Bax Activates Endophilin B1 Oligomerization and Lipid Membrane Vesiculation

Endophilins participate in membrane scission events that occur during endocytosis and intracellular organelle biogenesis through the combined activity of an N-terminal BAR domain that interacts with membranes and a C-terminal SH3 domain that mediates protein binding. Endophilin B1 (Endo B1) was identified to bind Bax, a Bcl-2 family member that promotes apoptosis, through yeast two-hybrid protein screens. Although Endo B1 does not bind Bax in healthy cells, during apoptosis, Endo B1 interacts transiently with Bax and promotes cytochrome c release from mitochondria. To explore the molecular mechanism of action of Endo B1, we have analyzed its interaction with Bax in cell-free systems. Purified recombinant Endo B1 in solution displays a Stokes radius indicating a tetrameric quarternary structure. However, when incubated with purified Bax, it assembles into oligomers more than 4-fold greater in molecular weight. Although Endo B1 oligomerization is induced by Bax, Bax does not stably associate with the high molecular weight Endo B1 complex. Endo B1 oligomerization requires its C-terminal Src homology 3 domain and is not induced by Bcl-xL. Endo B1 combined with Bax reduces the size and changes the morphology of giant unilamellar vesicles by inducing massive vesiculation of liposomes. This activity of purified Bax protein to induce cell-free assembly of Endo B1 may reflect its activity in cells that regulates apoptosis and/or mitochondrial fusion.     

After Embedding in Membranes Antiapoptotic Bcl-XL Protein Binds Both Bcl-2 Homology Region 3 and Helix 1 of Proapoptotic Bax Protein to Inhibit Apoptotic Mitochondrial Permeabilization

Bcl-XL binds to Bax, inhibiting Bax oligomerization required for mitochondrial outer membrane permeabilization (MOMP) during apoptosis. How Bcl-XL binds to Bax in the membrane is not known. Here, we investigated the structural organization of Bcl-XL·Bax complexes formed in the MOM, including the binding interface and membrane topology, using site-specific cross-linking, compartment-specific labeling, and computational modeling. We found that one heterodimer interface is formed by a specific interaction between the Bcl-2 homology 1–3 (BH1–3) groove of Bcl-XL and the BH3 helix of Bax, as defined previously by the crystal structure of a truncated Bcl-XL protein and a Bax BH3 peptide (Protein Data Bank entry 3PL7). We also discovered a novel interface in the heterodimer formed by equivalent interactions between the helix 1 regions of Bcl-XL and Bax when their helical axes are oriented either in parallel or antiparallel. The two interfaces are located on the cytosolic side of the MOM, whereas helix 9 of Bcl-XL is embedded in the membrane together with helices 5, 6, and 9 of Bax. Formation of the helix 1·helix 1 interface partially depends on the formation of the groove·BH3 interface because point mutations in the latter interface and the addition of ABT-737, a groove-binding BH3 mimetic, blocked the formation of both interfaces. The mutations and ABT-737 also prevented Bcl-XL from inhibiting Bax oligomerization and subsequent MOMP, suggesting that the structural organization in which interactions at both interfaces contribute to the overall stability and functionality of the complex represents antiapoptotic Bcl-XL·Bax complexes in the MOM.     

Clusterin inhibits apoptosis by interacting with activated Bax

Clusterin is an enigmatic glycoprotein that is overexpressed in several human cancers such as prostate and breast cancers, and squamous cell carcinoma. Because the suppression of clusterin expression renders human cancer cells sensitive to chemotherapeutic drug-mediated apoptosis, it is currently an antisense target in clinical trials for prostate cancer. However, the molecular mechanisms by which clusterin inhibits apoptosis in human cancer cells are unknown. Here we report that intracellular clusterin inhibits apoptosis by interfering with Bax activation in mitochondria. Intriguingly, in contrast to other inhibitors of Bax, clusterin specifically interacts with conformation-altered Bax in response to chemotherapeutic drugs. This interaction impedes Bax oligomerization, which leads to the release of cytochrome c from mitochondria and caspase activation. Moreover, we also find that clusterin inhibits oncogenic c-Myc-mediated apoptosis by interacting with conformation-altered Bax. Clusterin promotes c-Myc-mediated transformation in vitro and tumour progression in vivo. Taken together, our results suggest that the elevated level of clusterin in human cancers may promote oncogenic transformation and tumour progression by interfering with Bax pro-apoptotic activities.     

Bcl-XL inhibits membrane permeabilization by competing with Bax

Although Bcl-XL and Bax are structurally similar, activated Bax forms large oligomers that permeabilize the outer mitochondrial membrane, thereby committing cells to apoptosis, whereas Bcl-XL inhibits this process. Two different models of Bcl-XL function have been proposed. In one, Bcl-XL binds to an activator, thereby preventing Bax activation. In the other, Bcl-XL binds directly to activated Bax. It has been difficult to sort out which interaction is important in cells, as all three proteins are present simultaneously. We examined the mechanism of Bax activation by tBid and its inhibition by Bcl-XL using full-length recombinant proteins and measuring permeabilization of liposomes and mitochondria in vitro. Our results demonstrate that Bcl-XL and Bax are functionally similar. Neither protein bound to membranes alone. However, the addition of tBid recruited molar excesses of either protein to membranes, indicating that tBid activates both pro- and antiapoptotic members of the Bcl-2 family. Bcl-XL competes with Bax for the activation of soluble, monomeric Bax through interaction with membranes, tBid, or t-Bid-activated Bax, thereby inhibiting Bax binding to membranes, oligomerization, and membrane permeabilization. Experiments in which individual interactions were abolished by mutagenesis indicate that both Bcl-XL–tBid and Bcl-XL–Bax binding contribute to the antiapoptotic function of Bcl-XL. By out-competing Bax for the interactions leading to membrane permeabilization, Bcl-XL ties up both tBid and Bax in nonproductive interactions and inhibits Bax binding to membranes. We propose that because Bcl-XL does not oligomerize it functions like a dominant-negative Bax in the membrane permeabilization process.     

Bcl-XL Inhibits Membrane Permeabilization by Competing with Bax

Although Bcl-XL and Bax are structurally similar, activated Bax forms large oligomers that permeabilize the outer mitochondrial membrane, thereby committing cells to apoptosis, whereas Bcl-XL inhibits this process. Two different models of Bcl-XL function have been proposed. In one, Bcl-XL binds to an activator, thereby preventing Bax activation. In the other, Bcl-XL binds directly to activated Bax. It has been difficult to sort out which interaction is important in cells, as all three proteins are present simultaneously. We examined the mechanism of Bax activation by tBid and its inhibition by Bcl-XL using full-length recombinant proteins and measuring permeabilization of liposomes and mitochondria in vitro. Our results demonstrate that Bcl-XL and Bax are functionally similar. Neither protein bound to membranes alone. However, the addition of tBid recruited molar excesses of either protein to membranes, indicating that tBid activates both pro- and antiapoptotic members of the Bcl-2 family. Bcl-XL competes with Bax for the activation of soluble, monomeric Bax through interaction with membranes, tBid, or t-Bid-activated Bax, thereby inhibiting Bax binding to membranes, oligomerization, and membrane permeabilization. Experiments in which individual interactions were abolished by mutagenesis indicate that both Bcl-XL–tBid and Bcl-XL–Bax binding contribute to the antiapoptotic function of Bcl-XL. By out-competing Bax for the interactions leading to membrane permeabilization, Bcl-XL ties up both tBid and Bax in nonproductive interactions and inhibits Bax binding to membranes. We propose that because Bcl-XL does not oligomerize it functions like a dominant-negative Bax in the membrane permeabilization process.     

E1B 19K Blocks Bax Oligomerization and Tumor Necrosis Factor Alpha-Mediated Apoptosis

Tumor necrosis factor alpha (TNF-alpha)-mediated death signaling causes the recruitment of monomeric pro- apoptotic Bax into a 500-kDa protein complex. The adenovirus Bcl-2 homologue, E1B 19K, inhibits TNF-alpha-mediated apoptosis, interacts with Bax, and blocked the formation of the 500-kDa Bax complex. TNF-alpha and truncated Bid induced Bax-Bax cross-linking, indicative of oligomerization, and E1B 19K expression during infection inhibited this TNF-alpha-mediated Bax oligomerization. TNF-alpha signaled conformation changes at the Bax amino and carboxy termini. Exposure of the Bax amino terminus facilitates E1B 19K-Bax binding, which prevented exposure of the carboxy-terminal Bax Bcl-2 homology region 2 epitope. Inhibition of Bax oligomerization by E1B 19K is an activity that bears striking similarity to the means by which bacterial immunity proteins block pore formation by bacterial toxins which have structural homology to Bax.     

Bax activation and stress-induced apoptosis delayed by the accumulation of cholesterol in mitochondrial membranes

Activation of Bax or Bak is essential for the completion of many apoptotic programmes. Under cytotoxic conditions, these proteins undergo a series of conformational rearrangements that end up with their oligomerization. We found that unlike inactive monomeric Bax, active oligomerized Bax is partially resistant to trypsin digestion, providing a convenient read out to monitor Bax activation. Using this assay, we studied how the lipid composition of membranes affects tBid-induced Bax activation in vitro with pure liposomes. We report that Bax activation is inhibited by cholesterol and by decreases in membrane fluidity. This observation was further tested in vivo using the drug U18666A, which we found increases mitochondrial cholesterol levels. When incubated with tBid, mitochondria isolated from U18666A-treated cells showed a delay in the release of Smac/Diablo and Cytochrome c, as well as in Bax oligomerization. Moreover, pre-incubation with U18666A partially protected cells from stress-induced apoptosis. As many tumours display high mitochondrial cholesterol content, inefficient Bax oligomerization might contribute to their resistance to apoptosis-inducing agents.     

Biophysical characterization of the oligomeric state of Bax and its complex formation with Bcl-XL

The overexpression of Bax, a member of the Bcl-2 family, promotes cell death and the dimerization (or oligomerization) of Bax has been shown to be important for its function. Using size-exclusion chromatography and in vitro cross-linking experiments, we demonstrated that Bax exists mainly as a large oligomer of approximately 30 monomeric units. Furthermore, several binding assays demonstrated that Bcl-XL, an anti-apoptotic member of the Bcl-2 family, can bind to the oligomeric form of Bax without requiring Bax to dissociate to monomers.     

Loss of Bif-1 suppresses Bax/Bak conformational change and mitochondrial apoptosis

Bif-1, a member of the endophilin B protein family, interacts with Bax and promotes interleukin-3 withdrawal-induced Bax conformational change and apoptosis when overexpressed in FL5.12 cells. Here, we provide evidence that Bif-1 plays a regulatory role in apoptotic activation of not only Bax but also Bak and appears to be involved in suppression of tumorigenesis. Inhibition of endogenous Bif-1 expression in HeLa cells by RNA interference abrogated the conformational change of Bax and Bak, cytochrome c release, and caspase 3 activation induced by various intrinsic death signals. Similar results were obtained in Bif-1 knockout mouse embryonic fibroblasts. While Bif-1 did not directly interact with Bak, it heterodimerized with Bax on mitochondria in intact cells, and this interaction was enhanced by apoptosis induction and preceded the Bax conformational change. Moreover, suppression of Bif-1 expression was associated with an enhanced ability of HeLa cells to form colonies in soft agar and tumors in nude mice. Taken together, these findings support the notion that Bif-1 is an important component of the mitochondrial pathway for apoptosis as a novel Bax/Bak activator, and loss of this proapoptotic molecule may contribute to tumorigenesis.     

BARD1 translocation to mitochondria correlates with Bax oligomerization, loss of mitochondrial membrane potential, and apoptosis

The breast cancer regulatory protein-1 (BRCA1)-associated RING domain 1 (BARD1) gene is mutated in a subset of breast/ovarian cancers. BARD1 functions as a heterodimer with BRCA1 in nuclear DNA repair. BARD1 also has a BRCA1-independent apoptotic activity. Here we investigated the link between cytoplasmic localization and apoptotic function of BARD1. We used immunofluorescence microscopy and deconvolution analysis to resolve BARD1 cytoplasmic staining patterns and detected endogenous BARD1 at mitochondria. BARD1 was also detected in mitochondrial cell fractions by immunoblotting. The targeting of BARD1 to mitochondria was modestly stimulated by DNA damage and did not require BRCA1 as indicated by RNA interference and peptide-competition experiments. Transiently expressed yellow fluorescence protein-BARD1 localized to mitochondria, and the targeting sequences were mapped to both the N and C terminus of BARD1. Ectopic yellow fluorescence protein-BARD1 induced apoptosis and loss of mitochondrial membrane potential in MCF-7 breast tumor cells. BARD1 apoptotic function was associated with stimulation of Bax oligomerization at mitochondria. This distinguishes it from BRCA1, which is pro-apoptotic but did not induce Bax oligomerization. The cancer-associated BARD1 splice-variant DeltaRIN (lacks the BRCA1 binding domain and ankyrin repeats) was recruited to mitochondria but did not stimulate apoptosis or alter membrane permeability. We propose that BARD1 has two main sites of action in its cellular response to DNA damage, the nucleus, where it promotes cell survival through DNA repair, and the mitochondria, where BARD1 regulates apoptosis.     

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.     

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.     

TIEG1 induces apoptosis through mitochondrial apoptotic pathway and promotes apoptosis induced by homoharringtonine and velcade

Overexpression of TGFbeta inducible early gene (TIEG1) mimics TGFbeta action and induces apoptosis. In this study, we found that TIEG1 was significantly up-regulated during apoptosis induced by homoharringtonine or velcade. Overexpression of TIEG1 could induce apoptosis in K562 cells and promote apoptosis induced by HHT or velcade. TIEG1-induced apoptosis was shown to involve Bax and Bim up-regulation, Bcl-2 and Bcl-XL down-regulation, release of cytochrome c from mitochondria into the cytosol, activation of caspase 3 and disruption of the mitochondrial membrane potential (DeltaPsim). We concluded that TIEG1 is a key regulator which induces and promotes apoptosis through the mitochondrial apoptotic pathway.     

Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation

Nutlins, the newly developed small molecule antagonists of MDM2, activate p53 and induce apoptosis in cancer cells, offering a novel strategy of chemotherapy. Recent studies have further suggested synergistic effects of nutlins with other chemotherapeutic drugs. However, it is unclear whether nutlins increase or decrease the side effects of these drugs in normal non-malignant cells or tissues. Cisplatin is a widely used chemotherapy drug, which has a major side effect of kidney injury. Here we show that Nutlin-3 protected kidney cells against cisplatin-induced apoptosis. The cytoprotective effects of Nutlin-3 were not related to its regulation of p53 or consequent gene expression during cisplatin treatment. Moreover, the protective effects were shown in MDM2-, MDM4-, or p53-deficient cells. On the other hand, Nutlin-3 suppressed mitochondrial events of apoptosis during cisplatin incubation, including Bax activation and cytochrome c release. Nutlin-3 attenuated cisplatin-induced oligomerization of Bax and Bak but not their interactions with Bcl-XL. In isolated mitochondria, Nutlin-3 inhibited cytochrome c release induced by Ca2+, Bim peptide, and recombinant tBid. Importantly, it blocked both Bax and Bak oligomerization under these conditions. Together, the results have uncovered a new pharmacological function of nutlins, i.e. suppression of Bax and Bak, two critical mediators 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.     

14-3-3 Interacts directly with and negatively regulates pro-apoptotic Bax

The Bcl-2 family of proteins comprises well characterized regulators of apoptosis, consisting of anti-apoptotic members and pro-apoptotic members. Pro-apoptotic members possessing BH1, BH2, and BH3 domains (such as Bax and Bak) act as a gateway for a variety of apoptotic signals. Bax is normally localized to the cytoplasm in an inactive form. In response to apoptotic stimuli, Bax translocates to the mitochondria and undergoes oligomerization to induce the release of apoptogenic factors such as cytochrome c, but it is still largely unknown how the mitochondrial translocation and pro-apoptotic activity of Bax is regulated. Here we report that cytoplasmic protein 14-3-3 theta binds to Bax and, upon apoptotic stimulation, releases Bax by a caspase-independent mechanism, as well as through direct cleavage of 14-3-3 theta by caspases. Unlike Bad, the interaction with 14-3-3 theta is not dependent on the phosphorylation of Bax. In isolated mitochondria, we found that 14-3-3 theta inhibited the integration of Bax and Bax-induced cytochrome c release. Bax-induced apoptosis was inhibited by overexpression of either 14-3-3 theta or its mutant (which lacked the ability to bind to various phosphorylated targets but still bound to Bax), whereas overexpression of 14-3-3 theta was unable to inhibit apoptosis induced by a Bax mutant that did not bind to 14-3-3 theta. These findings indicate that 14-3-3 theta plays a crucial role in negatively regulating the activity of Bax.     

Bax and Bcl-2 interaction in a transgenic mouse model of familial amyotrophic lateral sclerosis

It has been proposed that mutations in copper/zinc-superoxide dismutase (SOD1), the only proven cause of amyotrophic lateral sclerosis (ALS), induce the disease by a toxic property that promotes apoptosis. Consistent with this, we have demonstrated that overexpression of Bcl-2, a protein that inhibits apoptosis, attenuates neurodegeneration produced by the familial ALS-linked SOD1 mutant G93A (mSOD1). Herein, we assessed the status of key members of the Bcl-2 family in the spinal cord of transgenic mSOD1 mice at different stages of the disease. In asymptomatic transgenic mSOD1 mice, expression of Bcl-2, Bcl-XL, Bad, and Bax does not differ from that in nontransgenic mice. In contrast, in symptomatic mice, expression of Bcl-2 and Bcl-XL, which inhibit apoptosis, is reduced, whereas expression of Bad and Bax, which stimulate apoptosis, is increased. These alterations are specific to affected brain regions and are caused by the mutant and not by the normal SOD1 enzyme. Relevant to the neuroprotective effects of Bcl-2 in transgenic mSOD1 mice, overexpression of Bcl-2 increases the formation of Bcl-2:Bax heterodimers, which abolish the Bax proapoptotic property. This study demonstrates significant alterations in the expression of key members of the Bcl-2 family associated with mSOD1 deleterious effects. That these changes contribute to the neurodegenerative process in this model of ALS is supported by our observations in double transgenic mSOD1/Bcl-2 mice in which the pernicious increase of Bax is tempered by an increase in formation of Bcl-2:Bax heterodimers. Based on these findings, it may be concluded that Bcl-2 family members appear as invaluable targets for the development of new neuroprotective therapies in ALS.     

L166P mutant DJ-1 promotes cell death by dissociating Bax from mitochondrial Bcl-XL

ABSTRACT: BACKGROUND: Mutations or deletions in DJ-1/PARK7 gene are causative for recessive forms of early onset Parkinson's disease (PD). Wild-type DJ-1 has cytoprotective roles against cell death through multiple pathways. The most commonly studied mutant DJ-1(L166P) shifts its subcellular distribution to mitochondria and renders cells more susceptible to cell death under stress stimuli. We previously reported that wild-type DJ-1 binds to Bcl-XL and stabilizes it against ultraviolet B (UVB) irradiation-induced rapid degradation. However, the mechanisms by which mitochondrial DJ-1(L166P) promotes cell death under death stimuli are largely unknown. RESULTS: We show that DJ-1(L166P) is more prone to localize in mitochondria and it binds to Bcl-XL more strongly than wild-type DJ-1. In addition, UVB irradiation significantly promotes DJ-1(L166P) translocation to mitochondria and binding to Bcl-XL. DJ-1(L166P) but not wild-type DJ-1 dissociates Bax from Bcl-XL, thereby leading to Bax enrichment at outer mitochondrial membrane and promoting mitochondrial apoptosis pathway in response to UVB irradiation. CONCLUSION: Our findings suggest that wild-type DJ-1 protects cells and DJ-1(L166P) impairs cells by differentially regulating mitochondrial Bax/Bcl-XL functions.     

The vaccinia virus protein F1L interacts with Bim and inhibits activation of the pro-apoptotic protein Bax

Vaccinia virus, the prototypic member of the orthopoxvirus genus, encodes the mitochondrial-localized protein F1L that functions to protect cells from apoptotic death and inhibits cytochrome c release. We previously showed that F1L interacts with the pro-apoptotic Bcl-2 family member Bak and inhibits activation of Bak following an apoptotic stimulus (Wasilenko, S. T., Banadyga, L., Bond, D., and Barry, M. (2005) J. Virol. 79, 14031-14043). In addition to Bak, the pro-apoptotic protein Bax is also capable of initiating cytochrome c release suggesting that vaccinia virus infection could also inhibit Bax activity. Here we show that F1L inhibits the activity of the pro-apoptotic protein Bax by inhibiting oligomerization and N-terminal activation of Bax. F1L expression also inhibited the subcellular redistribution of Bax to the mitochondria and the insertion of Bax into the outer mitochondrial membrane. The ability of F1L to inhibit Bax activation does not require Bak, because F1L expression inhibited cytochrome c release and Bax activation in Bak-deficient cells. No interaction between Bax and F1L was detected during infection, suggesting that F1L functions upstream of Bax activation. Notably, F1L was capable of interacting with the BH3-only protein BimL as shown by co-immunoprecipitation, and F1L expression inhibited apoptosis induced by BimL. These studies suggest that, in addition to interacting with the pro-apoptotic protein Bak, F1L also functions to indirectly inhibit the activation of Bax, likely by interfering with the pro-apoptotic activity of BH3-only proteins such as BimL.     

Viral pro-survival proteins block separate stages in Bax activation but changes in mitochondrial ultrastructure still occur

Mitochondrial dysfunction mediated by Bax and Bak is a critical step in mammalian cell apoptosis. However, the molecular mechanism of Bax activation remains unknown and has been difficult to investigate due to its rapid and stochastic nature. It is currently unclear whether mitochondria play a passive role in the initiation of apoptosis, remaining unaffected by cell stresses until Bax and Bak are active, or whether they actively participate in Bax/Bak activation. Here, two viral proteins, E1B19K and BHRF1, are examined for their ability to block Bax activation at different steps and thereby reveal the timing of mitochondrial changes during apoptosis. We demonstrate that BHRF1 strongly inhibits Bax activation but not upstream apoptotic signaling events, while E1B19K permits initial stages of Bax activation but prevents the subsequent oligomerization of Bax that is required for mitochondrial dysfunction. In this defined system we show that changes in mitochondrial ultrastructure, characteristic of cells undergoing apoptosis, precede Bax activation and are not blocked by E1B19K and BHRF1. We suggest that the ability of mitochondria to respond to apoptotic stress prior to Bax activation indicates that these organelles may play a direct role in activating Bax.