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.     

Differential regulation of Bax and Bak by anti-apoptotic Bcl-2 family proteins Bcl-B and Mcl-1

The pro-apoptotic members of the Bcl-2 family include initiator proteins that contain only BH3 domains and downstream effector multi-BH domain-containing proteins, including Bax and Bak. In this report, we compared the ability of the six human anti-apoptotic Bcl-2 family members to suppress apoptosis induced by overexpression of Bax or Bak, correlating findings with protein interactions measured by three different methods: co-immunoprecipitation, glutathione S-transferase pulldown, and fluorescence polarization assays employing synthetic BH3 peptides from Bax and Bak. Bcl-B and Mcl-1 showed strong preferences for binding to and suppression of Bax and Bak, respectively. In contrast, the other anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-X(L), Bcl-W, and Bfl-1) suppressed apoptosis induced by overexpression of either Bax or Bak, and they displayed an ability to bind both Bax and Bak by at least one of the three protein interaction methods. Interestingly, however, full-length Bax and Bak proteins and synthetic Bax and Bak BH3 peptides exhibited discernible differences in their interactions with some anti-apoptotic members of the Bcl-2 family, cautioning against reliance on a single method for detecting protein interactions of functional significance. Altogether, the findings reveal striking distinctions in the behaviors of Bcl-B and Mcl-1 relative to the other anti-apoptotic Bcl-2 family members, where Bcl-B and Mcl-1 display reciprocal abilities to bind and neutralize Bax and Bak.     

Direct Interaction of Bax and Bak Proteins with Bcl-2 Homology Domain 3 (BH3)-only Proteins in Living Cells Revealed by Fluorescence Complementation

The key event in the mitochondrial pathway of apoptosis is the activation of Bax and Bak by BH3-only proteins through a molecular mechanism that is still a matter of debate. Here we studied interactions among anti- and proapoptotic proteins of the Bcl-2 family in living cells by using bimolecular fluorescence complementation analysis. Our results indicate that the antiapoptotic proteins Mcl-1 and Bcl-x_L bind preferably to the BH3-only proteins Bim, PUMA, and Noxa but can also bind to Bak and Bax. We also found a direct interaction between Bim, PUMA, or Noxa with either Bax or Bak during apoptosis induction. In HeLa cells, interaction of Bim with Bax occurs in cytosol, and then Bim-Bax complexes translocate to mitochondria. Complexes of either PUMA or Noxa with Bax or Bak were always detected at mitochondria. Overexpression of Bcl-x_L or Mcl-1 delayed Bim/Bax translocation to mitochondria. These results reveal the ability of main BH3-only proteins to directly activate Bax and Bak in living cells and suggest that a complex network of interactions regulate the function of Bcl-2 family members during apoptosis.     

Mitochondrial permeabilization relies on BH3 ligands engaging multiple prosurvival Bcl-2 relatives, not Bak

The Bcl-2 family regulates apoptosis by controlling mitochondrial integrity. To clarify whether its prosurvival members function by sequestering their Bcl-2 homology 3 (BH3)-only ligands or their multidomain relatives Bak and Bax, we analyzed whether four prosurvival proteins differing in their ability to bind specific BH3 peptides or Bak could protect isolated mitochondria. Most BH3 peptides could induce temperature-dependent cytochrome c release, but permeabilization was prevented by Bcl-x(L), Bcl-w, Mcl-1, or BHRF1. However, their protection correlated with the ability to bind Bak rather than the added BH3 peptide and could be overcome only by BH3 peptides that bind directly to the appropriate prosurvival member. Mitochondria protected by both Bcl-x(L)-like and Mcl-1 proteins were disrupted only by BH3 peptides that engage both. BH3-only reagents freed Bak from Bcl-x(L) and Mcl-1 in mitochondrial and cell lysates. The findings support a model for the control of apoptosis in which certain prosurvival proteins sequester Bak/Bax, and BH3-only proteins must neutralize all protective prosurvival proteins to allow Bak/Bax to induce mitochondrial disruption.     

BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and anti-apoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein Bim(S). Regulated expression of Bim(S) in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of Bim(S) showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, Bim(S) enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting.     

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.     

Mcl-1 determines the Bax dependency of Nbk/Bik-induced apoptosis

B cell lymphoma 2 (Bcl-2) homology domain 3 (BH3)–only proteins of the Bcl-2 family are important functional adaptors that link cell death signals to the activation of Bax and/or Bak. The BH3-only protein Nbk/Bik induces cell death via an entirely Bax-dependent/Bak-independent mechanism. In contrast, cell death induced by the short splice variant of Bcl-x depends on Bak but not Bax. This indicates that Bak is functional but fails to become activated by Nbk. Here, we show that binding of myeloid cell leukemia 1 (Mcl-1) to Bak persists after Nbk expression and inhibits Nbk-induced apoptosis in Bax-deficient cells. In contrast, the BH3-only protein Puma disrupts Mcl-1–Bak interaction and triggers cell death via both Bax and Bak. Targeted knockdown of Mcl-1 overcomes inhibition of Bak and allows for Bak activation by Nbk. Thus, Nbk is held in check by Mcl-1 that interferes with activation of Bak. The finding that different BH3-only proteins rely specifically on Bax, Bak, or both has important implications for the design of anticancer drugs targeting Bcl-2.     

BH3-only proteins trigger cytochrome c release, but how?

The mitochondrial apoptosis pathway has been neatly ordered. Mitochondrial apoptosis is governed by Bcl-2 family proteins, and their respective contributions determine the release of cytochrome c. It is clear that, among the Bcl-2 family, BH3-only proteins are the triggers: activation of BH3-only proteins by apoptotic stimuli initiates the process. BH3-only proteins cause cytochrome c release by activating Bax and/or Bak, and the anti-apoptotic group of Bcl-2-like proteins prevents this. However, it is curiously uncertain how BH3-only proteins activate Bax/Bak. Current models suggest that this is either through direct interaction--although this interaction is not detectable experimentally--or by the neutralisation of Bcl-2-like proteins. Here we discuss the context in which these models are placed and attempt to weigh the evidence.     

Human Bop is a novel BH3-only member of the Bcl-2 protein family

One group of Bcl-2 protein family, which shares only the BH3 domain (BH3-only), is critically involved in the regulation of programmed cell death. Herein we demonstrated a novel human BH3-only protein (designated as Bop) which could induce apoptosis in a BH3 domain-dependent manner. Further analysis indicated that Bop mainly localized to mitochondria and used its BH3 domain to contact the loop regions of voltage dependent anion channel 1 (VDAC1) in the outer mitochondrial membrane. In addition, purified Bop protein induced the loss of mitochondrial transmembrane potential (ΔΨm) and the release of cytochrome c. Furthermore, Bop used its BH3 domain to contact pro-survival Bcl-2 family members (Bcl-2, Bcl-X_L, Mcl-1, A1 and Bcl-w), which could inhibit Bop-induced apoptosis. Bop would be constrained by pro-survival Bcl-2 proteins in resting cells, because Bop became released from phosphorylated Bcl-2 induced by microtubule-interfering agent like vincristine (VCR). Indeed, knockdown experiments indicated that Bop was partially required for VCR induced cell death. Finally, Bop might need to function through Bak and Bax, likely by releasing Bak from Bcl-X_L sequestration. In conclusion, Bop may be a novel BH3-only factor that can engage with the regulatory network of Bcl-2 family members to process intrinsic apoptotic signaling.     

The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis in a Bax- and Bim-dependent manner

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.     

Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival

Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins.     

Structural biology of the Bcl-2 family and its mimicry by viral proteins

Intrinsic apoptosis in mammals is regulated by protein–protein interactions among the B-cell lymphoma-2 (Bcl-2) family. The sequences, structures and binding specificity between pro-survival Bcl-2 proteins and their pro-apoptotic Bcl-2 homology 3 motif only (BH3-only) protein antagonists are now well understood. In contrast, our understanding of the mode of action of Bax and Bak, the two necessary proteins for apoptosis is incomplete. Bax and Bak are isostructural with pro-survival Bcl-2 proteins and also interact with BH3-only proteins, albeit weakly. Two sites have been identified; the in-groove interaction analogous to the pro-survival BH3-only interaction and a site on the opposite molecular face. Interaction of Bax or Bak with activator BH3-only proteins and mitochondrial membranes triggers a series of ill-defined conformational changes initiating their oligomerization and mitochondrial outer membrane permeabilization. Many actions of the mammalian pro-survival Bcl-2 family are mimicked by viruses. By expressing proteins mimicking mammalian pro-survival Bcl-2 family proteins, viruses neutralize death-inducing members of the Bcl-2 family and evade host cell apoptosis during replication. Remarkably, structural elements are preserved in viral Bcl-2 proteins even though there is in many cases little discernible sequence conservation with their mammalian counterparts. Some viral Bcl-2 proteins are dimeric, but they have distinct structures to those observed for mammalian Bcl-2 proteins. Furthermore, viral Bcl-2 proteins modulate innate immune responses regulated by NF-κB through an interface separate from the canonical BH3-binding groove. Our increasing structural understanding of the viral Bcl-2 proteins is leading to new insights in the cellular Bcl-2 network by exploring potential alternate functional modes in the cellular context. We compare the cellular and viral Bcl-2 proteins and discuss how alterations in their structure, sequence and binding specificity lead to differences in behavior, and together with the intrinsic structural plasticity in the Bcl-2 fold enable exquisite control over critical cellular signaling pathways.     

Activation of Bax by BH3 Domains during Apoptosis:The zunfolding of a Deadly Plot

The BH3-only proteins are intracellular death ligands that are critical for initiating apoptosis. Their mode of action is dependent upon the ability of their BH3 domain to interact with pro- and anti-apoptotic multi-domain members of the Bcl-2 family. Direct agonists of the pro-apoptotic multi-domain protein Bax, and other BH3-only proteins, induce Bax activation and apoptosis by independent, yet cooperative, pathways.     

How the Bcl-2 family of proteins interact to regulate apoptosis

Commitment of cells to apoptosis is governed largely by protein-protein interactions between members of the Bcl-2 protein family. Its three sub-families have distinct roles： the BH3-only proteins trigger apoptosis by binding via their BH3 domain to pro-survival relatives, while the pro-apoptotic Bax and Bak have an essential downstream role involving disruption of organellar membranes and induction of caspase activation. The BH3-only proteins act as damage sensors, held inert until their activation by stress signals. Once activated, they were thought to bind promiscuously to pro-survival protein targets but unexpected selectivity has recently emerged from analysis of their interactions. Some BH3-only proteins also bind to Bax and Bak. Whether Bax and Bak are activated directly by these BH3-only proteins, or indirectly as a consequence of BH3-only proteins neutralizing their pro-survival targets is the subject of intense debate. Regardless of this, a detailed understanding of the interactions between family members, which are often selective, has notable implications for designing anti-cancer drugs to target the Bcl-2 family.     

The Bcl-2 Homology Domain 3 (BH3)-only Proteins Bim and Bid Are Functionally Active and Restrained by Anti-apoptotic Bcl-2 Family Proteins in Healthy Liver

An intrinsic pathway of apoptosis is regulated by the B-cell lymphoma-2 (Bcl-2) family proteins. We previously reported that a fine rheostatic balance between the anti- and pro-apoptotic multidomain Bcl-2 family proteins controls hepatocyte apoptosis in the healthy liver. The Bcl-2 homology domain 3 (BH3)-only proteins set this rheostatic balance toward apoptosis upon activation in the diseased liver. However, their involvement in healthy Bcl-2 rheostasis remains unknown. In the present study, we focused on two BH3-only proteins, Bim and Bid, and we clarified the Bcl-2 network that governs hepatocyte life and death in the healthy liver. We generated hepatocyte-specific Bcl-xL- or Mcl-1-knock-out mice, with or without disrupting Bim and/or Bid, and we examined hepatocyte apoptosis under physiological conditions. We also examined the effect of both Bid and Bim disruption on the hepatocyte apoptosis caused by the inhibition of Bcl-xL and Mcl-1. Spontaneous hepatocyte apoptosis in Bcl-xL- or Mcl-1-knock-out mice was significantly ameliorated by Bim deletion. The disruption of both Bim and Bid completely prevented hepatocyte apoptosis in Bcl-xL-knock-out mice and weakened massive hepatocyte apoptosis via the additional in vivo knockdown of mcl-1 in these mice. Finally, the hepatocyte apoptosis caused by ABT-737, which is a Bcl-xL/Bcl-2/Bcl-w inhibitor, was completely prevented in Bim/Bid double knock-out mice. The BH3-only proteins Bim and Bid are functionally active but are restrained by the anti-apoptotic Bcl-2 family proteins under physiological conditions. Hepatocyte integrity is maintained by the dynamic and well orchestrated Bcl-2 network in the healthy liver.     

Vesicular Stomatitis Virus Induces Apoptosis Primarily through Bak Rather than Bax by Inactivating Mcl-1 and Bcl-XL

Vesicular stomatitis virus (VSV) induces apoptosis via the mitochondrial pathway. The mitochondrial pathway is regulated by the Bcl-2 family of proteins, which consists of both pro- and antiapoptotic members. To determine the relative importance of the multidomain proapoptotic Bcl-2 family members Bak and Bax, HeLa cells were transfected with Bak and/or Bax small interfering RNA (siRNA) and subsequently infected with recombinant wild-type VSV. Our results showed that Bak is more important than Bax for the induction of apoptosis in this system. Bak is regulated by two antiapoptotic Bcl-2 proteins, Mcl-1, which is rapidly turned over, and Bcl-X_L, which is relatively stable. Inhibition of host gene expression by the VSV M protein resulted in the degradation of Mcl-1 but not Bcl-X_L. However, inactivation of both Mcl-1 and Bcl-X_L was required for cells to undergo apoptosis. While inactivation of Mcl-1 was due to inhibition of its expression, inactivation of Bcl-X_L indicates a role for one or more BH3-only Bcl-2 family members. VSV-induced apoptosis was inhibited by transfection with siRNA against Bid, a BH3-only protein that is normally activated by the cleavage of caspase-8, the initiator caspase associated with the death receptor pathway. Similarly, treatment with an inhibitor of caspase-8 inhibited VSV-induced apoptosis. These results indicate a role for cross talk from the death receptor pathway in the activation of the mitochondrial pathway by VSV.The induction of cell death is a major mechanism by which many viruses cause disease in the tissues they infect (23). In addition, the cytolytic activity of viruses has the potential for therapeutic applications, such as the development of oncolytic viruses for the treatment of cancer (27). Vesicular stomatitis virus (VSV) is well studied as a prototype for negative-strand RNA viruses and is an exceptionally potent inducer of apoptosis in a wide variety of cell types (4, 20, 21). Due to its particularly rapid cytopathic effects, VSV is one of the major viruses being developed as an oncolytic agent (27). VSV is capable of inducing apoptosis by activation of multiple apoptotic pathways. It is important to determine how these pathways are activated and the role that they play in apoptosis induced by VSV in order to understand the virulence and oncolytic activity of the virus, as well as to provide a model to which other viruses can be compared.Previous work showed that wild-type (wt) VSV induces apoptosis via the mitochondrial (intrinsic) pathway through the initiator caspase caspase-9 (4, 19). This is due in part to the inhibition of host gene expression by the VSV M protein (19). The inhibition of host gene expression by M protein is the mechanism by which VSV inhibits the host antiviral response (2, 31) and leads to induction of apoptosis, similar to that induced by pharmacologic inhibitors of host gene expression (19). Additionally, M protein mutants of VSV that are deficient in the ability to inhibit new host gene expression are effective inducers of apoptosis (12, 13, 19, 20). However, in contrast to wt VSV, induction of apoptosis by M protein mutant virus occurs primarily via the extrinsic pathway through the initiator caspase caspase-8 (12, 13). Infection with M protein mutant VSV results in the expression of proapoptotic genes that are suppressed during infection with wt VSV (12). Therefore, in the case of VSV with wt M protein, the induction of apoptosis is most likely mediated by proteins already present in the host cell. Since it has previously been shown that wt VSV activates the intrinsic pathway, we focused on the Bcl-2 family of proteins to determine the role of Bcl-2 family members in apoptosis induced by wt VSV.Bcl-2 family proteins function to either suppress or promote mitochondrial outer membrane permeabilization, thereby regulating the release of proapoptotic factors into the cytosol, such as cytochrome c, apoptosis-inducing factor (AIF), and Smac/Diablo (5). Bcl-2 family proteins are subdivided into three groups, depending on the conservation of Bcl-2 homology (BH) domains and function (reviewed in references 8 and 38). The multidomain antiapoptotic Bcl-2 proteins contain BH domains BH1 to BH4 and function to inhibit apoptosis by binding to proapoptotic Bcl-2 family members. Members of this group include Bcl-2, Bcl-X_L, Mcl-1, Bcl-w, and BFL-1/A1. The proapoptotic Bcl-2 proteins are comprised of two groups, the multidomain proteins and the BH3-only proteins. Bax and Bak are the two main members of the multidomain group, containing BH domains BH1 to BH3. These proteins are primarily responsible for the permeabilization of the mitochondrial outer membrane, if their activity is not suppressed by antiapoptotic Bcl-2 family members. The BH3-only proteins contain only one Bcl-2 homology domain (BH3) and include Bid, Bad, Bim, Puma, Noxa, and Bik, among others. These proteins function as upstream sensors of signaling pathways and convey to other Bcl-2 family proteins the signals to initiate apoptosis. These death signals can be transmitted from the BH3-only proteins by either binding to antiapoptotic proteins, causing the release of Bak and Bax, or binding to Bak and Bax, thereby causing their activation (6).The pathways leading to activation of Bak differ from those that activate Bax. Interestingly, only two antiapoptotic Bcl-2 proteins, Mcl-1 and Bcl-X_L, have been shown to interact with Bak, while Bax appears to be able to interact with all of the antiapoptotic proteins, with the exception of Mcl-1 (7, 35). BH3-only proteins have strong binding affinities to the antiapoptotic proteins, suggesting that their primary role may be to derepress Bak and Bax by binding and inhibiting the antiapoptotic proteins (36). In addition, BH3-only proteins may play a role in activation of Bak and Bax by binding and inducing an activated conformation (6, 34). For some stimuli, such as the protein kinase inhibitor staurosporine (SSP), the topoisomerase II inhibitor etoposide, and UV radiation, Bak and Bax appear to be redundant, in that the deletion of both is required to render cells resistant to these agents (33). In contrast, Bak and Bax were nonredundant in the induction of apoptosis by Neisseria gonorrhoeae and cisplatin, such that both were required for apoptosis to occur (18).In the experiments reported here, the silencing of Bak or Bax expression with small interfering RNA (siRNA) showed that Bak is more important than Bax for the induction of apoptosis in HeLa cells infected with wt VSV. Overexpression of both of the antiapoptotic Bcl-2 family proteins known to interact with Bak, Mcl-1 and Bcl-X_L, delayed the onset of apoptosis, while depletion of Mcl-1 or Bcl-X_L by siRNA transfection prior to infection increased the rate of apoptosis. Furthermore, M protein inhibition of new host gene expression led to the depletion of Mcl-1, enabling the rapid activation of apoptosis. However, inhibition of Bcl-X_L was also required for the initiation of apoptosis, indicating a role for one or more BH3-only proteins. Bid, a BH3-only protein that is normally activated by the cleavage of caspase-8, was shown to be important for induction of apoptosis by VSV. Likewise, treatment with an inhibitor of caspase-8 inhibited VSV-induced apoptosis. These results indicate a role for cross talk from the death receptor pathway in the activation of the mitochondrial pathway by VSV.     

BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly

Using a Bax-dependent membrane-permeabilization assay, we show that peptides corresponding to the BH3 domains of Bcl-2 family "BH3-only" proteins have dual functions. Several BH3 peptides relieved the inhibition of Bax caused by the antiapoptotic Bcl-x(L) and/or Mcl-1 proteins, some displaying a specificity for either Bcl-x(L) or Mcl-1. Besides having this derepression function, the Bid and Bim peptides activated Bax directly and were the only BH3 peptides tested that could potently induce cytochrome c release from mitochondria in cultured cells. Furthermore, Bax activator molecules (cleaved Bid protein and the Bim BH3 peptide) synergistically induced cytochrome c release when introduced into cells along with derepressor BH3 peptides. These observations support a unified model of BH3 domain function, encompassing both positive and negative regulation of other Bcl-2 family members. In this model, the simple inhibition of antiapoptotic functions is insufficient to induce apoptosis unless a direct activator of Bax or Bak is present.     

Regulation of apoptosis by BH3 domains in a cell-free system

Background: The Bcl-2 family of proteins plays a key role in the regulation of apoptosis. Some family members prevent apoptosis induced by a variety of stimuli, whereas others promote apoptosis. Competitive dimerisation between family members is thought to regulate their function. Homologous domains within individual proteins are necessary for interactions with other family members and for activity, although the specific mechanisms might differ between the pro-apoptotic and anti-apoptotic proteins.Results: Using a cell-free system based on extracts of Xenopus eggs, we have investigated the role of the Bcl-2 homology domain 3 (BH3) from different members of the Bcl-2 family. BH3 domains from the pro-apoptotic proteins Bax and Bak, but not the BH3 domain of the anti-apoptotic protein Bcl-2, induced apoptosis in this system, as determined by the rapid activation of specific apoptotic proteases (caspases) and by DNA fragmentation. The apoptosis-inducing activity of the BH3 domains requires both membrane and cytosolic fractions of cytoplasm, involves the release of cytochrome c from mitochondria and is antagonistic to Bcl-2 function. Short peptides, corresponding to the minimal sequence of BH3 domains required to bind anti-apoptotic Bcl-2 family proteins, also trigger apoptosis in this system.Conclusions: The BH3 domains of pro-apoptotic proteins are sufficient to trigger cytochrome c release, caspase activation and apoptosis. These results support a model in which pro-apoptotic proteins, such as Bax and Bak, bind to Bcl-2 via their BH3 domains, inactivating the normal ability of Bcl-2 to suppress apoptosis. The ability of synthetic peptides to reproduce the effect of pro-apoptotic BH3 domains suggests that such peptides may provide the basis for engineering reagents to control the initiation of apoptosis.     

唯BH3域蛋白与神经细胞凋亡

细胞凋亡在神经细胞的生理性和病理性死亡中起着重要作用。唯BH3域蛋白是Bcl-2家族中的一类仅含有BH3同源结构域的促凋亡分子,它们通过抑制Bcl-2抗凋亡成员的活性或激活Bax/Bak样促凋亡成员的活性来调节细胞凋亡。最近研究表明,唯BH3域蛋白在凋亡的启动及凋亡通路的沟通中发挥着极其重要的作用。