Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner

Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.Mammalian cell death proceeds through a highly regulated program called apoptosis that is highly dependent on the mitochondria.¹ Mitochondrial outer membrane (MOM) multiple apoptotic stresses permeabilize the MOM, resulting in the release of apoptogenic factors including cytochrome c, Smac, AIF, and endoG.^{2, 3, 4} Released cytochrome c activates Apaf-1, which assists in caspase activation. Then, activated caspases cleave cellular proteins and contribute to the morphological and biochemical changes associated with apoptosis. Bcl-2 family proteins control a crucial apoptosis checkpoint in the mitochondria.^{2, 5, 6, 7} Multidomain proapoptotic Bax and Bak are essential effectors responsible for the permeabilization of the MOM, whereas anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 preserve mitochondrial integrity and prevent cytochrome c efflux triggered by apoptotic stimuli. The third Bcl-2 subfamily of proteins, BH3-only molecules (BH3s), promotes apoptosis by either activating Bax/Bak or inactivating Bcl-2/Bcl-xL/Mcl-1.^{8, 9, 10, 11, 12} Upon apoptosis, the ‘activator'' BH3s, including truncated Bid (tBid), Bim, and Puma, activate Bax and Bak to mediate cytochrome c efflux, leading to caspase activation.^{8, 11, 12} Conversely, antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 sequester activator BH3s into inert complexes, which prevents Bax/Bak activation.^{8, 9} Although it has been proposed that Bax and Bak activation occurs by default as long as all of the anti-apoptotic Bcl-2 proteins are neutralized by BH3s,¹³ liposome studies clearly recapitulate the direct activation model in which tBid or BH3 domain peptides derived from Bid or Bim induce Bax or Bak oligomerization and membrane permeabilization.^{12, 14, 15}Numerous studies have demonstrated a critical role for Bax in determining tumor cell sensitivity to drug induction and in tumor development. Bax has been reported to be mutated in colon^{16, 17} and prostate cancers,^{18, 19} contributing to tumor cell survival and promoting clonal expansion. Bax has been shown to restrain tumorigenesis²⁰ and is necessary for tBid-induced cancer cell apoptosis.²¹ Loss of Bax has been reported to promote tumor development in animal models.²² Bax knockout (KO) renders HCT116 cells resistant to a series of apoptosis inducers.^{23, 24, 25} p53 has been reported to be a tumor suppressor,²⁶ and its mutant can cause chemoresistance in cancer cells.^{27, 28, 29} Moreover, p53 is often inactivated in solid tumors via deletions or point mutations.^{30, 31} Thus, it is necessary to find an efficient approach or agent to overcome chemoresistance caused by Bax and/or p53 mutants.Few studies have focused on the role of Bak in tumor cell apoptosis and cancer development. Bak mutations have only been shown in gastric and colon cancer cells.³² Some studies have revealed that Bak is a determinant of cancer cell apoptosis.^{33, 34} Some studies have even demonstrated that Bak renders Bax KO cells sensitive to drug induction.^{33, 35} In this study, we are the first group to show that tBid induces Bak activation and the release of AIF and endoG in colon cancer cells, which causes cellular apoptosis independent of Bax/p53. We also found that caspase-3 is activated in apoptosis. Interestingly, downstream caspase-3 can strengthen Bak activation and the release of AIF and endoG during apoptosis via a feedback loop. Furthermore, we reveal that Akt upregulates apoptosis progression. These results will help us to better understand the function of mitochondrial apoptotic protein members in apoptosis and cancer therapies. Furthermore, our experiments may provide a theoretical basis for overcoming chemoresistance in cancer cells.     

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.     

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore.Mitochondrial permeabilization during apoptosis is regulated by the Bcl-2 family of proteins.^{1, 2, 3} Although the Bcl-2 homology 3 (BH3)-only members such as Bid and Bim trigger apoptosis by binding to other family members, the prosurvival members block apoptosis by sequestering their pro-apoptotic relatives. Two remaining members, Bak and Bax, form the apoptotic pore within the mitochondrial outer membrane (MOM).Bak and Bax are globular proteins comprising nine α-helices.^{4, 5} They are activated by BH3-only proteins binding to the α2–α5 surface groove,^{6, 7, 8, 9, 10, 11, 12} or for Bax, to the α1/α6 ‘rear pocket''.¹³ Binding triggers dissociation of the latch domain (α6–α8) from the core domain (α2–α5), together with exposure of N-terminal epitopes and the BH3 domain.^{6, 7, 14, 15, 16} The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers.^{6, 7, 14, 17, 18} In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer.¹⁹ In Bax, the α5 and α6 helices may insert into the MOM,²⁰ although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface.^{7, 21, 22, 23} The dimers can be linked via cysteine residues placed in α6,^{18, 24, 25} and more recently via cysteine residues in either α3 or α5,^{6, 21} allowing detection of the higher-order oligomers associated with pore formation.^{26, 27} However, whether these interactions are required for high-order oligomers and pore formation remains unclear.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the MOM in healthy cells,²⁸ whereas the Bax C terminus is either exposed²⁹ or sequestered within the hydrophobic groove until apoptotic signals trigger Bax translocation.^{5, 30, 31} The hydrophobic stretch is important, as substituting polar or charged residues decreased targeting of Bak and Bax.^{10, 32} Mitochondrial targeting is also controlled by basic residues at the far C termini,^{32, 33, 34} and by interaction with VDAC2^{35, 36} via the Bak and Bax C termini.^{37, 38} Retrotranslocation of Bak and Bax was also altered by swapping the C termini.³⁹The membrane topology of the Bak and Bax C termini before and after apoptosis has not been examined directly, due in part to difficulty in reconstituting oligomers of full-length Bak in artificial membranes. Nor is it known whether the C termini contribute to pore formation by promoting oligomerization or disturbing the membrane. To address these questions synthetic peptides based on the Bak and Bax C termini have been studied in model membranes. The peptides adopt a predominantly α-helical secondary structure,^{40, 41, 42, 43} with orientation affected by lipid composition.^{42, 44, 45} The peptides could also permeabilize lipid vesicles,^{41, 43, 46, 47} suggesting that the C termini in full-length Bak and Bax may contribute to pore formation.Here we examined the membrane topology of the C termini within full-length Bak and Bax in the MOM, both before and after apoptotic pore formation. After pore formation the α9 helices of Bak (and of Bax) became juxtaposed but did not line the surface of a pore. The α9:α9 interaction occurred after Bak activation and conformation change, but was promoted by formation of BH3:groove dimers. Combining linkage at more than one interface indicated that the Bak α9:α9 interface can link BH3:groove dimers to high-order oligomers, and moreover, that the α6–α9 region is flexible in oligomerized Bak.     

Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function

In non-apoptotic cells, Bak constitutively resides in the mitochondrial outer membrane. In contrast, Bax is in a dynamic equilibrium between the cytosol and mitochondria, and is commonly predominant in the cytosol. In response to an apoptotic stimulus, Bax and Bak change conformation, leading to Bax accumulation at mitochondria and Bak/Bax oligomerization to form a pore in the mitochondrial outer membrane that is responsible for cell death. Using blue native-PAGE to investigate how Bax oligomerizes in the mitochondrial outer membrane, we observed that, like Bak, a proportion of Bax that constitutively resides at mitochondria associates with voltage-dependent anion channel (VDAC)2 prior to an apoptotic stimulus. During apoptosis, Bax dissociates from VDAC2 and homo-oligomerizes to form high molecular weight oligomers. In cells that lack VDAC2, constitutive mitochondrial localization of Bax and Bak was impaired, suggesting that VDAC2 has a role in Bax and Bak import to, or stability at, the mitochondrial outer membrane. However, following an apoptotic stimulus, Bak and Bax retained the ability to accumulate at VDAC2-deficient mitochondria and to mediate cell death. Silencing of Bak in VDAC2-deficient cells indicated that Bax required either VDAC2 or Bak in order to translocate to and oligomerize at the mitochondrial outer membrane to efficiently mediate apoptosis. In contrast, efficient Bak homo-oligomerization at the mitochondrial outer membrane and its pro-apoptotic function required neither VDAC2 nor Bax. Even a C-terminal mutant of Bax (S184L) that localizes to mitochondria did not constitutively target mitochondria deficient in VDAC2, but was recruited to mitochondria following an apoptotic stimulus dependent on Bak or upon over-expression of Bcl-x_L. Together, our data suggest that Bax localizes to the mitochondrial outer membrane via alternate mechanisms, either constitutively via an interaction with VDAC2 or after activation via interaction with Bcl-2 family proteins.Bax and Bak are the key effectors of the intrinsic apoptotic pathway initiated in response to diverse stimuli including anoikis, DNA damage and growth factor withdrawal.¹ Both proteins are normally dormant in healthy cells, but upon reception of an apoptotic stimulus, they undergo conformation change that allows their self-association to form pores in the mitochondrial outer membrane (MOM).^{2, 3, 4, 5, 6, 7} The consequence of disruption of the MOM is twofold; it impairs the ability of mitochondria to generate ATP by oxidative phosphorylation and it allows the release of intermembrane proteins including cytochrome c that agonizes caspases that dismantle the cell.Bak and Bax share significant structural homology in their inactive states and have conserved mechanism of conformation change and oligomerization.^{3, 8, 9, 10} Further, genetic studies reveal that Bak and Bax perform at least partially overlapping function, with deficiency in both necessary to perturb apoptosis during embryonic development and in response to toxic insult.^{1, 11} However, whether Bak and Bax are regulated similarly is unclear. Whereas Bak is constitutively anchored in the MOM via its hydrophobic C-terminal transmembrane domain, Bax is predominantly cytosolic in the majority of non-apoptotic cells.¹² Recent evidence indicates that Bax is in a dynamic equilibrium between cytosol and mitochondria and is constantly trafficked away from the MOM in non-apoptotic cells.^{13, 14} In response to apoptotic stress this ‘retrotranslocation'' is disrupted causing Bax to accumulate at mitochondria; a hallmark of most apoptotic cells. The mechanism governing the dynamic distribution of Bax in healthy and apoptotic cells is unclear with interactions with pro-survival proteins debated.^{13, 14}Voltage-dependent anion channels (VDACs) are the major channels responsible for ion passage across the MOM. Studies have also implicated an additional role for the VDACs in the regulation of Bak or Bax apoptotic function or potentially even constituting a component of the Bak/Bax apoptotic pore.^{15, 16, 17, 18} However, these studies have provided contrasting findings relating to whether VDACs might positively or negatively regulate Bak/Bax apoptotic function.We used blue native-PAGE (BN-PAGE) to investigate how Bax oligomerizes in the MOM during apoptosis. We observed that VDAC2 is a determinant of the constitutive association of both Bax and Bak with the MOM. The defect in Bax mitochondrial localization can be bypassed by Bak-dependent recruitment during apoptosis. Thus, our data suggest that mitochondrial localization of Bax occurs via distinct mechanisms in healthy and apoptotic cells and that either VDAC2 or Bak is required for the efficient translocation of Bax and hence for the oligomerization at the MOM and Bax apoptotic function.     

Puma strikes Bax

The commitment to programmed cell death via apoptosis is largely made upon activation of the proapoptotic mitochondrial proteins Bax or Bak. In this issue, Gallenne et al. (Gallenne, C., F. Gautier, L. Oliver, E. Hervouet, B. Noël, J.A. Hickman, O. Geneste, P.-F. Cartron, F.M. Vallette, S. Manon, and P. Juin. 2009. J. Cell Biol. 185:279–290) provide evidence that the p53 up-regulated modulator of apoptosis (Puma) protein can directly activate Bax.The Bcl-2 family of proteins participates in the control of the cell''s commitment to programmed cell death via the mitochondrial or intrinsic apoptotic pathway. Certain proteins in this family, including Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and Bfl-1/A1, inhibit apoptosis, whereas others in this family promote apoptosis. Proapoptotic Bax and Bak appear to be indispensible for apoptosis (Lindsten et al., 2000; Wei et al., 2001). How does the cell determine fate in the face of competing pro- and antiapoptotic proteins? The rheostat model proposed that when there were more antiapoptotic proteins than proapoptotic proteins, the cell survived and vice versa. However, in many cases, the conversion of a living cell to one committed to death occurs without significant change in the levels of pro- and antiapoptotic proteins. The participation of a third class of proapoptotic proteins largely explained this riddle. These proteins, so-called BH3-only as they share homology only in the proapoptotic Bcl-2 homology 3 domain, appear to act as sentinels of cell damage, which convert initial perturbations into death signals, that act in the mitochondrial pathway. Now, Gallenne et al. (see p. 279 of this issue) provide mechanistic insight into how the BH3-only protein Puma promotes apoptosis. The authors find that Puma, like the BH3-only proteins Bim and Bid, directly activates Bax.A key event in the commitment to apoptosis is Bax- and Bak-mediated permeabilization of the outer mitochondrial membrane. For this to occur, Bax and Bak alter their conformation from an inactive to an active form, form homo-oligomers in the membrane, and contribute to the formation of pores, which allows the egress of proapoptotic proteins to the cytosol (Fig. 1). Although there is consensus that Bax and Bak must shift from an inactive to an active state for this to occur, there is less consensus about what specific factors cause this crucial switch (Willis et al., 2007). Bid and Bim have been shown to cause activation (conformational change and oligomerization) of Bax and Bak in cellular, mitochondrial, and liposomal systems (Wei et al., 2000; Kuwana et al., 2002; Cartron et al., 2004; Certo et al., 2006). Direct interaction between these activators and Bax has been established experimentally (Gavathiotis et al., 2008; Lovell et al., 2008). Additional studies have suggested that p53 itself may translocate to the mitochondria and activate Bax after select stimuli (Mihara et al. 2003; Chipuk et al., 2004). Even heat has been indicted as a potential activating factor (Pagliari et al., 2005). It is quite possible that many activating factors remain to be discovered.Open in a separate windowFigure 1.Control of mitochondrial permeabilization by Bcl-2 family proteins. Activated Bax or Bak are available to oligomerize either when they are directly activated by activating factors, including activator BH3-only proteins (top), or when preactivated Bax or Bak are displaced from antiapoptotic proteins by either activator or sensitizer BH3-only proteins (bottom). Gallenne et al. (2009) provide evidence that Puma is an activator rather than a sensitizer. Oligomerized Bax or Bak participate in forming a pore that allows egress of proapoptotic factors like cytochrome c. Cytochrome c promotes formation of the apoptosome complex, which causes activation of effector caspases. These proteases cleave many key cellular proteins to bring about the apoptotic phenotype. Figure adapted with permission from the Journal of Cell Science (Brunelle, J.K., and A. Letai. 2009. J. Cell Sci. 122:437–441).Antiapoptotic proteins inhibit apoptosis by binding proapoptotic factors. In many cases, the proapoptotic factors are activator BH3-only proteins like Bid and Bim. However, in some cases, the proapoptotic factors may also include activated monomeric Bax and Bak, which are intercepted before they can oligomerize and form pores. Cells have been described in which antiapoptotic proteins are loaded with abundant prodeath proteins as being “primed for death.” Such cells are particularly sensitive to treatment with chemotherapy and antagonists of antiapoptotic proteins like ABT-737 (Certo et al., 2006; Deng et al., 2007). In most cells, the vast majority of Bax and Bak are in the inactive form, and activated Bax and Bak can be difficult to detect in the absence of toxic perturbation. Nonetheless, BH3-only molecules, which lack the ability to directly activate Bax or Bak, can cause apoptosis by competing for binding to antiapoptotic proteins (Fig. 1). If this competition frees sufficient activator proteins (or activated Bax and Bak), oligomerization of Bax and Bak ensues, committing the cell to death. Based on performance in assays on mitochondria and artificial liposomes spiked with Bax, the BH3-only family has thus been segregated into two subfamilies: the sensitizers and the activators.Where does Puma fit in? Puma was initially identified as a p53-regulated gene that was induced after DNA damage (Nakano and Vousden, 2001). It has subsequently been found that Puma is responsible for much of the proapoptotic effect of p53 induction but that Puma can also cause apoptosis in a p53-independent fashion (Jeffers et al., 2003; Villunger et al., 2003). The assignment of Puma as either a sensitizer or an activator has been somewhat contentious. The BH3 domains of BH3-only proteins are both necessary and sufficient to interact with Bcl-2 family members and seem to largely recapitulate function of the entire protein. For instance, the BH3 domains of Bid and Bim can activate Bax and Bak in liposomal or mitochondrial settings. The Puma BH3 domain lacked this function in several studies, leading many to classify Puma as a sensitizer (Kuwana et al., 2005; Certo et al., 2006). However, experiments with the full-length protein translated in vitro show an ability to activate Bax comparable with that of Bim and Bid (Kim et al., 2006).Cartron et al. (2004) has previously found that the BH3 domains of Bim and Puma but not the sensitizer Bad interact with Bax and cause its activation. In Gallenne et al. (2009), the role of Puma as an activator is further supported by three main pieces of evidence. First, Bax preincubated with the Puma BH3 peptide is more toxic to microinjected cells than is Bax alone. This enhancement is blocked by coincubation with a peptide mimicking the putative interaction site on Bax, the Hα1 C-terminal peptide. This suggests that the interaction of the Puma BH3 domain with a site on the first α helix of Bax is necessary for Puma''s enhancement of Bax killing. It is worth noting that this interaction site on Bax, first identified by this group 4 yr ago, overlaps with an interaction site of the activator Bim BH3 peptide with Bax recently demonstrated by nuclear magnetic resonance in solution (Gavathiotis et al., 2008). The fact that two groups independently identified a similar and unexpected site for interaction of activating BH3 domains with Bax lends some confidence to this finding.Additionally, because the Bcl-2 family is absent from the yeast genome, the authors exploit yeast to study Puma and Bax in a setting uncontaminated by the contribution of unmeasured Bcl-2 family proteins. Again, they find that coexpression of Puma is necessary for efficient killing by Bax. Finally, the authors investigate the participation of Puma in killing human colorectal cancer cells with ABT-737. ABT-737 is a BH3 mimetic that promotes apoptosis by binding antiapoptotic proteins and displacing select prebound prodeath proteins. Thus, ABT-737 can only kill cells that are primed with either activators or preactivated Bax or Bak. They find that ABT-737 treatment results in the freeing of Puma, which then interacts with Bax, correlating with the death of the cell. This finding suggests that Puma can play the priming function that is likely critical to sensitivity to many chemotherapeutic agents as well as ABT-737 (Deng et al., 2007). This role may be particularly important in cells in which Bim and Bid are not expressed at high levels.Some questions remain. It is not clear why several laboratories have consistently failed to observe an activating function for the BH3 domain of Puma in either mitochondrial or liposomal systems. It is possible that even if Puma can play an activating role, the efficiency of this function may vary considerably according to context and perhaps be much less in many contexts than that of Bid or Bim. In a full-length Puma protein, perhaps interactions of the Puma BH3 domain with Bax are enhanced. It is also possible that unknown posttranslational modifications of Puma or Bax, varying according to cellular context, significantly influence the ability of Puma to activate Bax. In any case, Gallenne et al. (2009) have strengthened the case for Puma as an activator so that its potential contribution to this function cannot be ignored. One must now wonder: what other activators might still be out there waiting to be discovered?     

CHCHD2 inhibits apoptosis by interacting with Bcl-x L to regulate Bax activation

Mitochondrial outer membrane permeabilization (MOMP) is a critical control point during apoptosis that results in the release of pro-apoptotic mitochondrial contents such as cytochrome c. MOMP is largely controlled by Bcl-2 family proteins such as Bax, which under various apoptotic stresses becomes activated and oligomerizes on the outer mitochondrial membrane. Bax oligomerization helps promote the diffusion of the mitochondrial contents into the cytoplasm activating the caspase cascade. In turn, Bax is regulated primarily by anti-apoptotic Bcl-2 proteins including Bcl-xL, which was recently shown to prevent Bax from accumulating at the mitochondria. However, the exact mechanisms by which Bcl-xL regulates Bax and thereby MOMP remain partially understood. In this study, we show that the small CHCH-domain-containing protein CHCHD2 binds to Bcl-xL and inhibits the mitochondrial accumulation and oligomerization of Bax. Our data show that in response to apoptotic stimuli, mitochondrial CHCHD2 decreases prior to MOMP. Furthermore, when CHCHD2 is absent from the mitochondria, the ability of Bcl-xL to inhibit Bax activation and to prevent apoptosis is attenuated, which results in increases in Bax oligomerization, MOMP and apoptosis. Collectively, our findings establish CHCHD2, a previously uncharacterized small mitochondrial protein with no known homology to the Bcl-2 family, as one of the negative regulators of mitochondria-mediated apoptosis.Apoptosis is a tightly regulated form of programmed cell death that is critical for proper embryonic development, tissue homeostasis and immune response. Aberrant regulation of apoptosis contributes to a wide range of ailments including autoimmune disorders, neurodegenerative diseases and cancer. Unlike necrotic cell death, apoptosis is a genetic program that is characterized by distinct morphological features such as membrane blebbing, chromatin condensation, DNA fragmentation and cell shrinkage.¹ In vertebrates, apoptosis can occur through two pathways: extrinsic, or receptor-mediated apoptosis, and intrinsic, or mitochondria-mediated apoptosis. Intrinsic apoptosis is induced by cellular stressors such as DNA damage, which lead to mitochondrial outer membrane permeabilization (MOMP), cytochrome c release from the mitochondrial intermembrane space, activation of cysteine proteases (caspases) and induction of apoptosis. Once MOMP occurs, cell death is thought to be inevitable. Therefore, much research has been devoted to elucidating the mechanisms and signaling pathways that govern this critical regulatory point in apoptosis.MOMP is controlled largely by the B-cell lymphoma 2 (Bcl-2) family of proteins,² all of which contain at least one of four BH (Bcl-2 homology) domains designated BH1–4. During apoptosis, the pro-apoptotic Bcl-2 proteins Bax and/or Bak become activated and oligomerize on the mitochondrial outer membrane³ increasing mitochondrial membrane permeabilization through a mechanism that is not entirely clear. Bax and Bak are activated by BH3-only Bcl-2 family proteins such as Bim, t-Bid and Puma.^{4, 5, 6, 7, 8, 9, 10, 11, 12, 13} Conversely, Bax and Bak are inhibited by pro-survival Bcl-2 family proteins such as Bcl-2, Mcl-1 and Bcl-xL.^{2, 14, 15, 16} Of the pro-survival Bcl-2 family proteins, Bcl-2 is found at the outer mitochondrial membrane, whereas Bcl-xL and Mcl-1 localize to the outer mitochondrial membrane and the mitochondrial matrix.^{17, 18} Matrix-localized Bcl-xL and Mcl-1 have been shown to promote mitochondrial respiration,¹⁹ suggesting that crosstalk exists between apoptotic pathways and other mitochondria-based biological events. Based on this recent discovery, one might reason that other mitochondrial proteins previously characterized as structural proteins or metabolism-associated enzymes could play an additional intermediate role in the regulation of apoptosis by interacting with Bcl-2 family proteins.We identified CHCHD2 in a mass spectrometry-based screen for binding partners of p32, a mitochondrial protein previously shown by our lab to bind and mediate the apoptotic effects of the tumor suppressor p14ARF.²⁰ CHCHD2 was subsequently detected in independent screens for proteins that regulate cellular metabolism and migration;^{21, 22} however, the functions of CHCHD2 remain unknown. CHCHD2 is encoded by the chchd2 gene (coiled-coil helix coiled-coil helix domain-containing 2), which spans 4921 base pairs, contains 4 exons, and is located on human chromosome 7p11.2, a chromosomal region that is often amplified in glioblastomas.²³ The protein encoded by the chchd2 gene is ubiquitously expressed²⁴ and is relatively small, as it codes for only 151 amino acids. CHCHD2 is well-conserved among different species from humans to yeast, and mouse and human CHCHD2 share 87% amino acid sequence identity (Supplementary Figures S1A and S1B). CHCHD2 contains a C-terminal CHCH (coiled-coil helix coiled-coil helix) domain, which is characterized primarily by four cysteine residues spaced 10 amino acids apart from one another (CX(9)C motif).²⁵ The function of the CHCH domain is not well understood, and the few characterized proteins that harbor this domain have diverse functions. Many CHCH domain-containing proteins localize to the mitochondrial inner membrane or the intermembrane space, including Cox12, Cox17, Cox19, Cox23, Mia40 (yeast homolog of human CHCHD4), CHCHD3 and CHCHD6. Cox17 and Cox19 aid in the assembly of the COX complex,^{26, 27} whereas Mia40/Tim40 has been shown to transport proteins into the mitochondrial intermembrane space.^{28, 29} Furthermore, CHCHD3 and CHCHD6 are essential for maintaining the integrity of mitochondrial cristae and thus mitochondrial function.^{30, 31, 32} Interestingly, a recent report has shown that CHCHD6 is regulated by DNA damage stress, and alterations in CHCHD6 expression affect the viability of breast cancer cells in response to genotoxic anticancer drugs.³²Despite advances in our understanding of how MOMP and apoptosis are regulated by the Bcl-2 family of proteins, much remains unknown with respect to the mechanisms that lead to Bax activation and oligomerization particularly concerning the roles that mitochondria-associated proteins play in the process. In this study, we characterize the small, mitochondria-localized protein CHCHD2 as a novel regulator of Bax oligomerization and apoptosis. Furthermore, we show evidence that CHCHD2 binds to Bcl-xL at the mitochondria under unstressed conditions. In response to apoptotic stimuli, CHCHD2 decreases and loses its mitochondria localization, which is accompanied by decreased Bcl-xL–Bax interaction and increased Bax homo-oligomerization and Bax–Bak hetero-oligomerization. Collectively, our results suggest that CHCHD2 negatively regulates the apoptotic cascade upstream of Bax oligomerization.     

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.     

The Fowlpox Virus BCL-2 Homologue,FPV039, Interacts with Activated Bax and a Discrete Subset of BH3-Only Proteins To Inhibit Apoptosis

Apoptosis is a potent immune barrier against viral infection, and many viruses, including poxviruses, encode proteins to overcome this defense. Interestingly, the avipoxviruses, which include fowlpox and canarypox virus, are the only poxviruses known to encode proteins with obvious Bcl-2 sequence homology. We previously characterized the fowlpox virus protein FPV039 as a Bcl-2-like antiapoptotic protein that inhibits apoptosis by interacting with and inactivating the proapoptotic cellular protein Bak. However, both Bak and Bax can independently trigger cell death. Thus, to effectively inhibit apoptosis, a number of viruses also inhibit Bax. Here we show that FPV039 inhibited apoptosis induced by Bax overexpression and prevented both the conformational activation of Bax and the subsequent formation of Bax oligomers at the mitochondria, two critical steps in the induction of apoptosis. Additionally, FPV039 interacted with activated Bax in the context of Bax overexpression and virus infection. Importantly, the ability of FPV039 to interact with active Bax and inhibit Bax activity was dependent on the structurally conserved BH3 domain of FPV039, even though this domain possesses little sequence homology to other BH3 domains. FPV039 also inhibited apoptosis induced by the BH3-only proteins, upstream activators of Bak and Bax, despite interacting detectably with only two: BimL and Bik. Collectively, our data suggest that FPV039 inhibits apoptosis by sequestering and inactivating multiple proapoptotic Bcl-2 proteins, including certain BH3-only proteins and both of the critical “gatekeepers” of apoptosis, Bak and Bax.Apoptosis is a highly conserved form of programmed cell death that plays an important role in the immune defense against pathogens. The controlled and deliberate destruction of virally infected cells comprises a potent innate immune barrier against rampant viral replication and infection. As such, many viruses, including poxviruses, encode numerous proteins that inhibit a variety of steps in the biochemical pathways that lead to cell death (29, 69).The mitochondria, and the Bcl-2 family of proteins that preside over them, serve as an important control point in the regulation of apoptosis (87). United by the presence of one to four highly conserved Bcl-2 homology (BH) domains, the Bcl-2 family regulates the integrity of the outer mitochondrial membrane (OMM) and controls the release of apoptogenic molecules from the mitochondrial intermembrane space. Bak and Bax, the two proapoptotic Bcl-2 proteins, possess BH domains 1 to 3 and, upon activation, commit the cell to death (53, 77). Whereas Bak resides constitutively at the OMM, Bax exists in an inactive form in the cytoplasm and, upon apoptotic insult, undergoes a conformational change that exposes its C-terminal transmembrane domain and results in its relocalization to the OMM (10, 34, 41, 56). The attendant exposure of the N termini of both Bak and Bax precedes Bak and Bax homooligomerization, which facilitates mitochondrial damage and, ultimately, the release of cytochrome c (3, 4, 36, 37, 76). Cytochrome c, in turn, triggers the activation of caspases, a group of cysteine proteases responsible for dismantling the apoptotic cell (59). Bak and Bax are therefore crucial for the induction of apoptosis and, because either Bak or Bax alone is sufficient to facilitate the release of cytochrome c, both must be inactivated to effectively inhibit apoptosis (53, 77, 90). The activation of Bak and Bax is counteracted by the antiapoptotic members of the Bcl-2 family, including Bcl-2, Bcl-X_L, and Mcl-1. These three proteins, which possess all four BH domains, reside at the mitochondria and prevent apoptosis by directly interacting with and inhibiting Bak and Bax or the BH3-only proteins (87). The BH3-only proteins, which possess only the BH3 domain, act as sentinels responsive to a variety of cellular stresses, including virus infection (79). Upon receipt of an apoptotic stimulus, BH3-only proteins become activated and subsequently activate Bak and Bax or inhibit the antiapoptotic function of Bcl-2, Bcl-X_L, and Mcl-1 (15). Of the eight BH3-only proteins that are directly involved in the induction of apoptosis—namely, Bim, Bid, Puma, Bik, Bmf, Bad, Noxa, and Hrk—each displays a specific and characteristic ability to bind and inhibit Bcl-2 proteins (79).Like cellular antiapoptotic Bcl-2 proteins, viral inhibitors of apoptosis have evolved especially to interfere with the activation of Bak and Bax (18, 40). For example, E1B 19K, encoded by adenovirus, and M11L, encoded by myxoma virus, bind and inactivate both Bak and Bax to inhibit apoptosis (26, 49, 65, 67, 72). Similarly, ORF125, the antiapoptotic protein encoded by the poxvirus Orf virus, also inactivates Bak and Bax, but exactly how ORF125 mediates this inactivation remains unknown (78). Although interacting with Bak and Bax is ostensibly the most direct way to prevent apoptosis, several viral antiapoptotic proteins appear to inhibit apoptosis by functioning upstream of Bak and Bax at the level of the BH3-only proteins. The vaccinia virus protein F1L, for example, interacts with Bak but not Bax, yet F1L is nonetheless capable of inactivating Bax, likely a result of F1L interacting with the BH3-only protein and Bax activator, Bim (61, 70, 74). Moreover, the Bcl-2 homolog encoded by Kaposi''s sarcoma-associated herpesvirus, and BHRF-1, encoded by Epstein-Barr virus, each interact with a specific and distinct array of BH3-only proteins, yet neither protein interacts detectably with Bak or Bax (14, 27, 44). Thus, to effectively inhibit apoptosis, it may not be necessary for viral proteins to directly target Bak and Bax but, instead, to prevent the activation of Bak and Bax by interfering with the upstream BH3-only proteins (15).Recently, our lab has shown that FPV039, encoded by fowlpox virus, localizes to the mitochondria, where it inhibits apoptosis induced by a variety of stimuli (6). Interestingly, FPV039 is the only characterized poxvirus protein that shares obvious, albeit limited, sequence homology with cellular Bcl-2 proteins (1, 6). FPV039 possesses a highly conserved BH1 and BH2 domain but lacks an obvious BH3 and BH4 domain. Importantly, however, we predicted structural homology between the Bcl-2 BH3 domain and a corresponding region in FPV039, and we validated the prediction by showing that this cryptic FPV039 BH3 domain is functionally important (6). Indeed, the ability of FPV039 to interact with the proapoptotic protein Bak is dependent on this cryptic BH3 domain (6). Thus, despite lacking sequence conservation of a highly conserved BH3 domain, FPV039 is able to interact with, and inactivate, the proapoptotic protein Bak. Nevertheless, to completely inhibit apoptosis, both Bak and Bax must be inactivated.Accordingly, we wanted to determine whether FPV039, in addition to inactivating Bak, could inactivate Bax. We report here that FPV039 inhibited Bax activity and prevented critical steps in Bax activation. FPV039 did not appear to interact with endogenous inactive Bax; however, FPV039 was able to interact with active Bax. Moreover, FPV039 inhibited apoptosis induced by the BH3-only proteins despite interacting with only BimL and Bik. Together, these data strongly suggest FPV039 inhibits apoptosis by inactivating multiple proapoptotic Bcl-2 proteins, including the critical Bak and Bax, as well as a discrete subset of BH3-only proteins.     

Crystal structure of Bax bound to the BH3 peptide of Bim identifies important contacts for interaction

The BH3-only protein Bim is a potent direct activator of the proapoptotic effector protein Bax, but the structural basis for its activity has remained poorly defined. Here we describe the crystal structure of the BimBH3 peptide bound to BaxΔC26 and structure-based mutagenesis studies. Similar to BidBH3, the BimBH3 peptide binds into the cognate surface groove of Bax using the conserved hydrophobic BH3 residues h1–h4. However, the structure and mutagenesis data show that Bim is less reliant compared with Bid on its ‘h0'' residues for activating Bax and that a single amino-acid difference between Bim and Bid encodes a fivefold difference in Bax-binding potency. Similar to the structures of BidBH3 and BaxBH3 bound to BaxΔC21, the structure of the BimBH3 complex with BaxΔC displays a cavity surrounded by Bax α1, α2, α5 and α8. Our results are consistent with a model in which binding of an activator BH3 domain to the Bax groove initiates separation of its core (α2–α5) and latch (α6–α8) domains, enabling its subsequent dimerisation and the permeabilisation of the mitochondrial outer membrane.The intrinsic pathway to apoptosis is regulated by interactions between members of three factions of the Bcl-2 protein family: the BH3-only proteins such as Bim and Bid, which initiate the process, the essential effectors Bax and Bak, and the prosurvival members, which oppose the action of both other factions.¹ The interactions between prosurvival Bcl-2 family members and BH3 peptides have been well characterised as the earliest studies with Bcl-x_L and a BakBH3 peptide.² Such complexes are readily formed in solution by incubating the C-terminally (ΔC) truncated prosurvival Bcl-2 protein with a BH3 peptide. The absence of the C-terminal segment that can anchor the Bcl-2 protein in a membrane apparently has little effect on the ensuing complex. That complex is believed to be responsible for the antiapoptotic function of Bcl-2, by sequestration of the BH3 motif either of the so-called BH3-only proteins such as Bim (''mode 1'') or of Bax or Bak (''mode 2'').³Although proapoptotic Bax and Bak have very similar three-dimensional structures to their prosurvival relatives,^{4, 5, 6} until recently^{7, 8} no structure of a complex of either Bax or Bak with a BH3 peptide had been captured, despite an accumulation of evidence that Bax and Bak could be activated directly by interaction with the BH3-only proteins Bid, Bim and possibly others.^{9, 10, 11, 12, 13}Unlike Bak, which is constitutively anchored in the mitochondrial outer membrane (MOM) via its C-terminal segment, Bax is largely cytosolic in healthy cells and accumulates at the MOM only upon a death signal.^{14, 15} There it is believed to display at least two different conformers,^{16, 17} one loosely associated with the MOM and another in which its membrane anchor (helix α9) is inserted into the MOM. In striking contrast to the antiapoptotic relatives of Bcl-2, a construct of Bax lacking its C-terminal membrane anchor, BaxΔC21, has no measurable interaction with BH3 peptides. However, in the presence of the detergent octylglucoside binding is detected by surface plasmon resonance (SPR) for the BH3 peptides of Bim, Bid, Bak and Bax itself with IC50s in the range of 0.1–1μM,^{7, 18} some 100-fold weaker compared with those measured similarly with (for example) Bcl-x_LΔC, where no detergent is required. Weaker interactions between BidBH3 or BimBH3 and BaxΔC as compared with Bcl-x_LΔC are not inconsistent with various models for the function of the Bcl-2 protein family whereby the prosurvival molecules sequester BH3 motifs with high affinity and long half-lives, but proapoptotic Bax and Bak are activated by transient (‘hit-and-run'') interactions with BH3 motifs.^{19, 20, 21}Complexes of BaxΔC21 bound to BH3 peptides from Bid and Bax have been prepared by coincubation of the protein with CHAPS and an excess of the peptides.⁷ Under these conditions, the protein undergoes a conformational change and dimerises via domain swapping of helical segments α2–α5 and α6–α8, dubbed ‘core'' and ‘latch'' domains, respectively. Although this ‘core/latch dimer'' is thought to be an in vitro artefact, its formation is diagnostic for the core and latch separation, which is required for membrane-associated Bax to dimerise via its core domains and then to permeabilise the MOM.⁷ If the latch domain is absent, as in a recombinant construct of GFP fused to Bax α2–α5, the core domain forms BH3:groove symmetric dimers,⁷ which, consistent with a wide body of evidence,^{21, 22, 23, 24, 25} are present in apoptotic pores.Previous work⁷ highlighted the importance of two hydrophobic ‘h0'' residues (Figure 1) in the peptide (I82/I83 in BidBH3) in governing Bid''s ability to activate Bax. Similar to Bid, Bim is also a potent direct activator of Bax, and the ‘h0'' amino acids in Bim are proline and glutamic acid. In the absence of a structure of BimBH3:BaxΔC, it remained unclear how these ‘h0'' residues were accommodated. Here we describe the crystal structures of BimBH3 26- and 20-mer peptides bound to BaxΔC26. Comparison with the structure of BidBH3:BaxΔC21 allows a dissection of the critical contacts between these two peptides and BaxΔC. The binding profiles of mutant BH3 peptides illustrate that BimBH3 binding to Bax is less dependent on the ‘h0'' residues compare with that in the case for BidBH3. The BimBH3 complex displays a similar cavity adjacent to Bax α1, α2, α5 and α8 as seen in the BidBH3 complex. We also describe a structure of BidBH3 bound to a BaxΔC21 mutant, I66A, which is more typical of the BH3 signature of antiapoptotic Bcl-2 family proteins^{7, 26}Open in a separate windowFigure 1BimBH3 binds BaxΔC. (a) BH3 peptide sequences used in this study, indicating the 5 hydrophobic amino-acid positions ‘h0''–‘h4''. (b) The core/latch dimer of BaxΔC26 bound to BimBH3. The two Bax polypeptides, shown here as cartoons, are coloured yellow and grey, and the two Bim peptides cyan and orange. A crystallographic dyad symmetry axis passes through the centre of this particle. (c) Structure of BimBH3:BaxΔC26 complex. The globular unit depicted comprises Bax residues 1–128 from one polypeptide and 129–166 from the other, together with the associated Bim peptide. Bax is represented by its surface and colour coded according to surface charge (blue, positive potential (4kT/e); red, negative potential (−2kT/e); calculated using the Adaptive Poisson–Boltzmann Solver.⁴¹ The trace of the Bim peptide (cyan) is shown with ‘h0'' (P144, E145), ‘h1'' (I148), ‘h2'' (L152), ‘h3'' (I155) and ‘h4'' (F159) represented as sticks. (d) Overlay of BimBH3:BaxΔC26 with BidBH3:BaxΔC21 (PDB:4BD2). Structures represented as cartoon ribbons, yellow for Bax in the Bim complex and magenta for Bax in the Bid complex. The peptides (Bim cyan and Bid blue) stand vertically in the foreground in this view (similar to Figure 1c), with their N termini at the bottom of the figure     

The ratio of Mcl-1 and Noxa determines ABT737 resistance in squamous cell carcinoma of the skin

Tumour progression and therapy resistance in squamous cell carcinoma of the skin (SCC) is strongly associated with resistance to intrinsic mitochondrial apoptosis. We thus investigated the role of various anti-apoptotic Bcl-2 proteins for apoptosis protection in SCC using the BH3 agonist ABT737 that can overcome multidomain Bcl-2 protein protection. Sensitive SCC cells underwent rapid loss of mitochondrial membrane potential (MMP), subsequent apoptosis concomitant with caspase-3 activation and an early release of mitochondria-derived cytochrome c and smac/DIABLO. In contrast, ABT737 resistance in subsets of SCC cells was not explained by XIAP, important for protection from DR-induced apoptosis in SCC. Of note, ABT737 did not prime SCC cells to DR-induced apoptosis. Interestingly, the ratio of Mcl-1 and Noxa determined sensitivity to ABT737: loss of Mcl-1 rendered resistant cells sensitive to ABT737, whereas loss of Noxa promoted resistance in sensitive cells. In line, suppression of Mcl-1 by the pan-Bcl-2 inhibitor Obatoclax or overexpression of Noxa rendered resistant SCC cells sensitive to BH3 mimetics. Our data indicate that targeting of the Mcl-1/Noxa axis is important to overcome resistance to mitochondrial apoptosis in SCC. Therefore, combination treatment of ABT737 or derivatives with Mcl-1 inhibitors, or inducers of Noxa, may represent a novel option of targeted therapy in metastatic SCC of the skin.Apoptosis is an indispensible process to maintain cellular homeostasis, in particular in highly dynamic tissues. Apoptosis can be induced by activation of death receptors (DRs; such as TRAIL-R1/R2 or cluster of differentiation 95 (CD95)) or by intrinsic disturbance of mitochondria.¹ Death ligands (DLs; TNF-related apoptosis-inducing ligand (TRAIL) or CD95L), when bound to their respective DRs, induce apoptosis by activation of procaspase-8 within the death-inducing signalling complex (DISC).² Caspase-8 activation is followed by proteolytic cleavage of caspase-3.³ Extrinsic and intrinsic cell death is negatively controlled by caspase inhibitors such as X-linked inhibitor of apoptosis protein (XIAP)⁴ or by B-cell lymphoma 2 (Bcl-2) proteins that suppress the mitochondria outer membrane permeability (MOMP) by limiting Bax (Bcl-2-associated X protein)/Bak (Bcl-2 homologous antagonist/killer) translocation into the mitochondrial outer membrane.⁵ The extrinsic signalling cascade communicates with the intrinsic death pathway by cleavage of Bid (BH3 interacting-domain death agonist), a pro-apoptotic member of the BH3 (Bcl-2 homology domain 3)-only subfamily of Bcl-2 proteins.¹ Other stimuli such as genotoxic stress allow for translocation and pore formation of pro-apoptotic multidomain Bcl-2 proteins Bax and Bak in the outer mitochondrial membrane.^{6, 7, 8} This process promotes release of mitochondria-derived apoptogenic proteins, in particular cytochrome c,⁹ or smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP binding protein with low pI).¹⁰ Within the apoptosome,¹¹ active caspase-9 finally leads to activation of caspase-3,¹² and subsequent cell death.Anti-apoptotic multidomain Bcl-2 proteins (Bcl-2, Bcl-2-like protein 2 (Bcl-w), B-cell lymphoma-extra large (Bcl-X_L), induced myeloid leukaemia cell differentiation protein (Mcl-1) and Bcl-2-related protein A1 (A1)) with four Bcl-2 homology domains (BH1, BH2, BH3 and BH4) suppress the pro-apoptotic function of Bax-like proteins such as Bax, Bak and Bok (that contain BH1–BH3 domains) or the BH3-only proteins Bad (Bcl-2-associated death promoter), Bim (Bcl-2-like protein 11), Bid, Noxa (phorbol-12-myristate-13-acetate-induced protein 1) and Puma (p53 upregulated modulator of apoptosis).¹³ Regulation of mitochondria-mediated apoptosis is determined by the balance between pro- and anti-apoptotic Bcl-2 proteins.¹⁴In a variety of cancer types, a decrease of BH3-only protein or upregulation of pro-survival Bcl-2 proteins is associated with poor prognosis.¹⁵ In metastatic squamous cell carcinoma (SCC) of the skin or the so-called ‘head and neck SCC'' (HNSCC), high expression of pro-survival Bcl-2 proteins conferred radio- and chemotherapy resistance.^{16, 17} These findings mark Bcl-2 proteins as regulators of SCC apoptosis and indicate that BH3 mimetics may hold therapeutic potential for metastatic SCC. The BH3 mimetics navitoclax (ABT263) and ABT199 are currently under investigation in clinical studies.^{18, 19, 20} Mechanistically, their lead compound ABT737 suppresses Bcl-2 activity by binding to the hydrophobic groove of Bcl-2, Bcl-w and Bcl-X_L.¹⁸ As ABT263 upregulates Mcl-1, resistance to a number of Bcl-2 inhibitors (ABT737 and ABT263) has been described.²¹ Another compound, Obatoclax, was developed to block all anti-apoptotic Bcl-2 proteins including Mcl-1.²² Obatoclax blocks the interaction of Bim or Bax with Mcl-1.²³ In this report, we have studied the effect of ABT737 for cell death in SCC of the skin and investigated the molecular mechanisms of resistance to different BH3 mimetics.     

Intramitochondrial recruitment of endolysosomes mediates Smac degradation and constitutes a novel intrinsic apoptosis antagonizing function of XIAP E3 ligase

Intrinsic apoptosis involves BH3-only protein activation of Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). Consequently, cytochrome c is released from the mitochondria to activate caspases, and Smac (second mitochondria-derived activator of caspases) to inhibit XIAP-mediated caspase suppression. Dysfunctional mitochondria can be targeted for lysosomal degradation via autophagy (mitophagy), or directly through mitochondria-derived vesicle transport. However, the extent of autophagy and lysosomal interactions with apoptotic mitochondria remains largely unknown. We describe here a novel pathway of endolysosomal processing of mitochondria, activated in response to canonical BH3-only proteins and mitochondrial depolarization. We report that expression of canonical BH3-only proteins, tBid, Bim_EL, Bik, Bad, and mitophagy receptor mutants of atypical BH3-only proteins, Bnip3 and Bnip3L/Nix, leads to prominent relocalization of endolysosomes into inner mitochondrial compartments, in a manner independent of mitophagy. As an upstream regulator, we identified the XIAP E3 ligase. In response to mitochondrial depolarization, XIAP actuates Bax-mediated MOMP, even in the absence of BH3-only protein signaling. Subsequently, in an E3 ligase-dependent manner, XIAP rapidly localizes inside all the mitochondria, and XIAP-mediated mitochondrial ubiquitylation catalyses interactions of Rab membrane targeting components Rabex-5 and Rep-1 (RFP-tagged Rab escort protein-1), and Rab5- and Rab7-positive endolysosomes, at and within mitochondrial membrane compartments. While XIAP-mediated MOMP permits delayed cytochrome c release, within the mitochondria XIAP selectively signals lysosome- and proteasome-associated degradation of its inhibitor Smac. These findings suggest a general mechanism to lower the mitochondrial apoptotic potential via intramitochondrial degradation of Smac.The intrinsic mitochondrial apoptotic pathway is required for efficient chemotherapeutic killing of cancer cells,¹ and is initiated through BH3-only protein activation of Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). MOMP releases cytochrome c to activate effector caspases.² Conversely, inhibitor of apoptosis protein (IAP) family members suppress initiator and effector caspases via direct binding and E3 ligase activities.^{3, 4, 5} Consequently, MOMP-induced release of Smac (second mitochondria-derived activator of caspases) from the mitochondria, to inhibit XIAP (X-chromosome-linked IAP)-mediated caspase suppression, can be required for apoptosis.⁶Autophagy, a lysosomal degradative mechanism undergoing extensive crosstalk with cell death and survival pathways,⁷ degrades damaged mitochondria in a process termed mitophagy.^{8, 9} Damaged mitochondria are targeted by lysosomal degradation through the recruitment of autophagy receptors to the outer mitochondrial membrane (OMM),⁸ or via delivery of mitochondrial-derived vesicles (MDVs) directly to the lysosome.¹⁰ The E3 ubiquitin ligase Parkin targets and ubiquitylates mitochondria, mediating both MDV degradation¹¹ and autophagy receptor-dependent mitophagy.^{12, 13} Alternatively, Fundc1¹⁴ and atypical BH3-only Nip family proteins Bnip3 and Bnip3L/Nix localize to the OMM and act as mitophagy receptors via their LC3-interacting region (LIR).^{15, 16, 17, 18} While targeting of damaged mitochondria suggests that mitophagy may counter apoptotic mitochondria, mitophagy occurs progressively over days,^{12, 14, 16, 17, 19} a rate that is likely insufficient to alter intracellular propagation of mitochondrial apoptosis, which can occur within minutes.^{20, 21} Indeed, Bnip3- and Fundc1-induced mitophagy have no direct effect on apoptosis,^{14, 18} and we determined that Bnip3-mediated mitophagy was cytoprotective if activated before apoptosis.¹⁷ While MDV delivery of mitochondria to lysosomes operates at a higher rate, minutes to hours,¹⁰ this process is regulated by Parkin and restricted to specific mitochondrial components.¹¹ Overall, for most intrinsic apoptosis scenarios it remains unknown whether lysosomal processing of mitochondria influences their capacity to activate or enhance apoptosis.Here, we used high-resolution imaging to evaluate the behavior of apoptosis, autophagy, lysosomal and ubiquitylation pathways in response to canonical (tBid, Bim_EL, Bik, Bad) and atypical (Bnip3, Bnip3L/Nix) BH3-only protein expression. We report that, in parallel to intrinsic apoptosis signaling, canonical BH3-only proteins induce the recruitment of endolysosomal machinery, in the absence of mitophagy. We determined that mitochondrial depolarization rapidly translocates the caspase inhibitor XIAP to the mitochondria. There, XIAP actuates MOMP within all mitochondria, concomitant with ubiquitylation at the OMM and inside OMM-bound regions, and triggers ubiquitin-dependent recruitment of Rab5 and its binding partners, as well as late endosomes into the mitochondria. Consequently, in a manner dependent on lysosome- and proteasome-activities, XIAP degrades its inhibitor Smac. We propose that in response to bioenergetic stress, the functional integration between lysosomes and mitochondria, mediated by XIAP and independent of autophagy, offers a novel mechanism to modulate mitochondrial apoptosis.     

Context-dependent Bcl-2/Bak Interactions Regulate Lymphoid Cell Apoptosis

The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by interactions of Bax and Bak with antiapoptotic Bcl-2 family members. The factors that regulate these interactions are, at the present time, incompletely understood. Recent studies showing preferences in binding between synthetic Bcl-2 homology domain 3 and antiapoptotic Bcl-2 family members in vitro have suggested that the antiapoptotic proteins Mcl-1 and Bcl-x_L, but not Bcl-2, restrain proapoptotic Bak from inducing mitochondrial membrane permeabilization and apoptosis. Here we show that Bak protein has a much higher affinity than the 26-amino acid Bak Bcl-2 homology domain 3 for Bcl-2, that some naturally occurring Bcl-2 allelic variants have an affinity for full-length Bak that is only 3-fold lower than that of Mcl-1, and that endogenous levels of these Bcl-2 variants (which are as much as 40-fold more abundant than Mcl-1) restrain part of the Bak in intact lymphoid cells. In addition, we demonstrate that Bcl-2 variants can, depending on their affinity for Bak, substitute for Mcl-1 in protecting cells. Thus, the ability of Bcl-2 to protect cells from activated Bak depends on two important contextual variables, the identity of the Bcl-2 present and the amount expressed.The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by Bcl-2 family members (1–5). This group of proteins consists of three subgroups: Bax and Bak, which oligomerize upon death stimulation to form a putative pore in the outer mitochondrial membrane, thereby allowing efflux of cytochrome c and other mitochondrial intermembrane space components; Bcl-2, Bcl-x_L, Mcl-1, and other antiapoptotic homologs, which antagonize the effects of Bax and Bak; and BH3-only proteins² such as Bim, Bid, and Puma, which are proapoptotic Bcl-2 family members that share only limited homology with the other two groups in a single 15-amino acid domain (the BH3 domain, see Ref. 6). Although it is clear that BH3-only proteins serve as molecular sensors of various stresses and, when activated, trigger apoptosis (3, 6–11), the mechanism by which they do so remains incompletely understood. One current model suggests that BH3-only proteins trigger apoptosis solely by binding and neutralizing antiapoptotic Bcl-2 family members, thereby causing them to release the activated Bax and Bak that are bound (reviewed in Refs. 9 and 10; see also Refs. 12 and 13), whereas another current model suggests that certain BH3-only proteins also directly bind to and activate Bax (reviewed in Ref. 3; see also Refs. 14–17). Whichever model turns out to be correct, both models agree that certain antiapoptotic Bcl-2 family members can inhibit apoptosis, at least in part, by binding and neutralizing activated Bax and Bak before they permeabilize the outer mitochondrial membrane (13, 18, 19).Much of the information about the interactions between pro- and antiapoptotic Bcl-2 family members has been derived from the study of synthetic peptides corresponding to BH3 domains. In particular, these synthetic peptides have been utilized as surrogates for the full-length proapoptotic proteins during structure determinations (20–22) as well as in functional studies exploring the effect of purified BH3 domains on isolated mitochondria (14, 23) and on Bax-mediated permeabilization of lipid vesicles (15).Recent studies using these same peptides have suggested that interactions of the BH3 domains of Bax, Bak, and the BH3-only proteins with the “BH3 receptors” of the antiapoptotic Bcl-2 family members are not all equivalent. Surface plasmon resonance, a technique that is widely used to examine the interactions of biomolecules under cell-free conditions (24–26), has demonstrated that synthetic BH3 peptides of some BH3-only family members show striking preferences, with the Bad BH3 peptide binding to Bcl-2 and Bcl-x_L but not Mcl-1, and the Noxa BH3 peptide binding to Mcl-1 but not Bcl-2 or Bcl-x_L (27). Likewise, the Bak BH3 peptide exhibits selectivity, with high affinity for Bcl-x_L and Mcl-1 but not Bcl-2 (12). The latter results have led to a model in which Bcl-x_L and Mcl-1 restrain Bak and inhibit Bak-dependent apoptosis, whereas Bcl-2 does not (10).Because the Bak protein contains multiple recognizable domains in addition to its BH3 motif (28, 29), we compared the binding of Bak BH3 peptide and Bak protein to Bcl-2. Surface plasmon resonance demonstrated that Bcl-2 binds Bak protein with much higher affinity than the Bak 26-mer BH3 peptide. Further experiments demonstrated that the K_D for Bak differs among naturally occurring Bcl-2 sequence variants but is only 3-fold higher than that of Mcl-1 in some cases. In light of previous reports that Bcl-2 overexpression contributes to neoplastic transformation (30–33) and drug resistance (34–36) in lymphoid cells, we also examined Bcl-2 expression and Bak binding in a panel of neoplastic lymphoid cell lines. Results of these experiments demonstrated that Bcl-2 expression varies among different lymphoid cell lines but is up to 40-fold more abundant than Mcl-1. In lymphoid cell lines with abundant Bcl-2, Bak is detected in Bcl-2 as well as Mcl-1 immunoprecipitates; and Bak-dependent apoptosis induced by Mcl-1 down-regulation can be prevented by Bcl-2 overexpression. Collectively, these observations shed new light on the role of Bcl-2 in binding and neutralizing Bak.     

Conversion of cell-survival activity of Akt into apoptotic death of cancer cells by two mutations on the BIM BH3 domain

Survival and proliferation of cancer cells are often associated with hyperactivity of the serine/threonine kinase, Akt. Herein, we show that prosurvival activity of Akt can be converted into prodeath activity by embedding an Akt recognition sequence in the apoptogenic BH3 domain of human BIM. The recognition sequence was created by introducing two mutations, I155R and E158S, into the core region of the BIM BH3 domain. Although a 21-mer BIM BH3 peptide containing these two mutations bound weakly to BCL-X_L and BCL-2, this peptide with phosphorylation of Ser158 bound to these proteins with a dissociation constant of <10 nM. The crystal structure of the phosphorylated peptide bound to BCL-X_L revealed that the phospho-Ser158 makes favorable interactions with two BCL-X_L residues, which cannot be formed with unphosphorylated Ser158. Remarkably, the designed peptide showed a cytotoxic effect on PTEN-null PC3 tumor cells whose Akt activity is aberrantly high. The cell-killing activity disappeared when the cellular Akt activity was lowered by ectopic PTEN expression. Thus, these results lay a foundation for developing a peptide or protein agent that is dormant in normal cells but is transformed into a potent apoptogenic molecule upon phosphorylation by hyperactivity of Akt in cancer cells.The interplay between the BCL-2 family proteins regulates mitochondrion-mediated apoptotic cell death.^{1, 2} The BCL-2 family proteins are characterized by having at least one BCL-2 homology (BH) domain, and they are classified into three distinct subgroups based on their functional and structural features. One subgroup consists of BAX and BAK, which contain the BH1-BH4 domains and mediate apoptosis by increasing the permeability of the mitochondrial outer membrane (MOM) and thus leading to the release of the apoptogenic factors, such as cytochrome c and Smac/Diablo.^{3, 4, 5, 6} Another subgroup is composed of antiapoptotic proteins, BCL-2, BCL-X_L, BCl-w, MCL-1, A1 and BCL-B, which contain the BH1-BH4 domains that are arranged to form an extended hydrophobic groove known as the BH3-binding groove.⁷ The remaining subgroup is composed of a diverse set of proteins that are unrelated to each other except for the possession of the BH3 domain.⁷ These BH3-only proteins sense and convey apoptotic cell death signals, ultimately leading to the activation of BAX and BAK.^{8, 9} The antiapoptotic BCL-2 subfamily proteins bind the BH3 domain of BAX/BAK and of the BH3-only proteins through their BH3-binding groove.^{10, 11, 12, 13, 14, 15}Biochemical studies have discovered that a number of the BH3-only proteins termed ‘activators'', such as BID and BIM, bind directly to BAX and induce its activation, whereas other BH3-only proteins termed ‘sensitizers'' induce apoptosis by releasing the activators sequestered by the antiapoptotic proteins.^{5, 16, 17} A recent crystallographic study revealed that the BID BH3 peptide binds to the canonical BH3-binding groove of BAX and induces a pronounced conformational change that exposes the BH3 domain of BAX.¹⁸ The activated BAX oligomerizes to induce the permeabilization of the MOM.⁶ The antiapoptotic BCL-2 proteins were suggested to sequester the BH3 domains of both BAX and the activator BH3-only proteins to prevent the BAX oligomerization.¹⁸Apoptosis is attenuated in cancer cells because of the abundance of antiapoptotic BCL-2 proteins and/or prevention of apoptosis induction. Anticancer BH3 peptides have been developed, especially those derived from BIM, which interacts with all of the antiapoptotic proteins with extremely high affinity.^{15, 19} These BH3 peptides exhibit a broad and multimodal targeting of the BCL-2 family proteins.^{20, 21, 22} Promising small molecular anticancer compounds have also been developed that mimic the BH3 peptides and bind to the surface groove of the antiapoptotic proteins.²³ ABT-737 and ABT-263 selectively bind to and lower the amounts of the functional BCL-2, BCL-X_L and BCL-w proteins to induce the apoptotic death of tumor cells that depend especially on the overexpression of the three proteins.^{24, 25} The BH3 peptides and the BH3 mimetics both bear an intrinsic shortcoming in that they inhibit the BCL-2 family proteins not only in cancer cells but also in normal cells as they cannot distinguish cancerous from normal cells.One of the hallmarks of many cancer and tumor cells is the hyperactivation of the serine/threonine (Ser/Thr) protein kinase Akt, which is a key signaling molecule in the cellular survival pathway.²⁶ In many types of cancers, including glioma, prostate cancer and breast cancer, Akt is required to maintain a proliferative state and for progression into a more malignant state in conjunction with genetic mutations.^{26, 27, 28}We set out to develop a molecule that can respond to the hyperactivity of Akt and can lead to the death of cancer cells. Herein, we describe the embedment of the Akt recognition sequence into the BIM BH3 peptide and the cancer cell-specific apoptogenic property of the resulting BIM BH3 peptide variant characterized by X-ray crystallography, calorimetry and cell-based biochemistry.     

JNK1/2 regulate Bid by direct phosphorylation at Thr59 in response to ALDH1L1

BH3 interacting-domain death agonist (Bid) is a BH3-only pro-apoptotic member of the Bcl-2 family of proteins. Its function in apoptosis is associated with the proteolytic cleavage to the truncated form tBid, mainly by caspase-8. tBid translocates to mitochondria and assists Bax and Bak in induction of apoptosis. c-Jun N-terminal kinase (JNK)-dependent alternative processing of Bid to jBid was also reported. We have previously shown that the folate stress enzyme 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) activates JNK1 and JNK2 in cancer cells as a pro-apoptotic response. Here we report that in PC-3 prostate cancer cells, JNK1/2 phosphorylate Bid at Thr59 within the caspase cleavage site in response to ALDH1L1. In vitro, all three JNK isoforms, JNK 1–3, phosphorylated Thr59 of Bid with JNK1 being the least active. Thr59 phosphorylation protected Bid from cleavage by caspase-8, resulting in strong accumulation of the full-length protein and its translocation to mitochondria. Interestingly, although we did not observe jBid in response to ALDH1L1 in PC-3 cells, transient expression of Bid mutants lacking the caspase-8 cleavage site resulted in strong accumulation of jBid. Of note, a T59D mutant mimicking constitutive phosphorylation revealed more profound cleavage of Bid to jBid. JNK-driven Bid accumulation had a pro-apoptotic effect in our study: small interfering RNA silencing of either JNK1/2 or Bid prevented Bid phosphorylation and accumulation, and rescued ALDH1L1-expressing cells. As full-length Bid is a weaker apoptogen than tBid, we propose that the phosphorylation of Bid by JNKs, followed by the accumulation of the full-length protein, delays attainment of apoptosis, and allows the cell to evaluate the stress and make a decision regarding the response strategy. This mechanism perhaps can be modified by the alternative cleavage of phospho-T59 Bid to jBid at some conditions.BH3 interacting-domain death agonist (Bid), a member of BH3-only group of proteins in the Bcl-2 family, functions as a sensor of cellular damage and activator of pro-apoptotic Bax and Bak.^{1, 2} Bid is a 23 kDa protein localized primarily in the cytosol, but upon apoptotic stimuli it is cleaved to yield a truncated 15 kDa C-terminal fragment tBid. tBid translocates to the mitochondrial membrane, where it interacts with Bax and Bak, enhancing their oligomerization and leading to outer membrane permeabilization, loss of membrane potential and release of mitochondrial apoptogens.^{3, 4} The canonical example of the activation of Bid cleavage is the FAS-mediated apoptosis, and Bid is viewed as the key molecule in the integration of death receptor and mitochondrial apoptotic pathways.^{5, 6} The interaction of tBid with Bax or Bak proceeds through the BH3 domain of Bid and occurs only after the protein is localized to mitochondria.⁷ In the full-length Bid, the BH3 domain can be masked by the N-terminal portion of the protein through the interaction with an α-helical BH-3-like region, the BH3-B domain.^{5, 8} The caspase-8 cleavage in the middle of the large flexible loop connecting the BH3 and BH3-B domains leads to structural rearrangements of the C-terminal portion of Bid enabling its insertion into mitochondrial membrane.⁹ The dissociation of the N-terminal fragment in the presence of the mitochondrial membrane and conformational changes of tBid molecule make the BH3 domain accessible for Bax or Bak.¹⁰ Other proteolytic enzymes can cleave Bid within the loop but caspase-8 appears to be a major factor generating tBid.⁸ Full-length Bid can also translocate to mitochondria and induce apoptosis^{11, 12, 13, 14} but its pro-apoptotic activity is weaker than the activity of tBid.¹⁵ It has been hypothesized that in contrast to tBid, the conformational changes enabling the translocation of full-length Bid to mitochondria are reversible.⁹Several studies have also indicated the cleavage-independent pro-survival function of Bid in S-phase checkpoint and highlighted the regulation of Bid by phosphorylation at several residues.^{16, 17} Thus, ATM/ATR protein kinases can phosphorylate Bid at Ser61, Ser64 and Ser78, which protects from caspase-8 cleavage.¹⁷ In response to DNA damage, Bid is phosphorylated by ATM protein kinase and translocates to the nucleus to contribute to the decision of cell fate.^{16, 17} Interestingly, the ablation of phosphorylation at Ser61 and Ser78 ATM sites caused accumulation of full-length Bid in the mitochondria of hematopoetic stem cells and increased cellular proliferation.¹⁸ Furthermore, the phosphorylation of murine Bid at Thr58, Ser61 and Ser64 near the caspase-8 cleavage site by casein kinase I and II protected the protein from cleavage, thus making it less active towards the induction of apoptosis.¹⁹ Moreover, the pro-survival function of Bid was suggested by the finding that its loss inhibited tumorigenesis of T cells.²⁰ Overall, phosphorylation of Bid can serve as a switch between the pro-apoptotic and pro-survival functions of the protein.Although phosphorylation of Bid by c-Jun N-terminal kinase (JNK) has not been demonstrated so far, it has been reported that the alternative processing of Bid, which generates jBid, is JNK-dependent.²¹ Interestingly, the accumulation of full-length Bid and its translocation to mitochondria was observed in HeLa cells in response to staurosporine,²² a known JNK activator.²³ Tight relationships between JNK and Bid have been also demonstrated in mouse models of TNFα-induced liver injury.²⁴ This study indicated that Bid is downstream of JNK in TNFα-induced apoptosis and the pro-apoptotic activity of JNK2 is mainly mediated by Bid. Here we report that in PC-3 cells, JNK1/2 phosphorylate Bid at Thr59 in response to folate stress enzyme 10-formyltetrahydrofolate dehydrogenase (ALDH1L1), thus protecting Bid from caspase-8 cleavage. This leads to apoptosis owing to a strong accumulation and mitochondrial translocation of full-length Bid.