Bcl-XL protects BimEL-induced Bax conformational change and cytochrome C release independent of interacting with Bax or BimEL

The Bcl-2 homology (BH) 3-only pro-apoptotic Bcl-2 family protein Bim plays an essential role in the mitochondrial pathway of apoptosis through activation of the BH1-3 multidomain protein Bax or Bak. To further understand how the BH3-only protein activates Bax, we provide evidence here that BimEL induces Bax conformational change and apoptosis through a Bcl-XL-suppressible but heterodimerization-independent mechanism. Substitution of the conserved leucine residue in the BH3 domain of BimEL for alanine (M1) inhibits the interaction of BimEL with Bcl-XL but does not abolish the ability of BimEL to induce Bax conformational change and apoptosis. However, removal of the C-terminal hydrophobic region from the M1 mutant (M1DeltaC) abolishes its ability to activate Bax and to induce apoptosis, although deletion of the C-terminal domain (DeltaC) alone has little if any effect on the pro-apoptotic activity of BimEL. Subcellular fractionation experiments show that the Bim mutant M1DeltaC is localized in the cytosol, indicating that both the C-terminal hydrophobic region and the BH3 domain are required for the mitochondrial targeting and pro-apoptotic activity of BimEL. Moreover, the Bcl-XL mutant (mt1), which is unable to interact with Bax and BimEL, blocks Bax conformational change and cytochrome c release induced by BimEL in intact cells and isolated mitochondria. BimEL or Bak-BH3 peptide induces Bax conformational change in vitro only under the presence of mitochondria, and the outer mitochondrial membrane fraction is sufficient for induction of Bax conformational change. Interestingly, native Bax is attached loosely on the surface of isolated mitochondria, which undergoes conformational change and insertion into mitochondrial membrane upon stimulation by BimEL, Bak-BH3 peptide, or freeze/thaw damage. Taken together, these findings indicate that BimEL may activate Bax by damaging the mitochondrial membrane structure directly, in addition to its binding and antagonizing Bcl-2/Bcl-XL function.     

Bcl-2 inhibits a Fas-induced conformational change in the Bax N terminus and Bax mitochondrial translocation

Members of the Bcl-2 family of proteins control the cellular commitment to apoptosis, although their role in Fas-induced apoptosis is ill-defined. In this report we demonstrate that activation of the Fas receptor present on a human breast epithelial cell line resulted in a conformational change in the N terminus of the pro-apoptotic protein Bax. This conformational change appeared to occur in the cytosol and precede Bax translocation to the mitochondria. Overexpression of the anti-apoptotic protein Bcl-2 inhibited both the conformational change of Bax as well as its relocalization to the mitochondria. Bcl-2 overexpression did not, however, inhibit Fas-induced cleavage of both procaspase-8 and the pro-apoptotic protein Bid, indicating that Bcl-2 functions downstream of these events. These results suggest that the mechanism by which Bcl-2 inhibits Bax mitochondrial translocation and subsequent amplification of the apoptotic cascade is not by providing a physical barrier to Bax, but rather by inhibiting an upstream event necessary for Bax conformational change.     

Bcl-2 changes conformation to inhibit Bax oligomerization

Bcl-2 inhibits apoptosis by regulating the release of cytochrome c and other proteins from mitochondria. Oligomerization of Bax promotes cell death by permeabilizing the outer mitochondrial membrane. In transfected cells and isolated mitochondria, Bcl-2, but not the inactive point mutants Bcl-2-G145A and Bcl-2-V159D, undergoes a conformation change in the mitochondrial membrane in response to apoptotic agonists such as tBid and Bax. A mutant Bcl-2 with two cysteines introduced at positions predicted to result in a disulfide bond that would inhibit the mobility of alpha5-alpha6 helices (Bcl-2-S105C/E152C) was only active in a reducing environment. Thus, Bcl-2 must change the conformation to inhibit tBid-induced oligomerization of integral membrane Bax monomers and small oligomers. The conformationally changed Bcl-2 sequesters the integral membrane form of Bax. If Bax is in excess, apoptosis resumes as Bcl-2 is consumed by the conformational change and in complexes with Bax. Thus, Bcl-2 functions as an inhibitor of mitochondrial permeabilization by changing conformation in the mitochondrial membrane to bind membrane-inserted Bax monomers and prevent productive oligomerization of Bax.     

The putative pore-forming domain of Bax regulates mitochondrial localization and interaction with Bcl-X(L)

Bax is a proapoptotic member of the Bcl-2 family of proteins which localizes to and uses mitochondria as its major site of action. Bax normally resides in the cytoplasm and translocates to mitochondria in response to apoptotic stimuli, and it promotes apoptosis in two ways: (i) by disrupting mitochondrial membrane barrier function by formation of ion-permeable pores in mitochondrial membranes and (ii) by binding to antiapoptotic Bcl-2 family proteins via its BH3 domain and inhibiting their functions. A hairpin pair of amphipathic alpha-helices (alpha5-alpha6) in Bax has been predicted to participate in membrane insertion and pore formation by Bax. We mutagenized several charged residues in the alpha5-alpha6 domain of Bax, changing them to alanine. These substitution mutants of Bax constitutively localized to mitochondria and displayed a gain-of-function phenotype when expressed in mammalian cells. Furthermore, substitution of 8 out of 10 charged residues in the alpha5-alpha6 domain of Bax resulted in a loss of cytotoxicity in yeast but a gain-of-function phenotype in mammalian cells. The enhanced function of this Bax mutant was correlated with increased binding to Bcl-X(L), through a BH3-independent mechanism. These observations reveal new functions for the alpha5-alpha6 hairpin loop of Bax: (i) regulation of mitochondrial targeting and (ii) modulation of binding to antiapoptotic Bcl-2 proteins.     

Expression and purification of full-length human Bax alpha

Bax is a proapoptotic ion channel forming protein of the Bcl-2 family. In cells the protein is found in the cytosol and in the mitochondria membrane where it presumably is involved during apoptosis in disruption of the mitochondrial membrane potential and release of cytochrome c. The protein has a hydrophobic domain at the C-terminus, which renders it a limited solubility. Thus, all studies on recombinant Bax has so far been performed on C-terminal truncated protein. We have expressed and purified the full-length human Bax alpha. The protein was expressed with a His tag at the N-terminus and purified by affinity chromatography on Ni-NTA-agarose followed by ion-exchange chromatography on Q-Sepharose. The protein was more than 98% pure on SDS-PAGE and in the presence of 1% (w/v) octyl glucoside it could be concentrated up to 0.5 mg/ml. Full-length Bax was 25-fold more efficient, compared to C-terminal truncated Bax, in forming ion channels and trigger carboxyfluorescein release from liposomes.     

BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation of Bax

During mitochondrial apoptosis, pro-apoptotic BH3-only proteins cause the translocation of cytosolic Bcl-2-associated X protein (Bax) to the outer mitochondrial membrane (OMM) where it is activated to release cytochrome c from the mitochondrial intermembrane space, but the mechanism is under dispute. We show that most BH3-only proteins are mitochondrial proteins that are imported into the OMM via a C-terminal tail-anchor domain in isolated yeast mitochondria, independently of binding to anti-apoptotic Bcl-2 proteins. This C-terminal domain acted as a classical mitochondrial targeting signal and was sufficient to direct green fluorescent protein to mitochondria in human cells. When expressed in mouse fibroblasts, these BH3-only proteins localised to mitochondria and were inserted in the OMM. The BH3-only proteins Bcl-2-interacting mediator of cell death (Bim), tBid and p53-upregulated modulator of apoptosis sensitised isolated mitochondria from Bax/Bcl-2 homologous antagonist/killer-deficient fibroblasts to cytochrome c-release by recombinant, extramitochondrial Bax. For Bim, this activity is shown to require the C-terminal-targeting signal and to be independent of binding capacity to and presence of anti-apoptotic Bcl-2 proteins. Bim further enhanced Bax-dependent killing in yeast. A model is proposed where OMM-tail-anchored BH3-only proteins permit passive 'recruitment' and catalysis-like activation of extra-mitochondrial Bax. The recognition of C-terminal membrane-insertion of BH3-only proteins will permit the development of a more detailed concept of the initiation of mitochondrial apoptosis.     

Mitochondria as the target of the pro-apoptotic protein Bax

During apoptosis, engagement of the mitochondrial pathway involves the permeabilization of the outer mitochondrial membrane (OMM), which leads to the release of cytochrome c and other apoptogenic proteins such as Smac/DIABLO, AIF, EndoG, Omi/HtraA2 and DDP/TIMM8a. OMM permeabilization depends on activation, translocation and oligomerization of multidomain Bcl-2 family proteins such as Bax or Bak. Factors involved in Bax conformational change and the function(s) of the distinct domains controlling the addressing and the insertion of Bax into mitochondria are described in this review. We also discuss our current knowledge on Bax oligomerization and on the molecular mechanisms underlying the different models accounting for OMM permeabilization during apoptosis.     

The C-terminal helix of Bcl-xL mediates Bax retrotranslocation from the mitochondria

The proapoptotic Bcl-2 protein Bax can commit a cell to apoptosis by translocation from the cytosol to the mitochondria and permeabilization of the outer mitochondrial membrane. Prosurvival Bcl-2 family members, such as Bcl-x_L, control Bax activity. Bcl-x_L recognizes Bax after a conformational change in the N-terminal segment of Bax on the mitochondria and retrotranslocates it back into the cytoplasm, stabilizing the inactive form of Bax. Here we show that Bax retrotranslocation depends on the C-terminal helix of Bcl-x_L. Deletion or substitution of this segment reduces Bax retrotranslocation and correlates with the accumulation of GFP-tagged or endogenous Bax on the mitochondria of non-apoptotic cells. Unexpectedly, the substitution of the Bcl-x_L membrane anchor by the corresponding Bax segment reverses the Bax retrotranslocation activity of Bcl-x_L, but not that of Bcl-x_L shuttling. Bax retrotranslocation depends on interaction to the Bcl-x_L membrane anchor and interaction between the Bax BH3 domain and the Bcl-x_L hydrophobic cleft. Interference with either interaction increases mitochondrial levels of endogenous Bax. In healthy cells, mitochondrial Bax does not permeabilize the outer mitochondrial membrane, but increases cell death after apoptosis induction.     

Interaction with a membrane surface triggers a reversible conformational change in Bax normally associated with induction of apoptosis

The Bcl-2 family member Bax is an apoptosis-promoting protein that normally resides in an inactive state within the cytoplasm of healthy cells. Upon induction of apoptosis by diverse stimuli, Bax undergoes a conformational change and translocates to mitochondria, where it oligomerizes and forms pores that allow the release of cytochrome c and other cytotoxic factors. Protein-protein interactions between Bax and other Bcl-2 family members are strongly implicated in Bax activation, but a compelling case has recently been made for the involvement of lipids in this process as well. Here we report that purified Bax undergoes a reversible conformational change upon incubation with lipid vesicles in the absence of other proteins. This Bax-liposome interaction does not depend on a specific lipid composition. Changes in Bax conformation were observed by immunoprecipitation with the conformation-specific antibody 6A7, circular dichroism spectroscopy, and differential scanning calorimetry. Although liposomes induced Bax to become 6A7-reactive (a feature normally associated with the onset of apoptosis), the protein did not insert into membranes, become oligomeric, or form pores, clearly indicating that other triggers are required for Bax to achieve its final pro-apoptotic state. Indeed, the lipid-induced Bax conformational change is shown to be required for tBid-induced Bax oligomerization and pore formation, putting it upstream of tBid activity in this molecular pathway to Bax activation. These data demonstrate that Bax is sensitized to activation by transient interaction with lipid membrane surfaces and provide evidence that Bax activation proceeds in a stepwise fashion, with multiple triggers and potential levels of regulation.     

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.     

The N-terminal conformation of Bax regulates cell commitment to apoptosis

The Bcl-2 protein Bax normally resides in the cytosol, but during apoptosis it translocates to mitochondria where it is responsible for releasing apoptogenic factors. Using anoikis as a model, we have shown that Bax translocation does not commit cells to apoptosis, and they can be rescued by reattachment to extracellular matrix within a specific time. Bax undergoes an N-terminal conformational change during apoptosis that has been suggested to regulate conversion from its benign, cytosolic form to the active, membrane bound pore. We now show that the Bax N-terminus regulates commitment and mitochondrial permeabilisation, but not the translocation to mitochondria. We identify Proline 13 within the N-terminus of Bax as critical for this regulation. The subcellular distribution of Proline 13 mutant Bax was identical to wild-type Bax in both healthy and apoptotic cells. However, Proline 13 mutant Bax induced rapid progression to commitment, mitochondrial permeabilisation and death. Our data identify changes in Bax controlling commitment to apoptosis that are mechanistically distinct from those controlling its subcellular localisation. Together, they indicate that multiple regulatory steps are required to activate the proapoptotic function of Bax.     

Hsp70 inhibits heat-induced apoptosis upstream of mitochondria by preventing Bax translocation

Hsp70 overexpression can protect cells from stress-induced apoptosis. Our previous observation that Hsp70 inhibits cytochrome c release in heat-stressed cells led us to examine events occurring upstream of mitochondrial disruption. In this study we examined the effects of heat shock on the proapoptotic Bcl-2 family member Bax because of its central role in regulating cytochrome c release in stressed cells. We found that heat shock caused a conformational change in Bax that leads to its translocation to mitochondria, stable membrane association, and oligomerization. All of these events were inhibited in cells that had elevated levels of Hsp70. Hsp70 did not physically interact with Bax in control or heat-shocked cells, indicating that Hsp70 acts to suppress signals leading to Bax activation. Hsp70 inhibited stress-induced JNK activation and inhibition of JNK with SP600125 or by expression of a dominant negative mutant of JNK-blocked Bax translocation as effectively as Hsp70 overexpression. Hsp70 did not protect cells expressing a mutant form of Bax that has constitutive membrane insertion capability or cells treated with a small molecule activator of apoptosome formation, indicating that it is unable to prevent cell death after mitochondrial disruption and caspase activation have occurred. These results indicate that Hsp70 blocks heat-induced apoptosis primarily by inhibiting Bax activation and thereby preventing the release of proapoptotic factors from mitochondria. Hsp70, therefore, inhibits events leading up to mitochondrial membrane permeabilization in heat-stressed cells and thereby controls the decision to die but does not interfere with cell death after this event has occurred.     

Contributions to Bax insertion and oligomerization of lipids of the mitochondrial outer membrane

Under many apoptotic conditions, Bax undergoes conformational rearrangements, leading to its insertion in the mitochondrial outer membrane as a transmembrane oligomer. At the same time, mitochondria undergo fragmentation and activated Bax was reported to localize to fission sites. We studied how lipid composition and membrane curvature regulate Bax activation. When isolated mitochondria were incubated with phospholipase A2, which led to phosphatidylethanolamine and cardiolipin hydrolysis, tBid and Bax insertion were hindered. We thus studied in liposomes how phosphatidylethanolamine, cardiolipin, and its hydrolysis products affect Bax activation. Whereas phosphatidylethanolamine, a lipid with negative curvature, did not affect Bax insertion, it inhibited Bax oligomerization. Conversely, Bax insertion required cardiolipin, and was not blocked by cardiolipin hydrolysis products. These experiments support a direct role for cardiolipin in the recruitment and activation of Bax. To examine if the increase in membrane curvature that accompanies mitochondrial fission participates in Bax activation, we studied how liposome size affects the process, and observed that it was inhibited in small liposomes (相似文献   

Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells

Bax is a Bcl-2 family protein with proapoptotic activity, which has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. In control HeLa cells, Bax is present in the cytosol and weakly associated with mitochondria as a monomer with an apparent molecular mass of 20,000 Da. After treatment of the HeLa cells with the apoptosis inducer staurosporine or UV irradiation, Bax associated with mitochondria is present as two large molecular weight oligomers/complexes of 96,000 and 260,000 Da, which are integrated into the mitochondrial membrane. Bcl-2 prevents Bax oligomerization and insertion into the mitochondrial membrane. The outer mitochondrial membrane protein voltage-dependent anion channel and the inner mitochondrial membrane protein adenosine nucleotide translocator do not coelute with the large molecular weight Bax oligomers/complexes on gel filtration. Bax oligomerization appears to be required for its proapoptotic activity, and the Bax oligomer/complex might constitute the structural entirety of the cytochrome c-conducting channel in the outer mitochondrial membrane.     

An anti-apoptotic viral protein that recruits Bax to mitochondria

The viral mitochondria-localized inhibitor of apoptosis (vMIA), encoded by the UL37 gene of human cytomegalovirus, inhibits apoptosis-associated mitochondrial membrane permeabilization by a mechanism different from that of Bcl-2. Here we show that vMIA induces several changes in Bax that resemble those found in apoptotic cells yet take place in unstimulated, non-apoptotic vMIA-expressing cells. These changes include the constitutive localization of Bax at mitochondria, where it associates tightly with the mitochondrial membrane, forming high molecular weight aggregates that contain vMIA. vMIA recruits Bax to mitochondria but delays relocation of caspase-8-activated truncated Bid-green fluorescent protein (GFP) (t-Bid-GFP) to mitochondria. The ability of vMIA and its deletion mutants to associate with Bax and to induce relocation of Bax to mitochondria correlates with their anti-apoptotic activity and with their ability to suppress mitochondrial membrane permeabilization. Taken together, our data indicate that vMIA blocks apoptosis via its interaction with Bax. vMIA neutralizes Bax by recruiting it to mitochondria and "freezing" its pro-apoptotic activity. These data unravel a novel strategy of subverting an intrinsic pathway of apoptotic signaling.     

The pleiotropic effects of heterologous Bax expression in yeast

The finding that the heterologous expression of Bcl-2 proteins in yeast elicits effects that resemble their roles in metazoan apoptosis has contributed to the increasing use of this organism as a model for the study of apoptotic regulation. The pro-apoptotic Bax protein, for example, localizes to the yeast mitochondria, where it acts to promote alterations in mitochondrial physiology and cell death, similar to its ascribed mode of action in higher organisms. These observations lead to the hypothesis that the heterologous Bcl-2 proteins impinge on conserved elements of the apoptotic machinery in yeast. We herein provide a retrospective of the studies aimed at both testing this general hypothesis and investigating the mechanisms of the Bcl-2 proteins using yeast, with a particular emphasis on Bax. We also discuss the evidence for pleiotropic roles of Bax in yeast apoptosis.     

Conformational Rearrangements in the Pro-apoptotic Protein,Bax, as It Inserts into Mitochondria: A CELLULAR DEATH SWITCH*

The B-cell lymphoma 2 (Bcl-2) family of proteins regulates the activation of apoptosis through the mitochondria pathway. Pro- and anti-apoptotic members of this family keep each other in check until the correct time to commit to apoptosis. The point of no return for this commitment is the permeabilization of the outer mitochondrial membrane. Translocation of the pro-apoptotic member, Bax, from the cytosol to the mitochondria is the molecular signature of this event. We employed a novel method to reliably detect Förster resonance energy transfer (FRET) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in Bax as this translocation occurs in live cells. In the cytosol, our FRET measurement indicated that the C-terminal helix is exposed instead of tucked away in the core of the protein. In addition fluorescence correlation spectroscopy (FCS) showed that cytosolic Bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. Cross-linking the C-terminal helix (α9) to helix α4 reduced the potential of those interactions to occur. After translocation, our FRET measurements showed that Bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the BH3 domain (helix α2) and the C-terminal helix. These findings have implications for possible contacts with other Bcl-2 proteins necessary for the regulation of apoptosis.     

Increase in the ratio of mitochondrial Bax/Bcl-XL induces Bax activation in human leukemic K562 cell line

The p53- and Bcl-2-negative leukemic K562 cell line showed resistant to DNA damage-induced Bax activation and apoptosis. The constitutive balanced ratio of Bax/Bcl-XL in K562 mitochondria allowed the formation of active Bax and cytochrome c release from mitochondria in the presence of a BH3-only protein, tBid, in a cell-free system. Bax transfection led to Bax undergoing a conformational change, translocation to mitochondria and homo-oligomerization but not apoptosis in the K562 cell line. After treatment with UV light, while Bcl-XL but not Bax translocated to mitochondria in K562, both Bax and Bcl-XL translocated to mitochondria in the Bax stable transfectant K/Bax cells. The increased ratio of Bax/Bcl-XL in K/Bax mitochondria led to an increased conformationally changed Bax, formation of the homo-multimer of Bax-Bax, and a reduced hetero-dimerization of Bax-Bcl-XL. Increased proportion of active Bax was accompanied with increased percentage of apoptosis. We therefore demonstrate that direct increase in the ratio of mitochondrial Bax/Bcl-XL can induce Bax activation in the p53- and Bcl-2-negative leukemic cells. Increased Bcl-XL translocation and failure in Bax translocation from cytosol to mitochondria play important roles in preventing Bax activation.     

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.     

tBid elicits a conformational alteration in membrane-bound Bcl-2 such that it inhibits Bax pore formation

During initiation of apoptosis, Bcl-2 family proteins regulate the permeability of mitochondrial outer membrane. BH3-only protein, tBid, activates pro-apoptotic Bax to release cytochrome c from mitochondria. tBid also activates anti-apoptotic Bcl-2 in the mitochondrial outer membrane, changing it from a single-spanning to a multispanning conformation that binds the active Bax and inhibits cytochrome c release. However, it is not known whether other mitochondrial proteins are required to elicit the tBid-induced Bcl-2 conformational alteration. To define the minimal components that are required for the functionally important Bcl-2 conformational alteration, we reconstituted the reaction using purified proteins and liposomes. We found that purified tBid was sufficient to induce a conformational alteration in the liposome-tethered, but not cytosolic Bcl-2, resulting in a multispanning form that is similar to the one found in the mitochondrial outer membrane of drug-treated cells. Mutations that abolished tBid/Bcl-2 interaction also abolished the conformational alteration, demonstrating that a direct tBid/Bcl-2 interaction at the membrane is both required and sufficient to elicit the conformational alteration. Furthermore, active Bax also elicited the Bcl-2 conformational alteration. Bcl-2 mutants that displayed increased or decreased activity in the conformational alteration assay showed corresponding activities in inhibiting pore formation by Bax in vitro and in preventing apoptosis in vivo. Thus, there is a strong correlation between the direct interaction of membrane-bound Bcl-2 and tBid with activation of Bcl-2 in vitro and in vivo.