InteractiveFly: GeneBrief

death executioner Bcl-2 homologue : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - death executioner Bcl-2 homologue

Synonyms - Rob-1

Cytological map position - 42E-43A

Function - Bax-like proapoptotic Bcl-2 family member

Keywords - programmed cell death, oncogene

Symbol - debcl

FlyBase ID: FBgn0029131

Genetic map position -

Classification - Bcl-2/CED-9 family protein

Cellular location - mitochondrial membrane

NCBI link: Entrez Gene
debcl orthologs: Biolitmine
Recent literature
M'Angale, P.G. and Staveley, B.E. (2016). Bcl-2 homologue Debcl enhances α-synuclein-induced phenotypes in Drosophila. PeerJ 4: e2461. PubMed ID: 27672511
The common hallmark for both sporadic and familial forms of Parkinson disease (PD) is mitochondrial dysfunction. Mammals have at least twenty proapoptotic and antiapoptotic Bcl-2 family members, in contrast, only two Bcl-2 family genes have been identified in Drosophila melanogaster, the proapoptotic mitochondrial localized Debcl and the antiapoptotic Buffy. The expression of the human transgene α-synuclein, a gene that is strongly associated with inherited forms of PD, in dopaminergic neurons (DA) of Drosophila, results in loss of neurons and locomotor dysfunction to model PD in flies. The altered expression of Debcl in the DA neurons and neuron-rich eye and along with the expression of α-synuclein offers an opportunity to highlight the role of Debcl in mitochondrial-dependent neuronal degeneration and death. The directed overexpression of Debcl using the Ddc-Gal4 transgene in the DA of Drosophila results in flies with severely decreased survival and a premature age-dependent loss in climbing ability. The inhibition of Debcl results in enhanced survival and improved climbing ability whereas the overexpression of Debcl in the α-synuclein-induced Drosophila model of PD results in more severe phenotypes. In addition, the co-expression of Debcl along with Buffy partially counteracts the Debcl-induced phenotypes, to improve the lifespan and the associated loss of locomotor ability observed. In complementary experiments, the overexpression of Debcl along with the expression of α-synuclein in the eye, enhances the eye ablation that results from the overexpression of Debcl. The co-expression of Buffy along with Debcl overexpression results in the rescue of the moderate developmental eye defects. The co-expression of Buffy along with inhibition of Debcl partially restores the eye to a roughened eye phenotype. Taken all together these results clarify on the role for Debcl in neurodegenerative disorders.

Studies that have demonstrated the presence of at least four caspases and an Apaf-1 homolog (Apaf-1-related-killer) in flies strongly argue the existence of a Drosophila caspase cascade, similar to the mammalian cell death machinery. These findings have also predicted a role for the Bcl-2/CED-9 family in Drosophila apoptotic cell death. The caspases comprise a family of cysteine proteases that participate in a proteolytic cascade, cleaving downstream caspases and a number of cellular proteins that ultimately execute apoptotic biochemical events, such as DNA fragmentation and chromatin condensation. Apaf-1 functions at the initial step in this cascade to activate the initiator caspase, caspase-9, in the presence of cytochrome c and ATP/dATP. The Bcl-2 family of proteins consists of antiapoptotic and proapoptotic members, both of which control the cell-death decision by regulating such processes as mitochondrial cytochrome c release and caspase activation through adapter protein Apaf-1, and/or by neutralizing the effects of opposing Bcl-2 family members. The first Drosophila Bcl-2 protein was described simultaneously in two different laboratories: Igaki, (2000) termed the protein Rob-1, for Drosophila ortholog of the Bcl-2 family-1; Colussi, (2000) termed it Debcl (pronounced debacle) for Death executioner Bcl-2 homolog. Rob-1/Debcl was discovered on the basis of sequence homology, by the presence of expressed sequence tags (ESTs) with sequence resemblance to Bcl-2s in the EST database of the Berkeley Drosophila Genome Project. Although it seems like that the name Death executioner Bcl-2 homologue will prevail, this essay uses both names interchangeably, depending on the study cited.

To investigate whether Debcl is a pro- or anti-apoptotic protein in vivo, transgenic flies were generated with debcl cDNA under control of the yeast UAS-GAL4 promoter. Ectopic expression was then achieved by crossing these flies to various GAL4 drivers. To express debcl in all tissues at various developmental stages, UAS-debcl flies were crossed to hsp70-GAL4 flies; embryos or larvae were then heat shocked. Heat shock-induced expression of debcl results in enhanced levels of TUNEL positive cells (indicating the presence of fragmented DNA accompanying programmed cell death) in the embryo and in larval tissues (Colussi, 2000).

Tissue specific drivers were then used to express debcl during larval development. Ectopic expression of debcl in the posterior region of the eye imaginal disc, using the GMR-GAL4 driver, results in increased acridine orange staining cells in the posterior region of the eye. Similarly, expression throughout the eye imaginal disc of 2nd instar larvae, using the eyeless-GAL4 driver, results in increased TUNEL positive cells in the anterior and posterior regions of the eye. It was predicted that expression of debcl from eye specific drivers would result in adults with ablated eyes, as does expression of reaper, head involution defective, and grim from the GMR enhancer. Surprisingly, and despite the increase in apoptotic cells seen in the imaginal discs, the adult flies from these crosses exhibit only a mild rough eye phenotype, possibly because of the excess number of cells that are normally generated during eye development. However, other UAS-debcl lines, which presumably have a much higher level of expression, result in adults with severely ablated eyes when crossed to GMR-GAL4. debcl has also been expressed in the larval salivary gland using a salivary gland specific driver, 109-88-GAL4; this expression results in a massive increase in acridine orange staining cells and a reduction in the size of the salivary glands. Thus, debcl induces cell death when ectopically expressed in a number of different tissue types during Drosophila development, indicating that Debcl is a proapoptotic protein of the Bcl-2 family (Colussi, 2000).

To further characterize the biological activity of Debcl, debcl was expressed in Drosophila SL2 cells under the control of an inducible insect promoter. Within 16 h of transfection, Debcl induces apoptosis in a majority of the transfected SL2 cells. By 48 h, all debcl transfected cells have been lost. This cell death is partially inhibited by the cell permeable peptide caspase inhibitor zVAD-fmk and much more effectively by baculovirus caspase inhibitor P35, indicating that Debcl-induced apoptosis is, at least in part, mediated by caspases. While zVAD-fmk is an efficient inhibitor of many mammalian caspases, it is not known whether it can inhibit Drosophila caspases as effectively. Therefore, the partial inhibition of Debcl-induced cell death by zVAD-fmk may reflect its inability to efficiently inhibit all Drosophila caspases. To confirm that the cell killing function of Debcl is dependent on caspase activity, debcl transgenic flies were crossed with GMR-p35 flies. In the resulting flies the effect of Debcl in eye ablation is significantly reduced (Colussi, 2000).

In several proapoptotic Bcl-2 members, the BH3 domain is essential for their killing function (Adams, 1998; Gross, 1999a). To determine whether the BH3 domain in Debcl is required for its proapoptotic function, two substitution mutants (L146G and E151G) of the Debcl BH3 domain were generated and their killing activity was analyzed in SL2 cells. The 146L residue is conserved in the BH3 domains of most proapoptotic Bcl-2 members, whereas 151E corresponds to an acidic residue in most BH3 domains. Whereas the L146G mutation partially inhibits apoptosis induction by Debcl, E151G mutation completely abrogates Debcl-mediated cell killing. Drosophila proteins Grim, Reaper, and Hid are able to induce apoptosis in mammalian cells, despite the fact that mammalian homologs of these proteins have not been found. To determine whether Debcl can also induce apoptosis in mammalian cells, debcl cDNA was cloned in a mammalian expression vector and transfected into NIH 3T3 cells. Most of the debcl-transfected cells undergo apoptosis. When Debcl is cotransfected with expression vectors carrying caspase inhibitors P35, MIHA, or IAP, a substantial decrease in apoptosis is evident. These results indicate that Debcl-induced killing is dependent on its BH3 domain and requires caspase function. In addition to caspase inhibitors, coexpression of prosurvival Bcl-2 and Bcl-xL proteins also significantly inhibits Debcl-induced apoptosis (Colussi, 2000).

Studies indicated that Rob-1 (aka Debcl) can induce apoptosis by a caspase independent mechanism. Although Rob-1 strongly stimulates caspase activation when overexpressed in S2 cells, its ability to kill cells is barely antagonized by the caspase-related apoptosis inhibitors p35 and DIAP2, or by an active-site mutant of a Drosophila apical caspase, Dronc (Nedd2-like caspase). Furthermore, in transgenic flies, rough-eye phenotype caused by overexpression of Rob-1 is not completely suppressed by coexpression of p35. Similarly, previous studies on the proapoptotic Bax, Bak, and Mtd suggest that they all induce apoptosis in the presence of broad caspase inhibitors. Moreover, both Bax and Bak can induce mitochondrial dysfunction and also kill yeast, which lack endogenous caspases. Both Rpr- and Hid-induced cell death are blocked by coexpression of baculovirus p35. In contrast, overexpression of p35 shows no effect on Rob-1-induced apoptosis. Higher levels of p35 expression exhibit only a slight protective effect against Rob-1-induced cell killing. Similarly, DIAP2, which inhibits both Rpr- and Hid-induced apoptosis, or an active-site mutant of the CARD (caspase recruitment domain)-containing Drosophila caspase, DRONC, shows little effect on Rob-1-induced apoptosis, suggesting that the Rob-1-stimulated cell-death pathway is downstream or independent of DIAP2 and DRONC. Thus, overexpression of Rob-1 induces apoptosis, probably through a caspase-independent pathway partly distinct from that used by other known Drosophila killer proteins (Rpr, Hid, and Grim). Rob-1 overexpressed in human embryonic kidney 293T cells also exhibits a proapoptotic activity that is only slightly inhibited by p35. Thus, Rob-1, like mammalian Bax, Bak, and Mtd, may activate cell death by inducing both caspase-dependent and -independent pathways. The latter pathways probably can be antagonized by an as-yet-unidentified antiapoptotic member of the Drosophila Bcl-2 family. In the nematode C. elegans, in which two Bcl-2/CED-9 family members, CED-9 and EGL-1, have been identified, all cell deaths occur in a caspase-dependent manner. The presence of a Bax-like protein, Rob-1, in Drosophila might indicate the acquisition of a caspase-independent cell death pathway through evolution (Igaki, 2000).

Screening of suppressors of bax-induced cell death identifies glycerophosphate oxidase-1 as a mediator of debcl-induced apoptosis in Drosophila

Members of the Bcl-2 family are key elements of the apoptotic machinery. In mammals, this multigenic family contains about twenty members, which either promote or inhibit apoptosis. The mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model. This study reports the results of the screening of a P[UAS]-element insertion library performed to identify gene products that modify the phenotypes induced by the expression of bax in Drosophila melanogaster. Seventeen putative modifiers involved in various function or process were isolated: the ubiquitin/proteasome pathway; cell growth, proliferation and death; pathfinding and cell adhesion; secretion and extracellular signaling; metabolism and oxidative stress. The brat gene belongs to a group of suppressors, which is implicated in cell growth, proliferation or death. Other identified genes are involved in carbohydrate metabolism, such as Gpo-1. This result is in agreement with the evidence that Bcl-2 family proteins, in addition to their well characterized function in cell death, also play roles in metabolic processes in particular at the level of energetic metabolism. Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways. Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation (Colin, 2015).

Major executioners of programmed cell death by apoptosis are relatively well conserved throughout evolution. However, the control of commitment to apoptosis exhibits some differences between organisms. During mammalian cells apoptosis, various key pro-apoptotic factors are released from the inter-membrane space of mitochondria. These factors include cytochrome c, Apoptosis Inducing Factor (AIF), Endonuclease G, Smac/DIABLO (Second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI) and the serine protease Omi/HtrA2. Once released in the cytosol, cytochrome c binds to the WD40 domain of Apaf-1 and leads to the formation of a cytochrome c/Apaf-1/caspase-9 complex called 'apoptosome', in which caspase-9 (a cysteinyl aspartase) auto-activates to initiate a caspase activation cascade that will lead to cell death. Mitochondrial permeabilization is under the control of the Bcl-2 family of proteins. These proteins share one to four homology domains with Bcl-2 (named BH1-4) and exhibit very similar tertiary structures. However, while some of these proteins (such as Bcl-2) are anti-apoptotic, the others are pro-apoptotic and assigned to one of the following sub-classes: BH3-only proteins (such as Bid) and multi-domain proteins (such as Bax). During apoptosis, Bax translocates to the mitochondrial outer membrane, undergoes conformational changes, oligomerizes and finally allows the release of pro-apoptotic factors from the intermembrane space. Anti-apoptotic proteins of the Bcl-2 family oppose this Bax-mediated mitochondrial release of apoptogenic factors while BH3-only proteins can activate Bax or inhibit anti-apoptotic proteins of the family (Colin, 2015 and references therein).

In C. elegans, activation of the caspase CED-3 requires CED-4, the homologue of Apaf-1 but no cytochrome c. The Bcl-2 family protein CED-9 constitutively interacts with CED-4 and thereby prevents the activation CED-3. This repression of cell death is released upon binding of CED-9 to the BH3-only protein EGL-1, which induces a conformational change in CED-9 that results in the dissociation of the CED-4 dimer from CED-9. Released CED-4 dimers form tetramers, which facilitate auto-activation of CED-3. Although CED-9 appears bound to mitochondria, these organelles seem to play a minor role in apoptosis in C. elegans, contrarily to mammals (Colin, 2015 and references therein).

The role of mitochondria in Drosophila programmed cell death remains more elusive. Cytochrome c does not seem crucial in the apoptosome activation, which is mediated by the degradation of the caspase inhibitor DIAP1 by proteins of the Reaper/Hid/Grim (RHG) family. The apoptotic cascade appears somehow inverted between flies and worm/mammals. In these two last organisms, apoptosis regulators are relocated from mitochondria to the cytosol. Contrarily, Drosophila apoptosis regulators are concentrated at or around mitochondria during apoptosis. Indeed, targeting the RHG proteins Reaper (Rpr) and Grim to mitochondria seems to be required for their pro-apoptotic activity. Furthermore, Hid possesses a mitochondrial targeting sequence and is required for Rpr recruitment to the mitochondrial membrane and for efficient induction of cell death in vivo (Colin, 2015).

The important role played in Drosophila by the mitochondria in apoptosis is also suggested by the mitochondrial subcellular localization of Buffy and Debcl, the only two members of the Bcl-2 family identified, so far, in this organism. Buffy was originally described as an anti-apoptotic Bcl-2 family member, but it can also promote cell death. Debcl (death executioner Bcl 2 homolog), is a multidomain death inducer that can be inhibited by direct physical interaction with Buffy. When overexpressed in mammalian cells, debcl induces both cytochrome c release from mitochondria and apoptosis. This protein interacts physically with anti-apoptotic members of the Bcl-2 family, such as Bcl-2 itself, in mammals. In Drosophila, Debcl is involved in the control of some developmental cell death processes as well as in irradiation-induced apoptosis (Colin, 2015).

Previous studies have shown in Drosophila that mammalian Bcl-2 inhibits developmental and irradiation-induced cell death as well as rpr- and bax-induced mitochondrial membrane potential collapse . Interestingly, bax-induced cell death has been shown to be mitigated by loss-of-function (LOF) mutations in genes encoding some components of the TOM complex which controls protein insertion in the outer mitochondrial membrane. These results suggest that Bax mitochondrial location remains important for its activity in Drosophila. Therefore, flies provide a good animal model system to study Bax-induced cell death in a simple genetic background and look for new regulators of Bcl-2 family members (Colin, 2015).

This study reports the results of the screening of P[UAS]-element insertion (UYi) library, performed in order to identify modifiers of bax-induced phenotypes in Drosophila. Among 1475 UYi lines screened, 17 putative modifiers were isolated, that include genes involved in various cellular functions. This paper presents a more detailed study of one of these modifiers, UY1039, and shows that glycerophosphate oxidase-1 (Gpo-1) [EC] participates in debcl-induced apoptosis by increasing reactive oxygen species (ROS) production (Colin, 2015).

This screen provided 17 suppressors of phenotypes induced by the expression of bax under control of the wing specific vg-GAL4 driver (lethality and wing notches). The possibility that these suppressors affect GAL4 synthesis or that the selected insertions titrate the GAL4 transcription factor is unlikely, since the number of suppressors is limited (1.6% of the collection). Moreover, UYi insertions were isolated that were not identified in other screens performed using the same collection and the UAS/Gal4 system. Finally, the specificity of one of the suppressors, UY3010, which corresponds to a gain-of-function of the Ubiquitin activating enzyme-encoding gene Uba1 has been reported. Indeed, Uba1 overexpression allows the degradation of Bax and Debcl, thanks to the activation of the ubiquitin/proteasome pathway. This study also showed that Debcl is targeted to the proteasome by the E3 ubiquitin ligase Slimb, the β-TrCP homologue (Colin, 2015).

Nine of the bax-modifiers also behaved as suppressors of debcl-induced wing phenotype while 4 showed no significant effect on this phenotype. Three hypotheses could explain this discrepancy. One possibility is that these bax modifiers are context artifacts and do not represent bona fide Bax interactors. The second possible explanation involves the difference in the driver used in each assay (vg-GAL versus ptc-GAL). Indeed, UY3010 did not significantly suppress debcl-induced apoptosis while another Uba1 overexpression mutant (Uba1EP2375) did. Third, although Bax and Debcl, share similarities in their mode of action and regulation, some signaling pathways could be specific of bax-induced apoptosis. Indeed, a LOF of brat mitigates neither debcl -- (this paper) nor hid -- or Sca3-induced cell death(Colin, 2015).

The brat gene belongs to a group of suppressors, which is implicated in cell growth, proliferation or death. Mutations in this type of genes could compensate cell loss due to ectopic apoptosis induction. Results observed for this group of modifiers can generally be easily interpreted with the literature data. UY1131 corresponds to an insertion in the brat (for brain tumor) gene that could allow the expression of a truncated form of the protein. To check whether this insertion leads to a LOF or a GOF of brat, the effect of the characterized LOF allele bratk0602 on bax-induced phenotypes was tested. This mutation strongly suppressed the wing phenotype showing that UY1131 is a LOF of brat. Brat belongs to the NHL family of proteins, represses translation of specific mRNAs and is a negative regulator of cell growth. The suppression of bax-induced phenotypes by a LOF of brat could suggest that this gene also regulates cell death, which seems unlikely according to its inability to suppress other cell death pathways. Alternatively brat could regulate somehow compensatory proliferation in this system (Colin, 2015).

Some candidate suppressors encode proteins involved in secretion or components of the extra-cellular matrix. The effect of these genes could rely on cell signaling. Change in levels of secreted proteins could modify cell-extracellular matrix interactions and thus affect viability via processes similar to anoikis (Colin, 2015).

Several suppressors are implicated in pathfinding (comm, comm3, hat, scratch and lola). Two hypotheses can be formulated. Either neurons are of particular importance in bax-induced phenotypes or a more general role of these proteins in signaling is responsible for these suppressions. If the neuronal death could explain the decreased survival of bax expressing flies, it could hardly explain the wing phenotypes. Therefore, these suppressor genes may have a more general role in signaling and in particular in cell death regulation. For example, UY2669 corresponds to a GOF mutant of scratch (scrt). This gene is a Drosophila homologue of C. elegans ces-1, which encodes a snail family zinc finger protein involved in controlling programmed death of specific neurons. Interestingly, a mammalian homologue of scratch, named Slug, is involved in a survival pathway that protects hematopoietic progenitors from apoptosis after DNA damage. Slug also antagonizes p53-mediated apoptosis by repressing the bcl-2-family pro-apoptotic gene puma. More recently, a regulatory loop linking p53/Puma with Scratch has been described in the vertebrate nervous system, not only controlling cell death in response to damage but also during normal embryonic development (Colin, 2015).

Another possibility is that these modifiers could affect some extracellular survival and/or death factors. For example, sugarless, which was found twice in the screen, has been shown to interact with several survival pathways such as Wingless, EGF and FGF pathways that can play a role in defining shape and size of tissues and organs. This result can be paralleled with the suppressive effect of mutations in hephaestus and lola, both of which interact with the Notch/Delta signaling. Notably, lola, a gene encoding a Polycomb group epigenetic silencer, has been shown to be required for programmed cell death in the Drosophila ovary. Lola has also been identified for its role in normal phagocytosis of bacteria in Drosophila S2 cells and as a component of the Drosophila Imd pathway that is key to immunity. In contrast, Lola is required for axon growth and guidance in the Drosophila embryo. This indicates that lola could play a role in cell adhesion and motility. Accordingly, when coupled with overexpression of Delta, misregulation of pipsqueak and lola induces the formation of metastatic tumors associated with a downregulation of the Rbf (Retinoblastoma-family) gene (Colin, 2015).

Bcl-2 family proteins, in addition to their well characterized function in cell death, also play roles in metabolic processes in particular at the level of energetic metabolism. In particular, Bcl-2 regulates mitochondrial respiration and the level of different ROS through a control of cytochrome c oxidase activity. Study of heterologous bax expression in yeast has provided clues on Bax function in relation to ROS and yeast LOF mutants of genes involved in oxidative phosphorylation show increased sensitivity to Bax cytotoxicity. In agreement, Bcl-xL complements Saccharomyces cerevisiae genes that facilitate the switch from glycolytic to oxidative metabolism. Furthermore, both the anti-apoptotic effect of LOF mutations in Gpo-1 and the GOF in transketolase genes can be related to a protective effect against oxidative stress. This result suggests that the cell death process induced by Bax involves, at least in part, the modulation of different ROS levels (Colin, 2015).

Indeed, this study reports that the suppressor effect of a null allele of Gpo-1 is associated with a decreased ability of Debcl to induce ROS production. This result is in agreement with the observation that 70% of the total cellular H2O2 production was estimated to stem from Gpo-1 in isolated Drosophila mitochondria. This enzyme has also been implicated in ROS production in mammalian brown adipose tissue mitochondria when glycerol-3-phosphate was used as the respiratory substrate and, more recently, in prostate cancer cells. In this latter case, ROS production seems to be beneficial to cancer cells, whereas this study show that it favors cell death in Drosophila wing disc cells. This apparent contradiction could be related to the abnormal ROS production occurring during the oncogenic transformation and the shift to a glycolytic metabolism (Colin, 2015).

In conclusion, this study shows that Gpo-1 contributes to debcl-induced apoptosis by increasing reactive oxygen species (ROS) production and provides a substantial resource that will aid efforts to understand the regulation of pro-apoptotic members of the Bcl-2 family proteins (Colin, 2015).


Protein Interactions

Since Debcl induces cell death, which is partly inhibited by the overexpression of Bcl-2 and Bcl-xL, an attractive hypothesis is that Debcl binds to and neutralizes prosurvival Bcl-2 homologs. Because no prosurvival Bcl-2–like proteins have been identified so far in Drosophila, a test was performed to see if Debcl can bind to any of the known mammalian or viral prosurvival homologs of Bcl-2. In transient overexpression experiments in mammalian cells, Debcl associates with Bcl-2 and most of its functional homologs, although the binding to Bcl-xL, Mcl-1, and adenovirus E1B19K protein is relatively weaker. For these interaction studies, the method involved radiolabeled cell extracts that allow the simultaneous detection of two interacting proteins in the same sample. Conventional immunoblotting of the immunoprecipitated proteins was also used and similar results were obtained. These data clearly show that Debcl can interact with most of the known prosurvival Bcl-2 proteins and is likely to induce cell death by the same molecular mechanisms as other proapoptotic Bcl-2-related proteins. These results further provide evidence for the functional conservation of Bax-like proteins in mammals and flies. A test was also performed to see whether Debcl interacts with the proapoptotic members of Bcl-2 family. In coimmunoprecipitation experiments, Debcl does not associate with any of the BH3-only proteins (Bik, Bid, Bad, and Bim) or the BH1-, BH2-, and BH3- containing proteins (Bax and Bak) (Colussi, 2000).

Bcl-2 family proteins are key regulators of apoptosis. Both pro-apoptotic and anti-apoptotic members of this family are found in mammalian cells, but only the pro-apoptotic protein Debcl has been characterized in Drosophila. Buffy, the second Drosophila Bcl-2-like protein, is a pro-survival protein. Ablation of Buffy by RNA interference leads to ectopic apoptosis, whereas overexpression of buffy results in the inhibition of developmental programmed cell death and gamma irradiation-induced apoptosis. Buffy interacts genetically and physically with Debcl to suppress Debcl-induced cell death. Genetic interactions suggest that Buffy acts downstream of Reaper, Grim and Head involution defective, and upstream of the apical caspase Dronc. Furthermore, overexpression of buffy inhibits ectopic cell death in diap1 (th5) mutants. Taken together these data suggest that Buffy can act downstream of Rpr, Grim and Hid to block caspase-dependent cell death. Overexpression of Buffy in the embryo results in inhibition of the cell cycle, consistent with a G1/early-S phase arrest. These data suggest that Buffy is functionally similar to the mammalian pro-survival Bcl-2 family of proteins (Quinn, 2003).

The mammalian pro-apoptotic Bcl-2 proteins function by binding and sequestering pro-survival Bcl-2 members. Debcl binds most mammalian pro-survival Bcl-2 proteins, including Bcl-2 and Bcl-XL, but not their pro-apoptotic counterparts. In order to determine whether Debcl heterodimerizes with Buffy, co-immunoprecipitation experiments were carried out. FLAG-tagged Buffy was coexpressed with HA-tagged Debcl in 293T cells. Immunoprecipitation was performed with anti-FLAG or anti-HA antibodies. The control immunoblot with anti-FLAG shows that FLAG-Buffy (33 kDa) is precipitated. Immunoblotting of the FLAG immunoprecipitates with anti-HA reveals the HA-Debcl protein, suggesting that the two proteins can co-immunoprecipitate. Therefore, like the pro- and anti-apoptotic members of the mammalian Bcl-2 family, Debcl and Buffy can physically interact (Quinn, 2003).

Debcl and cell death mechanism

A Drosophila homolog of the human killer protein Bok (termed in this study DBok but this Interactive Fly report will conform to the FlyBase term Debcl) was identified. The predicted structure of Debcl is similar to pore-forming Bcl-2/Bax family members. Debcl induces apoptosis in insect and human cells, which is suppressible by anti-apoptotic human Bcl-2 family proteins. A caspase inhibitor suppresses Debcl-induced apoptosis but does not prevent Debcl-induced cell death. Moreover, Debcl targets mitochondria and triggers cytochrome c release through a caspase-independent mechanism. These characteristics of Debcl reveal evolutionary conservation of cell death mechanisms in flies and humans (Zhang, 2000).

Using the human Bcl-2 sequence for data base searches, an EST clone (AI513093) was identified and cDNAs were subsequently cloned encoding a 214-amino acid protein, which contains regions sharing extensive amino acid sequence homology with the BH1, BH2, and BH3 domains of other Bcl-2 family proteins. Overall, the sequence of this protein is most similar to rat Bok, a pro-apoptotic Bcl-2 family member, with 30% sequence identity (52% similarity). The homology of Debcl to other Bcl-2 family proteins was further analyzed by modeling the protein on the structure of human Bcl-XL, confirming the prediction that Debcl adopts a highly similar fold of an irregular alpha-helical bundle, resembling the pore-forming domains of bacterial toxins. In particular, six alpha-helices are predicted, including a hairpin pair of largely hydrophobic alpha-helices in the center of the molecule, surrounded by a shell of four amphipathic alpha-helices (Zhang, 2000).

To explore its effects on apoptosis, the Debcl protein was overexpressed by transient transfection in Sf9 insect cells, along with a GFP marker. Alternatively, Debcl was expressed as a GFP fusion protein, providing a convenient means of verifying expression. Staining of the fixed cells with a DNA-binding fluorochrome (DAPI) 1 day later revealed the presence of multiple apoptotic cells (with condensed chromatin, fragmented nuclei, and rounded, shrunken cell bodies) in cultures of Debcl- but not control plasmid-transfected Sf9 cells. Debcl also induces apoptosis of mammalian cells, such as HEK293, HT1080, and COS-7, implying evolutionary conservation of its cytotoxic function. Debcl-induced apoptosis is consistently suppressed by co-expressing the anti-apoptotic Bcl-2 family protein Mcl-1. Immunoblot analysis confirms that Debcl protein is still produced, and indeed is present at higher levels when cells are co-transfected with Mcl-1, suggesting that the cytoprotective effects of this anti-apoptotic Bcl-2 family protein allows the Debcl protein to accumulate to higher levels. Bcl-XL also partially inhibits Debcl-induced apoptosis but is far more variable in its effects, inhibiting Debcl-induced apoptosis by 0%-50% (mean = 19%). The BH3 domain of many pro-apoptotic Bcl-2 family proteins is required for dimerization with anti-apoptotic proteins such as Bcl-2 and Bcl-XL and for induction of cell death. However, similar to mammalian Bok, deletion of the BH3 domain of Debcl does not interfere with apoptosis induction by Debcl, indicating a BH3-independent mechanism (Zhang, 2000).

Based on fluorescence UV microscopy analysis of cells transfected with a plasmid producing GFP-Debcl fusion protein, the Debcl protein was determined to be cytosolic, localizing to intracellular organelles in a pattern typical of Bcl-2 family proteins. Subcellular fractionation studies confirm that Debcl is associated at least in part with mitochondria-containing heavy-membrane fractions, despite the absence of the C-terminal transmembrane domain, which is commonly found in many other Bcl-2 family proteins (Zhang, 2000).

The mammalian pro-apoptotic Bcl-2 family members, which share structural similarity with pore-forming proteins (Bax, Bak, and Bid), induce release of cytochrome c from mitochondria. Similarly, in transfection experiments, expression of Debcl in 293T cells induces an increase in cytosolic and a concomitant decrease in mitochondrial cytochrome c. Moreover, similar to mammalian Bax, release of cytochrome c is caspase-independent, as evidenced by failure of a broad spectrum caspase inhibitor, zVAD-fmk, to suppress it. Under the same conditions, however, zVAD-fmk suppresses Debcl-induced caspase activation (determined by measuring cleavage of the caspase substrate acetyl-Asp-Glu-Val-Asp-aminomethyl-coumarin (Ac-DEVD-AFC) and apoptosis, as determined by DAPI staining of fixed cells. Thus, zVAD-fmk effectively blocks the caspase activation that occurs downstream of cytochrome c release in Debcl-expressing cells. However, analogous to previous reports for mammalian Bax, Debcl-induces non-apoptotic cell death (as determined by failure of cells to exclude trypan blue dye) in the presence of caspase inhibitor, presumably due to the deleterious consequences of cytochrome c release on mitochondrial metabolic function. In contrast to zVAD-fmk, anti-apoptotic Mcl-1 protein suppresses Debcl-induced cytochrome c release, as well as Debcl-induced apoptosis and cell death. Taken together, these findings suggest that Debcl induces cell death through mechanisms similar to those employed by its mammalian counterparts (Zhang, 2000).

To determine whether the C-terminal hydrophobic region of Rob-1 (AKA Debcl) is necessary for its intracytoplasmic membrane docking or proapoptotic function, an expression plasmid producing a truncated form of Rob-1 (Drob-deltaTM) lacking the last 39-aa residues, which contain the putative transmembrane domain, was constructed. In contrast to the membrane localization of full-length Rob-1, the Drob-deltaTM mutant displays a diffuse distribution throughout the cytosol, when overexpressed in either COS7 cells or S2 cells. A cell death assay revealed that this mutation dramatically reduces the ability of Rob-1 to kill S2 cells. Furthermore, the caspase-stimulating activity of the Drob-deltaTM mutant is completely eliminated. These results suggest that the cell killing and caspase-stimulating activities of Rob-1 both require that it be targeted to subcellular components, most likely to the mitochondria, through its C-terminal transmembrane domain (Igaki, 2000).

The Drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the beta-TrCP homologue Slimb

The ubiquitin-proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, this study identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. Uba1 was demonstrated to regulate apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, these apoptotic processes were shown to involve the same multimeric E3 ligase-an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of beta-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family (Colin, 2014).

This paper reports the regulation of bax- and debcl-induced apoptosis by the ubiquitin-proteasome pathway. The stimulation of this pathway by overexpressing Uba1, which encodes the ubiquitin activating enzyme, leads to an almost complete loss of bax-induced cell death. This regulation seems conserved through evolution, as debcl-induced apoptosis is also regulated in this way. However, since Bax seems to necessitate Debcl in order to kill Drosophila eye cells, one could wonder whether suppression of Bax-induced cell death depends on the direct effect of Uba1 on Debcl. Nevertheless, since both Bax and Debcl proteins are degraded when Uba1 is overexpressed, this seems unlikely unless Debcl stabilizes Bax (Colin, 2014).

Since Buffy inhibits autophagy in response to starvation, it is hypothesized that Debcl induces an autophagic cell death. Autophagy was monitored by using a UAS-Atg8-GFP transgene. It was found that actually no autophagy could be detected upon Debcl expression. Uba1 has been shown in the literature to be required for autophagy and reduction of cell size in the intestine. This study shows that Debcl-induced cell death in the wing disc is not only suppressed by Uba1 but also by proteasome mutants. These data suggest that the UPS pathway is the main proteolytic pathway involved in the suppression of Bax and Debcl-induced apoptosis by Uba1. However, given that slmb has been shown to regulate Wg and Dpp pathways, it cannot be excluded that these pathways are partially involved in phenotypic suppression (Colin, 2014).

Studies of the proteasome-dependent regulation of members of the Bcl-2 family in mammals have only rarely led to the identification of the specific E3 ligases. This study identified an SCFSlmb complex as the E3 ubiquitin ligase that regulates the Debcl pathway and may target it to proteasomal degradation. It would be interesting to determine whether the mammalian homologue of Slmb, β-TrCP, targets Bax to the proteasome in mammalian cells (Colin, 2014).

This study has show Debcl is a target of the UPS, thus finding a new regulation of apoptosis that differs from the control of Dronc, Drice and RHG protein levels by Diap1. Indeed, Diap1 is a key enzyme that decides of cell fate by degrading either pro-apoptotic regulators or itself, leading to either cell survival or apoptosis. Since Diap1 levels are downregulated by the F-box protein Morgue in presence of Rpr or Grim, it could be hypothesized that the Morgue/Diap1 pathway is involved in bax- and debcl-induced apoptosis regulation. This does not seem to be the case because a hypomorphic allele of morgue did not increase bax- and debcl-induced apoptosis. Furthermore, overexpression of diap1 does not inhibit bax- and debcl-induced apoptosis. Thus, the proteasome-dependent regulation that this study has identified is independent of Diap1 and differs from the Morgue/Diap1 regulation of cell death. The existence of different UPS-modulated cell death pathways is also supported by the reported absence of genetic interaction between RHG pathway components and Debcl (Colin, 2014).

The results indicate an anti-apoptotic role of Uba1 in the wing tissue. However, two other studies revealed a pro-apoptotic role of Uba1 in the eye; strong Uba1 loss-of-function alleles lead to apoptosis and compensatory proliferation in the developing eye. As previously shown in other systems, these processes seem to involve the RHG/Diap1/Dronc pathway. Hypomorphic alleles of Uba1 have shown opposite effects as they suppressed hid- or grim-induced apoptosis in the eye. These results are consistent with previous data indicating that Uba1 overexpression, using the Uba1 EP2375 allele, increases RHG-induced apoptosis. In principle, this apparent contradiction may result from either the cell death signal or the studied tissue. The use of a GMR-Gal4 driver shows that Uba1 overexpression also inhibits debcl-induced apoptosis in the eye tissue, which suggests that the Uba1 effect is specific of debcl-induced apoptosis. In contrast, rpr-induced cell death is enhanced by Uba1 overexpression in the eye, whereas it is suppressed by Uba1 in the wing. These results suggest that Debcl could be involved in rpr-induced cell death in the wing but not in the eye. RHG proteins are known to mediate their pro-apoptotic function by stimulating Diap1 degradation by the UPS while Debcl is a direct target of ubiquitination. Therefore, RHG-induced degradation of Diap1 through its ubiquitination by Morgue could explain the pro-apoptotic role of Uba1 in the eye whereas the anti-apoptotic Uba1 function mediated by Slmb in Debcl-induced cell death would rely on Debcl degradation. By showing that different pro-apoptotic pathways are regulated by the UPS in Drosophila, this work suggests that the tissue-dependent effect of the pleiotropic enzyme Uba1 must result from a change in the balance between UPS pro- and anti-apoptotic effects. It is proposed that this change relies on the availability in E3 enzymes, the incoming signals and relative amounts of pro- and anti-apoptotic regulators of cell fate (Colin, 2014).


Because of the low expression of debcl, tyramide amplification was used after hybridization to detect Debcl mRNA expression in situ. In early embryos, Debcl mRNA is present uniformly, but becomes more concentrated in the tissues of the gut later in embryogenesis. The relatively high levels of Debcl RNA in early embryos are likely to be derived maternally, since zygotic transcription does not begin until stage 5. From stage 14 embryos, Debcl mRNA is detected in regions in the head that correspond to the pharynx and clypeolabrum, where many TUNEL positive cells (cells undergoing apoptosis as reflected in DNA fragmentation) are detected. Expression can also be detected in a segmentally reiterated pattern in stage 14 embryos that may correlate with the TUNEL positive cells that are detected in the central nervous system at this stage. During 3rd instar larval development, Debcl expression is detected in the brain lobes in the outer proliferative center, in the posterior part of the eye imaginal disc, and in the gut, where TUNEL positive cells are clearly seen. Debcl expression is also clearly evident in the salivary glands, particularly in the ducts. Because of background staining problems in salivary glands, acridine orange staining instead of TUNEL was used to detect apoptotic cells in this tissue. Using this technique, no apoptotic cells were detected in 3rd instar salivary glands, suggesting that Debcl expression may precede cell death in this tissue. High levels of Debcl expression are detected in the nurse cell compartment of stage 10a ovaries, which undergo apoptosis at stage 10b. Thus, Debcl expression late in embryogenesis, during larval development, and during oogenesis significantly correlates with tissues undergoing apoptosis (Colussi, 2000).

Northern blot analysis of Rob-1 has detected a 1.3-kb transcript, consistent with the size of the cDNA. RT-PCR analysis demonstrates that Rob-1 mRNA is expressed at all stages of development. In addition, in situ hybridization with digoxigenin-labeled Rob-1 RNA probes reveals that Rob-1 is highly expressed in early-stage embryos. As development proceeds, expression levels of Rob-1 mRNA are reduced. In the third-instar larval stages, Rob-1 is expressed in leg discs, eye discs, wing discs, and midgut, but no signal was detected in salivary glands and brain (Igaki, 2000).

To determine subcellular localization, HA-tagged Rob-1 was overexpressed in S2 cells, and the cells were stained with an anti-HA antibody. Confocal microscopy reveals that Rob-1 has a granular cytoplasmic distribution, consistent with an association with intracellular organelles. To better identify the localization of Rob-1, transfected cells were colabeled with a mitochondria-specific fluorescent dye, MitoTracker. The pattern of mitochondrial staining is similar to that of Rob-1 . Merged images of Rob-1- and MitoTracker-stained cells show that the two proteins colocalize. This colocalization is also observed when HA-Rob-1 is overexpressed in mammalian COS7 cells. Subcellular fractionation studies in S2 cells reveals that HA-Rob-1 is enriched in the heavy-membrane fraction. HA-Rob-1 is also present in the low speed pellet and light membrane fractions with a smaller amount of staining but not in the cytosolic fraction. Together with the microscopic data, these results suggest that Rob-1 localizes to the intracytoplasmic membranes, predominantly to the mitochondrial membranes (Igaki, 2000).


After screening a number of UAS-debcl lines, two lines (UAS-debcl#26 on chromosome III and UAS-debcl#18 on chromosome II) were found that, when crossed to GMR-GAL4, give rise to adults with severely ablated eyes. To use this phenotype to examine genetic interactions, a stock was generated containing GMR-GAL4 (2nd chromosome) and UAS-debcl#26. To examine whether the rough eye phenotype is due to the activity of caspases, GMR-p35 was crossed to these flies and the eye phenotype of the progeny was examined. GMR-p35 significantly improves the severe rough eye phenotype of GMR-GAL4; UAS-debcl#26 eyes. These results confirm that Debcl functions in a caspase-dependent fashion upstream of caspase activation (Colussi, 2000).

To determine the involvement of rpr, hid, or grim in the GMR-GAL4; UAS-debcl#26 eye phenotype, these flies were crossed to a deficiency that removes all three genes (Df(3L)H99). If rpr, hid, or grim are rate limiting for Debcl function, then suppression of the GMR-GAL4; UAS-debcl#26 eye phenotype would be expected. However, no significant suppression of this phenotype was observed, suggesting that the GMR-GAL4; UAS-debcl#26 eye phenotype is not dependent on the gene dosage of rpr, hid, or grim (Colussi, 2000).

Next, a test was performed to see whether the inhibitor of apoptosis (IAP) homolog, diap1, genetically interacts with debcl, by examining the GMR-GAL4; UAS-debcl#26 eye phenotype when the dosage of diap1 is halved. Halving the dosage of diap1, using two different deficiencies, results in a strong enhancement of the GMR-GAL4; UAS-debcl#26 eye phenotype. Furthermore, there is a significant reduction in the number of flies expected to contain either of the diap1 deficiencies and GMR-GAL4; UAS-debcl#26. This is possibly due to leaky expression of the GMR-GAL4; UAS-debcl#26 construct in other tissues during development and the enhancement of this effect by reducing the dose of diap1. Thus, diap1 genetically interacts with debcl. No genetic interaction between debcl and diap2 was observed when a diap2 deficiency was crossed with GMR-GAL4; UAS-debcl#26 (Colussi, 2000).

Described recently has been a mutation in the Drosophila Apaf1/ced4 homolog dark, (Rodriguez, 1999). To assess the effect of reducing the dosage of dark on the GMR-GAL4; UAS-debcl#26 eye phenotype, a hypomorphic allele of dark (darkCD8) was crossed to GMR-GAL4/CyO; UAS-debcl#26/TM6B flies. Reducing the dosage of dark suppresses the rough eye phenotype of GMR-GAL4; UAS-debcl#26 flies. Therefore, dark genetically interacts with debcl (Colussi, 2000).

Currently, no specific debcl mutants are available. Therefore, to examine the in vivo function of Debcl, RNAi studies were carried out to inhibit debcl gene function. RNAi is a technique whose effects mimic mutation; it is a powerful technique used to disrupt the function of specific genes. Additionally, RNAi has the advantage of ablating maternally contributed mRNA; this is difficult to achieve genetically. Precellularized embryos were injected with debcl double-stranded RNA, aged until stage 16, and analyzed by TUNEL staining, in order to detect apoptotic cells. debcl RNAi results in a large reduction in TUNEL positive cells in the embryos. These RNAi results indicate that debcl gene function is required for programed cell death in the embryos. Additionally, these data suggest that maternally deposited debcl mRNA may be required for normal cell death in fly embryos (Colussi, 2000).

Removal of Debcl function during Drosophila embryonic development results in excess glial cells, demonstrating its pro-apoptotic function. In cell culture assays, Debcl efficiently induces apoptosis but, remarkably, also demonstrates protective activity when death stimuli are introduced. Ectopic expression of Debcl in the eye leads to subtle defects that are strongly potentiated by ultraviolet irradiation, resulting in a dramatic loss of retinal cells (Brachmann, 2000).

Two Bcl-2 family members, Debcl and dBorg-2, have been identified in Drosophila. Both proteins contain BH1, BH2 and BH3 domains and are most similar to Bok/Mtd, a pro- apoptotic family member. Apart from an amino-terminal extension in Debcl, residues conserved within the Bcl-2 family are present throughout the length of the sequences and are concentrated in the BH domains. Both debcl and dborg-2 are expressed between embryonic stages 11 and 16, stages of maximal cell death and dynamically throughout development of the fly. These results suggest a role in developmentally regulated cell death; however, the overall pattern of transcription does not distinguish between a pro- or anti-apoptotic function for these molecules. A series of cell culture assays were undertaken to investigate both possibilities. Overexpression of debcl promotes both death and survival in cell culture assays. Overexpression of debcl in BHK.21, HEK 293, Chinese hamster ovary (CHO) and Drosophila Schneider S2 cells resulted in reduced viability, indicating a strong conservation of killing function across species. A dose-response curve of the killing effect of Debcl in CHO cells demonstrates the potency of this pro-apoptotic Bcl-2 family member: when 1 µg of the debcl expression vector is used, 9% of the transfected cells are alive 48 hours later. Decreasing the amount of debcl expression vector to 50 ng increases the survival rate to only 50% (Brachmann, 2000).

Co-expression of the baculovirus caspase inhibitor p35 and debcl in CHO cells indicates that the majority of cell killing by Debcl is through a caspase-dependent mechanism. Surprisingly, Schneider S2 cells are relatively resistant to killing by debcl expression, since transfections with less than 0.5 µg of debcl expression vector does not appreciably kill S2 cells. COS cells are also refractory to killing by Debcl. Perhaps these two cell types contain high levels of a protective Bcl-2 family member or do not normally express activators or cofactors required for the pro-apoptotic activity of Debcl (Brachmann, 2000).

Since there is sequence similarity between Debcl and the anti-apoptotic protein Bcl-2, the possibility that Debcl might rescue cells from death was examined. Following transfection of Drosophila S2 cells with an amount of debcl expression plasmid that results in no appreciable cell killing, apoptosis was induced in two ways: (1) by expressing the C. elegans caspase CED-3 and (2) by serum deprivation. In both cases, a significant increase in the number of viable cells after 24 hours is consistently observed, demonstrating that this pro-apoptotic protein can also act as a pro-survival molecule. Such a functional conversion of a Bcl-2 family member has been described previously -- Bcl-2 becomes a killer molecule upon cleavage. Similarly, it is possible that a post-translational modification could turn Debcl into a protective molecule. Alternatively, overexpression of debcl might lead to the up-regulation of a protective pathway in S2 cells, resulting in resistance to apoptosis (Brachmann, 2000).

RNA-mediated interference of debcl results in excess glial cells. An RNA interference assay (RNAi) was used to mimic loss of debcl function and to inactivate the maternal contribution of debcl mRNA. Double-stranded debcl RNA (dsRNA) was injected into the posterior pole region of pre-cellular blastoderm embryos. TUNEL analysis of stage 12/13 embryos injected with debcl dsRNA shows a reduction in TUNEL-positive cells, as compared with a wild-type embryo, indicating that developmental PCD is inhibited. To further assess the development of embryos injected with debcl dsRNA, stage 16 embryos were examined with an antibody to a glial-specific marker. A dramatic expansion of glial cells into the posterior region is observed near the site of injection in 44% of the debcl dsRNA-injected embryos. This phenotype is never observed in control embryos injected with white dsRNA. Thus, reduction of Debcl activity leads to ectopic cells at the site of injection, including a large number of glia, suggesting that this excess tissue is contiguous with the central nervous system (CNS). Fusion of the most posterior embryonic segments, A8 and A9, requires PCD and aids contraction of the CNS. Inhibition of PCD may have resulted in A8 and A9 remaining distinct, leading to an extended CNS region wrapped around excess posterior tissue. Alternatively, excess glial cells may be a response to injection trauma and Debcl might be required not only for normal developmental apoptosis, but also to remove these glial cells (Brachmann, 2000).

To further assess the in vivo role of Debcl, GMR-debcl transgenic flies were created that express debcl exclusively in the developing retina. None of the independent transgenic lines exhibits the expected ablated eye phenotype seen upon overexpression of other death-inducers such as reaper or grim; however, occasional interommatidial pigment cells are missing and bristle groups are mispositioned, suggesting a failure in the correct regulation of interommatidial cell position or removal (Brachmann, 2000).

The subtle phenotype of the GMR-debcl flies suggests that simple overexpression of debcl is insufficient to kill and raises the possibility that Debcl requires appropriate activation before inducing apoptosis in vivo, an idea consistent with the broad expression of debcl in embryonic tissues. One candidate activator is the DNA damage response pathway. UV irradiation was used to determine the ability of DNA damage to potentiate Debcl function. High levels of irradiation (50,000 µJ/cm2)result in a nearly ablated eye. Lower doses (20,000 µJ/cm2) result in only minor loss of cells. Concomitant expression of debcl results in dramatically enhanced cell killing and a severely reduced eye; this phenotype is partially rescued by co-expression of the baculovirus protein p35, an inhibitor of caspase function. These results indicate a strong synergy between death induced by Debcl and the DNA damage response pathway. The potentiation of UV-irradiation-induced death by ectopically expressed debcl suggests that the killer function of Debcl is post-transcriptionally regulated. Previous work in cell culture has led to the idea that the balance of pro-and anti-apoptotic Bcl-2 family members is critical for determining whether a cell is permitted to survive. Therefore, a vast excess of an as yet unknown, anti-apoptotic Bcl-2 family member in the developing eye could, by hetero-dimerization, neutralize the Debcl pro-apoptotic function (as observed for Bax with Bcl-2). Alternatively, Debcl may require additional post-translational modifications to be fully activated, modifications that are not provided during the normal development of the fly eye. By analogy with known Bcl-2 family members, this could be through conformational change (as observed for Bax), cleavage (as described for Bcl-2) or by phosphorylation (as is the case for Bad) (Brachmann, 2000 and references therein).

Bax-like protein Drob-1 protects neurons from expanded polyglutamine-induced toxicity in Drosophila

Bcl-2 family proteins regulate cell death through the mitochondrial apoptotic pathway. This study showed that the Drosophila Bax-like Bcl-2 family protein Drob-1 (Debcl) maintains mitochondrial function to protect cells from neurodegeneration. A pan-neuronal knockdown of Drob-1 results in lower locomotor activity and a shorter lifespan in adult flies. Either the RNAi-mediated downregulation of Drob-1 or overexpression of Drob-1 antagonist Buffy strongly enhances the polyglutamine-induced accumulation of ubiquitinated proteins and subsequent neurodegeneration. Furthermore, ectopic expression of Drob-1 suppresses the neurodegeneration and premature death of flies caused by expanded polyglutamine. Drob-1 knockdown decreases cellular ATP levels, and enhances respiratory inhibitor-induced mitochondrial defects such as loss of membrane potential (Deltapsim), morphological abnormalities, and reductions in activities of complex I+III and complex II+III, as well as cell death. Taken together, these results suggest that Drob-1 is essential for neuronal cell function, and that Drob-1 protects neurons from expanded polyglutamine-mediated neurodegeneration through the regulation of mitochondrial homeostasis (Senoo-Matsuda, 2005).

This report shows that Drob-1 can either promote (cell death during embryogenesis) or inhibit (polyglutamine-induced neurodegeneration) cell death, depending on a variety of conditions. In addition, it was shown that Buffy also has dual functions, namely a survival function (for cell death during embryogenesis) and a proapoptotic function (for polyglutamine-induced neurodegeneration) (Senoo-Matsuda, 2005).

Certain members of the Bcl-2 family proteins can function as both anti- and prodeath factors. Antiapoptotic Bcl-2 and Bcl-xL can be converted into proapoptotic proteins when they are cleaved by caspases or by other proteases. The resulting C-terminal fragments have a 'Bax-like' prodeath activity that induces cytochrome c release from mitochondria and forms pores in synthetic membranes. C. elegans CED-9 also exhibits prodeath as well as antideath activity. Proapoptotic Bax and Bak may promote or inhibit neuronal death depending on the specific death stimulus, neuron subtype, and stage of postnatal development. Bax promotes the survival of trigeminal ganglia neurons during development in mice that are deficient in NGF or TrkA, while it promotes the death of superior cervical ganglia neurons in the same models. Bax potently protects mice and cultured hippocampal neurons from Sindbis virus-induced apoptosis, whereas it promotes the death of Sindbis virus-infected dorsal root ganglia neurons. Bak protects hippocampal neurons from the cell death caused by excitotoxicity or viral infection; however, as mice mature, Bak function is converted from anti- to prodeath in virus-infected spinal cord neurons. Bak also protects mice from kainate-induced seizures, suggesting a possible role in regulating synaptic activity. Drob-1 has also been shown to have a protective activity against serum-deprivation- or CED-3-induced S2 cell death. Thus, individual Bcl-2 family proteins can have a pro- or antiapoptotic function, depending on the cellular context or specific stimulus. These findings, combined with the observations in this study, suggest that the dual-function nature of Bcl-2 family proteins may be evolutionarily conserved from nematodes to mammals (Senoo-Matsuda, 2005 and references therein).

Mitochondrial function [i.e., the production of ATP, regulation of apoptosis, and production of reactive oxygen species (ROS)] is crucial for the maintenance of postmitotic tissues (e.g., muscles and brain) in normal aging, and plays a role in degenerative diseases in humans and in animal models. During normal aging and the progression of human degenerative diseases, a decrease in the total number of cells in some postmitotic tissues (e.g., heart, skeletal muscle, and brain) is associated with a reduction in mitochondrial metabolic activity. This study shows that Drob-1 plays an important role in the survival of postmitotic neurons under both physiological and pathological conditions. Importantly, the finding that downregulation of Drob-1 results in a decrease in cellular ATP levels suggests that Drob-1 may be involved in the maintenance of mitochondrial metabolism. In addition, Drob-1 protects cells from stresses that cause mitochondrial dysfunction. Thus, these results suggest that Drob-1 may regulate the homeostasis of neurons and the aging process by maintaining mitochondrial metabolism. The ubiquitin-proteasome system plays a crucial role in preventing the polyglutamine-induced accumulation of unfolded proteins. This system acts in an ATP-dependent manner. Inhibition of the mitochondrial respiratory chain by the complex I inhibitor rotenone reduces the ubiquitin-proteasomal activity in both rat primary dopaminergic neurons and human SH-SY5Y neuroblastoma cells. Expanded polyglutamine protein has been reported to cause mitochondrial dysfunction, ATP loss, a defect in complex II enzyme activity, and subsequent inhibition of the ATP-dependent ubiquitin-proteasome system. These results lead to the proposal of a model in which Drob-1 suppresses polyglutamine-induced ATP depletion, thereby facilitating the subsequent activation of the ubiquitin-proteasome system, which protects neurons from cell death and degeneration. Buffy can antagonize this survival function of Drob-1 in neurons (Senoo-Matsuda, 2005).

Downregulation of Drob-1 induces ATP depletion and a shorter lifespan in flies. In C. elegans, mitochondrial complex II deficiency causes a shorter lifespan, hypersensitivity to oxidative stress, energy depletion, ROS overproduction, and CED-3- and CED-4-dependent supernumerary cell death (Ishii, 1998; Senoo-Matsuda, 2001; Senoo-Matsuda, 2003). In the complex II-deficient C. elegans mutant mev-1, the shorter lifespan is partially rescued by a loss-of-function mutation of CED-3, suggesting that the supernumerary apoptosis may contribute to shortening the lifespan in C. elegans (Senoo-Matsuda, 2003). Interestingly, the shorter lifespan in the mev-1 mutant may be associated with a decrease in the mitochondrial localization of CED-9 and its downregulation (Senoo-Matsuda, 2003). In mammals, Bcl-xL can prevent the perturbation of mitochondrial ATP/ADP exchange caused by growth factor deprivation, and can maintain oxidative phosphorylation under the growth-factor-withdrawal condition. The proapoptotic Bcl-2 family protein Bad is required to assemble the mitochondria-based glucokinase complex, which regulates glycolysis. Thus, the regulation of mitochondrial homeostasis may be an evolutionarily conserved role of Bcl-2 family proteins (Senoo-Matsuda, 2005).

The findings also suggest that Bcl-2 family proteins may play a crucial role in the pathogenesis of polyglutamine diseases. It would be greatly informative to determine whether Bcl-2 family proteins also play a crucial role in mammalian systems that can be a therapeutic target for neurodegenerative disorders. Further study of Drob-1 should increase understanding of the universal roles of Bcl-2 family proteins and may contribute to the development of new therapeutic applications, not only for polyglutamine diseases, but also for other abnormal-protein-accumulating neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (Senoo-Matsuda, 2005).

The Bax/Bak ortholog in Drosophila, Debcl, exerts limited control over programmed cell death

Bcl-2 family members are pivotal regulators of programmed cell death (PCD). In mammals, pro-apoptotic Bcl-2 family members initiate early apoptotic signals by causing the release of cytochrome c from the mitochondria, a step necessary for the initiation of the caspase cascade. Worms and flies do not show a requirement for cytochrome c during apoptosis, but both model systems express pro- and anti-apoptotic Bcl-2 family members. Drosophila encodes two Bcl-2 family members, Debcl (pro-apoptotic) and Buffy (anti-apoptotic). To understand the role of Debcl in Drosophila apoptosis, authentic null alleles at this locus were produced. Although gross development and lifespans were unaffected, it was found that Debcl was required for pruning cells in the developing central nervous system. debcl genetically interacted with the ced-4/Apaf1 counterpart dark, but was not required for killing by RHG (Reaper, Hid, Grim) proteins. debclKO mutants were unaffected for mitochondrial density or volume but, surprisingly, in a model of caspase-independent cell death, heterologous killing by murine Bax required debcl to exert its pro-apoptotic activity. Therefore, although debcl functions as a limited effector of PCD during normal Drosophila development, it can be effectively recruited for killing by mammalian members of the Bcl-2 gene family (Galindo, 2009).

Using a targeted recombination strategy, seven allelic strains were recovered that were definitively amorphic for debcl. These studies exclude a general requirement for debcl as a global apoptotic effector, which had been suggested from gene silencing analyses. Nevertheless, three compelling lines of evidence establish that debcl does function to regulate a limited number of developmental cell deaths. (1) In every allelic combination tested, TUNEL labeling was consistently and markedly reduced. (2) In every allele tested, debcl genetically interacted with a hypomorphic allele of the apoptosomal gene dark. (3) Extra cells in debcl embryos were detected using markers that visualize persisting or 'undead' cells in canonical PCD mutants. Although the impact caused by eliminating debcl was modest, it is noted that reproducible and consistent PCD phenotypes were observed for all alleles tested. Furthermore, it is also worth noting that the data reflect counts of marked cell populations, only a small fraction of which actually die. Hence, if only the cells that are lost are considered and these are compared against benchmarks seen in animals completely defective for PCD, the effects caused by eliminating debcl are substantial. For example, in H99 animals where no PCD occurs, a 37% excess of Kr+ cells is seen in Bolwig's organ and, by comparison, an excess of up to 23% Kr+ cells was seen in debcl mutants. Graded effects along the anteroposterior axis is another familiar phenotype seen here, and, likewise, has been reported for persisting motoneurons in H99 mutants. Specifically, in cases where extra neuronal cells were observed (e.g. α-Kruppel, these cells tended to appear more commonly among the posterior segments. This trend of extra cells in more posterior segments is consistent with previous studies reporting the prominence of neuronal degeneration in the abdominal ganglion. Segments with supernumerary cells averaged 17% additional cells in debclKO animals and, by comparison, mutations in the apoptosomal genes dark and dronc produced a range from 33%-50% excess cells. From these combined results, it is speculated that perhaps debcl augments apoptotic signaling in certain cell types. Not all tissues were impacted in debclKO animals, however. Notable examples of Drosophila PCD that were unaffected by debcl status included PCD of interommatidial cells in the eye, PCD of the salivary gland and elimination of Crz+ neurons during larval to pupal transition, as well as of markers specific for motoneurons during embryogenesis. Hence, to the extent represented by the markers that were studied, the impact of debcl status on Drosophila cell death appears to be limited to certain embryonic neurons and glia, but not on the PCD of motoneurons. Taken together, these studies exclude a universal requirement for debcl in PCD, yet establish a limited role for this gene in the death of certain cell (Galindo, 2009).

Mitochondrial protein Preli-like is required for development of dendritic arbors and prevents their regression in the Drosophila sensory nervous system

Dynamic morphological changes in mitochondria depend on the balance of fusion and fission in various eukaryotes, and are crucial for mitochondrial activity. Mitochondrial dysfunction has emerged as a common theme that underlies numerous neurological disorders, including neurodegeneration. However, how this abnormal mitochondrial activity leads to neurodegenerative disorders is still largely unknown. This study shows that the Drosophila mitochondrial protein Preli-like (Prel), a member of the conserved PRELI/MSF1 family, contributes to the integrity of mitochondrial structures, the activity of respiratory chain complex IV and the cellular ATP level. When Prel function was impaired in neurons in vivo, the cellular ATP level decreased and mitochondria became fragmented and sparsely distributed in dendrites and axons. Notably, the dendritic arbors were simplified and downsized, probably as a result of breakage of proximal dendrites and progressive retraction of terminal branches. By contrast, abrogation of the mitochondria transport machinery per se had a much less profound effect on the arbor morphogenesis. Interestingly, overexpression of Drob-1 (Debcl), a Drosophila Bax-like Bcl-2 family protein, in the wild-type background produced dendrite phenotypes that were reminiscent of the prel phenotype. Moreover, expression of the Drob-1 antagonist Buffy in prel mutant neurons substantially restored the dendritic phenotype. These observations suggest that Prel-dependent regulation of mitochondrial activity is important for both growth and prevention of breakage of dendritic branches (Tsubouchi, 2009).

Mitochondria are important for multiple cellular events such as ATP production, Ca2+ regulation, axonal and dendritic transport of organelles, and the release and re-uptake of neurotransmitters at synapses. Mitochondria in most healthy cells exist as tubules of variable size and undergo dynamic morphological changes that depend on the balance of fusion and fission. This fusion-fission cycle ensures mixing of metabolites and mitochondrial DNA and influences organelle shape and bioenergetics functionality. The dynamin-related GTPases have been shown to have central roles in the fusion-fission dynamics of mammalian mitochondria. The mitofusins (MFNs) are proteins localized at the mitochondrial outer membrane that are required for the fusion of mitochondria, whereas OPA1 in the inner membrane mediates the fusion. The key component of the fission machinery is dynamin-related protein 1 (Drp1) (Tsubouchi, 2009 and references therein).

Dysfunction of mitochondria is highly connected to neurodegenerative diseases, and abrogation of the fusion machinery is an early and causal event in neurodegeneration. Mutations in MFN2 cause the autosomal dominant disease Charcot-Marie-Tooth (CMT) type 2A, a peripheral neuropathy of long motor and sensory neurons; and Purkinje neurons in the mouse model have aberrant mitochondrial distribution, ultrastructure and electron transport activity. Mutations in OPA1 cause autosomal dominant optic atrophy (ADOA), the most commonly inherited form of optic nerve degeneration. However, it is still largely unknown as to how the abnormal mitochondrial morphology leads to neurodegenerative disorders (Tsubouchi, 2009).

Other consequences of impaired mitochondrial fusion-fission dynamics in the nervous system model have also been studied. In Drosophila, observation of opa1 and drp1 mutants has revealed impaired mitochondrial fusion-fission dynamics in the nervous system. An eye-specific homozygous mutation of opa1 causes rough and glossy eye phenotypes in adult flies, suggesting that an increase in apoptosis is occurring. Mutations in drp1 result in elongated mitochondria that are mostly absent from the presynapses. It has been reported that dendritic mitochondria are more metabolically active than axonal mitochondria and that the dendritic distribution of mitochondria and their activity are essential and limiting for the development and morphological plasticity of dendritic spines in cultured hippocampal neurons. In Drosophila loss of mitochondrial complex II activity causes degeneration of photoreceptors and disruption of mitochondrial protein translation severely affects the maintenance of terminal arborization of dendrites. Nevertheless, it is not yet well understood how proper mitochondrial morphology, distribution and activity contribute to the formation and maintenance of dendritic arbors. This study addressed this question by using Drosophila dendritic arborization (da) neurons. Individually identified da neurons are classified into classes I-IV in order of increasing field size and arbor complexity, and they produce dendritic arbors of stereotypic patterns in a two-dimensional manner between the epidermis and muscles (Tsubouchi, 2009).

This study shows that Preli (protein of relevant evolutionary and lymphoid interest)-like (Prel), a Drosophila mitochondrial protein of the conserved PRELI/MSF1 family, contributes to the integrity of mitochondrial structure and activity, and to the morphogenesis of dendritic arbors. Mutant prel class IV neurons simplified and downsized their dendritic arbors, and showed breakages of their major branches without detectable signs of apoptosis. Furthermore, genetic interactions were observed between Prel and the Drosophila Bax-like Bcl-2 family proteins Drob-1 (also known as Debcl) and Buffy. All of these observations suggest that Prel-dependent control of mitochondrial activity has a pivotal role in the development and maintenance of dendritic arbors (Tsubouchi, 2009).

A screening was conducted by using the Gene Search (GS) system to hunt for genes that control complex morphology of dendritic arbors of the class IV neuron at larval stages. One of the aims of this system is to drive overexpression or misexpression of genes neighboring the GS-vector insertion site. Out of 3000 GS lines screened, focus was placed on one, GS9160, and its candidate gene, preli-like (prel), which is conserved throughout eukaryotes. The predicted product of prel is 236 amino acids in length and a member of the PRELI/MSF1 family in Drosophila. The closest human homolog is PRELI; and its amino acid sequence shows 46% identity to that of Preli-like through the entire length. In yeast, Ups1p, a member of this family, is localized in mitochondria and regulates their shape. To confirm whether the Prel protein is localized to mitochondria in Drosophila cells, lysates of Drosophila Schneider 2 (S2) cells were fractionated, and both endogenous and exogenously expressed Prel proteins were shown to be predominantly detected in the mitochondria-enriched fraction but hardly found in the cytoplasm. Under the light microscope, the expressed Prel was mostly colocalized with the mitochondrial marker Mitotracker Orange, and it appeared to be associated with the cristae when observed by immunoelectron microscopy (Tsubouchi, 2009).

The role of Prel in shaping mitochondria in S2 cells was examined by both light and transmission electron microscopy. More than 80% of the control S2 cells had a filamentous mitochondrial network, and parallel, accordion-like folds of cristae structures were observed in each mitochondrion. Both knockdown of prel and its exogenous expression caused significant fragmentation of mitochondria. At the ultrastructural level, prel-knockdown cells had many mitochondria with lower electron density. Those mitochondria took on a round shape, having an abnormally expanded matrix and only a few thin cristae. It is known that OPA1, one of the mitochondrial dynamin-related GTPases, is important for both fusion of inner membranes and maintenance of the structure of cristae. This study showed that knockdown of opa1 in S2 cells affected the mitochondrial structure in a very similar manner to that found with knockdown of prel (Tsubouchi, 2009).

Thus, both prel loss-of-function and its overexpression abrogates mitochondrial structures and activity in S2 cells and also in da neurons. Apparently an appropriate expression level of Prel is required for controlling mitochondrial shape, structure of the cristae, the activity of respiratory chain complex IV and the cellular ATP level. What then is the exact molecular function of Prel (Tsubouchi, 2009)?

The molecular function of the Preli family is largely unknown in multicellular organisms. It has been recently shown that Ups1p, the yeast homologue of Prel, regulates the level of cardiolipin (CL), a phospholipid of mitochondrial membranes. CL is known to be located predominantly in the mitochondria and has diverse mitochondrial functions including stabilization of the respiratory chain supercomplex. These reports imply that the reduction in the complex IV activity and the ATP level in the prel-knockdown cells could be attributed to the altered phospholipid composition. Further biochemical study is required to measure the complex IV activity and phospholipid composition in various genetic backgrounds including Buffy or Drob1 overexpression, which might help to find evidence for a molecular pathway that includes Prel and Buffy (Tsubouchi, 2009).

However, it has also been proposed that loss of Ups1p affects the function of the yeast OPA1 homolog Mgm1p, which is important for both fusion of inner membranes and maintenance of the structure of the cristae. Mgm1p is imported into mitochondria, and its function is regulated by proteolytic cleavage. This import and cleavage pattern is altered, and the mitochondria fragmented, in the yeast ups1p mutant. Knockdown of Drosophila opa1 in S2 cells affected the structure and activity of mitochondria similarly to prel knockdown, suggesting that Drosophila Prel is also required for organizing the mitochondrial inner membrane structure in concert with Drosophila Opa1. However, positive evidence could not be provided for any functional linkage between these two molecules. Neither prel knockdown nor its overexpression strongly affected the cleavage pattern of endogenous Opa1 in S2 cells. A better understanding of Prel function in the protein import into mitochondria requires further study. It should be noted that there seems to be a bidirectional relationship between mitochondrial shape and bioenergetics, as suggested by the fact that a decrease in the ATP level can also stimulate mitochondrial fragmentation. One possible interpretation of the current data might be that Prel is primarily required for maintaining the normal level of intracellular ATP and controls mitochondrial shape indirectly (Tsubouchi, 2009).

Mitochondria are abundant in regions of intense energy consumption, such as muscles, sperm and neurons; and OPA1 expression is high in the retina, brain, testis, heart and skeletal muscle. The autosomal dominant disease Charcot-Marie-Tooth (CMT) type 2A phenotype due to MFN2 mutations might reflect the extreme cell geometry, as shown by the fact that long peripheral nerves are particularly sensitive to perturbations produced by MFN2-mediated mitochondrial dysfunction. Class IV neurons, which develop expansive and complicated dendritic arbors, probably consume the highest amount of ATP; thus they are very vulnerable to a loss or reduction in the prel-dependent mitochondrial function. The differential Gal4 expression of the trap line and the cis-element fusion line suggests that Prel is expressed most strongly in class IV neurons among the four subclasses (Tsubouchi, 2009).

It had speculated that the severe dendritic phenotype of the prel class IV neurons could be primarily due to the misdistribution of mitochondria. However, attempts to correlate the mitochondrial localization and the dendritic phenotype suggested that such a view might be naive. Loss of function of milt dramatically reduced the mitochondrial density in neuronal processes; nevertheless, the dendritic phenotype of the milt mutant neuron was much less profound than that of the prel neurons. Similarly, milt mutant eyes are indistinguishable from the wild-type eyes in their external and photoreceptor morphology in spite of the paucity of mitochondria in photoreceptor axon terminals. These observations imply the possibility that the visible misdistribution of mitochondria per se might not necessarily lead to the severe morphological abnormality of dendritic arbors or axons (Tsubouchi, 2009).

How can these observations be interpreted? Overexpression of prel diminished the ATP level in vivo, where the local ATP concentration within the cell might fall below a threshold that is necessary for dendritic growth and maintenance. However, mitochondria that remain in the cell body of the milt neuron might maintain their ATP-producing activity, and at least a subpopulation of synthesized ATP molecules might diffuse a long distance to reach the distal region in the dendritic arbor. These hypotheses would be testable if the ATP level in the cell body and its diffusion inside dendritic branches could be visualized and measured quantitatively in various genetic backgrounds (Tsubouchi, 2009).

Dysfunction of mitochondria correlates with neurodegenerative diseases and is an early and causal event in neurodegeneration. The results of this study imply that the evolutionally conserved Prel might prevent neurodegeneration in other animal species. When Prel function was impaired, branches in the proximal region of the arbor were degraded, and terminal branches were eliminated at the mature larval stage; and in adult flies, overall arbors retracted. These phenotypes are reminiscent of breakage of neurite branches within and near amyloid deposits in the brain of a transgenic mouse model of Alzheimer disease, which is speculated to occur through mitochondrial dysfunction, oxidative stress and calcium deregulation. The branch destruction of the prel mutant neuron could also be due to a 'physical' reason. Transport of various cargos might be impaired when ATP is limited, leading to a defect in mechanical strength of the membrane. Such fragile branches could be ruptured during larval locomotion (Tsubouchi, 2009).

It has been intensively studied whether the mutant proteins that are associated with hereditary neurodegenerative diseases affect mitochondrial function. This study has provided cellular and genetic evidence that Prel is a novel target of such research. Future studies should be directed towards further characterization of the Prel protein by using both fly and vertebrate systems to clarify its function in mitochondria and its involvement in mechanisms that prevent the regression of dendritic arbors (Tsubouchi, 2009).


Bcl-2 and Bcl-xL promote cell survival

The Bcl-2 family of proteins regulates apoptosis: some members antagonize cell death while others facilitate it. It has recently been demonstrated that Bcl-2 not only inhibits apoptosis but also restrains cell cycle entry. These two functions can be genetically dissociated. Mutation of a tyrosine residue within the conserved N-terminal BH4 region has no effect on the ability of Bcl-2 or its closest homologs to enhance cell survival and does not prevent heterodimerization with death-enhancing family members Bax, Bak, Bad and Bik. Neither does this mutation override the growth-inhibitory effect of p53. However, upon stimulation with cytokine or serum, starved quiescent cells expressing the mutant proteins re-enter the cell cycle much faster than those expressing comparable levels of wild-type proteins. When wild-type and Y28 mutant Bcl-2 are co-expressed, the mutant is dominant. Although R-Ras p23 has been reported to bind to Bcl-2, no interaction is detectable in transfected cells and R-Ras p23 does not interfere with the ability of Bcl-2 to inhibit apoptosis or cell cycle entry. These observations provide evidence that the anti-apoptotic function of Bcl-2 is mechanistically distinct from its inhibitory influence on cell cycle entry (Huang, 1997).

Bcl-2 can inhibit apoptosis induced by a variety of stimuli, including radiation; its presence in tumor cells would be expected to indicate poor prognosis. However, Bcl-2-expressing tumors are often low-grade and highly responsive to therapy. To investigate this apparent paradox, the responses of Burkitt lymphoma (BL) cells were examined in vitro to gamma-irradiation in the presence and absence of Bcl-2. High-level expression of Bcl-2 promotes BL cell survival following irradiation. However, a significant proportion of Bcl-2-rescued cells subsequently undergo apoptosis after an extended period in culture. In different BL lines, Bcl-2 either promotes or inhibits long-term proliferative activity following gamma-irradiation. This differential regulation of proliferation correlates both with differential effects of Bcl-2 on the cell cycle and with differences in p53 status. Thus, by one week after irradiation, BL cells expressing only wild-type p53 (wt/wt) have arrested in G1, whereas those with a mutant allele (wt/mu) are arrested in all phases of the cell cycle. The proportion of Bcl-2-rescued cells that subsequently undergo apoptosis is reduced by ligation of CD40 at the time of irradiation in wt/wt BL cells, but not in wt/mu cells. CD40-ligation reduces both G1-arrest and apoptosis in parallel. These results indicate that while Bcl-2 can delay apoptosis in BL cells following gamma-irradiation, the protein can also cause growth-arrest and thereby promote apoptosis. Long-term survival following Bcl-2-mediated rescue of gamma-irradiated cells may depend on p53 status and require additional death-repressing or growth-promoting signals (Milner, 1997).

Bcl-2 plays a key role in regulating cell survival in the immune and nervous systems. Mice lacking the bcl-2 gene have markedly reduced numbers of B and T cells as a result of increased apoptosis, whereas mice with a transgene causing high levels of Bcl-2 expression in the immune system show extended survival of B and T cells. Overexpression of Bcl-2 in cultured neurons prevents their death following neurotrophin deprivation; mice with a bcl-2 transgene under the control of a neuron-specific enolase promoter have increased numbers of neurons in several regions. Cultured neurons expressing antisense bcl-2 RNA have an attenuated survival response to neurotrophins, and neurons of postnatal bcl-2-deficient mice die more rapidly following NGF deprivation in vitro and are present in reduced numbers in vivo. Bcl-2 also plays a role in regulating axonal growth rates in embryonic neurons. Sensory neurons from the trigeminal ganglia of bcl-2-deficient mouse embryos, removed from the embryo on embryonic day 11 or 12, extend axons more slowly in vitro than do neurons from wild-type embryos of the same age. Serial measurements of axonal length in the same neurons reveal that there are marked differences in axonal growth rate between bcl-2-deficient and wild-type neurons, irrespective of whether the neurons are grown with nerve growth factor, brain-derived neurotrophic factor or neurotrophin-3. Because there is no significant difference in the numbers of wild-type and bcl-2-deficient neurons surviving with each neurotrophin at this early stage of development, the effect of Bcl-2 on axonal growth rate is not a consequence of its well documented role in preventing apoptosis (Hilton, 1997).

Stimulation of the Fas or tumor necrosis factor receptor 1 (TNFR1) cell surface receptors leads to the activation of the death effector protease, caspase-8, and subsequent apoptosis. In some cells, Bcl-xL overexpression can inhibit anti-Fas- and tumor necrosis factor (TNF)-alpha-induced apoptosis. To address the effect of Bcl-xL on caspase-8 processing, Fas- and TNFR1-mediated apoptosis were studied in the MCF7 breast carcinoma cell line stably transfected with human Fas cDNA (MCF7/F) or transfected with both Fas and human Bcl-xL cDNAs (MCF7/FB). Bcl-xL strongly inhibits apoptosis induced by either anti-Fas or TNF-alpha. Bcl-xL prevents the change in cytochrome c immunolocalization induced by anti-Fas or TNF-alpha treatment. Using antibodies that recognize the p20 and p10 subunits of active caspase-8, proteolytic processing of caspase-8 was detected in MCF7/F cells following anti-Fas or TNF-alpha, but not during UV-induced apoptosis. In MCF7/FB cells, caspase-8 is processed normally, while processing of the downstream caspase-7 is markedly attenuated. Apoptosis induced by direct microinjection of recombinant, active caspase-8 is completely inhibited by Bcl-xL. These data demonstrate that Bcl-xL can exert an anti-apoptotic function in cells in which caspase-8 is activated. Thus, at least in some cells, caspase-8 signaling in response to Fas or TNFR1 stimulation is regulated by a Bcl-xL-inhibitable step (Srinivasan, 1998).

Apoptotic cell death is driven by ICE family proteases (caspases) and negatively regulated by Bcl-2 family proteins. Although it has been shown that Bcl-2 exerts anti-apoptotic activity by blocking a step(s) leading to the activation of caspases, a role for Bcl-2 and Bcl-xL downstream of the caspase cascade has remained unclear. Purified active caspase-3 (CPP32/Yama/apopain) and caspase-1 (ICE) induce apoptosis when microinjected into the cytoplasm of cells; the apoptosis is not at all prevented by Bcl-2 and Bcl-xL, which are overexpressed more than sufficiently to prevent Fas-mediated and overexpressed procaspase-1-mediated apoptosis. Thus, Bcl-2 and Bcl-xL do not act downstream of the caspase cascade (Yasuhara, 1997).

The gene MRIT possesses overall sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases. However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus sequence QAC(R/Q)G. Nonetheless MRIT activates caspase-dependent death. Using yeast two-hybrid assays, it has been demonstrated that MRIT associates with caspases possessing large and small prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD. In addition, MRIT simultaneously and independently interacts with BclXL and FLICE in mammalian cells. Thus, MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family member (Han, 1997).

Nuclear factor (NF) kappaB is a ubiquitously expressed transcription factor whose function is regulated by the cytoplasmic inhibitor protein, IkappaBalpha. IkappaBalpha activity is diminished in ventricular myocytes expressing Bcl-2. In view of the growing evidence that the conserved N-terminal BH4 domain of Bcl-2 plays a critical role in suppressing apoptosis, it was ascertained whether this region accounts for the underlying effects of Bcl-2 on IkappaBalpha activity. Transfection of human embryonic 293 cells with full length Bcl-2 results in a significant 1.9-fold reduction in IkappaBalpha activity with a concomitant increase in DNA binding and 3.4-fold increase in NFkappaB-dependent gene transcription compared with vector transfected control cells. In contrast, no significant change in IkappaBalpha activity is detected with either a BH4 domain deletion mutant (residues 10-30) or BH4 domain point substitution mutants, I14G, V15G, Y18G, K22G, and L23G. However, a small 0.60-fold decrease in IkappaBalpha activity is noted with the BH4 mutant I19G, suggesting that this residue may not be critical for IkappaBalpha regulation. Furthermore, adenovirus-mediated delivery of an IkappaBalpha mutant to prevent NFkappaB activation impairs the ability of Bcl-2 to suppress apoptosis provoked by TNFalpha plus cycloheximide in ventricular myocytes. The data provide the first evidence for the regulation of IkappaBalpha by Bcl-2 through a mechanism that requires the conserved BH4 domain that links Bcl-2 to the NFkappaB signaling pathway for suppression of apoptosis (de Moissac, 1999).

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, mice were generated 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 prevents all these disorders. Indeed, loss of even one bim allele restores 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 (Bouillet, 2001).

The Bcl-2 family proteins are key regulators of apoptosis in human diseases and cancers. Though known to block apoptosis, Bcl-2 promotes cell death through an undefined mechanism. Bcl-2 is shown to interacts with orphan nuclear receptor Nur77 (also known as TR3), which is required for cancer cell apoptosis induced by many antineoplastic agents. The interaction is mediated by the N-terminal loop region of Bcl-2 and is required for Nur77 mitochondrial localization and apoptosis. Nur77 binding induces a Bcl-2 conformational change that exposes its BH3 domain, resulting in conversion of Bcl-2 from a protector to a killer. These findings establish the coupling of Nur77 nuclear receptor with the Bcl-2 apoptotic machinery and demonstrate that Bcl-2 can manifest opposing phenotypes, induced by interactions with proteins such as Nur77, suggesting novel strategies for regulating apoptosis in cancer and other diseases (Lin, 2004).

Nur77 (TR3 or NGFI-B), an orphan member of the steroid/thyroid/retinoid nuclear receptor superfamily, plays roles in regulating growth and apoptosis. Nur77 expression is rapidly induced during apoptosis in immature thymocytes and T cell hybridomas, and cancer cells of lung, ovary, colon, and stomach. High levels of Nor1, a Nur77-family member, are associated with favorable responses to several chemotherapeutic agents in patients with diffuse large B-cell lymphoma (Lin, 2004 and references therein).

A paradigm in cellular apoptosis has been discovered, wherein Nur77 translocates from the nucleus to the cytoplasm, targeting to mitochondria and inducing cyt c release. Nur77 mitochondrial-targeting occurs during apoptosis of different types of cancer cells. Sindbis virus-induced apoptosis also involves Nur77 translocation to mitochondria. How Nur77 targets mitochondria and induces apoptosis however has been unclear. In this study, the mechanism by which Nur77 targets mitochondria and induces apoptosis was been investigated. The results demonstrate that Nur77 interacts with Bcl-2 through its ligand binding domain (LBD) and that the interaction is required for Nur77 mitochondrial targeting and Nur77-dependent apoptosis. Interestingly, Nur77 binds to the Bcl-2 N-terminal loop region, located between its BH4 and BH3 domains, resulting in a conformational change in Bcl-2, which converts it from a protector to a killer protein (Lin, 2004).

Bcl-xL deamidation is a critical switch in the regulation of the response to DNA damage

The therapeutic value of DNA-damaging antineoplastic agents is dependent upon their ability to induce tumor cell apoptosis while sparing most normal tissues. A component of the apoptotic response to these agents in several different types of tumor cells is the deamidation of two asparagines in the unstructured loop of Bcl-xL. Deamidation of these asparagines imports susceptibility to apoptosis by disrupting the ability of Bcl-xL to block the proapoptotic activity of BH3 domain-only proteins. Conversely, Bcl-xL deamidation is actively suppressed in fibroblasts, and suppression of deamidation is an essential component of their resistance to DNA damage-induced apoptosis. These results suggest that the regulation of Bcl-xL deamidation has a critical role in the tumor-specific activity of DNA-damaging antineoplastic agents (Deverman, 2002).

In addition to its tumor-suppressor activity, Rb is a potent antiapoptotic protein -- loss of Rb in normal fibroblasts confers sensitivity to DNA-damaging agents, and reintroduction of Rb into Rb null tumors confers resistance to these agents. Hence, it was reasoned that Rb must suppress proapoptotic signals. Indeed, Rb suppresses the inactivating deamidation of Bcl-xL, and these findings indicate that the antiapoptotic activity of Rb is dependent upon the ability of Rb to suppress Bcl-xL deamidation. Finally, the data suggest that the inactivation of Rb increases the susceptibility of tumor cells to DNA-damaging agents in part because inactivation of Rb is permissive for Bcl-xL deamidation (Deverman, 2002).

Proapoptotic Bcl-2 family members

The NSM cells of the nematode Caenorhabditis elegans differentiate into serotonergic neurons, while their sisters, the NSM sister cells, undergo programmed cell death during embryogenesis. The programmed death of the NSM sister cells is dependent on the cell-death activator EGL-1, a BH3-only protein required for programmed cell death in C. elegans, and can be prevented by a gain-of-function (gf) mutation in the cell-death specification gene ces-1, which encodes a Snail-like DNA-binding protein. The genes hlh-2 and hlh-3, which encode a Daughterless-like and an Achaete-scute-like bHLH protein, respectively, are required to kill the NSM sister cells. A heterodimer composed of HLH-2 and HLH-3, HLH-2/HLH-3, binds to Snail-binding sites/E-boxes in a cis-regulatory region of the egl-1 locus in vitro that is required for the death of the NSM sister cells in vivo. Hence, it is proposed that HLH-2/HLH-3 is a direct, cell-type specific activator of egl-1 transcription. Furthermore, the Snail-like CES-1 protein can block the death of the NSM sister cells by acting through the same Snail-binding sites/E-boxes in the egl-1 locus. In ces-1(gf) animals, CES-1 might therefore prevent the death of the NSM sister cells by successfully competing with HLH-2/HLH-3 for binding to the egl-1 locus (Thellmann, 2003).

Extracellular survival factors alter a cell's susceptibility to apoptosis, often through posttranslational mechanisms. However, no consistent relationship has been established between such survival signals and the BCL-2 family, where the balance of death agonists versus antagonists determines susceptibility. One distant member, BAD, heterodimerizes with BCL-X(L) or BCL-2, neutralizing their protective effect and promoting cell death. In the presence of survival factor IL-3, cells phosphorylated BAD on two serine residues embedded in 14-3-3 consensus binding sites. Only the nonphosphorylated BAD heterodimerized with BCL-X(L) at membrane sites to promote cell death. Phosphorylated BAD is sequestered in the cytosol bound to 14-3-3. Substitution of serine phosphorylation sites further enhances BAD's death-promoting activity. The rapid phosphorylation of BAD following IL-3 connects a proximal survival signal with the BCL-2 family, modulating this checkpoint for apoptosis (Zha, 1996).

Mtd, a novel regulator of apoptosis, has been cloned and characterized. Sequence analysis reveals that Mtd is a member of the Bcl-2 family of proteins containing conserved BH1, BH2, BH3, and BH4 regions and a carboxyl-terminal hydrophobic domain. In adult tissues, Mtd mRNA is predominantly detected in the brain, liver, and lymphoid tissues, while in the embryo Mtd mRNA is detected in the liver, thymus, lung, and intestinal epithelium. Expression of Mtd promotes the death of primary sensory neurons, 293T cells and HeLa cells, indicating that Mtd is a proapoptotic protein. Unlike all other known death agonists of the Bcl-2 family, Mtd does not bind significantly to the survival-promoting proteins Bcl-2 or Bcl-XL. Furthermore, apoptosis induced by Mtd is not inhibited by Bcl-2 or Bcl-XL. A Mtd mutant with glutamine substitutions of highly conserved amino acids in the BH3 domain retains its ability to promote apoptosis, further indicating that Mtd does not promote apoptosis by heterodimerizing with Bcl-2 or Bcl-XL. Mtd-induced apoptosis is not blocked by broad range synthetic caspase inhibitors z-VAD-fmk or a viral protein CrmA. Mtd is the first example of a naturally occurring Bcl-2 family member that can activate apoptosis independently of heterodimerization with survival-promoting Bcl-2 and Bcl-XL (Inohara, 1998).

In the intracellular death program, hetero- and homodimerization of different anti- and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell fate. From a rat ovarian fusion cDNA library, a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok) has been isolated. Bok has conserved Bcl-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacks the BH4 domain found only in anti-apoptotic Bcl-2 proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bfl-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In addition, negligible interaction is found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induces apoptosis that is blocked by the baculoviral-derived cysteine protease inhibitor P35. Cell killing induced by Bok is also suppressed following coexpression with Mcl-1 and BHRF1 but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok is highly expressed in the ovary, testis and uterus. In situ hybridization analysis localized Bok mRNA in granulosa cells, the cell type that undergoes apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover (Hsu, 1997).

The proapoptotic molecule BAX is required for death of sympathetic and motor neurons in the setting of trophic factor deprivation. Adult Bax-/- mice have more motor neurons than do their wild-type counterparts. These findings raise the possibility that BAX regulates naturally occurring cell death during development in many neuronal populations. To test this idea, apoptosis was assessed in several well-studied neural systems during embryonic and early postnatal development in Bax-/- mice. Remarkably, naturally occurring cell death is virtually eliminated in most peripheral ganglia, in motor pools in the spinal cord, and in the trigeminal brainstem nuclear complex between embryonic day 11.5 (E11.5) and postnatal day 1 (PN1). Reduction, although not elimination, of cell death is found throughout the developing cerebellum, in some layers of the retina, and in the hippocampus. Saving of cells was verified by axon counts of dorsal and ventral roots, as well as facial and optic nerves that reveal a 24%-35% increase in axon numbers. Interestingly, many of the supernumerary axons have very small cross-sectional areas, suggesting that the associated neurons are not normal. It is concluded that BAX is a critical mediator of naturally occurring death of peripheral and CNS neurons during embryonic life. However, rescue from naturally occurring cell death does not imply that the neurons will develop normal functional capabilities (White, 1998).

Bcl-2 family proteins and ICE/CED-3 family proteases (caspases) are regarded as the basic regulators of apoptotic cell death. They are evolutionarily conserved and implicated in a variety of apoptosis. However, the precise mechanism by which these two families interact to regulate cell death is not yet known. Overexpression of the Bcl-2 family member Bax induces apoptotic cell death in COS-7 cells through the activation of CPP32 (caspase-3)-like proteases that cleave the DEVD tetrapeptide. This apoptotic cell death is suppressed by the viral proteins CrmA and p35, as well as by the chemically synthesized caspase inhibitors Z-Asp-CH2-DCB and zVAD-fmk. The Bax-induced apoptosis of COS-7 cells is suppressed by Bcl-xL and Bcl-2, though both Bcl-xL and Bcl-2 similarly prevent etoposide-induced apoptosis in COS-7 cells. Bcl-xL inhibits the activation of caspase-3-like proteases accompanying Bax-induced COS-7 cell death, but Bcl-2 does not. These results indicate that the caspase activation is essential for Bax-induced apoptosis, and that the ability of Bcl-2 and Bcl-xL to prevent the Bax-induced caspase activation and apoptosis in COS-7 cells can be differentially regulated. These results also suggest that Bcl-2 family proteins function upstream of caspase activation and control apoptosis through the regulation of caspase activity (Kitanaka, 1997).

Expression of the pro-apoptotic molecule BAX has been shown to induce cell death. While BAX forms both homo- and heterodimers, questions remain concerning its native conformation in vivo and which moiety is functionally active. A physiologic death stimulus, the withdrawal of interleukin-3 (IL-3), is shown to result in the translocation of monomeric BAX from the cytosol to the mitochondria where it can be cross-linked as a BAX homodimer. In contrast, cells protected by BCL-2 demonstrate a block in this process: BAX does not redistribute or homodimerize in response to a death signal. To test the functional consequence of BAX dimerization, a chimeric FKBP-BAX molecule was expressed. Enforced dimerization of FKBP-BAX by the bivalent ligand FK1012 results in its translocation to mitochondria and induces apoptosis. Caspases are activated yet caspase inhibitors do not block death; cytochrome c is not released detectably despite the induction of mitochondrial dysfunction. Moreover, enforced dimerization of BAX overrides the protection by BCL-XL and IL-3 and kills cells. These data support a model in which a death signal results in the activation of BAX. This conformational change in BAX manifests in its translocation, mitochondrial membrane insertion and homodimerization, and a program of mitochondrial dysfunction that results in cell death (Gross, 1998).

Following exposure of cells to stimuli that trigger programmed cell death (apoptosis), cytochrome c is rapidly released from mitochondria into the cytoplasm, where it activates proteolytic molecules known as caspases that specifically cleave the amino-acid sequence DEVD and are crucial for the execution of apoptosis. The protein Bcl-2 interferes with this activation of caspases by preventing the release of cytochrome c. These molecular interactions have been studied during apoptosis induced by the protein Bax, a pro-apoptotic homolog of Bcl-2. In cells transiently transfected with bax, Bax localizes to mitochondria and induces the release of cytochrome c, activation of caspase-3, membrane blebbing, nuclear fragmentation, and cell death. Caspase inhibitors do not affect Bax-induced cytochrome c release but block caspase-3 activation and nuclear fragmentation. Unexpectedly, Bcl-2 also fails to prevent Bax-induced cytochrome c release, although it co-localizes with Bax to mitochondria. Cells overexpressing both Bcl-2 and Bax show no signs of caspase activation and survive with significant amounts of cytochrome c in the cytoplasm. These findings indicate that Bcl-2 can interfere with Bax killing, both downstream and independent of cytochrome c release. This activity mediated by Bcl-2 may be due to an interaction of Bcl-2 with the cytochrome c receptor Apaf-1/CED-4 (Drosophila homolog: Apaf-1-related-killer), a recently identified mediator of caspase-3 activation by cytochrome c (Rosse, 1998).

'BH3 domain only' members of the BCL-2 family including the pro-apoptotic molecule BID represent candidates to connect with proximal signal transduction. Tumor necrosis factor alpha (TNFalpha) treatment induces a caspase-mediated cleavage of cytosolic, inactive p22 BID at internal Asp sites to yield a major p15 and minor p13 and p11 fragments. p15 BID translocates to mitochondria as an integral membrane protein. p15 BID within cytosol targets normal mitochondria and releases cytochrome c. Immunodepletion of p15 BID prevents cytochrome c release. In vivo, anti-Fas Ab results in the appearance of p15 BID in the cytosol of hepatocytes which translocates to mitochondria where it releases cytochrome c. Addition of activated caspase-8 to normal cytosol generates p15 BID which is also required in this system for release of cytochrome c. In the presence of BCL-XL/BCL-2, TNFalpha still induces BID cleavage and p15 BID becomes an integral mitochondrial membrane protein. However, BCL-XL/BCL-2 prevents the release of cytochrome c, yet other aspects of mitochondrial dysfunction still transpires and cells die nonetheless. Thus, while BID appears to be required for the release of cytochrome c in the TNF death pathway, the release of cytochrome c may not be required for cell death (Gross, 1999b).

DP5, which contains a BH3 domain, was cloned as a neuronal apoptosis-inducing gene. To confirm that DP5 interacts with members of the Bcl-2 family, 293T cells were transiently co-transfected with DP5 and Bcl-xl cDNA constructs, and immunoprecipitation was carried out. The 30-kDa Bcl-xl was co-immunoprecipitated with Myc-tagged DP5, suggesting that DP5 physically interacts with Bcl-xl in mammalian cells. DP5 is induced during neuronal apoptosis in cultured sympathetic neurons. DP5 gene expression and the specific interaction of DP5 with Bcl-xl was analyzed during neuronal death induced by amyloid-beta protein (A beta). DP5 mRNA is induced 6 h after treatment with A beta in cultured rat cortical neurons. The protein encoded by DP5 mRNA shows a specific interaction with Bcl-xl. Induction of DP5 gene expression is blocked by nifedipine, an inhibitor of L-type voltage-dependent calcium channels, and dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. These results suggested that the induction of DP5 mRNA occurs downstream of the increase in cytosolic calcium concentration caused by A beta. Moreover, DP5 specifically interacts with Bcl-xl during neuronal apoptosis following exposure to A beta, and its binding can impair the survival-promoting activities of Bcl-xl. Thus, the induction of DP5 mRNA and the interaction of DP5 and Bcl-xl could play significant roles in neuronal degeneration following exposure to A beta (Imaizumi, 1999).

Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, has been deleted, do not undergo apoptosis in 5 mM potassium, yet do undergo an excitotoxic cell death in response to stimulation with 30 or 100 microM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decrease glucose uptake to <20% of control. Protein synthesis also decreases rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax -/- neurons increase mRNA levels of c-jun, and caspase 3 (CPP32) and increase phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increase cleavage of DEVD-aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells do not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases; they indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, block cleavage of DEVD-AMC and block an increase in DNA degradation. However, these inhibitors have only a marginal effect on the prevention of cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells (Miller, 1997).

BAD is a distant member of the Bcl-2 family that promotes cell death. Phosphorylation of BAD prevents this. BAD phosphorylation induced by interleukin-3 (IL-3) was inhibited by specific inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Akt, a survival-promoting serine-threonine protein kinase, was activated by IL-3 in a PI 3-kinase-dependent manner. Only active forms of Akt are found to phosphorylate BAD in vivo and in vitro at the same residues that are phosphorylated in response to IL-3. Thus, the proapoptotic function of BAD is regulated by the PI 3-kinase-Akt pathway (del Peso, 1997).

Growth factor deprivation is a physiological mechanism to regulate cell death. An interleukin-2 (IL-2)-dependent murine T-cell line was used to identify proteins that interact with Bad upon IL-2 stimulation or deprivation. Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, it was found that Bad interacts with protein phosphatase 1alpha (PP1alpha). Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with the appearance of apoptosis. IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation. This enzymatic activity also dephosphorylates in vivo 32P-labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1alpha. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1alpha phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1alpha phosphatase, whose enzymatic activity is regulated by Ras (Ayllon, 2000).

BAK is a pro-apoptotic BCL-2 family protein that localizes to mitochondria. The function of BAK has been evaluated in several mouse models of neuronal injury including neuronotropic Sindbis virus infection, Parkinson's disease, ischemia/stroke, and seizure. BAK promotes or inhibits neuronal death depending on the specific death stimulus, neuron subtype, and stage of postnatal development. BAK protects neurons from excitotoxicity and virus infection in the hippocampus. As mice mature, BAK is converted from anti- to pro-death function in virus-infected spinal cord neurons. In addition to regulating cell death, BAK also protects mice from kainate-induced seizures, suggesting a possible role in regulating synaptic activity. BAK can alter neurotransmitter release in a direction consistent with its protective effects on neurons and mice. These findings suggest that BAK inhibits cell death by modifying neuronal excitability (Fannjiang, 2003).

Bax (Bcl2-associated X protein) is an apoptosis-inducing protein that during normal development participates in cell death and also participates in various diseases. Bax resides in an inactive state in the cytosol of many cells. In response to death stimuli, Bax protein undergoes conformational changes that expose membrane-targeting domains, resulting in its translocation to mitochondrial membranes, where Bax inserts and causes release of cytochrome c and other apoptogenic proteins. It is unknown what controls conversion of Bax from the inactive to active conformation. This study shows that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria. Conversely, reducing HN expression by small interfering RNAs sensitizes cells to Bax and increases Bax translocation to membranes. HN peptides also block Bax association with isolated mitochondria, and suppress cytochrome c release in vitro. Notably, the mitochondrial genome contains an identical open reading frame, and the mitochondrial version of HN can also bind and suppress Bax. It is speculated therefore that HN arose from mitochondria and transferred to the nuclear genome, providing a mechanism for protecting these organelles from Bax (Guo, 2003).

The tumor suppressor p53 exerts its versatile function to maintain the genomic integrity of a cell, and the life of cancerous cells with DNA damage is often terminated by induction of apoptosis. The role of Noxa, one of the transcriptional targets of p53 that encodes a proapoptotic protein of the Bcl-2 family, was studied by the gene-targeting approach. Mouse embryonic fibroblasts deficient in Noxa [Noxa-/- mouse embryonic fibroblasts (MEFs)] show notable resistance to oncogene-dependent apoptosis in response to DNA damage, which is further increased by introducing an additional null zygosity for Bax. These MEFs also show increased sensitivity to oncogene-induced cell transformation in vitro. Furthermore, Noxa is also involved in the oncogene-independent gradual apoptosis induced by severe genotoxic stresses, under which p53 activates both survival and apoptotic pathways through induction of p21WAF1/Cip1 and Noxa, respectively. Noxa-/- mice show resistance to X-ray irradiation-induced gastrointestinal death, accompanied with impaired apoptosis of the epithelial cells of small intestinal crypts, indicating the contribution of Noxa to the p53 response in vivo (Shibue, 2003).

Interactions among pro- and anti-apoptotic Bcl-2 family members

The BCL-2 family of proteins consists of both antagonists (e.g., BCL-2) and agonists (e.g., BAX) that regulate apoptosis and compete by means of dimerization. The BH1 and BH2 domains of BCL-2 are required to heterodimerize with BAX and to repress cell death; conversely, the BH3 domain of BAX is required to heterodimerize with BCL-2 and to promote cell death. Interactive cloning was used to identify Bid , which encodes a novel death agonist that heterodimerizes with either agonists (BAX) or antagonists (BCL-2). BID possesses only the BH3 domain, lacks a carboxy-terminal signal-anchor segment, and is found in both cytosolic and membrane locations. BID counters the protective effect of BCL-2. Moreover, expression of BID, without another death stimulus, induces ICE-like proteases and apoptosis. Mutagenesis reveals that an intact BH3 domain of BID is required to bind the BH1 domain of either BCL-2 or BAX. A BH3 mutant of BID that still heterodimerizes with BCL-2 fails to promote apoptosis, dissociating these activities. In contrast, the only BID BH3 mutant that retains death promoting activity interacts with BAX, but not BCL-2. This BH3-only molecule supports BH3 as a death domain and favors a model in which BID represents a death ligand for the membrane-bound receptor BAX (Wang, 1996).

Bak has been shown to both promote apoptosis and to inhibit cell death; in contrast, two other members of the Bcl-2 family of proteins, Bcl-XL and Bcl-2 delay apoptosis induced by various stimuli, including chemotherapeutic agents. Clones with stable expression of Bak wild-type (wt) and Bak with its BH3 (delta78-86) domain deleted (deltaBH3) were generated in FL5.12 cells or FL5.12 cells expressing either Bcl-XL or Bcl-2 to determine if Bak could accelerate apoptosis and antagonize the death repressor activity of Bcl-XL and Bcl-2 during chemotherapy-induced apoptosis. Bak accelerates cell death in FL5.12 cells treated with either etoposide, fluorouracil or taxol. In FL5.12 cells expressing Bcl-XL and Bak wt or Bak deltaBH3, both Bak wt and Bak deltaBH3 are able to antagonize the protective effect of Bcl-XL when treated with etoposide or fluorouracil. Both Bak wt and Bak deltaBH3 are also able to abrogate the protective effect of Bcl-2 in cells expressing Bcl-2 and Bak wt or Bak deltaBH3 when challenged by etoposide or fluorouracil. Immunoprecipitation studies reveal that deletion of BH3 disrupts heterodimerization between Bak and Bcl-XL and that both Bak wt and Bak deltaBH3 fail to interact with Bcl-2. These results demonstrate that Bak does not require its BH3 domain to promote apoptosis in stably transfected cells. Bak can accelerate chemotherapy-induced cell death independently of its heterodimerization with Bcl-XL and Bcl-2 (Simonian, 1997).

Bcl-2 and close homologs 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), the role of this amphipathic helix has been explored for its survival function and for interactions with several agonists of apoptosis, including Bax and CED-4, an essential regulator in the nematode Caenorhabditis elegans. The BH4 of Bcl-2 can be replaced by BH4 of Bcl-x without perturbing function but this is not the case when it is replaced by a somewhat similar region near the N-terminus of Bax. Bcl-2 cell survival activity is reduced by substitutions in two of the ten conserved BH4 residues. Deletion of BH4 renders Bcl-2 (and Bcl-xL) inactive but does not impair either Bcl-2 homodimerization or its 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 loses 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 (Huang, 1998).

The Bcl-2 family proteins comprise pro-apoptotic as well as anti-apoptotic members. Heterodimerization between members of the Bcl-2 family proteins is a key event in the regulation of apoptosis. Bcl-2 protein is selectively cleaved by active caspase-3-like proteases in CTLL-2 cell apoptosis in response to interleukin-2 deprivation. Structural and functional analyses of the cleaved fragment revealed that the NH2-terminal region of Bcl-2 (1-34 amid acids) is required for its anti-apoptotic activity and heterodimerization with pro-apoptotic Bax protein. Site-directed mutagenesis of the NH2-terminal region showed that substitutions of hydrophobic residues of BH4 domain results in the loss of ability to form a heterodimer with Bax. Particularly instructive was that the V15E mutant of Bcl-2, which completely lost the ability to form a heterodimer with Bax, fails to inhibit Bax- and staurosporine-induced apoptosis. These results suggest that the BH4 domain of Bcl-2 is critical for its heterodimerization with Bax and for exhibiting anti-apoptotic activity. Therefore, agents interferring with the critical residues of the BH4 domain may provide a new strategy in cancer therapy by impairing Bcl-2 function (Hirotani, 1999).

Recent reports suggest that a cross-talk exists between apoptosis pathways mediated by mitochondria and cell death receptors. Mitochondrial events are required for apoptosis induced by the cell death ligand TRAIL (TNF-related apoptosis-inducing ligand) in human cancer cells. The Bax null cancer cells are resistant to TRAIL-induced apoptosis. Bax deficiency has no effect on TRAIL-induced caspase-8 activation and subsequent cleavage of Bid; however, it results in an incomplete caspase-3 processing because of inhibition by XIAP. Release of Smac/DIABLO from mitochondria through the TRAIL-caspase-8-tBid-Bax cascade is required to remove the inhibitory effect of XIAP and allow apoptosis to proceed. Inhibition of caspase-9 activity has no effect on TRAIL-induced caspase-3 activation and cell death, whereas expression of the active form of Smac/DIABLO in the cytosol is sufficient to reconstitute TRAIL sensitivity in Bax-deficient cells. These results show for the first time that Bax-dependent release of Smac/DIABLO, not cytochrome c, from mitochondria mediates the contribution of the mitochondrial pathway to death receptor-mediated apoptosis (Deng, 2002).

The signaling events leading to apoptosis can be divided into two distinct pathways, involving either mitochondria or death receptors. In the mitochondria pathway, death signals lead to changes in mitochondrial membrane permeability and the subsequent release of pro-apoptotic factors involved in various aspects of apoptosis. The released factors include cytochrome c (cyto c), apoptosis inducing factor (AIF), second mitochondria-derived activator of caspase (Smac/DIABLO), and endonuclease G. Cytosolic cyto c forms an essential part of the apoptosis complex 'apoptosome,' which is composed of cyto c, Apaf-1, and procaspase-9. Formation of the apoptosome leads to the activation of caspase-9, which then processes and activates other caspases to orchestrate the biochemical execution of cells. Smac/DIABLO is also released from the mitochondria along with cyto c during apoptosis, and it functions to promote caspase activation by inhibiting IAP (inhibitor of apoptosis) family proteins (Deng, 2002).

The IAP family proteins negatively regulate apoptosis by inhibiting caspase activity directly. Six human IAPs have been discovered. They regulate apoptosis by preventing the action of the central execution phase of apoptosis through direct inhibition of the effector caspase-3 and/or caspase-7. In addition, they prevent initiation of the intrinsic caspase activation cascade by directly inhibiting the apical caspase-9. Structural and biochemical dissection of XIAP, a widely expressed IAP member, reveals that the conserved BIR domains of XIAP mediate both its inhibitory activity on caspases and the protein-protein interaction with Smac/DIABLO. Binding of Smac/DIABLO to XIAP antagonizes caspase-XIAP interaction, thereby promoting apoptosis. Recent studies have shown that XIAP is highly expressed in most human cancer cells and that high levels of XIAP confer tumor resistance to chemotherapy or irradiation (Deng, 2002).

The key regulatory proteins of mitochondria-mediated apoptotosis are the Bcl-2 family of proteins, which can either promote cell survival, as do Bcl-2 and Bcl-xl, or induce cell death, as do Bax and Bak. Bcl-2 and Bcl-xl appear to directly or indirectly preserve the integrity of the outer mitochondrial membrane, thus preventing cyto c release and mitochondria-mediated cell death initiation, whereas the pro-apoptotic proteins Bax and Bak promote cyto c release from mitochondria. Bax has been implicated in apoptosis in many cell types under various conditions. More recently, studies using Bax-deficient human colon cancer cells have provided direct evidence that Bax plays a key role in mediating apoptosis induced by certain anti-cancer agents. The Bax protein exerts at least part of its activity by triggering cyto c release from mitochondria. Bax is in a predominantly cytosolic latent form in healthy cells and translocates to mitochondria after death signal stimulation. Accumulating evidence suggests that Bax translocation is required for its pro-apoptotic function and that regulation of Bax's association with the mitochondrial membrane represents a critical step in the transduction of apoptotic signals (Deng, 2002).

In the death receptor pathway, the apoptotic events are initiated by engaging the tumor necrosis factor (TNF)-family receptors, including TNFR1, Fas, DR-3, DR-4, and DR-5. Upon ligand binding or when overexpressed in cells, TNF receptor family members aggregate, resulting in the recruitment of an adapter protein called FADD. The receptor-FADD complex then recruits procaspase-8. This allows proteolytic processing and activation of the receptor-associated procaspase-8, thereby initiating the subsequent cascade of additional processing and activation of downstream effector caspases (Deng, 2002).

TRAIL/Apo2L (TNF-related apoptosis-inducing ligand TRAIL or Apo2 ligand) is an apoptosis-inducing member of the TNF gene superfamily. Unlike TNF-alpha and FasL, TRAIL appears to specifically kill transformed and cancer cells while leaving normal cells intact. Preclinical experiments in mice and nonhuman primates have shown that administration of TRAIL suppresses tumor growth without apparent systematic cytotoxicity. Therefore, TRAIL represents a promising anti-cancer agent. TRAIL interacts with four cellular receptors that form a distinct subgroup within the TNFR superfamily. Most recent experiments have shown that FADD and procaspase-8 associate with the endogenous TRAIL receptors DR4 and DR5. FADD and caspase-8 are required for TRAIL-induced apoptosis. Thus, TRAIL/Apo2L and FasL appear to engage similar pathways to apoptosis (Deng, 2002).

Although the extrinsic pathway (through the death receptors) and the intrinsic pathway (through the mitochondria) for apoptosis are capable of operating independently, accumulating evidence suggests that a cross-talk between the two pathways exists in cells. The link between death receptor signaling and the mitochondrial pathway comes from the finding that a BH3-domain-only subfamily protein, Bid, is cleaved by active caspase-8. The truncated Bid (tBid) translocates to mitochondria and triggers cyto c release. It has been proposed that tBid regulates cyto c release by inducing the homo-oligomerization of pro-apoptotic family members Bak or Bax. Cells lacking both Bax and Bak, but not cells lacking just one of these components, are completely resistant to tBid-induced cyto c release and apoptosis (Deng, 2002).

Bid appears to link the intrinsic pathway to the cell death receptor-mediated apoptosis. However, the precise mitochondrial events required for this cross-talk remain unclear. The mechanisms of TRAIL-induced apoptosis and the role of mitochondria in the cell death receptor pathway also need further investigation. Using human colon cancer cells defective in Bax function, it has been shown that mitochondrial events are required for TRAIL-induced apoptosis. The reason for this requirement is the presence of negative regulation of caspase cascade by XIAP. Activation of the mitochondrial pathway leads to the release of Smac/DIABLO, which removes XIAP blockage of caspase activation. These results further show that release of Smac/DIABLO, not cyto c, is the key event mediating the contribution of the mitochondrial pathway to the death receptor-mediated apoptosis (Deng, 2002).

Commitment of cells to apoptosis is governed largely by the interaction between members of the Bcl-2 protein family. Its three subfamilies have distinct roles: The BH3-only proteins trigger apoptosis by binding via their BH3 domain to prosurvival relatives, while the proapoptotic Bax and Bak have an essential downstream role involving permeabilization of organellar membranes and induction of caspase activation. The regulation of Bak was investigated and it was found that, in healthy cells, Bak associates with Mcl-1 (a close relative of Bcl-2) and Bcl-xL but surprisingly not Bcl-2, Bcl-w, or A1. These interactions require the Bak BH3 domain, which is also necessary for Bak dimerization and killing activity. When cytotoxic signals activate BH3-only proteins that can engage both Mcl-1 and Bcl-xL (such as Noxa plus Bad), Bak is displaced and induces cell death. Accordingly, the BH3-only protein Noxa could bind to Mcl-1, displace Bak, and promote Mcl-1 degradation, but Bak-mediated cell death also requires neutralization of Bcl-xL by other BH3-only proteins. The results indicate that Bak is held in check solely by Mcl-1 and Bcl-xL and induces apoptosis only if freed from both. The finding that different prosurvival proteins have selective roles has notable implications for the design of anti-cancer drugs that target the Bcl-2 family (Willis, 2005).

Ribonucleases, antibiotics, bacterial toxins, and viruses inhibit protein synthesis, which results in apoptosis in mammalian cells. How the BCL-2 family of proteins regulates apoptosis in response to the shutoff of protein synthesis is not known. This study demonstrates that an Escherichia coli toxin, MazF, inhibits protein synthesis by cleavage of cellular mRNA and induces apoptosis in mammalian cells. MazF-induced apoptosis requires proapoptotic BAK and its upstream regulator, the proapoptotic BH3-only protein NBK/BIK, but not BIM, PUMA, or NOXA. Interestingly, in response to MazF induction, NBK/BIK activates BAK by displacing it from anti-apoptotic proteins MCL-1 and BCL-XL that sequester BAK. Furthermore, NBK/BIK- or BAK-deficient cells are resistant to cell death induced by pharmacologic inhibition of translation and by virus-mediated shutoff of protein synthesis. Thus, the BH3-only protein NBK/BIK is the apical regulator of a BAK-dependent apoptotic pathway in response to shutoff of protein synthesis that functions to displace BAK from sequestration by MCL1 and BCL-XL. Although NBK/BIK is dispensable for development, it is the BH3-only protein targeted for inactivation by viruses, suggesting that it plays a role in pathogen/toxin response through apoptosis activation (Shimazu, 2007).

Domain structure and function of proapoptotic Bcl-2 family members

The Bcl-2 related protein Bad is a promoter of apoptosis and has been shown to dimerize with the anti-apoptotic proteins Bcl-2 and Bcl-XL. Overexpression of Bad in murine FL5.12 cells demonstrates that the protein not only can abrogate the protective capacity of coexpressed Bcl-XL but can accelerate the apoptotic response to a death signal when it is expressed in the absence of exogenous Bcl-XL. Using deletion analysis, the minimal domain able to dimerize with Bcl-xL in the murine Bad protein has been identified. A 26-amino-acid peptide within this domain, which shows significant homology to the alpha-helical BH3 domains of related apoptotic proteins like Bak and Bax, is found to be necessary and sufficient to bind Bcl-xL. To determine the role of dimerization in regulating the death-promoting activity of Bad and the death-inhibiting activity of Bcl-xL, mutations within the hydrophobic BH3-binding pocket in Bcl-xL that eliminate the ability of Bcl-xL to form a heterodimer with Bad were tested for the ability to promote cell survival in the presence of Bad. Several of these mutants retain the ability to impart protection against cell death, regardless of the level of coexpressed Bad protein. These results suggest that BH3-containing proteins like Bad promote cell death by binding to antiapoptotic members of the Bcl-2 family and thus inhibiting their survival promoting functions (Kelekar, 1997).

Bax is a proapoptotic member of the Bcl-2 family of proteins which localizes to and uses mitochondria as its major site of action. Bax normally resides in the cytoplasm and translocates to mitochondria in response to apoptotic stimuli, and it promotes apoptosis in two ways: (1) by disrupting mitochondrial membrane barrier function by formation of ion-permeable pores in mitochondrial membranes and (2) by binding to antiapoptotic Bcl-2 family proteins via its BH3 domain and inhibiting their functions. A hairpin pair of amphipathic alpha-helices (alpha5-alpha6) in Bax has been predicted to participate in membrane insertion and pore formation by Bax. Several charged residues in the alpha5-alpha6 domain of Bax were mutagenized, changing them to alanine. These substitution mutants of Bax constitutively localize to mitochondria and display a gain-of-function phenotype when expressed in mammalian cells. Furthermore, substitution of 8 out of 10 charged residues in the alpha5-alpha6 domain of Bax results in a loss of cytotoxicity in yeast but a gain-of-function phenotype in mammalian cells. The enhanced function of this Bax mutant was correlated with increased binding to Bcl-X(L), through a BH3-independent mechanism. These observations reveal new functions for the alpha5-alpha6 hairpin loop of Bax: (1) regulation of mitochondrial targeting and (2) modulation of binding to antiapoptotic Bcl-2 proteins (Nouraini, 2000).

BNIP3 (formerly NIP3) is a pro-apoptotic, mitochondrial protein classified in the Bcl-2 family based on limited sequence homology to the Bcl-2 homology 3 (BH3) domain and COOH-terminal transmembrane (TM) domain. BNIP3 expressed in yeast and mammalian cells interacts with survival promoting proteins Bcl-2, Bcl-X(L), and CED-9. Typically, the BH3 domain of pro-apoptotic Bcl-2 homologues mediates Bcl-2/Bcl-X(L) heterodimerization and confers pro-apoptotic activity. Deletion mapping of BNIP3 excluded its BH3-like domain and identified the NH(2) terminus (residues 1-49) and TM domain as critical for Bcl-2 heterodimerization, and either region is sufficient for Bcl-X(L) interaction. Additionally, the removal of the BH3-like domain in BNIP3 does not diminish its killing activity. The TM domain of BNIP3 is critical for homodimerization, pro-apoptotic function, and mitochondrial targeting. Several TM domain mutants were found to disrupt SDS-resistant BNIP3 homodimerization but did not interfere with its killing activity or mitochondrial localization. Substitution of the BNIP3 TM domain with that of cytochrome b(5) directs protein expression to nonmitochondrial sites and still promotes apoptosis and heterodimerization with Bcl-2 and Bcl-X(L). It is proposed that BNIP3 represents a subfamily of Bcl-2-related proteins that functions without a typical BH3 domain to regulate apoptosis from both mitochondrial and nonmitochondrial sites by selective Bcl-2/Bcl-X(L) interactions (Ray, 2000).

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. 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 results 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 abolishes 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 resensitizes Bcl-xL-expressing cells to Fas-mediated apoptosis. BH3-induced apoptosis is blocked by caspase inhibitors, demonstrating a dependence on caspase activation, but is 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 (Holinger, 1999).

Bcl-2 family members as regulators of the cell death hierarchy: Bcl-2 interacts with Apaf-1

The C. elegans Bcl-2-like protein CED-9 prevents programmed cell death by antagonizing the Apaf-1-like cell-death activator CED-4. Endogenous CED-9 and CED-4 proteins localize to mitochondria in wild-type embryos, in which most cells survive. By contrast, in embryos in which cells have been induced to die, CED-4 assumes a perinuclear localization. CED-4 translocation induced by the cell-death activator EGL-1 (EGL-1 protein contains a Bcl-2 homology 3 domain and can physically interact with CED-9) is blocked by a gain-of-function mutation in ced-9 but is not dependent on ced-3 function, suggesting that CED-4 translocation precedes caspase activation and the execution phase of programmed cell death. Thus, a change in the subcellular localization of CED-4 may drive programmed cell death (Chen, 2000).

The death-promoting proteins Bax and BAD, which like EGL-1 contain BH3 domains, translocate to mitochondria and bind anti-apoptotic Bcl-2 family members in response to apoptotic signals. Whether and how this translocation promotes cell death is unknown. The results presented here suggest that Bax and BAD may act to release Apaf-1 or another CED-4-like protein, allowing it to activate caspase processing. Some caspase precursors, specifically procaspases-2, and -3, are present in mitochondria and upon activation translocate to nuclei. It is possible that this movement of caspases involves the translocation of a complex that includes a CED-4-like protein. By analogy, the translocation of a CED-4-CED-3 complex from mitochondria to the nuclear envelope could provide access for the active caspase to both the nucleus and the cytosol, thereby fulfilling the roles of the multiple, differentially localized mammalian caspases (Chen, 2000).

The Bcl-2 family of proteins regulates apoptosis, the cell death program triggered by activation of certain proteases (caspases). An attractive model for how Bcl-2 and its closest relatives prevent caspase activation is that they bind to and inactivate an adaptor protein required for procaspase processing. That model has been supported by reports that mammalian prosurvival Bcl-2 relatives bind the adaptor Apaf-1, which activates procaspase-9. However, the in vivo association studies reported here with both overexpressed and endogenous Apaf-1 challenge this notion. Apaf-1 can be immunoprecipitated together with procaspase-9, and the Apaf-1 caspase-recruitment domain is necessary and sufficient for their interaction. Apaf-1 does not bind, however, to any of the six known mammalian prosurvival family members (Bcl-2, Bcl-x(L), Bcl-w, A1, Mcl-1, or Boo), or their viral homologs adenovirus E1B 19K and Epstein-Barr virus BHRF-1. Endogenous Apaf-1 also fails to coimmunoprecipitate with endogenous Bcl-2 or Bcl-x(L), or with two proapoptotic relatives (Bax and Bim). Moreover, apoptotic stimuli do not induce Apaf-1 to bind to these family members. Thus, the prosurvival Bcl-2 homologs do not appear to act by sequestering Apaf-1 and probably instead constrain its activity indirectly (Moriishi, 1999).

In the initiation of apoptosis, Apaf-1, homologous to C. elegans CED-4, functions downstream of bcl-2 but upstream of caspase-3. Bcl-2 may function upstream of Apaf-1 by regulating the release of cytochrome c from mitochondria. Cytochrome c is a required cofactor for Apaf-1. Another protein factor, Apaf-3, has been identified that participates in caspase-3 activation in vitro. Apaf-3 was identified as a member of the caspase family, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. This N-terminal region of caspase-9 is termed a caspase recruitment domain (CARD). Activated caspase-9 in turn cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminished caspase-3 activation. Mutation of the active site of caspase-9 attenuates the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade that is triggered by cytochrome c and dATP (Li, 1997).

Bcl-2 family members are targets of caspases

There is a direct interaction between caspases and Bcl-xL. The loop domain of Bcl-xL is cleaved by caspases in vitro and in cells induced to undergo apoptotic death after Sindbis virus infection or interleukin 3 withdrawal. Mutation of the caspase cleavage site in Bcl-xL in conjunction with a mutation in the BH1 homology domain impairs the death-inhibitory activity of Bcl-xL, suggesting that interaction of Bcl-xL with caspases may be an important mechanism for inhibiting cell death. The BH1 and BH2 domains of Bcl-2 and Bcl-xL are important for heterodimerization with other Bcl-2 family members. Once Bcl-xL is cleaved, the C-terminal fragment of Bcl-xL potently induces apoptosis. Taken together, these findings indicate that the recognition/cleavage site of Bcl-xL may facilitate protection against cell death by acting at the level of caspase activation and that cleavage of Bcl-xL during the execution phase of cell death converts Bcl-xL from a protective to a lethal protein (Clem, 1998).

Caspases comprise a family of cysteine proteases implicated in the biochemical and morphological changes that occur during apoptosis (programmed cell death). The loop domain of Bcl-2 is cleaved at Asp34 by caspase-3 (CPP32) in vitro, in cells overexpressing caspase-3, and after induction of apoptosis by Fas ligation and interleukin-3 withdrawal. The carboxyl-terminal Bcl-2 cleavage product triggers cell death and accelerates Sindbis virus-induced apoptosis, which is dependent on the BH3 homology and transmembrane domains of Bcl-2. Inhibitor studies indicate that cleavage of Bcl-2 may further activate downstream caspases and contribute to amplification of the caspase cascade. Cleavage-resistant mutants of Bcl-2 have increased protection from interleukin-3 withdrawal and Sindbis virus-induced apoptosis. Thus, cleavage of Bcl-2 by caspases may ensure the inevitability of cell death (Cheng, 1997).

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. Endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax is only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolishes cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 has no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell (Kirsch, 1999).

Bcl-2 family members: transcriptional regulation

Binding of the proinflammatory cytokine tumor necrosis factor (TNFalpha) to its receptor triggers competing signaling pathways that determine whether a cell lives or dies. Whereas one pathway is conducive to cell death, the other leads to activation of Rel/NF-kappaB transcription factors and the coincident inhibition of apoptosis. Accumulating evidence supports a proactive role for NF-kappaB in the inhibition of cell death induced by TNFalpha and other death-causing agents. Whereas the activation of NF-kappaB blocks cell killing, its inhibition enhances the cytotoxicity of TNFalpha and promotes apoptosis in various cell systems, demonstrating the need for NF-kappaB function for cell survival. Bcl-2-family proteins are key regulators of the apoptotic response. The pro-survival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kappaB. bfl-1 gene expression is dependent on NF-kappaB activity and it can substitute for NF-kappaB to suppress TNFalpha-induced apoptosis. bfl-1 promoter analysis has identified an NF-kappaB site responsible for its Rel/NF-kappaB-dependent induction. The expression of bfl-1 in immune tissues supports the protective role of NF-kappaB in the immune system. The activation of Bfl-1 may be the means by which NF-kappaB functions in oncogenesis and promotes cell resistance to anti-cancer therapy (Zong, 1999).

Activation of CD40 is essential for thymus-dependent humoral immune responses and rescuing B cells from apoptosis. Many of the effects of CD40 are believed to be achieved through altered gene expression. In addition to Bcl-x, a known CD40-regulated antiapoptotic molecule, a related antiapoptotic molecule, A1/Bfl-1, has been identified as a CD40-inducible gene. Inhibition of the NF-kappaB pathway by overexpression of a dominant-active inhibitor of NF-kappaB abolishes CD40-induced up-regulation of both the Bfl-1 and Bcl-x genes and also eliminates the ability of CD40 to rescue Fas-induced cell death. Within the upstream promoter region of Bcl-x, a potential NF-kappaB-binding sequence was found to support NF-kappaB-dependent transcriptional activation. Furthermore, expression of physiological levels of Bcl-x protects B cells from Fas-mediated apoptosis in the absence of NF-kappaB signaling. Thus, these results suggest that CD40-mediated cell survival proceeds through NF-kappaB-dependent up-regulation of Bcl-2 family members (Lee, 1999).

The Brn-3a POU family transcription factor has been shown to strongly activate expression of the Bcl-2 proto-oncogene and thereby protect neuronal cells from programmed cell death (apoptosis). This activation of the Bcl-2 promoter by Brn-3a is strongly inhibited by the p53 anti-oncogene protein. This inhibitory effect of p53 on Brn-3a-mediated transactivation is observed with nonoverlapping gene fragments containing either the Bcl-2 p1 or p2 promoters but is not observed with other Brn-3a-activated promoters such as in the gene encoding alpha-internexin or with an isolated Brn-3a binding site from the Bcl-2 promoter linked to a heterologous promoter. In contrast, p53 mutants, which are incapable of binding to DNA, do not affect Brn-3a-mediated activation of the Bcl-2 p1 and p2 promoters. Moreover, Brn-3a and p53 have been shown to bind to adjacent sites in the p2 promoter and to directly interact with one another, both in vitro and in vivo, with this interaction being mediated by the POU domain of Brn-3a and the DNA binding domain of p53. The significance of these effects is discussed in terms of the antagonistic effects of Bcl-2 and p53 on the rate of apoptosis and the overexpression of Brn-3a in specific tumor cell types (Budhram-Mahadeo, 1999).

Nerve growth factor (NGF) and other neurotrophins support survival of neurons through processes that are incompletely understood. The transcription factor CREB is a critical mediator of NGF-dependent gene expression, but whether CREB family transcription factors regulate expression of genes that contribute to NGF-dependent survival of sympathetic neurons is unknown. To determine whether CREB-mediated gene expression is necessary for NGF-dependent neuronal survival, this study monitored survival of sympathetic neurons after expression of either of two distinct inhibitors of CREB. One CREB inhibitor, A-CREB, is a potent and selective inhibitor of CREB DNA binding activity. The other, CREBm1, binds to CREB binding sites in DNA but is not activated because the transcriptional regulatory residue, serine 133, is mutated to alanine. CREB-mediated gene expression is both necessary for NGF-dependent survival and sufficient on its own to promote survival of sympathetic neurons. Moreover, expression of Bcl-2 is activated by NGF and other neurotrophins by a CREB-dependent transcriptional mechanism. A region of the bcl-2 gene between 1640 and 1337 relative to the translation start site is required for NGF-sensitive transcription. This region contains a near-perfect consensus CRE. Activated CREB can bind to this region of the bcl-2 promoter, and this interaction is critical for expression of Bcl-2 in a B lymphocyte cell line. Thus, a test was performed to see if the integrity of the bcl-2 CRE is necessary for the NGF-induced expression of bcl-2. A bcl-2 reporter construct harboring a two-base pair mutation of the CRE, rendering it unable to bind CREB, is impaired in its responsiveness to NGF. Overexpression of Bcl-2 reduces the death-promoting effects of CREB inhibition. Together, these data support a model in which neurotrophins promote survival of neurons, in part through a mechanism involving CREB family transcription factor-dependent expression of genes encoding prosurvival factors (Riccio, 1999).

The ETS family transcriptional repressor TEL is frequently disrupted by chromosomal translocations, including the t(12;21) in which the second allele of TEL is deleted in up to 90% of the cases. Consistent with its role as a putative tumor suppressor, TEL expression inhibits colony formation by Ras-transformed NIH 3T3 cells and hinders proliferation of a variety of cell types. Although no alteration is observed in the cell cycle of TEL-expressing cells, a marked increase in apoptosis of serum-starved TEL-expressing NIH 3T3 cells was found. This decrease in cell survival requires the DNA binding domain of TEL, suggesting that TEL represses an anti-apoptotic gene. These observations prompted a search for genes regulated by ETS family proteins that regulate apoptosis. The anti-apoptotic molecule Bcl-XL contains multiple ets-factor binding sites within its promoters, and TEL represses a Bcl-XL promoter-linked reporter gene. Moreover, the enforced expression of TEL decreases the endogenous expression of both Bcl-XL mRNA and protein. TEL-mediated repression of Bcl-XL likely affects cell survival via regulation of the apoptotic pathway (Irvin, 2003).

The pattern of programmed cell death was studied in the neural crest and how it is controlled by the activity of the transcription factors Slug and msx1 was examined. The results indicate that apoptosis is more prevalent in the neural folds than in the rest of the neural ectoderm. Through gain- and loss-of-function experiments with inducible forms of both Slug and msx1 genes, it was shown that Slug acts as an anti-apoptotic factor whereas msx1 promotes cell death, either in the neural folds of the whole embryos, in isolated or induced neural crest and in animal cap assays. The protective effect of expressing Slug can be reversed by expressing the apoptotic factor Bax, while the apoptosis promoted by msx1 can be abolished by expressing the Xenopus homologue of Bcl2 (XR11). Furthermore, Slug and msx1 control the transcription of XR11 and several caspases required for programmed cell death. In addition, expression of Bax or Bcl2 produced similar effects on the survival of the neural crest and on the development of its derivatives as those produced by altering the activity of Slug or msx1. Finally, it was shown that in the neural crest, the region of the neural folds where Slug is expressed, cells undergo less apoptosis, than in the region where the msx1 gene is expressed; this region corresponds to cells adjacent to the neural crest. The expression of Slug and msx1 controls cell death in certain areas of the neural folds, and how this equilibrium is necessary to generate sharp boundaries in the neural crest territory and to precisely control cell number among neural crest derivatives is discussed (Tribulo, 2004).

Signaling upstream of Bcl-2 family members

BAD is a distant member of the Bcl-2 family that promotes cell death. Phosphorylation of BAD prevents this. BAD phosphorylation induced by interleukin-3 (IL-3) is inhibited by specific inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Akt, a survival-promoting serine-threonine protein kinase, is activated by IL-3 in a PI 3-kinase-dependent manner. Active, but not inactive, forms of Akt are found to phosphorylate BAD in vivo and in vitro at the same residues that are phosphorylated in response to IL-3. Thus, the proapoptotic function of BAD is regulated by the PI 3-kinase-Akt pathway (del Paso, 1997).

Growth factors can promote cell survival by activating the phosphatidylinositide-3'-OH kinase and its downstream target, the serine-threonine kinase Akt. However, the mechanism by which Akt functions to promote survival is not understood. Growth factor activation of the PI3'K/Akt signaling pathway culminates in the phosphorylation of the BCL-2 family member BAD, thereby suppressing apoptosis and promoting cell survival. Akt phosphorylates BAD in vitro and in vivo, and blocks the BAD-induced death of primary neurons in a site-specific manner. These findings define a mechanism by which growth factors directly inactivate a critical component of the cell-intrinsic death machinery (Datta, 1997).

The initiation of apoptosis often transpires in the presence of agents that regulate cell survival. This study evaluated the effects of stress-induced ceramide on the anti-apoptotic activity of the phosphoinositide-3 kinase [PI(3)K] pathway. PI(3)K activity is directly down-regulated by stress-induced ceramide in a dose-dependent manner with rapid kinetics and high specificity. Ceramide inhibition of PI(3)K is dependent on acid-sphingomyelinase. Down-regulation of PI(3)K by ceramide results in inhibition of the kinase Akt and decreased phosphorylation of the death effector, Bad. Thus, ceramide levels could act as a general apoptotic rheostat controlling cell survival by regulating PI(3)K anti-apoptotic effector mechanisms. Ceramide contributes to apoptosis not only by regulating effector mechanisms such as caspases and c-Jun, but by deregulation of the anti-apoptotic PI(3)K Akt/Bad pathway. It has been shown that the interaction of ceramide with PI(3)K is independent of its p110 catalytic subunit. Regulatory subunit (p85) knockouts are resistent to oxidative apoptosis in a PI(3)K-independent, p53-dependent fashion. Because ceramide modulates the PI(3)K response at extremely early times following stress, it is feasible that the effect of ceramide on p85 could modulate p85's affinity for p110 and thus make it available for binding other potential targets that could be proapoptotic (Zundel, 1998).

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K that is necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. Granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity; when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. In contrast, Interleukin 3 (IL-3) requires PI3K for survival, and blocking PI3K partially inhibits Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras-mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it does not stimulate Bad phosphorylation. Finally, although these data suggest that the MAPK pathway is not required for inhibition of apoptosis, evidence is provided that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. These data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect (Scheid, 1998).

The ratio of proapoptotic versus antiapoptotic Bcl-2 members is a critical determinant that plays a significant role in altering susceptibility to apoptosis. Therefore, a reduction of antiapoptotic protein levels in response to proximal signal transduction events may switch on the apoptotic pathway. In endothelial cells, tumor necrosis factor alpha (TNF-alpha) induces dephosphorylation and subsequent ubiquitin-dependent degradation of the antiapoptotic protein Bcl-2. The roles of different putative phosphorylation sites to facilitate Bcl-2 degradation were investigation. Mutation of the consensus protein kinase B/Akt site or of potential protein kinase C or cyclic AMP-dependent protein kinase sites does not affect Bcl-2 stability. In contrast, inactivation of the three consensus mitogen-activated protein (MAP) kinase sites leads to a Bcl-2 protein that is ubiquitinated and subsequently degraded by the 26S proteasome. Inactivation of these sites within Bcl-2 revealed that dephosphorylation of Ser87 appears to play a major role. A Ser-to-Ala substitution at this position results in 50% degradation, whereas replacement of Thr74 with Ala leads to 25% degradation, as assessed by pulse-chase studies. It was further demonstrated that incubation with TNF-alpha induces dephosphorylation of Ser87 of Bcl-2 in intact cells. Furthermore, MAP kinase triggers phosphorylation of Bcl-2, whereas a reduction in Bcl-2 phosphorylation was observed in the presence of MAP kinase-specific phosphatases or the MAP kinase-specific inhibitor PD98059. Moreover, oxidative stress mediates TNF-alpha-stimulated proteolytic degradation of Bcl-2 by reducing MAP kinase activity. Taken together, these results demonstrate a direct protective role for Bcl-2 phosphorylation by MAP kinase against apoptotic challenges to endothelial cells and other cells (Breitschopf, 2000).

Bad is a critical regulatory component of the intrinsic cell death machinery that exerts its death-promoting effect upon heterodimerization with the antiapoptotic proteins Bcl-2 and Bcl-x(L). Growth factors promote cell survival through phosphorylation of Bad, resulting in its dissociation from Bcl-2 and Bcl-x(L) and its association with 14-3-3tau. Survival of interleukin 3 (IL-3)-dependent FL5.12 lymphoid progenitor cells is attenuated upon treatment with the Rho GTPase-inactivating toxin B from Clostridium difficile. p21-activated kinase 1 (PAK1) is activated by IL-3 in FL5.12 cells, and this activation is reduced by the phosphatidylinositol 3-kinase inhibitor LY294002. Overexpression of a constitutively active PAK mutant (PAK1-T423E) promoted cell survival of FL5.12 and NIH 3T3 cells, while overexpression of the autoinhibitory domain of PAK (amino acids 83 to 149) enhanced apoptosis. PAK phosphorylates Bad in vitro and in vivo on Ser112 and Ser136, resulting in a markedly reduced interaction between Bad and Bcl-2 or Bcl-x(L) and the increased association of Bad with 14-3-3tau. These findings indicate that PAK inhibits the proapoptotic effects of Bad by direct phosphorylation and that PAK may play an important role in cell survival pathways (Schurmann, 2000).

Phosphorylation of the Bcl-2 family protein Bad may represent an important bridge between survival signaling by growth factor receptors and the prevention of apoptosis. Bad phosphorylation was examined following cytokine stimulation, which revealed phosphorylation on a critical residue, serine 112, in a MEK-dependent manner. Furthermore, Bad phosphorylation also increases on several sites distinct from serine 112 but could not be detected on serine 136, previously thought to be a protein kinase B/Akt-targeted residue. Serine 112 phosphorylation is be absolutely required for dissociation of Bad from Bcl-x(L). These results demonstrate for the first time in mammalian cells the involvement of the Ras-MAPK pathway in the phosphorylation of Bad and the regulation of its function (Scheid, 1999).

Growth factors activate an array of cell survival signaling pathways. Mitogen-activated protein (MAP) kinases transduce signals emanating from their upstream activators: MAP kinase kinases (MEKs). The MEK-MAP kinase signaling cassette is a key regulatory pathway promoting cell survival. The downstream effectors of the mammalian MEK-MAP kinase cell survival signal have not been previously described. Identified here is a pro-survival role for the serine/threonine kinase S6 kinase p90 ribosomal S6 kinase Rsk1 ( (see Drosophila RSK)), a downstream target of the MEK-MAP kinase signaling pathway. In cells that are dependent on interleukin-3 (IL-3) for survival, pharmacological inhibition of MEKs antagonize the IL-3 survival signal. In the absence of IL-3, a kinase-dead Rsk1 mutant eliminates the survival effect afforded by activated MEK. Conversely, a novel constitutively active Rsk1 allele restores the MEK-MAP kinase survival signal. Experiments in vitro and in vivo have demonstrated that Rsk1 directly phosphorylates the pro-apoptotic protein Bad at the serine residues that, when phosphorylated, abrogate Bad's pro-apoptotic function. Constitutively active Rsk1 causes constitutive Bad phosphorylation and protection from Bad-modulated cell death. Kinase-inactive Rsk1 mutants antagonize Bad phosphorylation. Bad mutations that prevent phosphorylation by Rsk1 also inhibit Rsk1-mediated cell survival. These data support a model in which Rsk1 transduces the mammalian MEK-MAP kinase signal in part by phosphorylating Bad (Shimamura, 2000).

Insulin-like growth factor-I (IGF-I) is known to prevent apoptosis induced by diverse stimuli. The present study examined the effect of IGF-I on the promoter activity of bcl-2, a gene with antiapoptotic function. A luciferase reporter driven by the promoter region of bcl-2 from -1640 to -1287 base pairs upstream of the translation start site containing a cAMP-response element was used in transient transfection assays. Treatment of PC12 cells with IGF-I enhances the bcl-2 promoter activity by 2.3-fold, which is inhibited significantly (p < 0.01) by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Cotransfection of the bcl-2 promoter with MAPK kinase 6 and the beta isozyme of p38 MAPK results in 2-3-fold increase in the reporter activity. The dominant negative form of MAPKAP-K3, a downstream kinase activated by p38 MAPK, and the dominant negative form of cAMP-response element-binding protein, inhibited the reporter gene activation by IGF-I and p38beta MAPK significantly. IGF-I increases the activity of p38beta MAPK introduced into the cells by adenoviral infection. Thus, a novel signaling pathway (MAPK kinase 6/p38beta MAPK/MAPKAP-K3) has been characterized that defines a transcriptional mechanism for the induction of the antiapoptotic protein Bcl-2 by IGF-I through the nuclear transcription factor cAMP-response element-binding protein in PC12 cells (Pugazhenthi, 1999).

The familial Alzheimer's disease gene products, presenilin-1 and presenilin-2, have been reported to be functionally involved in amyloid precursor protein processing, notch receptor signaling, and programmed cell death or apoptosis. However, the molecular mechanisms by which presenilins regulate these processes remain unknown. With regard to the latter, a molecular link is described between presenilins and the apoptotic pathway. Bcl-X(L), an anti-apoptotic member of the Bcl-2 family has been shown to interact with the carboxyl-terminal fragments of PS1 and PS2 by the yeast two-hybrid system. In vivo interaction analysis reveals that both PS2 and its naturally occurring carboxyl-terminal products, PS2short and PS2Ccas, associated with Bcl-X(L), whereas the caspase-3-generated amino-terminal PS2Ncas fragment do not. This interaction has been corroborated by demonstrating that Bcl-X(L) and PS2 partially co-localized to sites of the vesicular transport system. Functional analysis revealed that presenilins can influence mitochondrial-dependent apoptotic activities, such as cytochrome c release and Bax-mediated apoptosis. Together, these data support a possible role of the Alzheimer's presenilins in modulating the anti-apoptotic effects of Bcl-X(L) (Passer, 1999).

The p75 neurotrophin receptor (p75NTR) has been shown to mediate neuronal death through an unknown pathway. p75NTR expression plasmids were microinjected into sensory neurons in the presence of growth factors and the effect of the expressed proteins on cell survival were assessed. Unlike other members of the TNFR family, p75NTR signals death through a unique caspase-dependent death pathway that does not involve the "death domain" and is differentially regulated by Bcl-2 family members: the anti-apoptotic molecule Bcl-2 both promotes, and is required for, p75NTR killing, whereas killing is inhibited by its homologue Bcl-xL. These results demonstrate that Bcl-2, through distinct molecular mechanisms, either promotes or inhibits neuronal death depending on the nature of the death stimulus (Coulson, 1999).

IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here, a mechanism is reported by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrates into the mitochondrial membrane. This translocation is attributable to a conformational change in the Bax protein itself. A rise in intracellular pH precedes mitochondrial translocation and triggers the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rises steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax is directly altered by pH of 7.8 or higher and has been demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induces a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, it has been shown that Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death (Khaled, 1999).

Neutrophils are important effector cells in immunity to microorganisms, particularly bacteria. The process of neutrophil apoptosis is delayed in several inflammatory diseases, suggesting that this phenomenon may represent a general feature contributing to the development of neutrophilia, and, therefore, in many cases to host defense against infection. The delay of neutrophil apoptosis is associated with markedly reduced levels of Bax, a pro-apoptotic member of the Bcl-2 family. Such Bax-deficient cells are also observed upon stimulation of normal neutrophils with cytokines present at sites of neutrophilic inflammation, such as granulocyte and granulocyte-macrophage colony-stimulating factors, in vitro. Moreover, Bax-deficient neutrophils generated by using Bax antisense oligodeoxynucleotides demonstrated delay apoptosis, providing direct evidence for a role of Bax as a pro-apoptotic molecule in these cells. Interestingly, the Bax gene is reexpressed in Bax-deficient neutrophils under conditions of cytokine withdrawal. Thus, both granulocyte expansion and the resolution of inflammation appear to be regulated by the expression of the Bax gene in neutrophils (Dibbert, 1999).

Plakoglobin is a vertebrate cytoplasmic protein and a homolog of beta-catenin and Armadillo in Drosophila, with similar adhesive and signaling functions. These proteins interact with cadherins to mediate cell-cell adhesion and associate with transcription factors to induce changes in the expression of genes involved in cell fate determination and proliferation. Unlike the relatively well characterized role of beta-catenin in cell proliferation via activation of c-MYC and cyclin D1 gene expression, the signaling function of plakoglobin in regulation of cell growth is undefined. High levels of plakoglobin expression in plakoglobin-deficient human SCC9 cells leads to uncontrolled growth and foci formation. Concurrent with the change in growth characteristics is observed a pronounced inhibition of apoptosis. This correlates with an induction of expression of BCL-2, a prototypic member of apoptosis-regulating proteins. The BCL-2 expression coincides with decreased proteolytic processing and activation of caspase-3, an executor of programmed cell death. These data suggest that the growth regulatory function of plakoglobin is independent of its role in mediating cell-cell adhesion. These observations clearly implicate plakoglobin in pathways regulating cell growth and provide initial evidence of its role as a pivotal molecular link between pathways regulating cell adherence and cell death (Hakimelahi, 2000).

Nitric oxide is a chemical messenger implicated in neuronal damage associated with ischemia, neurodegenerative disease, and excitotoxicity. Excitotoxic injury leads to increased NO formation, as well as stimulation of the p38 mitogen-activated protein (MAP) kinase in neurons. In the present study, it was determined if NO-induced cell death in neurons is dependent on p38 MAP kinase activity. Sodium nitroprusside (SNP), a NO donor, elevates caspase activity and induces death in human SH-SY5Y neuroblastoma cells and primary cultures of cortical neurons. Concomitant treatment with SB203580, a p38 MAP kinase inhibitor, diminishes caspase induction and protects SH-SY5Y cells and primary cultures of cortical neurons from NO-induced cell death, whereas the caspase inhibitor zVAD-fmk does not provide significant protection. A role for p38 MAP kinase is further substantiated by the observation that SB203580 blocks translocation of the cell death activator, Bax, from the cytosol to the mitochondria after treatment with SNP. Moreover, expressing a constitutively active form of MKK3, a direct activator of p38 MAP kinase promotes Bax translocation and cell death in the absence of SNP. Bax-deficient cortical neurons are resistant to SNP, further demonstrating the necessity of Bax in this mode of cell death. These results demonstrate that p38 MAP kinase activity plays a critical role in NO-mediated cell death in neurons by stimulating Bax translocation to the mitochondria, thereby activating the cell death pathway (Ghatan, 2000).

Sympathetic neurons require nerve growth factor for survival and die by apoptosis in its absence. Key steps in the death pathway include c-Jun activation, mitochondrial cytochrome c release, and caspase activation. Neurons rescued from NGF withdrawal-induced apoptosis by expression of dominant-negative c-Jun do not release cytochrome c from their mitochondria. Furthermore, mRNA for BIMEL, a proapoptotic BCL-2 family member, increases in level after NGF withdrawal and this is reduced by dominant-negative c-Jun. Overexpression of BIMEL in neurons induces cytochrome c redistribution and apoptosis in the presence of NGF, and neurons injected with Bim antisense oligonucleotides or isolated from Bim-/- knockout mice die more slowly after NGF withdrawal (Whitfield, 2001).

BH3-only proapoptotic proteins of the Bcl-2 family such as Bad, Bid, Bim, or Bik transduce death stimuli from the cell surface to the central death machinery. Following apoptosis stimulation, these molecules translocate from the cytosol to mitochondria where they bind to membrane-based Bcl-2 family members. Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In type II cells, such as Jurkat cells or hepatocytes, death-inducing signaling complex (DISC) formation is strongly reduced compared to type I cells in which activation of large amounts of caspase 8 by the DISC enables direct activation of downstream caspases leading to irreversible cell damage. In type II cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerates Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delays apoptosis. When phosphorylated, Bid is insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis (Desagher, 2001).

A mechanism that triggers neuronal apoptosis has been characterized. The cell cycle-regulated protein kinase Cdc2 is expressed in postmitotic granule neurons of the developing rat cerebellum and Cdc2 mediates apoptosis of cerebellar granule neurons upon the suppression of neuronal activity. Cdc2 catalyzes the phosphorylation of the BH3-only protein BAD at a distinct site, serine 128, and thereby induces BAD-mediated apoptosis in primary neurons by opposing growth factor inhibition of the apoptotic effect of BAD. The phosphorylation of BAD serine 128 inhibits the interaction of growth factor-induced serine 136-phosphorylated BAD with 14-3-3 proteins. These results suggest that a critical component of the cell cycle couples an apoptotic signal to the cell death machinery via a phosphorylation-dependent mechanism that may generally modulate protein-protein interactions (Konishi, 2002).

Growth factor suppression of apoptosis correlates with the phosphorylation and inactivation of multiple proapoptotic proteins, including the BCL-2 family member BAD. However, the physiological events required for growth factors to block cell death are not well characterized. To assess the contribution of BAD inactivation to cell survival, mice were made with point mutations in the BAD gene that abolish BAD phosphorylation at specific sites. BAD phosphorylation protects cells from the deleterious effects of apoptotic stimuli and attenuates death pathway signaling by raising the threshold at which mitochondria release cytochrome c to induce cell death. These findings establish a function for endogenous BAD phosphorylation, and elucidate a mechanism by which survival kinases block apoptosis in vivo (Datta, 2002).

p53 is a well-known tumor suppressor and is also involved in processes of organismal aging and developmental control. A recent exciting development in the p53 field is the discovery of various p53 isoforms. One p53 isoform is human Delta133p53 and its zebrafish counterpart Delta113p53. These N-terminal-truncated p53 isoforms are initiated from an alternative p53 promoter, but their expression regulation and physiological significance at the organismal level are not well understood. This study shows here that zebrafish Delta113p53 is directly transactivated by full-length p53 in response to developmental and DNA-damaging signals. More importantly, Delta113p53 functions to antagonize p53-induced apoptosis via activating bcl2L [closest to human Bcl-x(L)], and knockdown of Delta113p53 enhances p53-mediated apoptosis under stress conditions. Thus, it was demonstrated that the p53 genetic locus contains a new p53 response gene and that Delta113p53 does not act in a dominant-negative manner toward p53 but differentially modulates p53 target gene expression to antagonize p53 apoptotic activity at the physiological level in zebrafish. These results establish a novel feedback pathway that modulates the p53 response and suggest that modulation of the p53 pathway by p53 isoforms might have an impact on p53 tumor suppressor activity (Chen, 2009).

JNK regulates FoxO-dependent autophagy in neurons

The cJun N-terminal kinase (JNK) signal transduction pathway is implicated in the regulation of neuronal function. JNK is encoded by three genes that play partially redundant roles. This study reports the creation of mice with targeted ablation of all three Jnk genes in neurons. Compound JNK-deficient neurons are dependent on autophagy for survival. This autophagic response is caused by FoxO-induced expression of Bnip3 that displaces the autophagic effector Beclin-1 from inactive Bcl-XL complexes. These data identify JNK as a potent negative regulator of FoxO-dependent autophagy in neurons (Xu, 2011).

Studies of nonneuronal cells have implicated JNK in the induction of autophagy. Indeed, this study confirmed the conclusion that JNK can contribute to increased autophagy by examining primary mouse embryonic fibroblasts (MEFs) with compound JNK deficiency. The mechanism of JNK-induced autophagy may be mediated by phosphorylation of Bcl2 by JNK and the subsequent release of the autophagic effector Beclin-1. The sites of JNK phosphorylation on Bcl2 are conserved in the related protein Bcl-XL. This conservation suggests that phosphorylation of Bcl2 and Bcl-XL is functionally important. Phosphorylation of Bcl2 and Bcl-XL by JNK and other protein kinases may represent an important mechanism of autophagy regulation. Indeed, the properties of JNK as a stress-responsive kinase provide an elegant mechanism for coupling stress exposure to the induction of autophagy (Xu, 2011).

Studies of nonneuronal cells demonstrate that JNK is markedly activated from a low basal state when cells are exposed to stress. However, JNK is regulated very differently in neurons. JNK1 remains constitutively activated under basal conditions, while JNK2 and JNK3 exhibit low basal activity and are stress-responsive. The proautophagy role of JNK in nonneuronal cells has been reported to be mediated by JNK1. It is therefore intriguing that JNK1 is constitutively activated in neurons. Based on studies of nonneuronal cells, the constitutive activation of JNK1 in neurons should cause autophagy. A mechanism must therefore exist to prevent autophagy activation by constitutively activated JNK1 in neurons. Although the mechanism is unclear, these considerations indicate that neurons are refractory to the proautophagy JNK1 signaling pathway that has been identified in nonneuronal cells (Xu, 2011).

This analysis of compound JNK-deficient neurons demonstrates that JNK regulates neuronal autophagy. In contrast to the proautophagy role of JNK nonneuronal cells, neuronal JNK acts to suppress autophagy. Loss of neuronal JNK function causes engagement of a transcriptional program that leads to increased expression of autophagy-related genes and the induction of an autophagic response. One consequence of autophagy induction caused by JNK deficiency is improved neuronal survival (Xu, 2011).

FoxO transcription factors are implicated in the induction of both cell death (apoptosis) and cell survival (autophagy) responses. The results of this study identify JNK as a signaling molecule that may contribute to the coordination of these divergent responses to FoxO transcription factor activation (Xu, 2011).

FoxO activation in neurons leads to the expression of the target gene Bim, a proapoptotic BH3-only protein, and causes cell death. JNK activation in neurons promotes expression of Bim, most likely because JNK-dependent AP-1 activity is required for Bim expression. Moreover, JNK phosphorylates Bim on an activating site, and also causes the release of Bim from complexes with the anti-apoptotic Bcl2 family protein Mcl-1. Together, these processes initiate JNK-dependent apoptosis. JNK inhibition can therefore prevent neuronal cell death. Indeed, small molecule inhibitors of JNK cause neuroprotection in models of neurodegenerative disease (Xu, 2011).

Activation of FoxO transcription factors can also cause increased expression of autophagy-related genes, including Atg8/Lc3b, Atg12, and Bnip3. While JNK cooperates with FoxO to increase proapoptotic Bim expression, JNK deficiency prevents induction of Bim expression and promotes a survival response that is mediated by increased FoxO-dependent expression of the autophagy-related target genes Atg8/Lc3b, Atg12, and Bnip3. Indeed, inhibition of autophagy in JNK-deficient neurons causes rapid death. This neuronal survival response is relevant to stroke models in which neuronal death is mediated by a JNK-dependent mechanism (Xu, 2011).

Together, these data demonstrate that cross-talk between the FoxO and JNK signaling pathways leads to neuronal death. In contrast, loss of JNK promotes FoxO-induced survival mediated by increased autophagy. JNK therefore acts as a molecular switch that defines the physiological consequence of FoxO activation in neurons (Xu, 2011).

Thus, JNK is implicated in the induction of autophagy in nonneuronal cells. However, JNK1 is constitutively activated in neurons, and these cells are refractory to JNK-induced autophagy. Instead, JNK acts to suppress autophagy in neurons by inhibiting FoxO-induced expression of autophagy-related genes (e.g., Atg8/Lc3b, Atg12, and Bnip3) and increasing the expression of proapoptotic genes (e.g., Bim). JNK inhibition causes neuroprotection that is mediated by loss of proapoptotic gene expression and increased autophagy (Xu, 2011).

Bcl-2 family members as regulators of the cell death hierarchy: Bcl-2 proteins as ion channels and regulators of mitochondrial membrane permiability

The crystal and solution structures of a Bcl-2 family member, Bcl-xL, have been solved. The structures consist of two central, primarily hydrophobic alpha-helices, which are surrounded by amphipathic helices. A 60-residue loop connecting helices alpha1 and alpha2 was found to be flexible and non-essential for anti-apoptotic activity. The three functionally important Bcl-2 homology regions (BH1, BH2 and BH3) are in close spatial proximity and form an elongated hydrophobic cleft that may represent the binding site for other Bcl-2 family members. The arrangement of the alpha-helices in Bcl-xL is reminiscent of the membrane translocation domain of bacterial toxins, in particular diphtheria toxin and the colicins. The structural similarity may provide a clue to the mechanism of action of the Bcl-2 family of proteins (Muchmore, 1996).

The BCL-2 family of proteins is composed of both pro- and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-XL monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion efflux. Release is maximum at approximately pH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings reveal a minimal channel conductance for BAX of 22 pS that evolve to channel currents with at least three subconductance levels. The final, apparently stable BAX channel has a conductance of 0.731 nS at pH 4. 0, which changes to 0.329 nS when shifted to pH 7.0, but which remains mildly Cl- selective and predominantly open. When BAX-incorporated lipid vesicles are fused to planar lipid bilayers at pH 7.0, a Cl- selective (PK/PCl = 0.3) 1.5-nS channel displaying mild inward rectification is noted. In contrast, BCL-2 forms mildly K+-selective (PK/PCl = 3.9) channels with a most prominent initial conductance of 80 pS, which increases to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also reveals mild K+ selectivity (PK/PCl = 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics, including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites (Schlesinger, 1997).

Mitochondrial physiology is disrupted in either apoptosis or necrosis. A wide variety of apoptotic and necrotic stimuli induce progressive mitochondrial swelling and outer mitochondrial membrane rupture. Discontinuity of the outer mitochondrial membrane results in cytochrome c redistribution from the intermembrane space to the cytosol, followed by subsequent inner mitochondrial membrane depolarization. The mitochondrial membrane protein Bcl-xL can inhibit these changes in cells treated with apoptotic stimuli. Bcl-xL-expressing cells adapt to growth factor withdrawal or staurosporine treatment by maintaining a decreased mitochondrial membrane potential. Bcl-xL expression also prevents mitochondrial swelling in response to agents that inhibit oxidative phosphorylation, in particular, oligomycin and antimycin A. Oligomycin functions to block the inner membrane F1F0-ATPase, which utilizes the H+ ion gradient to generate ATP. If H+ ions are not consumed and electron transport continues, the mitochondria become acutely hyperpolarized and subsequently undergo the osmotic swelling associated with necrosis. Antimycin A inhibits complex III of the electron transport chain. Cells treated with antimycin A are unable to maintain mitochondrial osmotic homeostasis following the inhibition of electron transport. These data suggest that Bcl-xL promotes cell survival by regulating the electrical and osmotic homeostasis of mitochondria (Vander Heiden, 1997).

Bcl-2-related proteins are critical regulators of cell survival that are localized to the outer mitochondrial, outer nuclear and endoplasmic reticulum membranes. Despite their physiological importance, the biochemical function of Bcl-2-related proteins has remained elusive. The three-dimensional structure of Bcl-xL, an inhibitor of apoptosis, is similar to the structures of the pore-forming domains of bacterial toxins. A key feature of these pore-forming domains is the ability to form ion channels in biological membranes. Bcl-xL shares this functional feature. Like the bacterial toxins, Bcl-xL can insert into either synthetic lipid vesicles or planar lipid bilayers and form an ion-conducting channel. This channel is pH-sensitive and becomes cation-selective at physiological pH. The ion-conducting channel(s) formed by Bcl-xL displays multiple conductance states that have identical ion selectivity. Together, these data suggest that Bcl-xL may maintain cell survival by regulating the permeability of the intracellular membranes to which it is distributed (Minn, 1997).

Bax is a pro-apoptotic member of the Bcl-2 protein family that resides in the outer mitochondrial membrane. It is controversial whether Bax promotes cell death directly through its putative function as a channel protein versus indirectly by inhibiting cellular regulators of the cell death proteases (caspases). Addition of submicromolar amounts of recombinant Bax protein to isolated mitochondria can induce cytochrome c (Cyt c) release, whereas a peptide representing the Bax BH3 domain is inactive. When placed into purified cytosol, neither mitochondria nor Bax individually induce proteolytic processing and activation of caspases. In contrast, the combination of Bax and mitochondria triggered release of Cyt c from mitochondria and induce caspase activation in cytosols. Supernatants from Bax-treated mitochondria also induce caspase processing and activation. Recombinant Bcl-XL protein abrogates Bax-induced release of Cyt c from isolated mitochondria and prevented caspase activation. In contrast, the broad-specificity caspase inhibitor benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(0-methyl)- fluoromethylketone (zVAD-fmk) and the caspase-inhibiting protein X-IAP have no effect on Bax-induced release of Cyt c from mitochondria in vitro but prevent the subsequent activation of caspases in cytosolic extracts. Unlike Ca2+, a classical inducer of mitochondrial permeability transition, Bax does not induce swelling of mitochondria in vitro. Because the organellar swelling caused by permeability transition causes outer membrane rupture, the findings, therefore, dissociate these two events, implying that Bax uses an alternative mechanism for triggering release of Cyt c from mitochondria (Jurgensmeier, 1998).

A hydrophobic cleft formed by the BH1, BH2 and BH3 domains of Bcl-xL is responsible for interactions between Bcl-xL and BH3-containing death agonists. Bcl-xL primarily counters the proapoptotic effect of Bax by forming an inactivating heterodimer with Bax in solution. Mutants of Bcl-xL were constructed that do not bind to Bax but retain anti-apoptotic activity. Since Bcl-xL can form an ion channel in synthetic lipid membranes, the possibility that this property has a role in heterodimerization-independent cell survival was tested by replacing amino acids within the predicted channel-forming domain, the region surrounding the Bcl-xL helix 5 and helix 6 hairpin, with the corresponding amino acids from Bax. This chimera is referred to as XB. The resulting XB chimera shows a reduced ability to adopt an open conductance state over a wide range of membrane potentials. Although this construct retains the ability to heterodimerize with Bax and to inhibit apoptosis, when a mutation is introduced that renders the chimera incapable of heterodimerization, the resulting protein fails to prevent both apoptosis in mammalian cells and Bax-mediated growth defect in yeast. Similar to mammalian cells undergoing apoptosis, yeast cells expressing Bax exhibit changes in mitochondrial properties that are inhibited by Bcl-xL through heterodimerization-dependent and -independent mechanisms. These data suggest that Bcl-xL regulates cell survival by at least two distinct mechanisms; one is associated with heterodimerization and the other with the ability to form a sustained ion channel. Bax is able to disrupt mitochondrial function, leading to apoptosis in mammalian cells and growth inhibition in yeast. Bcl-xL primarily counters this effect by forming an inactivating heterodimer with Bax in solution. However, when Bcl-xL is prevented from interacting with Bax, it may form an ion channel that establishes a permeability pathway that counters the effects of the Bax ion channel. The establishment and/or properties of this Bcl-xL-mediated permeability pathway may be disrupted in the XB mutants. Alternatively, this counter mechanism may involve interactions within the membrane between the central hydrophobic helices of membrane-inserted Bcl-xL and membrane-inserted Bax, resulting in Bax channel inactivation or the formation of a hybrid, non-toxic channel. In this scenario, the XB mutation prevents proper intermembrane helical interactions. Interactions involving membrane-inserted helices are thought to be the mechanism by which immunity proteins prevent the toxicity of bactericidal colicins, which contain a pore-forming domain structurally similar to that of Bcl-xL (Minn, 1999).

Loss of the mitochondrial membrane potential (Deltapsi) precedes apoptosis and chemical-hypoxia-induced necrosis; this is prevented by the expression of Bcl-2. An examination was carried out of the biochemical mechanism involved when Bcl-2 prevents Deltapsi loss. Mitochondria were isolated either from a cell line overexpressing human Bcl-2 or from livers of Bcl-2 transgenic mice. Although Bcl-2 has no effect on the respiration rate of isolated mitochondria, it prevents both Deltapsi loss and the permeability transition (PT) induced by various reagents, including Ca2+, H2O2, and tert-butyl hydroperoxide. Even under conditions that did not allow PT, Bcl-2 maintains Deltapsi, suggesting that the functional target of Bcl-2 is regulation of Deltapsi but not PT. Bcl-2 also maintains Deltapsi in the presence of the protonophore SF6847, which induces proton influx, suggesting that Bcl-2 regulates ion transport to maintain Deltapsi. Although treatment with SF6847 in the absence of Ca2+ causes massive H+ influx in control mitochondria, the presence of Bcl-2 induces H+ efflux after transient H+ influx. In this case, Bcl-2 does not enhance K+ efflux. Bcl-2 enhances H+ efflux but not K+ flux after treatment of mitochondria with Ca2+ or tert-butyl hydroperoxide. These results suggest that Bcl-2 maintains Deltapsi by enhancing H+ efflux in the presence of Deltapsi-loss-inducing stimuli (Shimizu, 1998).

During transduction of an apoptotic (death) signal into the cell, there is an alteration in the permeability of the membranes of the cell's mitochondria that causes the translocation of the apoptogenic protein cytochrome c into the cytoplasm. In turn, this activates death-driving proteolytic proteins known as caspases. The Bcl-2 family of proteins, whose members may be anti-apoptotic or pro-apoptotic, regulates cell death by controlling this mitochondrial membrane permeability during apoptosis, but how that is achieved is unclear. Liposomes that carry the mitochondrial porin channel (also called the voltage-dependent anion channel, or VDAC) have been created to show that the recombinant pro-apoptotic proteins Bax and Bak accelerate the opening of VDAC, whereas the anti-apoptotic protein Bcl-x(L) closes VDAC by binding to it directly. Bax and Bak allow cytochrome c to pass through VDAC out of liposomes, but passage is prevented by Bcl-x(L). In agreement with this, VDAC1-deficient mitochondria from a mutant yeast do not exhibit a Bax/Bak-induced loss in membrane potential and cytochrome c release, both of which are inhibited by Bcl-x(L). These results indicate that the Bcl-2 family of proteins bind to the VDAC in order to regulate the mitochondrial membrane potential and the release of cytochrome c during apoptosis (Shimizu, 1999).

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic Bcl-2 family members such as Bax and Bak induce apoptogenic mitochondrial cytochrome c release and membrane potential (Deltapsi) loss in isolated mitochondria. Using isolated mitochondria, it has been shown that Bid and Bik, BH3-only proteins from the Bcl-2 family, induce cytochrome c release but not Deltapsi loss. Unlike Bax/Bak, the cytochrome c release induced by Bid/Bik is Ca(2+)-independent, cyclosporin A-insensitive, and respiration-independent. Furthermore, in contrast to Bax/Bak, Bid/Bik neither interacte with VDAC nor directly affected the VDAC activity in liposomes. Consistently, Bid/Bik induces apoptosis without Deltapsi loss, whereas Bax induces apoptosis with Deltapsi loss. These findings indicate the involvement of a different mechanism in BH3-only, protein-induced apoptogenic cytochrome c release (Shimizu, 2000).

The proapoptotic protein BAX contains a single predicted transmembrane domain at its COOH terminus. In unstimulated cells, BAX is located in the cytosol and in peripheral association with intracellular membranes including mitochondria, but inserts into mitochondrial membranes after a death signal. This failure to insert into mitochondrial membrane in the absence of a death signal correlates with repression of the transmembrane signal-anchor function of BAX by the NH2-terminal domain. Targeting can be instated by deleting the domain or by replacing the BAX transmembrane segment with that of BCL-2. In stimulated cells, the contribution of the NH2 terminus of BAX correlates with further exposure of this domain after membrane insertion of the protein. The peptidyl caspase inhibitor zVAD-fmk partly blocks the stimulated mitochondrial membrane insertion of BAX in vivo, which is consistent with the ability of apoptotic cell extracts to support mitochondrial targeting of BAX in vitro, dependent on activation of caspase(s). Taken together, these results suggest that regulated targeting of BAX to mitochondria in response to a death signal is mediated by discrete domains within the BAX polypeptide. The contribution of one or more caspases may reflect an initiation and/or amplification of this regulated targeting (Goping, 1998).

Release of proteins through the outer mitochondrial membrane can be a critical step in apoptosis, and the localization of apoptosis-regulating Bcl-2 family members in this membrane suggests that Bcl-2 proteins control this process. Planar phospholipid membranes were used to test the effect of full-length Bax and Bcl-xL synthesized in vitro and native Bax purified from bovine thymocytes. Instead of forming pores with reproducible conductance levels expected for ionic channels, Bax, but not Bcl-xL, creates arbitrary and continuously variable changes in membrane permeability and decreases the stability of the membrane, regardless of whether the source of the protein is synthetic or native. This breakdown of the membrane permeability barrier and destabilization of the bilayer was quantified by using membrane lifetime measurements. Bax decreases membrane lifetime in a voltage- and concentration-dependent manner. Bcl-xL does not protect against Bax-induced membrane destabilization, supporting the idea that these two proteins function independently of one another. Corresponding to a physical theory for lipidic pore formation, Bax potently diminishes the linear tension of the membrane (i.e., the energy required to form the edge of a new pore). It is suggested that Bax acts directly by destabilizing the lipid bilayer structure of the outer mitochondrial membrane, promoting the formation of a pore (the apoptotic pore) large enough to allow mitochondrial proteins such as cytochrome c to be released into the cytosol. Bax could then enter and permeabilize the inner mitochondrial membrane through the same hole (Basanez, 1999).

Bcl-2 family members either promote or repress programmed cell death. Bax, a death-promoting member, is a pore-forming, mitochondria-associated protein whose mechanism of action is still unknown. During apoptosis, cytochrome C is released from the mitochondria into the cytosol where it binds to APAF-1, a mammalian homolog of Ced-4, and participates in the activation of caspases. The release of cytochrome C has been postulated to be a consequence of the opening of the mitochondrial permeability transition pore (PTP). Bax is reported to be sufficient to trigger the release of cytochrome C from isolated mitochondria. This pathway is distinct from the previously described calcium-inducible, cyclosporin A-sensitive PTP. Rather, the cytochrome C release induced by Bax is facilitated by Mg2+ and cannot be blocked by PTP inhibitors. These results strongly suggest the existence of two distinct mechanisms leading to cytochrome C release: one stimulated by calcium and inhibited by cyclosporin A; the other Bax dependent, Mg2+ sensitive but cyclosporin insensitive (Eskes, 1998).

In many types of apoptosis, the proapoptotic protein Bax undergoes a change in conformation at the level of the mitochondria. This event always precedes the release of mitochondrial cytochrome c, which, in the cytosol, activates caspases through binding to Apaf-1. The mechanisms by which Bax triggers cytochrome c release are unknown. This study shows that following binding to the BH3-domain-only proapoptotic protein Bid, Bax oligomerizes and then integrates in the outer mitochondrial membrane, where it triggers cytochrome c release. Bax mitochondrial membrane insertion triggered by Bid may represent a key step in pathways leading to apoptosis (Eskes, 2000).

Members of the BCL-2 family of proteins either promote or repress programmed cell death. Neonatal sympathetic neurons undergoing apoptosis after nerve growth factor (NGF) deprivation exhibit a protein synthesis-dependent, caspase-independent subcellular redistribution of BAX from cytosol to mitochondria, followed by a loss of mitochondrial cytochrome c and cell death. Treatment with elevated concentrations of the neuroprotectants KCl or cAMP at the time of deprivation prevents BAX translocation and cytochrome c release. However, administration of KCl or cAMP 12 hr after NGF withdrawal prevents acute loss of mitochondrial cytochrome c, but not redistribution of BAX; rescue with NGF prevents both events. Overexpression of Bcl-2 neither alters the normal subcellular localization of BAX nor prevents its redistribution with deprivation but does inhibit the subsequent release of cytochrome c, caspase activation, and cell death. Bcl-2 overexpression does not prevent cell death induced by cytoplasmic microinjection of cytochrome c into NGF-deprived competent-to-die neurons. These observations suggest that the subcellular redistribution of BAX is a critical event in neuronal apoptosis induced by trophic factor deprivation. BCL-2 acts primarily, if not exclusively, at the level of mitochondria to prevent BAX-mediated cytochrome c release, whereas NGF, KCl, or cAMP may abort the apoptotic program at multiple checkpoints (Putcha, 1999).

Unlike apoptosis induced by NGF deprivation in sympathetic neurons, which requires de novo protein synthesis, Fas- or TNF-mediated cell death is facilitated by the inhibition of macromolecular synthesis. It has been reported that activation of the Fas and TNF-R1 receptors causes receptor oligomerization, recruitment and activation of procaspase-8, and N-terminal cleavage of BID; this is followed by translocation of the C-terminal fragment to mitochondria, cytochrome c release, and cell death It is proposed that a critical difference between macromolecular synthesis-dependent (e.g., NGF deprivation in sympathetic neurons) and independent (e.g., Fas or TNF treatment in non-neuronal cells) paradigms of cell death may be that the former require the expression of new gene products to induce the translocation of a BH3-domain-containing, proapoptotic BCL-2 family member to mitochondria, whereas the latter do not. In NGF-deprived sympathetic neurons, BAX alone appears to serve the role of this proapoptotic, BH3-containing protein that mediates the loss of cytochrome c, caspase activation, and cell death. In other models of cell death the identity of the proapoptotic, BH3-containing protein responsible for the loss of mitochondrial cytochrome c may vary according to cell type and apoptotic stimulus (Putcha, 1999 and references).

The mechanism of cytochrome c release in response to apoptotic stimuli and its regulation by the Bcl2 family of proteins is unclear. Inasmuch as the structure of Bcl-xL is reminiscent of pore-forming proteins of bacterial toxins such as diphtheria toxin and colicins, it has been hypothesized that Bcl-xL may function as an ion channel that regulates the permeability of mitochondria. Such an ion channel could minimize osmotic stress, and in doing so, the release of cytochrome c would be prevented due to mitochondrial matrix swelling and outer membrane disruption. Indeed, both swelling of the mitochondrial matrix and bursting of the outer membrane were observed in cells treated with agonistic antibody against Fas. However, whether such a phenomenon is the cause of cytochrome c release or an effect of the apoptotic program is unclear. Activation of cell surface receptor Fas leads to rapid inactivation of the electron transfer activity of cytochrome c and subsequent release of cytochrome c from mitochondria. The inactivation and release of cytochrome c induced by Fas activation is sensitive to z-VAD-fmk, a broad range caspase inhibitor. Since activation of cell surface death receptor leads to rapid activation of caspase-8, the apical caspase in the Fas-induced apoptotic pathway, the loss of cytochrome c from mitochondria is likely a result of caspase-8 activation. Indeed, addition of active caspase-8 to a Xenopus cell-free system induces rapid cytochrome c release from mitochondria. The activation of caspase-8, therefore, initiates two pathways leading to the activation of downstream caspases. Caspase-8 can activate downstream caspases (like caspase-3, caspase-6, and caspase-7) by directly cleaving them. Caspase-8 activates these downstream caspases indirectly by causing cytochrome c release from mitochondria that triggers caspase activation through Apaf1. The latter pathway is regulated by Bcl2 or Bcl-xL while a caspase-8 inhibitor like CrmA blocks both pathways. The contributions of these two pathways to Fas-induced cell death vary between different cell types, presumably due to different levels of activity (Luo, 1998 and references).

The target of Caspase-8, the apical caspase activated by cell surface death receptors such as Fas and TNF, has now been identifed. A cytosolic protein has been purified that induces cytochrome c release from mitochondria in response to caspase-8. Peptide mass fingerprinting identified this protein as Bid, a BH3 domain-containing protein known to interact with both Bcl2 and Bax. Caspase-8 cleaves Bid, and the COOH-terminal part translocates to mitochondria where it triggers cytochrome c release. Immunodepletion of Bid from cell extracts eliminates the cytochrome c releasing activity. The cytochrome c releasing activity of Bid is antagonized by Bcl2. A mutation at the BH3 domain diminishes its cytochrome c releasing activity. Bid, therefore, relays an apoptotic signal from the cell surface to mitochondria (Luo, 1998). The role of mitochondrial Ca2+ [Ca(m)] homeostasis in cell survival has been investigated. Disruption of Ca(m) homeostasis via depletion of the mitochondrial Ca(2+) store is the earliest event that occurs during staurosporine-induced apoptosis in neuroblastoma cells (SH-SY5Y). The decrease of Ca(m) precedes activation of the caspase cascade and DNA fragmentation. Overexpression of the anti-apoptosis protein Bcl-2 leads to increased Ca(m) load, increased mitochondrial membrane potential (DeltaPsi(m)), and inhibition of staurosporine-induced apoptosis. On the other hand, ectopic expression of the pro-apoptotic protein Bik leads to decreased Ca(m) load and decreased DeltaPsi(m). Inhibition of calcium uptake into mitochondria by ruthenium red induces a dose-dependent apoptosis as determined by nuclear staining and DNA ladder assay. Similarly, reducing the Ca(m) load by lowering the extracellular calcium concentration also leads to apoptosis. It is suggested that the anti-apoptotic effect of Bcl-2 is related to its ability to maintain a threshold level of Ca(m) and DeltaPsi(m) while the pro-apoptotic protein Bik has the opposite effect. Furthermore, both ER and mitochondrial Ca(2+) stores are important, and the depletion of either one will result in apoptosis. Thus, these results, for the first time, provide evidence that the maintenance of Ca(m) homeostasis is essential for cell survival (Zhu, 1999).

Bcl-2 family proteins regulate the release of proteins like cytochrome c from mitochondria during apoptosis. Cell-free systems and ultimately a vesicular reconstitution from defined molecules have been used to show that outer membrane permeabilization by Bcl-2 family proteins requires neither the mitochondrial matrix, the inner membrane, nor other proteins. Bid, or its BH3-domain peptide, activates monomeric Bax to produce membrane openings that allow the passage of very large (2 megadalton) dextran molecules. This explains the translocation of large mitochondrial proteins during apoptosis. This process requires cardiolipin and is inhibited by antiapoptotic Bcl-xL. It is concluded that mitochondrial protein release in apoptosis can be mediated by supramolecular openings in the outer mitochondrial membrane, promoted by BH3/Bax/lipid interaction and directly inhibited by Bcl-xL (Kuwana, 2002).

The data show that both pro- and anti-apoptotic Bcl-2 family proteins can regulate macromolecular efflux directly at the mitochondrial outer membrane. Bax forms supramolecular openings in mitochondrial outer membranes and liposomes, suggesting that the ion channel activity of Bcl-2 family proteins may be irrelevant for mitochondrial protein release in apoptosis. Furthermore, these results show that neither swelling of the mitochondrial matrix and inner membrane nor, indeed, any other process requiring ANT or the inner membrane is required for Bid/Bax-induced membrane permeabilization. Similarly, prior studies on cells and mitochondria concluded that cytochrome c release can take place in the absence of permeability transition, matrix swelling, or outer membrane rupture. It is concluded that permeabilization requires only the interaction of Bcl-2 family proteins such as Bax and Bid with the outer membrane. Other mitochondrial proteins, including VDAC, are not required for protein efflux. However, in principle other proteins could modulate the function or membrane localization of Bax, for example by altering lipid microdomains in the outer membrane or by modifying Bax postsynthetically (Kuwana, 2002).

Mitochondrial fusion and division play important roles in the regulation of apoptosis. Mitochondrial fusion proteins attenuate apoptosis by inhibiting release of cytochrome c from mitochondria, in part by controlling cristae structures. Mitochondrial division promotes apoptosis by an unknown mechanism. This study addressed how division proteins regulate apoptosis, using inhibitors of mitochondrial division identified in a chemical screen. The most efficacious inhibitor, mdivi-1 (for mitochondrial division inhibitor) attenuates mitochondrial division in yeast and mammalian cells by selectively inhibiting the mitochondrial division dynamin. In cells, mdivi-1 retards apoptosis by inhibiting mitochondrial outer membrane permeabilization. In vitro, mdivi-1 potently blocks Bid-activated Bax/Bak-dependent cytochrome c release from mitochondria. These data indicate the mitochondrial division dynamin directly regulates mitochondrial outer membrane permeabilization independent of Drp1-mediated division. These findings raise the interesting possibility that mdivi-1 represents a class of therapeutics for stroke, myocardial infarction, and neurodegenerative diseases (Cassidy-Stone, 2008).

Signaling downstream of Bcl-2 family members

The anti-apoptosis protein Bcl-2 potently inhibits p53-dependent transcriptional activation of various p53-responsive promoters in reporter gene co-transfection assays in human embryonic kidney 293 and MCF7 cells, without affecting nuclear accumulation of p53 protein. In contrast, Bcl-2(Deltatransmembrane (TM)), which lacks a hydrophobic membrane-anchoring domain, has no effect on p53 activity. Similarly, in MCF7 cells stably expressing either Bcl-2 or Bcl-2(DeltaTM), nuclear levels of p53 protein are up-regulated upon treatment with the DNA-damaging agents doxorubicin and UV radiation, whereas p53-responsive promoter activity and expression of p21(CIP1/WAF1) are strongly reduced in MCF7-Bcl-2 cells but not in MCF7-Bcl-2(DeltaTM) or control MCF7 cells. The issue of membrane anchoring was further explored by testing the effects of Bcl-2 chimeric proteins that contained heterologous transmembrane domains from the mitochondrial protein ActA or the endoplasmic reticulum protein cytochrome b5. Both Bcl-2(ActA) and Bcl-2(Cytob5) suppresses p53-mediated transactivation of reporter gene plasmids with efficiencies comparable to wild-type Bcl-2. These results suggest that (a) Bcl-2 not only suppresses p53-mediated apoptosis but also interferes with the transcriptional activation of p53 target genes at least in some cell lines, and (b) membrane anchoring is required for this function of Bcl-2. It is speculated that membrane-anchored Bcl-2 may sequester an unknown factor necessary for p53 transcriptional activity (Froesch, 1999).

Cytolytic granule-mediated target cell killing is effected in part through the synergistic action of the membrane-acting protein perforin and serine proteases such as granzymes (Gr) A and B. In this study, the subcellular distribution of granzymes in the presence of perforin and the induction of apoptosis in mouse FDC-P1 myeloid and YAC-1 lymphoma cells that express the proto-oncogene bcl2 were examined. Using confocal laser scanning microscopy to visualize and quantitate subcellular transport of fluoresceinated granzyme, it was found that granzyme entry into the cytoplasm in the absence of perforin is not impaired in the bcl2-expressing lines. However, perforin-dependent enhancement of granzyme cellular uptake and, importantly, granzyme redistribution to the nucleus are strongly inhibited in the bcl2-expressing lines, concomitant with greatly increased resistance to granzyme/perforin-induced cell death. DNA fragmentation induced by granzyme/perforin is severely reduced in the bcl2-expressing lines, implying that prevention of granzyme nuclear translocation blocks the nuclear events of apoptosis. The kinetics of GrB nuclear uptake and induction of apoptosis are faster than for GrA, whereas YAC-1 cells show greater resistance to granzyme nuclear uptake and apoptosis than FDC-P1 cells. In all cases, granzyme nuclear accumulation in the presence of perforin correlates precisely with ensuing apoptosis. All results supported the idea that GrA and GrB share a common, specific nuclear targeting pathway that contributes significantly to the nuclear changes of apoptosis (Jans, 1999).

Apoptosis and autophagy are both tightly regulated biological processes that play a central role in tissue homeostasis, development, and disease. The anti-apoptotic protein, Bcl-2, interacts with the evolutionarily conserved autophagy protein, Beclin 1 (Coiled-coil myosin-like BCL2-interacting protein: Drosophila homolog - CG5429). However, little is known about the functional significance of this interaction. Wild-type Bcl-2 antiapoptotic proteins, but not Beclin 1 binding defective mutants of Bcl-2, inhibit Beclin 1-dependent autophagy in yeast and mammalian cells and cardiac Bcl-2 transgenic expression inhibits autophagy in mouse heart muscle. Furthermore, Beclin 1 mutants that cannot bind to Bcl-2 induce more autophagy than wild-type Beclin 1 and, unlike wild-type Beclin 1, promote cell death. Thus, Bcl-2 not only functions as an antiapoptotic protein, but also as an antiautophagy protein via its inhibitory interaction with Beclin 1. This antiautophagy function of Bcl-2 may help maintain autophagy at levels that are compatible with cell survival, rather than cell death (Pattingre, 2005).

Interaction between mutant superoxide dismutase and Bcl-2

Familial amyotrophic lateral sclerosis (ALS)-linked mutations in the copper-zinc superoxide dismutase (SOD1) gene cause motor neuron death in about 3% of ALS cases. While the wild-type (wt) protein is anti-apoptotic, mutant SOD1 promotes apoptosis. Both wt and mutant SOD1 bind the anti-apoptotic protein Bcl-2, providing evidence of a direct link between SOD1 and an apoptotic pathway. This interaction is evident in vitro and in vivo in mouse and human spinal cord. In mice and humans, Bcl-2 binds to high molecular weight SDS-resistant mutant SOD1 containing aggregates that are present in mitochondria from spinal cord but not liver. These findings provide new insights into the anti-apoptotic function of SOD1 and suggest that entrapment of Bcl-2 by large SOD1 aggregates may deplete motor neurons of this anti-apoptotic protein (Belford, 2004).

Bcl-2 family members as regulators of the cell death hierarchy: ced-9 in C. elegans

ced-9, a member of the bcl-2 gene family in Caenorhabditis elegans plays a central role in preventing cell death in worms. Overexpression of human bcl-2 can partially prevent cell death in C. elegans. However, it remains to be elucidated whether ced-9 can regulate cell death when expressed in other organisms. The CED-9 protein is co-localized with BCL-2 in COS cells and Drosophila Schneider's L2 (SL2) cells, suggesting that the site of CED-9 action is located to specific cytoplasmic compartments. Overexpression of ced-9 only poorly protects cells from the death induced by ced-3 in HeLa cells, but ced-9 significantly reduces the cell death induced by ced-3 in Drosophila SL2 cells. Apoptosis of SL2 cells that is induced by a Drosophila cell-death gene, reaper, is partially prevented by ced-9, bcl-2 and bcl-xL. These results suggest that the signaling pathway that is required for the anti-apoptotic function of bcl-2 family members, including ced-9, is conserved in Drosophila cells. SL2 cells provide a unique systems for dissecting the main machinery of cell death (Hisahara, 1998).

Gain-of-function mutations in the Caenorhabditis elegans gene egl-1 cause the HSN neurons to undergo programmed cell death. By contrast, a loss-of-function egl-1 mutation prevents most if not all somatic programmed cell deaths. The egl-1 gene negatively regulates the ced-9 gene, which protects against cell death and is a member of the bcl-2 family. Searches of current nucleotide and protein databases using various BLAST programs have identified no known sequences or proteins with significant similarity to the egl-1 gene or the EGL-1 protein. egl-1 therefore encodes a novel protein of 91 amino acids with no apparent hydrophobic stretches (indicative of transmembrane domains). The EGL-1 protein contains a nine amino acid region similar to the Bcl-2 homology region 3 (BH3) domain but does not contain a BH1, BH2, or BH4 domain, suggesting that EGL-1 may be a member of a family of cell death activators that includes the mammalian proteins Bik, Bid, Harakiri, and Bad. The EGL-1 and CED-9 proteins interact physically. This paper proposes that EGL-1 activates programmed cell death by binding to and directly inhibiting the activity of CED-9, perhaps by releasing the cell death activator CED-4 from a CED-9/CED-4-containing protein complex (Conradt, 1998).

The Caenorhabditis elegans gene ced-9 prevents cells from undergoing programmed cell death and encodes a protein similar to the mammalian cell-death inhibitor Bcl-2. The CED-9 protein is a substrate for the C. elegans cell-death protease CED-3, which is a member of a family of cysteine proteases first defined by CED-3 and human interleukin-1beta converting enzyme (ICE). CED-9 can be cleaved by CED-3 at two sites near its amino terminus: the presence of at least one of these sites is important for complete protection by CED-9 against cell death. Cleavage of CED-9 by CED-3 generates a carboxy-terminal product that resembles Bcl-2 in sequence and in function. Bcl-2 and the baculovirus protein p35 (which inhibits cell death in different species through a mechanism that depends on the presence of its cleavage site for the CED-3/ICE family of proteases) inhibit cell death additively in C. elegans. These results indicate that CED-9 prevents programmed cell death in C. elegans through two distinct mechanisms: (1) CED-9 may, by analogy with p35, directly inhibit the CED-3 protease by an interaction involving the CED-3 cleavage sites in CED-9 and (2) CED-9 may directly or indirectly inhibit CED-3 by means of a protective mechanism similar to that used by mammalian Bcl-2. The second mechanism involves physical interaction with CED-4 in C. elegans and a CED-4 homolog in mammals (Xue, 1997).

Examining the effects of overexpressing cell-death-related genes in specific C. elegans neurons that normally live, it was demonstrated that the cell-death genes ced-3, ced-4, and ced-9 all can act cell autonomously to control programmed cell death. Not only the protective activity of ced-9 but also the killer activities of ced-3 and ced-4 are likely to be present in cells that normally live. Killing by overexpression of ced-3 does not require endogenous ced-4 function, whereas killing by overexpression of ced-4 is at least in part dependent on endogenous ced-3 function. These results suggest either that ced-4 acts upstream of ced-3 and ced-4 function can be bypassed by high levels of ced-3 activity or that ced-3 and ced-4 act in parallel, with ced-3 perhaps having a greater ability to kill. The finding that ced-4 appears to facilitate the inhibition of ced-3 by ced-9 suggests that ced-9 acts to negatively regulate ced-4. It is proposed that both in C. elegans and in other organisms a competition between antagonistic protective and killer activities determines whether specific cells will live or die. These results suggest a genetic pathway for programmed cell death in C. elegans in which ced-4 acts upstream of or in parallel withced-3, and ced-9 negatively regulates the activity of ced-4 (Shaham, 1996a).

ced-4 encodes two transcripts; whereas the major transcript can cause programmed cell death, the minor transcript can act oppositely and prevent programmed cell death, thus defining a novel class of cell death inhibitors. That ced-4 has both cell-killing and cell-protective functions is consistent with previous genetic studies. The dual protective and killer functions of the C. elegans bcl-2-like gene ced-9 are mediated by inhibition of the killer and protective ced-4 functions, respectively. It is proposed that a balance between opposing ced-4 functions influences the decision of a cell to live or to die by programmed cell death and that both ced-9 and ced-4 protective functions are required to prevent programmed cell death (Shaham, 1996b).

Three principal genes are involved in developmental programmed cell death in C. elegans: ced-3 and ced-4 genes are both required for PCD, whereas ced-9 acts to prevent the death-promoting actions of these genes. ced-9 is homologous to the Bcl-2 family, whose role in protecting PCD is illusive; no vertebrate homolog of ced-4 is known. This paper describes the effect of expression of C. elegans ced-4 in yeast. Induction of wild type ced-4 results in rapid focal chromatin condensation and lethality. Mutation of a putative nucleotide binding P-loop motif of CED-4 eliminates the lethal phenotype. Immunolocalization of CED-4 to the condensed chromatin suggests that the phenotype may result from an intrinsic ability of CED-4 to interact with chromatin. Co-expression of ced-9 prevents CED-4-induced chromatin condensation and lethality, and causes the relocalization of CED-4 to endoplasmic reticulum and outer mitochondrial membranes. A direct interaction between CED-4 and CED-9 was confirmed by yeast two-hybrid analysis. It is concluded that CED-4 has a direct role in chromatin condensation. Chromatin condensation is a ubiquitous feature of metazoan apoptosis that has yet to be linked to an effector. Further studies are required to establish whether the CED-9/CED-4 interaction is required for the activation of CED-3, the Caspase cysteine protease (James, 1997).

Genetic studies suggest that ced-9 controls programmed cell death by regulating ced-4 and ced-3. However, the mechanism by which CED-9 controls the activities of CED-4 and the cysteine protease CED-3, the effector arm of the cell-death pathway, remains poorly understood. Immunoprecipitation analysis demonstrates that in vivo CED-9 forms a multimeric protein complex with CED-4 and CED-3. Expression of wild-type CED-4 promotes the ability of CED-3 in mammalian cells to induce apoptosis otherwise inhibited by CED-9. The pro-apoptotic activity of CED-4 requires the expression of a functional CED-3 protease. Significantly, loss-of-function CED-4 mutants are impaired in their ability to promote CED-3-mediated apoptosis. Expression of CED-4 enhances the proteolytic activation of CED-3. CED-9 inhibits the formation of p13 and p15, two cleavage products of CED-3 associated with its proteolytic activation in vivo. Moreover, CED-9 inhibits the enzymatic activity of CED-3 promoted by CED-4. Thus, these results provide evidence that CED-4 and CED-9 regulate the activity of CED-3 through physical interactions, which may provide a molecular basis for the control of programmed cell death in C. elegans (Wu, 1997).

ced-9 is an element of a polycistronic locus that also contains the gene cyt-1, which encodes a protein similar to cytochrome b560 of complex II of the mitochondrial respiratory chain. ced-9 encodes a 280 amino acid protein showing sequence and structural similarities to the mammalian proto-oncogene bcl-2. Overexpression of bcl-2 can mimic the protective effect of ced-9 on C. elegans cell death and can prevent the ectopic cell deaths that occur in ced-9 loss-of-function mutants. These results suggest that ced-9 and bcl-2 are homologs and that the molecular mechanism of programmed cell death has been conserved from nematodes to mammals (Hengartner, 1994).

Programmed cell death (PCD) is regulated by multiple evolutionarily conserved mechanisms to ensure the survival of the cell. This study describes pvl-5, a gene that likely regulates PCD in Caenorhabditis elegans. In wild-type hermaphrodites at the L2 stage there are 11 Pn.p hypodermal cells in the ventral midline arrayed along the anterior-posterior axis and 6 of these cells become the vulval precursor cells. In pvl-5(ga87) animals, there are fewer Pn.p cells (average of 7.0) present at this time. Lineage analysis reveals that the missing Pn.p cells die around the time of the L1 molt in a manner that often resembles the programmed cell deaths that occur normally in C. elegans development. This Pn.p cell death is suppressed by mutations in the caspase gene ced-3 and in the bcl-2 homolog ced-9, suggesting that the Pn.p cells are dying by PCD in pvl-5 mutants. Surprisingly, the Pn.p cell death is not suppressed by loss of ced-4 function. ced-4 (Apaf-1) is required for all previously known apoptotic cell deaths in C. elegans. This suggests that loss of pvl-5 function leads to the activation of a ced-3-dependent, ced-4-independent form of PCD and that pvl-5 may normally function to protect cells from inappropriate activation of the apoptotic pathway (Joshi, 2004).

Genetic analyses in C. elegans have been instrumental in the elucidation of the central cell-death machinery, which is conserved from C. elegans to mammals. One possible difference that has emerged is the role of mitochondria. By releasing cytochrome c, mitochondria are involved in the activation of caspases in mammals. However, there has previously been no evidence that mitochondria are involved in caspase activation in C. elegans. This study shows that mitochondria fragment in cells that normally undergo programmed cell death during C. elegans development. Mitochondrial fragmentation is induced by the BH3-only protein EGL-1 and can be blocked by mutations in the bcl-2-like gene ced-9, indicating that members of the Bcl-2 family might function in the regulation of mitochondrial fragmentation in apoptotic cells. Mitochondrial fragmentation is independent of CED-4/Apaf-1 and CED-3/caspase, indicating that it occurs before or simultaneously with their activation. Furthermore, DRP-1/dynamin-related protein, a key component of the mitochondrial fission machinery, is required and sufficient to induce mitochondrial fragmentation and programmed cell death during C. elegans development. These results assign an important role to mitochondria in the cell-death pathway in C. elegans (Jagasia, 2005).

Temporal control of programmed cell death is necessary to ensure that cells die at only the right time during animal development. How such temporal regulation is achieved remains poorly understood. In some Caenorhabditis elegans somatic cells, transcription of the egl-1/BH3-only gene promotes cell-specific death. The EGL-1 protein inhibits the CED-9/Bcl-2 protein, resulting in the release of the caspase activator CED-4/Apaf-1. Subsequent activation of the CED-3 caspase by CED-4 leads to cell death. Despite the important role of egl-1 transcription in promoting CED-3 activity in cells destined to die, it remains unclear whether the temporal control of cell death is mediated by egl-1 expression. This study show shows that egl-1 and ced-9 play only minor roles in the death of the C. elegans tail-spike cell, demonstrating that temporal control of tail-spike cell death can be achieved in the absence of egl-1. The timing of the onset of tail-spike cell death is controlled by transcriptional induction of the ced-3 caspase. The developmental expression pattern of ced-3 has been characterized; in the tail-spike cell, ced-3 expression is induced shortly before the cell dies, and this induction is sufficient to promote the demise of the cell. Both ced-3 expression and cell death are dependent on the transcription factor PAL-1, the C. elegans homolog of the mammalian tumor suppressor gene Cdx2. PAL-1 can bind to the ced-3 promoter sites that are crucial for tail-spike cell death, suggesting that it promotes cell death by directly activating ced-3 transcription. These results highlight a role that has not been described previously for the transcriptional regulation of caspases in controlling the timing of cell death onset during animal development (Maurer, 2007).

To obtain insight into the role of the retinoblastoma susceptibility gene (Rb; also known as Rb1) in apoptosis, Caenorhabditis elegans mutants lacking a functional lin-35 RB gene were analyzed. The loss of lin-35 function results in a decrease in constitutive germ cell apoptosis. Evidence is presented that lin-35 promotes germ cell apoptosis by repressing the expression of ced-9, an anti-apoptotic C. elegans gene that is orthologous to the human proto-oncogene BCL2. Furthermore, the genes dpl-1 DP, efl-1 E2F and efl-2 E2F were also shown to promote constitutive germ cell apoptosis. However, in contrast to lin-35, dpl-1 (and probably also efl-1 and efl-2) promotes germ cell apoptosis by inducing the expression of the pro-apoptotic genes ced-4 and ced-3, which encode an APAF1-like adaptor protein and a pro-caspase, respectively. Based on these results, it is proposed that C. elegans orthologs of components of the RB tumor suppressor complex have distinct pro-apoptotic functions in the germ line and that the transcriptional regulation of components of the central apoptosis machinery is a critical determinant of constitutive germ cell apoptosis in C. elegans. Finally, lin-35, dpl-1 and efl-2, but not efl-1, function either downstream of or in parallel to cep-1 p53 (also known as TP53) and egl-1 BH3-only were shown to cause DNA damage-induced germ cell apoptosis. These results have implications for the general mechanisms through which RB-like proteins control gene expression, the role of RB-, DP- and E2F-like proteins in apoptosis, and the regulation of apoptosis (Schertel, 2007).

The developmental control of apoptosis is fundamental and important. The Caenorhabditis elegans Bar homeodomain transcription factor CEH-30 is required for the sexually dimorphic survival of the male-specific CEM (cephalic male) sensory neurons; the homologous cells of hermaphrodites undergo programmed cell death. It is proposed that the cell-type-specific anti-apoptotic gene ceh-30 is transcriptionally repressed by the TRA-1 transcription factor, the terminal regulator of sexual identity in C. elegans, to cause hermaphrodite-specific CEM death. The established mechanism for the regulation of specific programmed cell deaths in C. elegans is the transcriptional control of the BH3-only gene egl-1, which inhibits the Bcl-2 homolog ced-9; similarly, most regulation of vertebrate apoptosis involves the Bcl-2 superfamily. In contrast, ceh-30 acts within the CEM neurons to promote their survival independently of both egl-1 and ced-9. Mammalian ceh-30 homologs can substitute for ceh-30 in C. elegans. Mice lacking the ceh-30 homolog Barhl1 show a progressive loss of sensory neurons and increased sensory-neuron cell death. Based on these observations, it is suggested that the function of Bar homeodomain proteins as cell-type-specific inhibitors of apoptosis is evolutionarily conserved (Schwartz, 2007).

Ceramide biogenesis, CED-4, CED-9 and radiation induced apoptosis in C. elegans

Ceramide engagement in apoptotic pathways has been a topic of controversy. To address this controversy, loss-of-function (lf) mutants of conserved genes of sphingolipid metabolism were tested in C. elegans. Although somatic (developmental) apoptosis was unaffected, ionizing radiation-induced apoptosis of germ cells was obliterated upon inactivation of ceramide synthase and restored upon microinjection of long-chain natural ceramide. Radiation-induced increase in the concentration of ceramide localized to mitochondria and was required for BH3-domain protein EGL-1-mediated displacement of CED-4 (an APAF-1-like protein) from the CED-9 (a Bcl-2 family member)/CED-4 complex, an obligate step in activation of the CED-3 caspase. These studies define CEP-1 (the worm homolog of the tumor suppressor p53)-mediated accumulation of EGL-1 and ceramide synthase-mediated generation of ceramide through parallel pathways that integrate at mitochondrial membranes to regulate stress-induced apoptosis (Deng, 2008).

Although studies that use genetic deficiency in ceramide production support it as essential for apoptosis in diverse models, many have questioned whether ceramide functions as a bona fide transducer of apoptotic signals. One reason for skepticism is that, despite delineation of a number of ceramide-activated proteins, no single protein has been identified as mediator of ceramide-induced apoptosis. Recent studies have suggested an alternate mode of ceramide action, based on its capacity to self-associate and locally rearrange membrane bilayers into ceramide-rich macrodomains (1 to 5 µm in diameter), which are sites of protein concentration and oligomerization. Ceramide may thus mediate apoptosis through its ability to reconfigure membranes, coordinating protein complexation at critical junctures of signaling cascades (Deng, 2008).

To establish the role of ceramide definitively, a model of radiation-induced apoptosis was used in C. elegans germ cells. Germline stem cells, located at the distal gonad tip, divide incessantly throughout adult life, with daughter cells arresting in meiotic prophase. Upon exiting prophase, germ cells become sensitive to radiation-induced apoptosis, detected morphologically just proximal to the bend of the gonadal arm. This apoptotic pathway is antagonized by the ABL-1 tyrosine kinase, requiring sequentially the cell cycle checkpoint genes rad-5, hus-1, and mrt-2; the C. elegans p53 homolog cep-1; and the genes making up the conserved apoptotic machinery, the caspase ced-3, the apoptotic protease activating factor 1-like protein ced-4, the Bcl-2 protein ced-9, and the BH3-domain protein egl-1. This pathway differs from apoptotic somatic cell death, which is not subject to upstream checkpoint regulation via the CEP-1 pathway (Deng, 2008).

This study has identified conserved genes that regulate C. elegans sphingolipid intermediary metabolism and has tested deletion alleles. Screening for mutants resistant to radiation-induced germ cell apoptosis revealed apoptosis suppression in only deletion mutants of hyl-1 and lagr-1, two of the three ceramide synthase (CS) genes. CS gene products regulate de novo ceramide biosynthesis, acylating sphinganine to form dihydroceramide that is subsequently converted to ceramide by a desaturase. CSs contain six to seven putative transmembrane domains and a Lag1p motif [which confers enzyme activity, regions conserved in the C. elegans orthologs. The deleted CS sequences in hyl-1(ok976) and lagr-1(gk327) result in frameshifts that disrupt the Lag1p motifs. An ~1.6-kb hyl-1 transcript was detected in wild-type (WT) worms and a smaller ~1.35-kb transcript in hyl-1(ok976), whereas an ~1.4-kb lagr-1 transcript was detected in WT worms and a ~1.25-kb transcript in lagr-1(gk327). In contrast, a deletion mutant of the third C. elegans CS, hyl-2(ok1766), lacking a 1626-base pair fragment of the hyl-2 gene locus that eliminates exons 2 to 5 corresponding to 74% of the coding sequence, displayed no defect in germ cell death (Deng, 2008).

In N2 WTstrain young adults, apoptotic germ cells gradually increased in abundance with age from a baseline of 0.7 ± 0.1 to 1.8 ± 0.2 corpses per distal gonad arm over 48 hours. Exposure to a 120-gray (Gy) ionizing radiation dose increased germ cell apoptosis to 5.2 ± 0.3 cells 36 to 48 hours after treatment. In contrast, in hyl-1 (ok976) and lagr-1(gk327) animals, age-dependent and radiation-induced germ cell apoptosis were nearly abolished. Similar effects were observed in the lagr-1(gk327);hyl-1(ok976) double mutant. The rate of germ cell corpse removal was unaffected in CS mutants, excluding the possibility that defective corpse engulfment elevated corpse numbers. In contrast, loss-of-function (lf) mutations of hyl-1 or lagr-1 did not affect developmental somatic cell death, nor did the lf hyl-2(ok1766) mutation. These studies indicate a requirement for two C. elegans CS genes for radiation-induced germline apoptosis (Deng, 2008).

To confirm ceramide as critical for germline apoptosis, C16-ceramide was injected into gonads of young adult WT worms. C16-ceramide is the predominant ceramide species in apoptosis induction by diverse stresses in multiple organisms. C16-ceramide microinjection resulted in time- and dose-dependent increases in germ cell apoptosis, with a median effective dose of ~0.05 µM gonadal ceramide. Peak effect occurred at ~0.1 µM gonadal ceramide at 36 hours, qualitatively and quantitatively mimicking the 120-Gy effect in WT worms. In contrast, C16-dihydroceramide, which differs from C16-ceramide in a trans double bond at sphingoid base position four to five, was without effect, indicating specificity for ceramide in apoptosis induction. Furthermore, C16-ceramide microinjection into lagr-1(gk327);hyl-1(ok976) animals (~1 µM gonadal ceramide) resulted in a 5.7-fold increase in germ cell apoptosis. Note that the baseline level of apoptosis in lagr-1(gk327);hyl-1(ok976) was less than one-half that in WT worms. Moreover, ~0.005 µM gonadal ceramide, a concentration without impact on germ cell apoptosis, completely restored radiation (120 Gy)-induced apoptosis, an effect inhibitable in a lf ced-3 background. C16-ceramide's ability to bypass the genetic defect and restore the radiation-response phenotype is strong evidence that hyl-1 and lagr-1 represent legitimate C. elegans CS genes. Animals with sphk-1(ok1097), a null allele of sphingosine kinase (SPHK), which prevents conversion of ceramide to its anti-apoptotic derivative sphingosine 1-phosphate (S1P), displayed high baseline germ cell death and were hypersensitive to radiation-induced germ cell apoptosis, inhibitable (by 85 ± 9%) in a lagr-1(gk327);sphk-1(ok1097) double mutant. Collectively, these studies identify ceramide as a critical effector of radiation-induced germ cell apoptosis, although they do not define its mode of engaging the apoptotic pathway (Deng, 2008).

Inactivation of the C. elegans ABL-1 ortholog in the lf mutant abl-1(ok171) (or by RNA interference) increases baseline and post-radiation germ cell apoptosis, modeling radiation hypersensitivity phenotypes. To order CS action relative to ABL-1, hyl-1(ok976);abl-1(ok171) and lagr-1(gk327);abl-1(ok171) and a triple mutant lagr-1(gk327);hyl-1(ok976);abl-1(ok171) were generated. lf hyl-1 or lagr-1 in an abl-1(ok171) genetic background prevented the time-dependent increase in physiologic germ cell apoptosis and completely blocked radiation-induced apoptosis. Similarly, lagr-1(gk327);hyl-1(ok976);abl-1(ok171) displayed inhibition of baseline and radiation-induced germ cell apoptosis. Thus, increased germ cell apoptosis in irradiated abl-1(ok171) depends on the CS genes hyl-1 and lagr-1 (Deng, 2008).

In C. elegans, DNA damage activates the p53 homolog CEP-1, which is required for transcriptional up-regulation of the BH3-only proteins, EGL-1 and CED-13, that in turn activate the core apoptotic machinery (CED-9, CED-4, and CED-3). Exposure of hyl-1(ok976) and lagr-1 (gk327) to 120 Gy increased egl-1 transcripts four- to fivefold at 9 hours after irradiation, whereas ced-13 expression was enhanced five- to sixfold -- levels comparable to those detected in irradiated WTworms. Thus, the loss of CS did not affect CEP-1 activation upon irradiation, suggesting that ceramide and CEP-1 might function in parallel, coordinately conferring radiation-induced germ cell death (Deng, 2008).

It was reasoned that in contrast to radiation-induced germ cell apoptosis, which apparently requires increased abundance of both BH3-only proteins and ceramide, C16-ceramide provided exogenously might act independent of p53-mediated egl-1 expression by maximizing the effect of baseline EGL-1. In fact, microinjected C16-ceramide partially restored germ cell death in cep-1(gk138) from 0.4 ± 0.13 to 2.5 ± 0.32 corpses per distal gonad arm. Since C16-ceramide is inactive in the lf egl-1 mutant egl-1(n1084n3082), it appears that there is a requirement for at least a baseline level of BH3-only proteins for ceramide-induced apoptosis. Consistent with this notion, C16-ceramide administration did not increase egl-1 and ced-13 transcription. Furthermore, inactivating the core apoptotic machinery in lf ced-3(n717) and ced-4(n1162) or in gain-of-function ced-9(n1950) animals, which abolish radiation-induced germline apoptosis, similarly abolished C16-ceramide-induced death. Collectively, these data indicate that ceramide acts in conjunction with BH3-only proteins upstream of the mitochondrial commitment step of apoptosis in the C. elegans germ line (Deng, 2008).

Since these studies point to a mitochondrial site of ceramide action, an immune histochemical approach was devised to evaluate whether ceramide might increase in the mitochondria of C. elegans germ cells. Advantage was taken of the increased frequency of germ cell apoptosis in abl-1(ok171), anticipating a maximized ceramide signal upon irradiation in this strain. Gonads from unirradiated or irradiated worms were dissected, opened by freeze-cracking, and then stained with MID15B4, a specific anti-ceramide antibody. Mitochondria were localized with an antibody to the mitochondrial marker protein OxPhos Complex IV subunit I (COX-IV) or by Rhodamine B staining. COX-IV staining (green) before and after irradiation displayed a prominent perinuclear distribution reminiscent of mitochondrial topography in some mammalian cell systems. Ceramide staining (red) displayed a similar profile and at baseline was faint, increasing 2.4-fold at 24 hours post-irradiation. Merging the two signals (red and green) revealed that ceramide accumulation was distinctively mitochondrial (yellow). Radiation-induced ceramide accumulation was abrogated in lagr1(gk327);hyl-1(ok976);abl-1(ok171) animals. Similarly, ceramide increase was abrogated in irradiated lagr-1(gk327);hyl-1(ok976) as compared with WT animals. These results define ionizing radiation-induced ceramide accumulation in the C. elegans germ line as mitochondrial in origin, mediated via the classic ceramide biosynthetic pathway (Deng, 2008).

Whether mitochondrial ceramide accumulation was required for CED-4 redistribution to nuclear membranes was examined. In nonapoptotic somatic cells, CED-4 is sequestered to mitochondria by binding CED-9. When displaced by EGL-1, CED-4 targets nuclear membranes and activates caspase CED-3, necessary for the effector phase of apoptosis. For these studies, abl-1(ok171) and lagr-1(gk327);hyl-1(ok976);abl-1(ok171) animals were exposed to 120 Gy, and germ cells were released from gonads and stained with antibodies against C. elegans CED-4 and Ce-lamin, a nuclear membrane marker. CED-4 and Ce-lamin colocalization by confocal microscopy (yellow merged signal) served as readout for nuclear CED-4 redistribution. After irradiation nuclear CED-4 staining intensity increased 4.3-fold in abl-1(ok171). Consistent with reduced germ cell apoptosis, nuclear CED-4 staining is significantly reduced in lagr-1(gk327);hyl-1(ok976);abl-1(ok171). Specifically, baseline CED-4 intensity at the nuclear membrane is lower in lagr-1(gk327);hyl-1(ok976);abl-1(ok171) than in abl-1(ok171), increasing post-irradiation only to the control level of unirradiated abl-1(ok171) worms, an effect probably of biologic relevance as the biophysical effects of ceramide on membrane structure are concentration-dependent (Deng, 2008).

This study also used opls219 worms, a strain expressing a CED-4::GFP fusion protein (where GFP is green fluorescent protein), which permits in vivo detection of CED-4 trafficking. opls219 worms were cultured on plates containing Rhodamine B to stain mitochondria (red). Merged images detect mitochondrial CED-4 as a yellow signal (red and green overlay), whereas nonmitochondrial CED-4 appears green. Although a low-intensity green CED-4 signal was detected in nuclear membranes of unirradiated germ cells, the large majority of CED-4 was present in mitochondria before irradiation. At 36 hours postradiation, the CED-4 signal was markedly reduced in mitochondria, relocalizing primarily to nuclear membranes as bright green platformlike structures. In eight worms, overall reduction in CED-4 mitochondrial colocalization upon irradiation was ~50%, abrogated in lagr-1(gk327);opls219. Consistent with the anti-CED-4 antibody staining, the loss of mitochondrial CED-4 signal in opls219 was accompanied by a twofold increase in nuclear CED-4 signal, blocked entirely in lagr-1(gk327);opls219. These results indicate that mitochondrial ceramide contributes substantively to CED-4 displacement from mitochondrial membranes during radiation-induced germ cell apoptosis (Deng, 2008).

These data indicate that the ceramide synthetic pathway is required for radiation-induced apoptosis of C. elegans germ cells. The most parsimonious molecular ordering suggests that CS (as well as its enzymatic product ceramide) functions on a pathway that is parallel to the CEP-1/p53-EGL-1 system. The coordinated function of these two pathways occurs at the mitochondrial commitment step of the apoptotic process. It is hypothesized that ceramide may recompartmentalize the mitochondrial outer membrane, yielding a permissive microenvironment for EGL-1-mediated displacement of CED-4, the trigger for the effector stage of the apoptotic process (Deng, 2008).

Bcl-2 and inflamation

Caspases are intracellular proteases that cleave substrates involved in apoptosis or inflammation. In C. elegans, a paradigm for caspase regulation exists in which caspase CED-3 is activated by nucleotide-binding protein CED-4, which is suppressed by Bcl-2-family protein CED-9. A mammalian analog of this caspase-regulatory system has been identified in the NLR-family protein NALP1, a nucleotide-dependent activator of cytokine-processing protease caspase-1, which responds to bacterial ligand muramyl-dipeptide (MDP). Antiapoptotic proteins Bcl-2 and Bcl-XL bind and suppress NALP1, reducing caspase-1 activation and interleukin-1β (IL-1β) production. When exposed to MDP, Bcl-2-deficient macrophages exhibit more caspase-1 processing and IL-1β production, whereas Bcl-2-overexpressing macrophages demonstrate less caspase-1 processing and IL-1β production. The findings reveal an interaction of host defense and apoptosis machinery (Bruey, 2007).

Bcl-2, development, cell growth, and entry into the cell cycle

Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, expression of the human bcl-2 gene was targeted in mice by using the promoter of the vav gene, which is active throughout this compartment but rarely outside it. The vav-bcl-2 transgene is expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice display increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen is enlarged and the lymphoid follicles expanded. Although total thymic cellularity is normal, T cell development is altered: cells at the very immature and most mature stages are increased, whereas those at the intermediate stage are decreased. Unexpectedly, blood platelets are reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remain cytokine dependent, and the frequency of most progenitor and preprogenitor cells is normal. Macrophage progenitors are less frequent and yield smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhances clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment (Ogilvy, 1999).

Proteins of the Bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult. The recently isolated bcl-w gene encodes a pro-survival member of the Bcl-2 family, which is widely expressed. To explore its physiological role, the bcl-w gene in the mouse was inactivated by homologous recombination. Mice that lack Bcl-w are viable, healthy, and normal in appearance. Most tissues exhibit typical histology, and hematopoiesis is unaffected, presumably due to redundant function with other pro-survival family members. Although female reproductive function is normal, the males are infertile. The testes developed normally, and the initial, prepubertal wave of spermatogenesis is largely unaffected. The seminiferous tubules of adult males, however, are disorganized, contained numerous apoptotic cells, and produce no mature sperm. Both Sertoli cells and germ cells of all types are reduced in number, the most mature germ cells being the most severely depleted. The bcl-w-/- mouse provides a unique model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility (Print, 1998).

Bcl-x is a member of the Bcl2 family and has been suggested to be important for the survival and maturation of various cell types including the erythroid lineage. To define the consequences of Bcl-x loss in erythroid cells and other adult tissues, mice conditionally deficient in the Bcl-x gene were generated using the Cre-loxP recombination system. The temporal and spatial excision of the floxed Bcl-x locus was achieved by expressing the Cre recombinase gene under control of the MMTV-LTR. By the age of five weeks, Bcl-x conditional mutant mice exhibit hyperproliferation of megakaryocytes and a decline in the number of circulating platelets. Three-month-old animals suffer from severe hemolytic anemia, hyperplasia of immature erythroid cells and profound enlargement of the spleen. Bcl-x is only required for the survival of erythroid cells at the end of maturation, which includes enucleated reticulocytes in circulation. The extensive proliferation of immature erythroid cells in the spleen and bone marrow might be the result of a fast turnover of late red blood cell precursors and accelerated erythropoiesis in response to tissue hypoxia. The increase in cell death of late erythroid cells is independent from the proapoptotic factor Bax, as demonstrated in conditional double mutant mice for Bcl-x and Bax. Mice conditionally deficient in Bcl-x permitted a study of the effects of Bcl-x deficiency on cell proliferation, maturation and survival under physiological conditions in an adult animal (Wagner, 2000).

It is suggested that the function of Bcl-x as a cell survival factor might not only be restricted to nucleated cells where classical markers of apoptotic cell death can be analyzed. Increased numbers of reticulocytes in the Bcl-x mutants indicate that the bone marrow and spleen are responding to the anemic situation with accelerated erythropoiesis and increased release of enucleated erythrocytes. However, Bcl-x-deficient mice still suffer from severe anemia, which could suggest that these maturing erythrocytes have a shorter half-life and hemolyze prematurely. Similarly, erythroid hyperplasia is more pronounced in individuals with hemolytic anemia than non-hemolytic anemia. The expansion of Bcl-x function as a survival factor for cells without a nucleus would demand a new definition for apoptosis, which is generally believed to be a phenomenon for nucleated cells. The hypothesis that Bcl-x is important for the survival of reticulocytes is supported by earlier findings on in vitro differentiated mouse and human erythroid cells. The translation of the Bcl-x protein is sharply increased at the time of maximal hemoglobin synthesis and remains to accumulate when the majority of erythroblasts have undergone enucleation to form reticulocytes (Wagner, 2000).

It is known from recent studies that Bcl-x regulates cell survival by at least two distinct mechanisms: heterodimerization with other Bcl2 family members and sustained ion-channel formation. The configuration of ion channels might be the more potent function in erythroid cells since the Bcl-x/Bax counteractive mechanism does not appear to regulate cell survival. These ion channels might control processes such as mitochondrial ATP/ADP exchange or cytochrome C release. It can therefore be assumed, that Bcl-x is important until the reticulocyte stage when mitochondria are still present. Mitochondria are progressively eliminated from mature erythrocytes as they meet their energy needs by anaerobic glycolysis instead of the Krebs cycle. If Bcl-x function is largely restricted to mitochondria in RBCs it can be predicted that its role as a survival factor, pore-channel-forming unit or countermeasure against free radicals has to diminish at the very end of erythrocyte maturation (Wagner, 2000).

Proapoptotic Bcl-2 family members have been proposed to play a central role in regulating apoptosis. However, mice lacking bax display limited phenotypic abnormalities. bak-/- mice are developmentally normal and reproductively fit and fail to develop any age-related disorders. However, when Bak-deficient mice are mated to Bax-deficient mice to create mice lacking both genes, the majority of bax-/-bak-/- animals die perinatally with fewer than 10% surviving into adulthood. bax-/-bak-/- mice display multiple developmental defects, including persistence of interdigital webs, an imperforate vaginal canal, and accumulation of excess cells within both the central nervous and hematopoietic systems. Thus, Bax and Bak have overlapping roles in the regulation of apoptosis during mammalian development and tissue homeostasis (Lindsten, 2000).

A study has been carried out of roles of two anti-apoptotic members of the Bcl2 family, Bcl-w and Bcl-xL, in regulating the survival of sensory neurons during development. Microinjection was used to introduce expression plasmids containing Bcl-w and Bcl-xL cDNAs in the sense and antisense orientations into the nuclei of BDNF-dependent nodose neurons and NGF-dependent trigeminal neurons at stages during and after the period of naturally occurring neuronal death. While overexpression of either protein promotes neuronal survival in the absence of neurotrophins and microinjection of antisense constructs reduce neuronal survival in the presence of neurotrophins, the magnitude of these effects changes with age. Whereas Bcl-w overexpression becomes more effective in promoting neuronal survival with age, Bcl-xL overexpression becomes less effective, and whereas antisense Bcl-w becomes much more effective in killing neurotrophin-supplemented neurons with age, antisense Bcl-xL becomes much less effective in killing these neurons. There is a marked increased in Bcl-w mRNA and Bcl-w immunoreactive neurons and a decrease in Bcl-xL mRNA and Bcl-xL immunoreactive neurons in the trigeminal and nodose ganglia over this period of development. These results demonstrate that both Bcl-w and Bcl-xL play an important anti-apoptotic role in regulating the survival of NGF- and BDNF-dependent neurons, and that reciprocal changes occur in the relative importance of these proteins with age. Whereas Bcl-xL plays a more important role during the period of naturally occurring neuronal death, Bcl-w plays a more important role at later stages (Middleton, 2001).

Male mice deficient in BCLW, a death-protecting member of the BCL2 family, are sterile due to an arrest in spermatogenesis that is associated with a gradual loss of germ cells and Sertoli cells from the testis. Since Bclw is expressed in both Sertoli cells and diploid male germ cells, it has been unclear which of these cell types requires BCLW in a cell-autonomous manner for survival. To determine whether death of Sertoli cells in Bclw mutants is influenced by the protracted loss of germ cells, testes from Bclw/c-kit double mutant mice, which lack germ cells from birth, were examined. Loss of BCLW-deficient Sertoli cells occurs in the absence of germ cells, indicating that germ cell death is not required to mediate loss of Sertoli cells in BCLW-deficient mice. This suggests that Sertoli cells require BCLW in a cell-intrinsic manner for long-term survival. The loss of Sertoli cells in Bclw mutants commences shortly after Sertoli cells have become postmitotic. In situ hybridization analysis indicates that Bclw is expressed in Sertoli cells both before and after exit from mitosis. Therefore, Bclw-independent pathways promote the survival of undifferentiated, mitotic Sertoli cells. BAX and BAK, two closely related death-promoting members of the BCL2 family, are expressed in Sertoli cells. To determine whether either BAX or BAK activity is required for Sertoli cell death in Bclw mutant animals, survival of Sertoli cells was analyzed in Bclw/Bax and Bclw/Bak double homozygous mutant mice. While mutation of Bak has no effect, ablation of Bax suppresses the loss of Sertoli cells in Bclw mutants. Thus, BCLW mediates survival of postmitotic Sertoli cells in the mouse by suppressing the death-promoting activity of BAX (Ross, 2001).

In the mouse embryo, significant numbers of primordial germ cells (PGCs) fail to migrate correctly to the genital ridges early in organogenesis. These usually die in ectopic locations. In humans, 50% of pediatric germ line tumors arise outside the gonads, and these are thought to arise from PGCs that fail to die in ectopic locations. The pro-apoptotic gene Bax, previously shown to be required for germ cell death during later stages of their differentiation in the gonads, is also expressed during germ cell migration, and is required for the normal death of germ cells left in ectopic locations during and after germ cell migration. In addition, Bax is shown to be downstream of the known cell survival signaling interaction mediated by the Steel factor/Kit ligand/receptor interaction. Together, these observations identify the major mechanism that removes ectopic germ cells from the embryo at early stages (Stallock, 2003).

A significantly increased number of ectopic germ cells is present in Bax-/- embryos. The ectopic germ cells are developmentally delayed. They retain expression of early PGC markers and retain motility. This shows that signals from the gonad regulate expression of PGC markers and inhibit their motility. Bax-/- ectopic germ cells occupy many positions in the embryo. However, they do not grow, and their numbers dwindle until by E18.5 very few can be found. Since these mice have not been reported to have an increased incidence of germline tumors, the data suggest that mice have a back-up mechanism for removing embryonic migratory germ cells in ectopic locations. Inactivation of Bax protects germ cells against rapid cell death in culture, and against removal of the Steel/Kit signaling interaction in culture. This shows that Bax is downstream of the Kit receptor. However, protection against cell death in culture is a short-term effect, showing that other apoptotic pathways exist in germ cells (Stallock, 2003).

During inner ear development, programmed cell death occurs in specific areas of the otic epithelium but the significance of this death and the molecules involved have remained unclear. An analysis was undertaken of mouse mutants in which genes encoding apoptosis-associated molecules have been inactivated. Disruption of the Apaf1 gene leads to a dramatic decrease in apoptosis in the inner ear epithelium, severe morphogenetic defects and a significant size reduction of the membranous labyrinth, demonstrating that an Apaf1-dependent apoptotic pathway is necessary for normal inner ear development. This pathway most probably operates through the apoptosome complex because caspase 9 mutant mice suffer similar defects. Inactivation of the Bcl2-like (Bcl2l) gene leads to an overall increase in the number of cells undergoing apoptosis but does not cause any major morphogenetic defects. In contrast, decreased apoptosis is observed in specific locations that suffer from developmental deficits, indicating that proapoptotic isoform(s) produced from Bcl2l might have roles in inner ear development. In Apaf1-/-/Bcl2l-/- double mutant embryos, no cell death could be detected in the otic epithelium, demonstrating that the cell death regulated by the anti-apoptotic Bcl2l isoform (Bcl-XL) in the otic epithelium is Apaf1-dependent. Furthermore, the otic vesicle fails to close completely in all double mutant embryos analyzed. These results indicate important roles for both Apaf1 and Bcl2l in inner ear development (Cecconi, 2004).

Bcl-2 is required for appropriate development of retinal vasculature as well as its neovascularization during oxygen-induced ischemic retinopathy

Bcl-2 is a death repressor that protects cells from apoptosis mediated by a variety of stimuli. Bcl-2 expression is regulated by both pro- and anti-angiogenic factors; thus, it may play a central role during angiogenesis. However, the role of bcl-2 in vascular development and growth of new vessels requires further delineation. In this study, the physiological role of bcl-2 was investigated in development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR). Mice deficient in bcl-2 exhibit a significant decrease in retinal vascular density compared to wild-type mice. This was attributed to a decreased number of endothelial cells and pericytes in retinas from bcl-2-/- mice. In bcl-2-/- mice, delayed development of retinal vasculature and remodeling was observed, and a significant decrease in the number of major arteries, which branch off from near the optic nerve. Interestingly, hyaloid vessel regression, an apoptosis-dependent process, was not affected in the absence of bcl-2. The retinal vasculature of bcl-2-/- mice exhibits a similar sensitivity to hyperoxia-mediated vessel obliteration compared to wild-type mice during OIR. However, the degree of ischemia-induced retinal neovascularization is significantly reduced in bcl-2-/- mice. These results suggest that expression of bcl-2 is required for appropriate development of retinal vasculature as well as its neovascularization during OIR (Wang, 2005).

Bcl-2 and Lymphomagenesis: MYC activation and BCL2L11 silencing by a tumour virus through the large-scale reconfiguration of enhancer-promoter hubs

Lymphomagenesis in the presence of deregulated MYC (see Drosophila Myc) requires suppression of MYC-driven apoptosis, often through downregulation of the pro-apoptotic BCL2L11 gene (Bim; see Drosophila Death executioner Bcl-2). Transcription factors (EBNAs) encoded by the lymphoma-associated Epstein-Barr virus (EBV) activate MYC and silence BCL2L11. This study shows that the EBNA2 transactivator activates multiple MYC enhancers and reconfigures the MYC locus to increase upstream and decrease downstream enhancer-promoter interactions. EBNA2 recruits the BRG1 ATPase of the SWI/SNF remodeller (see Drosophila Brahma) to MYC enhancers, and BRG1 is required for enhancer-promoter interactions in EBV-infected cells. At BCL2L11, a haematopoietic enhancer hub was identified that is inactivated by the EBV repressors EBNA3A and EBNA3C through recruitment of the H3K27 methyltransferase EZH2 (see Drosophila Enhancer of zeste). Reversal of enhancer inactivation using an EZH2 inhibitor upregulates BCL2L11 and induces apoptosis. EBV therefore drives lymphomagenesis by hijacking long-range enhancer hubs and specific cellular co-factors. EBV-driven MYC enhancer activation may contribute to the genesis and localisation of MYC-Immunoglobulin translocation breakpoints in Burkitt's lymphoma (Wood, 2016).


Search PubMed for articles about Drosophila death executioner Bcl-2 homologue

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Biological Overview

date revised: 30 September 2016

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