The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CED-4.

Bcl-2 and close homologues such as Bcl-xL promote cell survival, while other relatives such as Bax antagonize this function. Since only the pro-survival family members possess a conserved N-terminal region denoted BH4, we have explored the role of this amphipathic helix for their survival function and for interactions with several agonists of apoptosis, including Bax and CED-4, an essential regulator in the nematode Caenorhabditis elegans. BH4 of Bcl-2 could be replaced by that of Bcl-x without perturbing function but not by a somewhat similar region near the N-terminus of Bax. Bcl-2 cell survival activity was reduced by substitutions in two of ten conserved BH4 residues. Deletion of BH4 rendered Bcl-2 (and Bcl-xL) inactive but did not impair either Bcl-2 homodimerization or ability to bind to Bax or five other pro-apoptotic relatives (Bak, Bad, Bik, Bid or Bim). Hence, association with these death agonists is not sufficient to promote cell survival. Significantly, however, Bcl-xL lacking BH4 lost the ability both to bind CED-4 and antagonize its pro-apoptotic activity. These results favour the hypothesis that the BH4 domain of pro-survival Bcl-2 family members allows them to sequester CED-4 relatives and thereby prevent apoptosis.     

Identification of novel isoforms of the BH3 domain protein Bim which directly activate Bax to trigger apoptosis

Bim (Bcl-2-interacting mediator of cell death) is a member of the BH3 domain-only subgroup of Bcl-2 family members, for which three splice variants have been described. Bim is expressed in many healthy cell types, where it is maintained in an inactive conformation through binding to the microtubule-associated dynein motor complex. Upon certain apoptotic stimuli, Bim is released from microtubules and mediates caspase-dependent apoptosis through a mechanism that is still unclear. Here, we have identified and characterized novel splice variants of human Bim mRNA. In particular, we show that a newly discovered, small protein isoform, BimAD, is also able to induce apoptosis strongly in several human cell lines. BimAD and the previously characterized isoform BimS are shown to be capable of heterodimerizing in vivo with both death antagonists (Bcl-2 and Bcl-X(L)) and death agonists (Bax). Mutants of BimAD that bind to Bax but not to Bcl-2 still promote apoptosis, indicating that Bim can regulate apoptosis through direct activation of the Bax-mediated cell death pathway without interaction with antiapoptotic Bcl-2 family members. Furthermore, we have shown that the interaction of the BimS and BimAD isoforms with Bax leads to a conformational change in this protein analogous to that triggered by the BH3-only protein Bid.     

The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex

Bcl-2 family members that have only a single Bcl-2 homology domain, BH3, are potent inducers of apoptosis, and some appear to play a critical role in developmentally programmed cell death. We examined the regulation of the proapoptotic activity of the BH3-only protein Bim. In healthy cells, most Bim molecules were bound to LC8 cytoplasmic dynein light chain and thereby sequestered to the microtubule-associated dynein motor complex. Certain apoptotic stimuli disrupted the interaction between LC8 and the dynein motor complex. This freed Bim to translocate together with LC8 to Bcl-2 and to neutralize its antiapoptotic activity. This process did not require caspase activity and therefore constitutes an initiating event in apoptosis signaling.     

Degenerative disorders caused by Bcl-2 deficiency prevented by loss of its BH3-only antagonist Bim.

Apoptosis is triggered when proapoptotic members of the Bcl-2 protein family bearing only the BH3 association domain bind to Bcl-2 or its homologs and block their antiapoptotic activity. To test whether loss of the BH3-only protein Bim could prevent the cellular attrition caused by Bcl-2 deficiency, we generated mice lacking both genes. Mice without Bcl-2 have a fragile lymphoid system, become runted, turn gray, and succumb to polycystic kidney disease. Concomitant absence of Bim prevented all these disorders. Indeed, loss of even one bim allele restored normal kidney development, growth, and health. These results demonstrate that Bim levels set the threshold for initiation of apoptosis in several tissues and suggest that degenerative diseases might be alleviated by blocking BH3-only proteins.     

Bfk: a novel weakly proapoptotic member of the Bcl-2 protein family with a BH3 and a BH2 region

Proteins of the Bcl-2 family are critical regulators of apoptosis. Proapoptotic members, like Bax, contain three of the four Bcl-2 homology regions (BH1-3), while BH3-only proteins, like Bim, possess only the short BH3 motif. Database searches revealed Bfk, an unusual novel member of the Bcl-2 family that contains a BH2 and BH3 region but not BH1 or BH4. Bfk is thus most closely related to Bcl-G(L). It lacks a C-terminal membrane anchor and is cytosolic. Enforced expression of Bfk weakly promoted apoptosis and antagonized Bcl-2's prosurvival function. Like Bcl-G(L), Bfk did not bind to any Bcl-2 family members, even though its BH3 motif can mediate association with prosurvival proteins. Low amounts of Bfk were found in stomach, ovary, bone marrow and spleen, but its level in the mammary gland rose markedly during pregnancy, suggesting that Bfk may play a role in mammary development.     

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

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

Glutathione binding to the Bcl-2 homology-3 domain groove: a molecular basis for Bcl-2 antioxidant function at mitochondria

Bcl-2 protects cells against mitochondrial oxidative stress and subsequent apoptosis. However, the mechanism underlying the antioxidant function of Bcl-2 is currently unknown. Recently, Bax and several Bcl-2 homology-3 domain (BH3)-only proteins (Bid, Puma, and Noxa) have been shown to induce a pro-oxidant state at mitochondria (1-4). Given the opposing effects of Bcl-2 and Bax/BH3-only proteins on the redox state of mitochondria, we hypothesized that the antioxidant function of Bcl-2 is antagonized by its interaction with the BH3 domains of pro-apoptotic family members. Here, we show that BH3 mimetics that bind to a hydrophobic surface (the BH3 groove) of Bcl-2 induce GSH-sensitive mitochondrial dysfunction and apoptosis in cerebellar granule neurons. BH3 mimetics displace a discrete mitochondrial GSH pool in neurons and suppress GSH transport into isolated rat brain mitochondria. Moreover, BH3 mimetics and the BH3-only protein, Bim, inhibit a novel interaction between Bcl-2 and GSH in vitro. These results suggest that Bcl-2 regulates an essential pool of mitochondrial GSH and that this regulation may depend upon Bcl-2 directly interacting with GSH via the BH3 groove. We conclude that this novel GSH binding property of Bcl-2 likely plays a central role in its antioxidant function at mitochondria.     

Proapoptotic BH3-only Bcl-2 family member Bik/Blk/Nbk is expressed in hemopoietic and endothelial cells but is redundant for their programmed death

The BH3-only members of the Bcl-2 protein family are essential for initiation of programmed cell death and stress-induced apoptosis. We have determined the expression pattern in mice of the BH3-only protein Bik, also called Blk or Nbk, and examined its physiological function by gene targeting. We found that Bik is expressed widely in the hematopoietic compartment and in endothelial cells of the venous but not arterial lineages. Nevertheless, its loss did not increase the numbers of such cells in mice or protect hematopoietic cells in vitro from apoptosis induced by cytokine withdrawal or diverse other cytotoxic stimuli. Moreover, whereas loss of the BH3-only protein Bim rescued mice lacking the prosurvival protein Bcl-2 from fatal polycystic kidney disease and lymphopenia, loss of Bik did not. These results indicate that any function of Bik in programmed cell death and stress-induced apoptosis must overlap that of other BH3-only proteins.     

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.     

BH3-only Bcl-2 family members are coordinately regulated by the JNK pathway and require Bax to induce apoptosis in neurons

The Bcl-2 family of proteins are key regulators of programmed cell death. A distinct subfamily of BH3-only molecules has been identified, but their exact mechanism of action remains unclear. Here we show that the BH3-only Bcl-2 family members, Dp5/Hrk and Bim, are induced upstream of the Bax checkpoint in neuronal apoptosis in a manner that shows significant dependence on JNK signaling. We also show that Dp5 and other BH3-only proteins kill cerebellar granule neurons in a Bax-dependent manner. These studies demonstrate that BH3-only members do not act independently in their proapoptotic activities but rather require the action of multidomain proapoptotic Bcl-2 family members to produce cell death.     

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.     

Molecular analysis of neutrophil spontaneous apoptosis reveals a strong role for the pro-apoptotic BH3-only protein Noxa

Neutrophils enter the peripheral blood from the bone marrow and die after a short time. Molecular analysis of spontaneous neutrophil apoptosis is difficult as these cells die rapidly and cannot be easily manipulated. We use conditional Hoxb8 expression to generate mouse neutrophils and test the regulation of apoptosis by extensive manipulation of B-cell lymphoma protein 2 (Bcl-2)-family proteins. Spontaneous apoptosis was preceded by downregulation of anti-apoptotic Bcl-2 proteins. Loss of the pro-apoptotic Bcl-2 homology domain (BH3)-only protein Bcl-2-interacting mediator of cell death (Bim) gave some protection, but only neutrophils deficient in both BH3-only proteins, Bim and Noxa, were strongly protected against apoptosis. Function of Noxa was at least in part neutralization of induced myeloid leukemia cell differentiation protein (Mcl-1) in neutrophils and progenitors. Loss of Bim and Noxa preserved neutrophil function in culture, and apoptosis-resistant cells remained in circulation in mice. Apoptosis regulated by Bim- and Noxa-driven loss of Mcl-1 is thus the final step in neutrophil differentiation, required for the termination of neutrophil function and neutrophil-dependent inflammation.     

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.     

Bim and the Pro-Survival Bcl-2 Proteins: Opposites Attract,ERK Repels

Bim (Bcl-2-interacting mediator of cell death) is a BH3-only protein (BOP), a pro-apoptotic member of the Bcl-2 protein family. The Bim mRNA undergoes alternate splicing to give rise to the short, long and extra long protein variants (BimS, BimL and BimEL). These proteins have distinct potency in promoting death and distinct modes of regulation conferred by their interaction with other proteins. Quite how Bim and other BOPs promote apoptosis has been the subject of some debate. Bim was isolated by it’s interaction with pro-survival proteins such as Bcl-2 and it has been suggested that this is key to the ability of Bim to induce apoptosis. However, an alternative model argues that some forms of Bim can bind directly to the pro-apoptotic Bax and Bak proteins to initiate apoptosis. A new study may finally put this debate to rest as it provides strong evidence to suggest that Bim and other BOPs act primarily by binding to pro-survival Bcl-2 proteins, thereby releasing Bax or Bak proteins to promote apoptosis. The importance of the interaction between Bim and the pro-survival Bcl-2 proteins is underlined by our demonstration that it is regulated by ERK1/2-dependent phosphorylation of BimEL. ERK1/2-dependent dissociation of BimEL from pro-survival proteins is the first step in a process by which the pro-survival ERK1/2 pathway promotes the destruction of this most abundant Bim splice variant. In this review we outline the significance of these new studies to our understanding of how BOPs such as Bim initiate apoptosis and how this process is regulated by growth factor-dependent signalling pathways.     

Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases

The Bcl-2 homology 3 (BH3) domain is crucial for the death-inducing and dimerization properties of pro-apoptotic members of the Bcl-2 protein family, including Bak, Bax, and Bad. Here we report that synthetic peptides corresponding to the BH3 domain of Bak bind to Bcl-xL, antagonize its anti-apoptotic function, and rapidly induce apoptosis when delivered into intact cells via fusion to the Antennapedia homeoprotein internalization domain. Treatment of HeLa cells with the Antennapedia-BH3 fusion peptide resulted in peptide internalization and induction of apoptosis within 2-3 h, as indicated by caspase activation and subsequent poly(ADP-ribose) polymerase cleavage, as well as morphological characteristics of apoptosis. A point mutation within the BH3 peptide that blocks its ability to bind to Bcl-xL abolished its apoptotic activity, suggesting that interaction of the BH3 peptide with Bcl-2-related death suppressors, such as Bcl-xL, may be critical for its activity in cells. While overexpression of Bcl-xL can block BH3-induced apoptosis, treatment with BH3 peptides resensitized Bcl-xL-expressing cells to Fas-mediated apoptosis. BH3-induced apoptosis was blocked by caspase inhibitors, demonstrating a dependence on caspase activation, but was not accompanied by a dramatic early loss of mitochondrial membrane potential or detectable translocation of cytochrome c from mitochondria to cytosol. These findings demonstrate that the BH3 domain itself is capable of inducing apoptosis in whole cells, possibly by antagonizing the function of Bcl-2-related death suppressors.     

Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity

Prosurvival Bcl-2-like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.