Assembly of the Bak Apoptotic Pore: A CRITICAL ROLE FOR THE BAK PROTEIN α6 HELIX IN THE MULTIMERIZATION OF HOMODIMERS DURING APOPTOSIS*

Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins.     

Conformational Changes in BAK,a Pore-forming Proapoptotic Bcl-2 Family Member,upon Membrane Insertion and Direct Evidence for the Existence of BH3-BH3 Contact Interface in BAK Homo-oligomers

During apoptosis, the pro-apoptotic Bcl-2 family proteins BAK and BAX form large oligomeric pores in the mitochondrial outer membrane. Apoptotic factors, including cytochrome c, are released through these pores from the mitochondrial intermembrane space into the cytoplasm where they initiate the cascade of events leading to cell death. To better understand this pivotal step toward apoptosis, a method was developed to induce membrane permeabilization by BAK in the membrane without using the full-length protein. Using a soluble form of BAK with a hexahistidine tag at the C terminus and a liposomal system containing the Ni²⁺-nitrilotriacetic acid lipid analog that can bind hexahistidine-tagged proteins, BAK oligomers were formed in the presence of the activator protein p7/p15Bid. In this system, we determined the conformational changes in BAK upon membrane insertion by applying the site-directed spin labeling method of EPR to 13 different amino acid locations. Upon membrane insertion, the BH3 domains were reorganized, and the α5-α6 helical hairpin structure was partially exposed to the membrane environment. The monomer-monomer interface in the oligomeric structure was also mapped by measuring the distance-dependent spin-spin interactions for each residue location. Spin labels attached in the BH3 domain were juxtaposed within 5–10 Å distance in the oligomeric form in the membrane. These results are consistent with the current hypothesis that BAK or BAX forms homodimers, and these homodimers assemble into a higher order oligomeric pore. Detailed analyses of the data provide new insights into the structure of the BAX or BAK homodimer.     

Organization of the Mitochondrial Apoptotic BAK Pore: OLIGOMERIZATION OF THE BAK HOMODIMERS*

The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX “BH3-in-groove homodimer.” Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a “worm hole.”     

Bcl-2 and Bax Interact via the BH1–3 Groove-BH3 Motif Interface and a Novel Interface Involving the BH4 Motif

The interaction of Bcl-2 family proteins at the mitochondrial outer membrane controls membrane permeability and thereby the apoptotic program. The anti-apoptotic protein Bcl-2 binds to the pro-apoptotic protein Bax to prevent Bax homo-oligomerization required for membrane permeabilization. Here, we used site-specific photocross-linking to map the surfaces of Bax and Bcl-2 that interact in the hetero-complex formed in a Triton X-100 micelle as a membrane surrogate. Heterodimer-specific photoadducts were detected from multiple sites in Bax and Bcl-2. Many of the interaction sites are located in the Bcl-2 homology 3 (BH3) region of Bax and the BH1–3 groove of Bcl-2 that likely form the BH3-BH1–3 groove interface. However, other interaction sites form a second interface that includes helix 6 of Bax and the BH4 region of Bcl-2. Loss-of-function mutations in the BH3 region of Bax and the BH1 region of Bcl-2 disrupted the BH3-BH1–3 interface, as expected. Surprisingly the second interface was also disrupted by these mutations. Similarly, a loss-of-function mutation in the BH4 region of Bcl-2 that forms part of the second interface also disrupted both interfaces. As expected, both kinds of mutation abolished Bcl-2-mediated inhibition of Bax oligomerization in detergent micelles. Therefore, Bcl-2 binds Bax through two interdependent interfaces to inhibit the pro-apoptotic oligomerization of Bax.     

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.     

BH3 domains other than Bim and Bid can directly activate Bax/Bak

Bcl-2 family proteins regulate a critical step in apoptosis referred to as mitochondrial outer membrane permeabilization (MOMP). Members of a subgroup of the Bcl-2 family, known as the BH3-only proteins, activate pro-apoptotic effectors (Bax and Bak) to initiate MOMP. They do so by neutralizing pro-survival Bcl-2 proteins and/or directly activating Bax/Bak. Bim and Bid are reported to be direct activators; however, here we show that BH3 peptides other than Bim and Bid exhibited various degrees of direct activation of the effector Bax or Bak, including Bmf and Noxa BH3s. In the absence of potent direct activators, such as Bim and Bid, we unmasked novel direct activator BH3 ligands capable of inducing effector-mediated cytochrome c release and liposome permeabilization, even when both Bcl-xL- and Mcl-1-type anti-apoptotic proteins were inhibited. The ability of these weaker direct activator BH3 peptides to cause MOMP correlated with that of the corresponding full-length proteins to induce apoptosis in the absence of Bim and Bid. We propose that, in certain contexts, direct activation by BH3-only proteins other than Bim and Bid may significantly contribute to MOMP and apoptosis.     

Three-dimensional structure of Bax-mediated pores in membrane bilayers

B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) is a member of the Bcl-2 protein family having a pivotal role in triggering cell commitment to apoptosis. Bax is latent and monomeric in the cytosol but transforms into its lethal, mitochondria-embedded oligomeric form in response to cell stress, leading to the release of apoptogenic factors such as cytochrome C. Here, we dissected the structural correlates of Bax membrane insertion while oligomerization is halted. This strategy was enabled through the use of nanometer-scale phospholipid bilayer islands (nanodiscs) the size of which restricts the reconstituted system to single Bax-molecule activity. Using this minimal reconstituted system, we captured structural correlates that precede Bax homo-oligomerization elucidating previously inaccessible steps of the core molecular mechanism by which Bcl-2 family proteins regulate membrane permeabilization. We observe that, in the presence of BH3 interacting domain death agonist (Bid) BH3 peptide, Bax monomers induce the formation of ∼3.5-nm diameter pores and significantly distort the phospholipid bilayer. These pores are compatible with promoting release of ions as well as proteinaceous components, suggesting that membrane-integrated Bax monomers in the presence of Bid BH3 peptides are key functional units for the activation of the cell demolition machinery.     

Mechanistic Issues of the Interaction of the Hairpin-Forming Domain of tBid with Mitochondrial Cardiolipin

Background

The pro-apoptotic effector Bid induces mitochondrial apoptosis in synergy with Bax and Bak. In response to death receptors activation, Bid is cleaved by caspase-8 into its active form, tBid (truncated Bid), which then translocates to the mitochondria to trigger cytochrome c release and subsequent apoptosis. Accumulating evidence now indicate that the binding of tBid initiates an ordered sequences of events that prime mitochondria from the action of Bax and Bak: (1) tBid interacts with mitochondria via a specific binding to cardiolipin (CL) and immediately disturbs mitochondrial structure and function idependently of its BH3 domain; (2) Then, tBid activates through its BH3 domain Bax and/or Bak and induces their subsequent oligomerization in mitochondrial membranes. To date, the underlying mechanism responsible for targeting tBid to mitochondria and disrupting mitochondrial bioenergetics has yet be elucidated.

Principal Findings

The present study investigates the mechanism by which tBid interacts with mitochondria issued from mouse hepatocytes and perturbs mitochondrial function. We show here that the helix αH6 is responsible for targeting tBid to mitochondrial CL and disrupting mitochondrial bioenergetics. In particular, αH6 interacts with mitochondria through electrostatic interactions involving the lysines 157 and 158 and induces an inhibition of state-3 respiration and an uncoupling of state-4 respiration. These changes may represent a key event that primes mitochondria for the action of Bax and Bak. In addition, we also demonstrate that tBid required its helix αH6 to efficiently induce cytochrome c release and apoptosis.

Conclusions

Our findings provide new insights into the mechanism of action of tBid, and particularly emphasize the importance of the interaction of the helix αH6 with CL for both mitochondrial targeting and pro-apoptotic activity of tBid. These support the notion that tBid acts as a bifunctional molecule: first, it binds to mitochondrial CL via its helix αH6 and destabilizes mitochondrial structure and function, and then it promotes through its BH3 domain the activation and oligomerization of Bax and/or Bak, leading to cytochrome c release and execution of apoptosis. Our findings also imply an active role of the membrane in modulating the interactions between Bcl-2 proteins that has so far been underestimated.     

The first alpha helix of Bax plays a necessary role in its ligand-induced activation by the BH3-only proteins Bid and PUMA

The mechanism by which some BH3-only proteins of the Bcl-2 family directly activate the "multidomain" proapoptotic member Bax is poorly characterized. We report that the first alpha helix (Halpha1) of Bax specifically interacts with the BH3 domains of Bid and PUMA but not with that of Bad. Inhibition of this interaction, by a peptide comprising Halpha1 or by a mutation in this helix, prevents ligand-induced activation of Bax by Bid, PUMA, or their BH3 peptides. Halpha1-mutated Bax, which can mediate death induced by Bad or its BH3 peptide, does not mediate that induced by Bid, PUMA, or their BH3 peptides. The response of Halpha1-mutated Bax to Bid can be restored by a compensating mutation in Bid BH3. Thus, a specific interaction between Bax Halpha1 and their BH3 domains allows Bid and PUMA to function as "death agonists" of Bax, whereas Bad recruits Bax activity through a distinct pathway.     

Bax activation and mitochondrial insertion during apoptosis

The mitochondrial apoptotic pathway is a highly regulated biological mechanism which determines cell fate. It is defined as a cascade of events, going from an apoptotic stimulus to the MOM permeabilization, resulting in the activation of the so-called executive phase. This pathway is very often altered in cancer cells.The mitochondrial permeabilization is under the control of the Bcl-2 family of proteins (pBcls). These proteins share one to four homology domains (designed BH1-4) with Bcl-2, and are susceptible of homo- and/or hetero-dimerization. In spite of a poor amino-acid sequence homology, these proteins exhibit very similar tertiary structures. Strikingly, while some of these proteins are anti-apoptotic, the others are pro-apoptotic. Pro-apoptotic proteins are further divided in two sub-classes: multi-domains proteins, among which Bax and Bak, which exhibit BH1-3 domains, and BH3-only proteins (or BOPs). Schematically, BOPs and anti-apoptotic proteins antagonistically regulate the activation of the multi-domain proteins Bax and Bak and their oligomerization in the MOM, the latter process being responsible for the apoptotic mitochondrial permeabilization.Considering the critical role of Bax in cancer cells apoptosis, we focus in this review on the molecular events of Bax activation through its interaction with the other proteins from the Bcl-2 family. The mechanism by which Bax triggers the MOM permeabilization once activated will be discussed in some other reviews in this special issue.     

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.     

Integration and Oligomerization of Bax Protein in Lipid Bilayers Characterized by Single Molecule Fluorescence Study

Bax is a pro-apoptotic Bcl-2 family protein. The activated Bax translocates to mitochondria, where it forms pore and permeabilizes the mitochondrial outer membrane. This process requires the BH3-only activator protein (i.e. tBid) and can be inhibited by anti-apoptotic Bcl-2 family proteins such as Bcl-x_L. Here by using single molecule fluorescence techniques, we studied the integration and oligomerization of Bax in lipid bilayers. Our study revealed that Bax can bind to lipid membrane spontaneously in the absence of tBid. The Bax pore formation undergoes at least two steps: pre-pore formation and membrane insertion. The activated Bax triggered by tBid or BH3 domain peptide integrates on bilayers and tends to form tetramers, which are termed as pre-pore. Subsequent insertion of the pre-pore into membrane is highly dependent on the composition of cardiolipin in lipid bilayers. Bcl-x_L can translocate Bax from membrane to solution and inhibit the pore formation. The study of Bax integration and oligomerization at the single molecule level provides new evidences that may help elucidate the pore formation of Bax and its regulatory mechanism in apoptosis.     

The use of a neutral peptide aptamer scaffold to anchor BH3 peptides constitutes a viable approach to studying their function

The B-cell CLL/lymphoma-2 (Bcl-2) family of proteins are important regulators of the intrinsic pathway of apoptosis, and their interactions, driven by Bcl-2 homology (BH) domains, are of great interest in cancer research. Particularly, the BH3 domain is of clinical relevance, as it promotes apoptosis through activation of Bcl-2-associated x protein (Bax) and Bcl-2 antagonist killer (Bak), as well as by antagonising the anti-apoptotic Bcl-2 family members. Although investigated extensively in vitro, the study of the BH3 domain alone inside cells is more problematic because of diminished secondary structure of the unconstrained peptide and a lack of stability. In this study, we report the successful use of a novel peptide aptamer scaffold – Stefin A quadruple mutant – to anchor and present the BH3 domains from Bcl-2-interacting mediator of cell death (Bim), p53 upregulated modulator of apoptosis (Puma), Bcl-2-associated death promoter (Bad) and Noxa, and demonstrate its usefulness in the study of the BH3 domains in vivo. When expressed intracellularly, anchored BH3 peptides exhibit much the same binding specificities previously established in vitro, however, we find that, at endogenous expression levels, Bcl-2 does not bind to any of the anchored BH3 domains tested. Nonetheless, when expressed inside cells the anchored PUMA and Bim BH3 α-helices powerfully induce cell death in the absence of efficient targeting to the mitochondrial membrane, whereas the Noxa helix requires a membrane insertion domain in order to kill Mcl-1-dependent myeloma cells. Finally, the binding of the Bim BH3 peptide to Bax was the only interaction with a pro-apoptotic effector protein observed in this study.     

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.     

The Juxtamembrane Linker of Full-length Synaptotagmin 1 Controls Oligomerization and Calcium-dependent Membrane Binding

Synaptotagmin 1 (Syt1) is the calcium sensor for synchronous neurotransmitter release. The two C2 domains of Syt1, which may mediate fusion by bridging the vesicle and plasma membranes, are connected to the vesicle membrane by a 60-residue linker. Here, we use site-directed spin labeling and a novel total internal reflection fluorescence vesicle binding assay to characterize the juxtamembrane linker and to test the ability of reconstituted full-length Syt1 to interact with opposing membrane surfaces. EPR spectroscopy demonstrates that the majority of the linker interacts with the membrane interface, thereby limiting the extension of the C2A and C2B domains into the cytoplasm. Pulse dipolar EPR spectroscopy provides evidence that purified full-length Syt1 is oligomerized in the membrane, and mutagenesis indicates that a glycine zipper/GXXXG motif within the linker helps mediate oligomerization. The total internal reflection fluorescence-based vesicle binding assay demonstrates that full-length Syt1 that is reconstituted into supported lipid bilayers will capture vesicles containing negatively charged lipid in a Ca²⁺-dependent manner. Moreover, the rate of vesicle capture increases with Syt1 density, and mutations in the GXXXG motif that inhibit oligomerization of Syt1 reduce the rate of vesicle capture. This work demonstrates that modifications within the 60-residue linker modulate both the oligomerization of Syt1 and its ability to interact with opposing bilayers. In addition to controlling its activity, the oligomerization of Syt1 may play a role in organizing proteins within the active zone of membrane fusion.     

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.     

Bax Forms an Oligomer via Separate,Yet Interdependent,Surfaces

Interactions of Bcl-2 family proteins regulate permeability of the mitochondrial outer membrane and apoptosis. In particular, Bax forms an oligomer that permeabilizes the membrane. To map the interface of the Bax oligomer we used Triton X-100 as a membrane surrogate and performed site-specific photocross-linking. Bax-specific adducts were formed through photo-reactive probes at multiple sites that can be grouped into two surfaces. The first surface overlaps with the BH1–3 groove formed by Bcl-2 Homology motif 1, 2, and 3; the second surface is a rear pocket located on the opposite side of the protein from the BH1–3 groove. Further cross-linking experiments using Bax BH3 peptides and mutants demonstrated that the two surfaces interact with their counterparts in neighboring proteins to form two separated interfaces and that interaction at the BH1–3 groove primes the rear pocket for further interaction. Therefore, Bax oligomerization proceeds through a series of interactions that occur at separate, yet allosterically, coupled interfaces.     

BH3-only activator proteins Bid and Bim are dispensable for Bak/Bax-dependent thrombocyte apoptosis induced by Bcl-xL deficiency: molecular requisites for the mitochondrial pathway to apoptosis in platelets

A pivotal step in the mitochondrial pathway of apoptosis is activation of Bak and Bax, although the molecular mechanism remains controversial. To examine whether mitochondrial apoptosis can be induced by just a lack of antiapoptotic Bcl-2-like proteins or requires direct activators of the BH3-only proteins including Bid and Bim, we studied the molecular requisites for platelet apoptosis induced by Bcl-xL deficiency. Severe thrombocytopenia induced by thrombocyte-specific Bcl-xL knock-out was fully rescued in a Bak and Bax double knock-out background but not with single knock-out of either one. In sharp contrast, deficiency of either Bid, Bim, or both did not alleviate thrombocytopenia in Bcl-xL knock-out mice. An in vitro study revealed that ABT-737, a Bad mimetic, induced platelet apoptosis in association with a conformational change of the amino terminus, translocation from the cytosol to mitochondria, and homo-oligomerization of Bax. ABT-737-induced Bax activation and apoptosis were also observed in Bid/Bim-deficient platelets. Human platelets, upon storage, underwent spontaneous apoptosis with a gradual decline of Bcl-xL expression despite a decrease in Bid and Bim expression. Apoptosis was attenuated in Bak/Bax-deficient or Bcl-xL-overexpressing platelets but not in Bid/Bim-deficient platelets upon storage. In conclusion, platelet lifespan is regulated by a fine balance between anti- and proapoptotic multidomain Bcl-2 family proteins. Despite residing in platelets, BH3-only activator proteins Bid and Bim are dispensable for Bax activation and mitochondrial apoptosis.     

In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins

Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak.The regulated elimination of cells by apoptosis is a key mechanism of development, tissue homeostasis and defense. In vertebrates, apoptosis is regulated through two pathways, the death receptor-mediated (extrinsic) and the mitochondrial (intrinsic) pathway, which is activated by numerous apoptotic stimuli. Mitochondrial apoptosis is characterized by loss of mitochondrial outer membrane integrity and the release of mitochondrial intermembrane space proteins, most notably cytochrome c, which leads to the activation of the caspase-9 and effector caspases.¹Release of cytochrome c is governed by proteins of the B-cell lymphoma 2 (Bcl-2) family.² The Bcl-2 family consists of three groups, whose expression and interaction decide cell survival. The anti-apoptotic Bcl-2 proteins include Bcl-2, Bcl-X_L (B-cell lymphoma-extra large), Bcl-w (Bcl-2-like protein 2), Mcl-1 (myeloid cell leukemia sequence 1) and A1 (Bcl-2-related protein A1). The pro-apoptotic group of BH3-only proteins (containing a BH3-domain: Bim (Bcl-2-interacting mediator of cell death), Bid (BH3-interacting domain death agonist), Puma (p53-upregulated modulator of apoptosis), Noxa (Phorbol-12-myristate-13-acetate-induced protein 1), Bad (Bcl-2-associated death promoter), Bik (Bcl-2-interacting killer) and Hrk (activator of apoptosis hara-kiri)) activate the pro-apoptotic effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak). Bax and Bak can replace each other in most situations, but the presence of one of them is required for mitochondrial apoptosis. Upon activation Bax and Bak form oligomers in the outer mitochondrial membrane and cause the release of cytochrome c. How Bax and Bak are activated is still under debate. Different activation models have been proposed and investigated.According to the direct activation model BH3-only proteins can directly, by physical interaction activate Bax and Bak.³ The model was derived in studies investigating synthetic BH3-domain peptides in in vitro systems, that is, isolated mitochondria or liposomes, where peptides encompassing the BH3-domains of Bim or Bid (‘activator'' BH3-only proteins) were able to activate Bax. Peptides derived from the BH3-only proteins Bad, Bik, Hrk, Noxa or Puma did not activate Bax directly. However, these peptides can bind to anti-apoptotic Bcl-2 proteins with varying preferences.⁴ As this may neutralize a combination of anti-apoptotic proteins it may facilitate Bax/Bak activation by activator BH3-only proteins. Consequently, this group of BH3-only proteins has been named ‘sensitizer'' or ‘derepressor'' BH3-only proteins.^{3, 5, 6, 7} The direct activation model has received recent support by structural studies of activator BH3-domains bound to Bax.⁸ That study also found that the BH3-only peptides used previously lacked a residue that is important in the activation of Bax, and the previous results may have to be reconsidered. Indeed, a recent study illustrates that placing the BH3-domain from the various BH3-only proteins into intact Bid protein enhances Bax/Bak-activating capacity of the BH3-domains of Bid, Bim, Puma, Bmf (Bcl-2-modifying factor), Bik and Hrk.⁹The displacement (or indirect activation) model on the other hand posits that Bax and Bak are held in check by anti-apoptotic Bcl-2 proteins and auto-activate when this interaction is broken by BH3-only proteins (displacement). BH3-only proteins can bind to anti-apoptotic Bcl-2 proteins and upon apoptotic stimulation may cause the displacement of these proteins from Bax and Bak, which may lead to the activation of effectors. BH3-peptides derived from Bim and Puma can bind to all anti-apoptotic Bcl-2 proteins and its corresponding proteins exert killing upon overexpression, whereas Bad, Bmf, Bid, Bik, Hrk and Noxa display binding patterns restricted to certain anti-apoptotic Bcl-2 proteins.⁴ It was therefore suggested that Bax/Bak activation requires the neutralization/displacement of several anti-apoptotic proteins, which may be achieved by one BH3-only protein with broadly binding characteristics (such as Bim) or by the combination of BH3-only proteins with restricted binding capabilities (for instance Bad plus Noxa).^{10, 11}The models have been further refined; the ‘embedded together'' model additionally considers the dynamic interaction of the proteins with the mitochondrial membrane,¹² and it has been proposed that the models can be unified by taking two ‘modes'' of inhibition into account: anti-apoptotic Bcl-2 proteins have a dual function in inactivating both, BH3-only proteins and effectors. Pro-apoptotic signals cause the release of activator BH3-only proteins from sequestration with anti-apoptotic Bcl-2 proteins. Free BH3-only proteins directly activate effectors, however, cell death may still not be initiated because the effectors are then held in check by anti-apoptotic Bcl-2 proteins. Free activator BH3-only proteins are required to activate effectors.¹³This model unifies the two above models in the sense that it incorporates aspects of both, inhibition and displacement as well as direct activation. However, the core difference between the (direct) activation and the displacement model appears to be irreconcilable: in the activation model Bax and Bak are inactive unless receiving a stimulus from BH3-only proteins whereas in the displacement model they are active unless bound to anti-apoptotic proteins. Thus, in the absence of all other proteins one model predicts that Bax/Bak are active, the other that they are inactive. Obviously they cannot be both.The direct activation model has initially been established with Bax and the displacement model with Bak. The data are very strong that Bax is activated by direct interaction with BH3-only proteins. Recombinant Bak can also be directly activated by recombinant tBid,¹⁴ and Bid/BH3-chimaeras can activate recombinant Bak missing its C terminus.⁹ However, since Bak is normally inserted into the outer mitochondrial membrane where it may be bound to numerous other Bcl-2-family members, it has been difficult directly to test activation of Bak in the physiological situation.One possibility to ‘unify'' the original models may be in a model where Bax is physiologically activated by direct activation (Bax is inactive until receiving a signal through BH3-only proteins) whereas Bak is activated indirectly (auto-activates when the inhibition by Bcl-2-like proteins is relieved). Here we test this possibility of indirect Bak activation. We targeted anti-apoptotic Bcl-2 family proteins using RNAi. In this setting, protein concentrations and conditions are physiological, which avoids some of the problems associated with overexpression or cell-free experiments. Non-malignant cells may respond differently to the loss of anti-apoptotic Bcl-2 proteins compared with tumor cells.¹⁵ In this study, using non-malignant cells, we targeted all anti-apoptotic Bcl-2 molecules in combinations of two. In the absence of apoptotic stimuli we observed that the combined loss of Bcl-X_L and Mcl-1 was sufficient to induce apoptosis. The direct activator proteins Bid, Bim and Puma were not needed. These observations provide evidence for indirect activation of Bak.     

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.