Bax-independent manner. HN peptide inhibits tBid-induced oligomerization of Bax and Bak in mitochondrial membranes, as shown by experiments with chemical cross-linkers or gel filtration. Gene transfection experiments showed that HN (but not an inactive mutant of HN) also protects intact cells from apoptosis induced by overexpression of tBid. We conclude that Bid represents an additional cellular target of HN, and we propose that HN-mediated suppression of Bid contributes to the antiapoptotic activity of this endogenous peptide.     

Inhibition of Bid-induced apoptosis by Bcl-2. tBid insertion,Bax translocation,and Bax/Bak oligomerization suppressed

Bcl-2 family proteins are important regulators of apoptosis. They can be pro-apoptotic (e.g. Bid, Bax, and Bak) or anti-apoptotic (e.g. Bcl-2 and Bcl-x(L)). The current study examined Bid-induced apoptosis and its inhibition by Bcl-2. Transfection of Bid led to apoptosis in HeLa cells. In these cells, Bid was processed into active forms of truncated Bid or tBid. Following processing, tBid translocated to the membrane-bound organellar fraction. Bcl-2 co-transfection inhibited Bid-induced apoptosis but did not prevent Bid processing or tBid translocation. On the other hand, Bcl-2 blocked the release of mitochondrial cytochrome c in Bid-transfected cells, suggesting actions at the mitochondrial level. Alkaline treatment stripped off tBid from the membrane-bound organellar fraction of Bid plus Bcl-2-co-transfected cells, but not from cells transfected with only Bid, suggesting inhibition of tBid insertion into mitochondrial membranes by Bcl-2. Bcl-2 also prevented Bid-induced Bax translocation from cytosol to the membrane-bound organellar fraction. Finally, Bcl-2 diminished Bid-induced oligomerization of Bax and Bak within the membrane-bound organellar fraction, shown by cross-linking experiments. In conclusion, Bcl-2 inhibited Bid-induced apoptosis at the mitochondrial level by blocking cytochrome c release, without suppressing Bid processing or activation. Critical steps blocked by Bcl-2 included tBid insertion, Bax translocation, and Bax/Bak oligomerization in the mitochondrial membranes.     

Fluorescence fluctuation spectroscopy in the presence of immobile fluorophores

Fluorescence contributions from immobile sources present a challenge for fluorescence fluctuation spectroscopy (FFS) because the absence of signal fluctuations from stationary fluorophores leads to a biased analysis. This is especially of concern for cellular FFS studies on proteins that interact with immobile structures. Here we present a method that correctly analyzes FFS experiments in the presence of immobile sources by exploiting selective photobleaching of immobile fluorophores. The fluorescence decay due to photobleaching of the immobile species is modeled taking into account the nonuniform illumination volume. The experimentally observed decay curve serves to separate the mobile and immobile fluorescence contribution, which is used to calculate the molecular brightness from the FFS data. We experimentally verify this approach in vitro using the fluorescent protein EGFP as our immobilized species and a diffusing dye of a different color as the mobile one. For this special case, we also use an alternative method of determining the brightness by spectrally resolving the two species. By conducting a dilution study, we show that the correct parameters are obtained using either technique for a wide range of mobile fractions. To demonstrate the application of our technique in living cells, we perform experiments using the histone core protein H2B fused with EGFP expressed in COS-1 cells. We successfully recovered the brightness of the mobile fraction of H2B-EGFP.     

Mcl-1 interacts with truncated Bid and inhibits its induction of cytochrome c release and its role in receptor-mediated apoptosis

Engagement of death receptors such as tumor necrosis factor-R1 and Fas brings about the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria to activate Bax/Bak, resulting in the release of cytochrome c. The mechanism underlying the activation, however, is not fully understood. Here, we have identified the anti-apoptotic Bcl-2 family member Mcl-1 as a potent tBid-binding partner. Site-directed mutagenesis reveals that the Bcl-2 homology (BH)3 domain of tBid is essential for binding to Mcl-1, whereas all three BH domains (BH1, BH2, and BH3) of Mcl-1 are required for interaction with tBid. In vitro studies using isolated mitochondria and recombinant proteins demonstrate that Mcl-1 strongly inhibits tBid-induced cytochrome c release. In addition to its ability to interact directly with Bax and Bak, tBid also binds Mcl-1 and displaces Bak from the Mcl-1-Bak complex. Importantly, overexpression of Mcl-1 confers resistance to the induction of apoptosis by both TRAIL and tumor necrosis factor-alpha in HeLa cells, whereas targeting Mcl-1 by RNA interference sensitizes HeLa cells to TRAIL-induced apoptosis. Therefore, our study demonstrates a novel regulation of tBid by Mcl-1 through protein-protein interaction in apoptotic signaling from death receptors to mitochondria.     

The pro-apoptotic Bcl-2 family member tBid localizes to mitochondrial contact sites

Background

Following cleavage by caspase 8, the C-terminus of Bid translocates from the cytosol to the mitochondria that is dependent upon structures formed by the mitochondrial-specific lipid cardiolipin. Once associated with mitochondria, truncated Bid (tBid) causes the potent release of cytochrome c, endonuclease G, and smac.

Results

We investigated whether tBid localizes specifically to the contact sites of mitochondria purported to be rich in cardiolipin. A point mutation changing the glycine at position 94 to glutamic acid in the BH3 domain of tBid (tBid_G94E) was principally used because mitochondria treated with this mutant tBid displayed better preservation of the outer membrane than those treated with wild type tBid. Additionally, tBid_G94E lowers the cytochrome c releasing activity of tBid without affecting its targeting to mitochondria. Electron microscope tomography coupled with immunogold labeling was used as a new hybrid technique to investigate the three-dimensional distributions of tBid and tBid_G94E around the mitochondrial periphery. The statistics of spatial point patterns was used to analyze the association of these proteins with contact sites.

Conclusions

Immunoelectron tomography with statistical analysis confirmed the preferential association of tBid with mitochondrial contact sites. These findings link these sites with cardiolipin in tBid targeting and suggest a role for Bcl-2 family members in regulating the activity of contact sites in relation to apoptosis. We propose a mechanism whereby Bcl-2 proteins alter mitochondrial function by disrupting cardiolipin containing contact site membranes.     

Real-time monitoring full length bid interacting with Bax during TNF-α-induced apoptosis

Bid, a member of the pro-apoptotic Bcl-2 protein family, is activated through caspase-8-mediated cleavage into a truncated form (p15 tBid) during TNF-α(tumor necrosis factor α)-induced apoptosis. Activated tBid can induce Bax oligomerization and translocation to mitochondria, triggering the release of cytochrome c, caspase-3 activation and cell apoptosis. However, it is debatable that whether Bid and tBid can interact directly with Bax in living cells. In this study, we used confocal fluorescence microscope, combined with both FRET (fluorescence resonance energy transfer) and acceptor photobleaching techniques, to study the dynamic interaction between Bid and Bax during TNF-α-induced apoptosis in single living cell. In ASTC-a-1 cells, full length Bid induced Bax translocation to mitochondria by directly interacting with Bax transiently in response to TNF-α treatment before cell shrinkage. Next, we demonstrated that, in both ASTC-a-1 and HeLa cells, Bid was not cleaved before cell shrinkage even under the condition that caspase-8 had been activated, but in MCF-7 cells Bid was cleaved. In addition, in ASTC-a-1 cells, caspase-3 activation was a biphasic process and Bid was cleaved after the second activation of caspase-3. In summary, these findings indicate that, FL-Bid (full length-Bid) directly regulated the activation of Bax during TNF-α-induced apoptosis in ASTC-a-1 cells and that the cleavage of Bid occurred in advanced apoptosis.     

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.     

Role of cardiolipin on tBid and tBid/Bax synergistic effects on yeast mitochondria

The apoptotic effector Bid regulates cell death at the level of mitochondria. Under its native state, Bid is a soluble cytosolic protein that undergoes proteolysis and yields a 15 kDa-activated form tBid (truncated Bid). tBid translocates to mitochondria and participates in cytochrome c efflux by a still unclear mechanism, some of them at least mediated by Bax. Using mitochondria isolated from wild-type and cardiolipin (CL)-synthase-less yeast strains, we observed that tBid perturbs mitochondrial bioenergetics by inhibiting state-3 respiration and ATP synthesis and that this effect was strictly dependent on the presence of CL. In a second set of experiments, heterologous coexpression of tBid and Bax in wild-type and CL-less yeast strains showed that (i) tBid binding and the subsequent alteration of mitochondrial bioenergetics increased Bax-induced cytochrome c release and (ii) the absence of CL favors Bax effects independently of the presence of t-Bid. These data support recent views suggesting a dual function of CL in mitochondria-dependent apoptosis.