Aven, a novel inhibitor of caspase activation, binds Bcl-xL and Apaf-1

Bcl-x(L), an antiapoptotic Bcl-2 family member, is postulated to function at multiple stages in the cell death pathway. The possibility that Bcl-x(L) inhibits cell death at a late (postmitochondrial) step in the death pathway is supported by this report of a novel apoptosis inhibitor, Aven, which binds to both Bcl-x(L) and the caspase regulator, Apaf-1. Identified in a yeast two-hybrid screen, Aven is broadly expressed and is conserved in other mammalian species. Only those mutants of Bcl-x(L)that retain their antiapoptotic activity are capable of binding Aven. Aven interferes with the ability of Apaf-1 to self-associate, suggesting that Aven impairs Apaf-1-mediated activation of caspases. Consistent with this idea, Aven inhibited the proteolytic activation of caspases in a cell-free extract and suppressed apoptosis induced by Apaf-1 plus caspase-9. Thus, Aven represents a new class of cell death regulator.     

Bcl-x(L) sequesters its C-terminal membrane anchor in soluble, cytosolic homodimers

Bcl-x(L) is a potent inhibitor of apoptosis. While Bcl-x(L) can be bound to mitochondria, a substantial fraction, depending on the cell type or tissue, is found in the cytosol of healthy cells. Gel filtration and crosslinking experiments reveal that, unlike monomeric Bax, Bcl-x(L) migrates in a complex of approximately 50 kDa in the cytosol. Co-immunoprecipitation experiments indicate that Bcl-x(L) in the cytosol forms homodimers. The C-terminal hydrophobic tails of two Bcl-x(L) molecules are involved in homodimer formation, and analysis of mutants demonstrates that the C-terminal lysine residue and the G138 residue lining the BH3-binding pocket are required for homodimerization. The flexible loop preceding the C-terminal tail in Bcl-x(L) is longer than that of several monomeric Bcl-2 family members and is a requisite for the homodimer formation. Bad binding to Bcl-x(L) dissociates the homodimers and triggers Bcl-x(L) binding to mitochondrial membranes. The C-terminal tail of Bcl-x(L) is also required to mediate Bcl-x(L)/Bax heterodimer formation. Both mitochondrial import and antiapoptotic activity of different Bcl-x(L) mutants correlate with their ability to form homodimers.     

Bax activation by the BH3-only protein Puma promotes cell dependence on antiapoptotic Bcl-2 family members

It is still unclear whether the BH3-only protein Puma (p53 up-regulated modulator of apoptosis) can prime cells to death and render antiapoptotic BH3-binding Bcl-2 homologues necessary for survival through its ability to directly interact with proapoptotic Bax and activate it. In this study, we provide further evidence, using cell-free assays, that the BH3 domain of Puma binds Bax at an activation site that comprises the first helix of Bax. We also show that, in yeast, Puma interacts with Bax and triggers its killing activity when Bcl-2 homologues are absent but not when Bcl-xL is expressed. Finally, endogenous Puma is involved in the apoptotic response of human colorectal cancer cells to the Bcl-2/Bcl-xL inhibitor ABT-737, even in conditions where the expression of Mcl-1 is down-regulated. Thus, Puma is competent to trigger Bax activity by itself, thereby promoting cellular dependence on prosurvival Bcl-2 family members.     

Peptides derived from BH3 domains of Bcl-2 family members: a comparative analysis of inhibition of Bcl-2, Bcl-x(L) and Bax oligomerization,induction of cytochrome c release,and activation of cell death

Overexpression of Bcl-2, an anti-apoptotic oncoprotein, is commonly observed in a variety of human malignancies and is associated with resistance to chemotherapy and radiotherapy. Although the precise mechanism of Bcl-2 action remains elusive, current evidence indicates that Bcl-2 inhibits apoptosis by binding and inhibiting pro-apoptotic molecules such as Bax. Therefore, agents that disrupt the ability of Bcl-2, or other anti-apoptotic molecules, to bind to pro-apoptotic molecules may have therapeutic value. Several studies have shown that the BH3 domains of Bcl-2 and Bax are critically important for Bax/Bcl-2 heterodimerization. In this report, we designed and synthesized peptides based on the BH3 domains of three distinct Bcl-2 family members, Bcl-2, Bax and Bad. In vitro interaction assays were used to compare the abilities of the different peptides to inhibit Bax/Bcl-2 and Bax/Bcl-x(L) heterodimerization, as well as Bcl-2 and Bax homodimerization. Bax BH3 peptide (20-amino acids) potently inhibited both Bax/Bcl-2 and Bax/Bcl-x(L) interactions, exhibiting IC(50) values of 15 and 9.5 microM, respectively. The Bad BH3 peptide (21 amino acids) was slightly more potent than Bax BH3 at inhibiting Bax/Bcl-x(L) but failed to disrupt Bax/Bcl-2. Bcl-2 BH3 peptide (20-amino acids) was inactive toward Bax/Bcl-2 and had only a weak inhibitory effect on Bax/Bcl-x(L) heterodimerization. All three BH3 peptides failed to significantly inhibit homodimerization of Bcl-2 or Bax. Consistent with its ability to disrupt Bax/Bcl-2 heterodimerization, Bax BH3 peptide was able to overcome Bcl-2 overexpression and induce cytochrome c release from mitochondria of Bcl-2-overexpressing Jurkat T leukemic cells. Bad BH3 peptide, while potently inducing cytochrome c release in wild-type Jurkat cells, only partially overcame the effects of Bcl-2 overexpression. Bcl-2 BH3 failed to induce cytochrome c release, even in wild-type cells. Delivery of the Bax BH3 and Bad BH3 peptides into wild-type Jurkat cells induced comparable levels of cell death. In cells overexpressing Bcl-2, the potency of Bax BH3 peptide was similar to that seen in wild-type cells, while the efficacy of Bad BH3 peptide was reduced. By contrast, in Bcl-x(L)-overexpressing cells, Bad BH3 exhibited greater cell-killing activity than Bax BH3. The Bcl-2 BH3 peptide and a mutant Bax BH3 peptide had no appreciable effect on Jurkat cells. Together, our data suggest that agents based on the Bax BH3 domain may have therapeutic value in cancers overexpressing Bcl-2, while agents based on the BH3 domain of Bad may be more useful for tumors overexpressing Bcl-x(L).     

p14(ARF)-induced apoptosis in p53 protein-deficient cells is mediated by BH3-only protein-independent derepression of Bak protein through down-regulation of Mcl-1 and Bcl-xL proteins

The p14(ARF) tumor suppressor plays a central role in regulating cell cycle arrest and apoptosis. We reported previously that p14(ARF) is capable of triggering apoptosis in a p53-independent manner. However, the mechanism remained unclear. Here we demonstrate that the p53-independent activation of the mitochondrial apoptosis pathway by p14(ARF) is primarily mediated by the pro-apoptotic Bax-homolog Bak. Expression of p14(ARF) exclusively triggers a N-terminal conformational switch of Bak, but not Bax, which allows for mitochondrial permeability shift, release of cytochrome c, activation of caspases, and subsequent fragmentation of genomic DNA. Although forced expression of Bak markedly sensitizes toward p14(ARF)-induced apoptosis, re-expression of Bax has no effect. Vice versa, knockdown of Bak by RNA interference attenuates p14(ARF)-induced apoptosis, whereas down-regulation of Bax has no effect. Bak activation coincides with a prominent, caspase-independent deprivation of the endogenous Bak inhibitors Mcl-1 and Bcl-x(L). In turn, mitochondrial apoptosis is fully blocked by overexpression of either Mcl-1 or Bcl-x(L). Taken together, these data indicate that in the absence of functional p53 and Bax, p14(ARF) triggers mitochondrial apoptosis signaling by activating Bak, which is facilitated by down-regulating anti-apoptotic Mcl-1 and Bcl-x(L). Moreover, our data suggest that the simultaneous inhibition of two central endogenous Bak inhibitors, i.e. Mcl-1 and Bcl-x(L), may be sufficient to activate mitochondrial apoptosis in the absence of BH3-only protein regulation.     

The ability of Bcl-x(L) and Bcl-2 to prevent apoptosis can be differentially regulated

Although expression of Bcl-2 has been shown to prevent apoptosis under many circumstances, there are several systems in which Bcl-2 fails to promote cell survival. We have previously reported that Bcl-2 and Bcl-x(L) display differential ability to protect WEHI-231 cells from multiple inducers of apoptosis. A possible explanation for this paradox was provided by the discovery of Bax. Bax is a Bcl-2-related protein which can inhibit the ability of Bcl-2 to enhance the survival of growth factor-dependent cell lines in the absence of growth factor. Consistent with the possibility that Bcl-2 function in WEHI-231 cells is inhibited by Bax, WEHI-231 cells were found to express a high level of Bax. To directly test the effects of Bax expression on Bcl-x(L) function, FL5.12 cells were transfected with both genes. Although Bax overexpression can inhibit Bcl-2 from prolonging cell survival upon growth factor withdrawal, Bax overexpression did not inhibit Bcl-x(L) from preventing apoptosis in this cell line. Although Bcl-2 and Bcl-x(L) were both found to be able to form heterodimers with Bax, the majority of Bax in both cases was not complexed to a partner. Our data suggest that Bcl-x(L) does not function by simply preventing the formation of Bax homodimers which promote cell death. Instead Bax appears to display selectivity in its ability to inhibit Bcl-2 but not Bcl-x(L) from prolonging survival. Furthermore, our data suggest that the abilities of Bcl-2 and Bcl-x(L) to promote cell survival are not identical and can be independently regulated within a cell. Regulation of a cell's apoptotic threshold is likely to result from a complex set of interactions among Bcl-2 family members and other, as yet uncharacterised, regulators of apoptosis.     

Bcl-x(L) complements Saccharomyces cerevisiae genes that facilitate the switch from glycolytic to oxidative metabolism

All eukaryotic organisms have mechanisms to adapt to changing metabolic conditions. The mammalian cell survival gene Bcl-x(L) enables cells to adapt to changes in cellular metabolism. To identify genes whose function can be substituted by Bcl-x(L) in a unicellular eukaryote, a genetic screen was performed using the yeast Saccharomyces cerevisiae. S. cerevisiae grows by anaerobic glycolysis when glucose is available, switching to oxidative phosphorylation when carbohydrate in the media becomes limiting (diauxic shift). Given that Bcl-x(L) appears to facilitate the switch from glycolytic to oxidative metabolism in mammalian cells, a library of yeast mutants was tested for the ability to efficiently undergo diauxic shift in the presence and absence of Bcl-x(L). Several mutants were identified that have a defect in growth when switched from a fermentable to a nonfermentable carbon source that is corrected by the expression of Bcl-x(L). These genes include the mitochondrial chaperonin TCM62, as well as previously uncharacterized genes. One of these uncharacterized genes, SVF1, promotes cell survival in mammalian cells in response to multiple apoptotic stimuli. The finding that TCM62 and the analogous human prohibitin gene also inhibit mammalian cell death following growth factor withdrawal implicates mitochondrial chaperones as regulators of apoptosis. Further characterization of the genes identified in this screen may enhance our understanding of Bcl-x(L) function in mammalian cells, and of cell survival pathways in general.     

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

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

Serum-nutrient starvation induces cell death mediated by Bax and Puma that is counteracted by p21 and unmasked by Bcl-x(L) inhibition

The cyclin-dependent kinase inhibitor p21 (p21WAF1/Cip1) is a multifunctional protein known to promote cell cycle arrest and survival in response to p53-dependent and p53 independent stimuli. We herein investigated whether and how it might contribute to the survival of cancer cells that are in low-nutrient conditions during tumour growth, by culturing isogenic human colorectal cancer cell lines (HCT116) and breast cancer cell lines in a medium deprived in amino acids and serum. We show that such starvation enhances, independently from p53, the expression of p21 and that of the pro-apoptotic BH3-only protein Puma. Under these conditions, p21 prevents Puma and its downstream effector Bax from triggering the mitochondrial apoptotic pathway. This anti-apoptotic effect is exerted from the cytosol but it is unrelated to the ability of p21 to interfere with the effector caspase 3. The survival function of p21 is, however, overcome by RNA interference mediated Bcl-x(L) depletion, or by the pharmacological inhibitor ABT-737. Thus, an insufficient supply in nutrients may not have an overt effect on cancer cell viability due to p21 induction, but it primes these cells to die, and sensitizes them to the deleterious effects of Bcl-x(L) inhibitors regardless of their p53 status.     

Mutual regulation of Bcl-2 proteins independent of the BH3 domain as shown by the BH3-lacking protein Bcl-x(AK)

The BH3 domain of Bcl-2 proteins was regarded as indispensable for apoptosis induction and for mutual regulation of family members. We recently described Bcl-x(AK), a proapoptotic splice product of the bcl-x gene, which lacks BH3 but encloses BH2, BH4 and a transmembrane domain. It remained however unclear, how Bcl-x(AK) may trigger apoptosis.For efficient overexpression, Bcl-x(AK) was subcloned in an adenoviral vector under Tet-OFF control. The construct resulted in significant apoptosis induction in melanoma and nonmelanoma cell lines with up to 50% apoptotic cells as well as decreased cell proliferation and survival. Disruption of mitochondrial membrane potential, and cytochrome c release clearly indicated activation of the mitochondrial apoptosis pathways. Both Bax and Bak were activated as shown by clustering and conformation analysis. Mitochondrial translocation of Bcl-x(AK) appeared as an essential and initial step. Bcl-x(AK) was critically dependent on either Bax or Bak, and apoptosis was abrogated in Bax/Bak double knockout conditions as well by overexpression of Bcl-2 or Bcl-x(L). A direct interaction with Bcl-2, Bax, Bad, Noxa or Puma was however not seen by immunoprecipitation. Thus besides BH3-mediated interactions, there exists an additional way for mutual regulation of Bcl-2 proteins, which is independent of the BH3. This pathway appears to play a supplementary role also for other proapoptotic family members, and its unraveling may help to overcome therapy resistance in cancer.     

Bcl-rambo, a novel Bcl-2 homologue that induces apoptosis via its unique C-terminal extension

The Bcl-2 family of proteins plays a central regulatory role in apoptosis. We have identified a novel, widely expressed Bcl-2 member which we have named Bcl-rambo. Bcl-rambo shows overall structural homology to the anti-apoptotic Bcl-2 members containing conserved Bcl-2 homology (BH) motifs 1, 2, 3, and 4. Unlike Bcl-2, however, the C-terminal membrane anchor region is preceded by a unique 250 amino acid insertion containing two tandem repeats. No interaction of Bcl-rambo with either anti-apoptotic (Bcl-2, Bcl-x(L), Bcl-w, A1, MCL-1, E1B-19K, and BHRF1) or pro-apoptotic (Bax, Bak, Bik, Bid, Bim, and Bad) members of the Bcl-2 family was observed. In mammalian cells, Bcl-rambo was localized to mitochondria, and its overexpression induces apoptosis that is specifically blocked by the caspase inhibitors, IAPs, whereas inhibitors controlling upstream events of either the 'death receptor' (FLIP, FADD-DN) or the 'mitochondrial' pro-apoptotic pathway (Bcl-x(L)) had no effect. Surprisingly, the Bcl-rambo cell death activity was induced by its membrane-anchored C-terminal domain and not by the Bcl-2 homology region. Thus, Bcl-rambo constitutes a novel type of pro-apoptotic Bcl-2 member that triggers cell death independently of its BH motifs.     

Sheeppox Virus SPPV14 Encodes a Bcl-2-Like Cell Death Inhibitor That Counters a Distinct Set of Mammalian Proapoptotic Proteins

Many viruses express inhibitors of programmed cell death (apoptosis), thereby countering host defenses that would otherwise rapidly clear infected cells. To counter this, viruses such as adenoviruses and herpesviruses express recognizable homologs of the mammalian prosurvival protein Bcl-2. In contrast, the majority of poxviruses lack viral Bcl-2 (vBcl-2) homologs that are readily identified by sequence similarities. One such virus, myxoma virus, which is the causative agent of myxomatosis, expresses a virulence factor that is a potent inhibitor of apoptosis. In spite of the scant sequence similarity to Bcl-2, myxoma virus M11L adopts an almost identical 3-dimensional fold. We used M11L as bait in a sequence similarity search for other Bcl-2-like proteins and identified six putative vBcl-2 proteins from poxviruses. Some are potent inhibitors of apoptosis, in particular sheeppox virus SPPV14, which inhibited cell death induced by multiple agents. Importantly, SPPV14 compensated for the loss of antiapoptotic F1L in vaccinia virus and acts to directly counter the cell death mediators Bax and Bak. SPPV14 also engages a unique subset of the death-promoting BH3-only ligands, including Bim, Puma, Bmf, and Hrk. This suggests that SPPV14 may have been selected for specific biological roles as a virulence factor for sheeppox virus.     

Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells.

Apoptosis as a form of programmed cell death (PCD) in multicellular organisms is a well-established genetically controlled process that leads to elimination of unnecessary or damaged cells. Recently, PCD has also been described for unicellular organisms as a process for the socially advantageous regulation of cell survival. The human Bcl-2 family member Bak induces apoptosis in mammalian cells which is counteracted by the Bcl-x(L) protein. We show that Bak also kills the unicellular fission yeast Schizosaccharomyces pombe and that this is inhibited by coexpression of human Bcl-x(L). Moreover, the same critical BH3 domain of Bak that is required for induction of apoptosis in mammalian cells is also required for inducing death in yeast. This suggests that Bak kills mammalian and yeast cells by similar mechanisms. The phenotype of the Bak-induced death in yeast involves condensation and fragmentation of the chromatin as well as dissolution of the nuclear envelope, all of which are features of mammalian apoptosis. These data suggest that the evolutionarily conserved metazoan PCD pathway is also present in unicellular yeast.     

Bcl-x(S) can form homodimers and heterodimers and its apoptotic activity requires localization of Bcl-x(S) to the mitochondria and its BH3 and loop domains

Proteins of the Bcl-2 family regulate apoptosis, some antagonizing cell death and others, such as Bcl-x(S), promoting it. We previously showed that expression of Bcl-x(S) in PC12 cells is a useful system for studying the mechanism of Bcl-x(S)-induced apoptosis. To further investigate this apoptotic effect and its prevention by anti-apoptotic agents, we assessed the role of distinct Bcl-x(S) domains, via the study of their mutations, on the ability of Bcl-x(S) to induce apoptosis and to localize to the mitochondria, as well as the ability of these domains to counteract the effects of anti-apoptotic agents on Bcl-x(S). Deletion of the transmembrane domain (DeltaTM) prevented the localization of Bcl-x(S) DeltaTM to the mitochondria and the ability of this mutant to induce apoptosis. Deletion of the amino acids GD 94-95 from the BH3 domain, or deletion of the loop region, impaired the ability of these mutants to induce apoptosis but not their localization to the mitochondria. Deletion of the BH4 domain or destruction of the caspase cleavage site in the loop region (by replacing amino acid D61 with A61) did not affect either the localization of these mutants to the mitochondria or their ability to induce cell death. It thus appears that Bcl-x(S)-induced apoptosis in PC12 cells is mediated by localization of Bcl-x(S) to the mitochondria by a process that requires the transmembrane domain. Furthermore, once localized to the mitochondria Bcl-x(S) requires the BH3 domain, and to a lesser extent the loop domain, for its subsequent activity. The anti-apoptotic agents Bcl-2 and Bcl-x(L), the caspase inhibitor Z-VAD-FMK, and nerve growth factor (NGF) did not prevent Bcl-x(S) localization to the mitochondria, and did not require the BH4 or the loop domains of Bcl-x(S) for their survival effect. Bcl-x(S) is capable of forming homodimers with itself and heterodimers with Bcl-x(L) or Bcl-2. Accordingly co-expression of Bcl-x(S) DeltaTM with Bcl-x(S), Bcl-2, or Bcl-x(L) leads to a change in the subcellular distribution of Bcl-x(S) DeltaTM, from a diffuse distribution throughout the cell to a more defined distribution. Moreover co-immunoprecipitation experiments directly demonstrated that Bcl-x(S) can associate with GFP-Bcl-x(S), Bcl-x(L), or Bcl-2. These results suggest that such Bcl-x(S) interactions may be important for the mechanism of action of this protein.     

IL-4 induces apoptosis in A549 lung adenocarcinoma cells: evidence for the pivotal role of 15-hydroxyeicosatetraenoic acid binding to activated peroxisome proliferator-activated receptor gamma transcription factor

The proinflammatory cytokine IL-4 is secreted in large amounts during allergic inflammatory response in asthma and plays a pivotal role in the airway inflammation. IL-4 has been shown to up-regulate 15-lipoxygenase and produce 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) in A549 cells via the Janus kinase/STAT6 pathway under coactivation of CREB binding protein/p300. IL-4 has also been shown to up-regulate peroxisome proliferator-activated receptor gamma (PPARgamma) nuclear receptors in macrophages and A549 cells. In this study we demonstrate that 15(S)-HETE binds to PPARgamma nuclear receptors and induces apoptosis in A549 cells. Moreover, pretreatment of cells with nordihydroguaiaretic acid, a 15-lipoxygenase inhibitor, prevented PPARgamma activation and apoptosis. The latter was accomplished by the interaction of the 15(S)-HETE/PPARgamma complex with the adapter protein Fas-associating protein with death domain and caspase-8, as shown by transfection of Fas-associating protein with death domain dominant negative vector and cleavage of caspase 8 to active subunits p41/42 and p18. Whereas IL-4 and PPARgamma ligands failed to induce cleavage of Bid and release of cytochrome c from mitochondria, they caused translocation of the proapoptotic protein Bax from cytoplasm to mitochondria with a concomitant decrease in the Bcl-x(L) level. We therefore believe that in unstimulated cells Bcl-x(L) and Bax form a heterodimer, in which Bcl-x(L) dominates and prevents the induction of apoptosis, whereas in IL-4-stimulated cells the 15(S)-HETE/PPARgamma complex down-regulates Bcl-x(L), and the resulting overweight of Bax commits the cell to apoptosis via caspase-3. However, this pathway does not rule out the direct caspase-8-mediated activation of caspase-3. In conclusion, IL-4-induced apoptosis may contribute to severe loss of alveolar structures and infiltration of eosinophils, mononuclear phagocytes, etc., into the lung tissue of chronic asthma patients.     

Production of reactive oxygen species and loss of viability in yeast mitochondrial mutants: protective effect of Bcl-xL

The capacity of yeast cells to produce reactive oxygen species (ROS), both as a response to manipulation of mitochondrial functions and to growth conditions, was estimated and compared with the viability of the cells. The chronological ageing of yeast cells (growth to late-stationary phase) was accompanied by increased ROS accumulation and a significantly higher loss of viability in the mutants with impaired mitochondrial functions than in the parental strain. Under these conditions, the ectopic expression of mammalian Bcl-x(L), which is an anti-apoptotic protein, allowed cells to survive longer in stationary phase. The protective effect of Bcl-x(L) was more prominent in respiratory-competent cells that contained defects in mitochondrial ADP/ATP translocation, suggesting a model for Bcl-x(L) regulation of chronological ageing at the mitochondria. Yeast can also be triggered into apoptosis-like cell death, at conditions leading to the depletion of the intramitochondrial ATP pool, as a consequence of the parallel inhibition of mitochondrial respiration and ADP/ATP translocation. If respiratory-deficient (rho(0)) cells were used, no correlation between the numbers of ROS-producing cells and the viability loss in the population was observed, indicating that ROS production may be an accompanying event. The protective effect of Bcl-x(L) against death of these cells suggests a mitochondrial mechanism which is different from the antioxidant activity of Bcl-x(L).