Tumor necrosis factor-alpha induces Bax-Bak interaction and apoptosis, which is inhibited by adenovirus E1B 19K

Tumor necrosis factor (TNF)-alpha-mediated death signaling induces oligomerization of proapoptotic Bcl-2 family member Bax into a high molecular mass protein complex in mitochondrial membranes. Bax complex formation is associated with the release of cytochrome c, which propagates death signaling by acting as a cofactor for caspase-9 activation. The adenovirus Bcl-2 homologue E1B 19K blocks TNF-alpha-mediated apoptosis by preventing cytochrome c release, caspase-9 activation, and apoptosis of virus-infected cells. TNF-alpha induces E1B 19K-Bax interaction and inhibits Bax oligomerization. Oligomerized Bax may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K can also bind to proapoptotic Bak, but the functional significance is not known. TNF-alpha signaling induced Bak-Bax interaction and both Bak and Bax oligomerization. E1B 19K was constitutively in a complex with Bak, and blocked the Bak-Bax interaction and oligomerization of both. The TNF-alpha-mediated cytochrome c and Smac/DIABLO release from mitochondria was inhibited by E1B 19K expression in adenovirus-infected cells. Since either Bax or Bak is essential for death signaling by TNF-alpha, the interaction between E1B 19K and both Bak and Bax may be required to inhibit their cooperative or independent oligomerization to release proteins from mitochondria which promote caspase activation and cell death.     

TNF-alpha signals apoptosis through a bid-dependent conformational change in Bax that is inhibited by E1B 19K

The adenovirus E1B 19K gene product is an inhibitor of apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) during viral infection. We report that E1B 19K inhibited neither caspase-8 activation nor caspase-8-dependent Bid cleavage by TNF-alpha. Rather, TNF-alpha induced a tBid-dependent conformational change in Bax that allowed an interaction between E1B 19K and conformationally altered Bax, which caused inhibition of cytochrome c release and caspase-9 activation. E1B 19K expression interrupted caspase-3 processing, permitting cleavage to remove the p12 subunit but not the prodomain consistent with caspase-8 and not caspase-9 enzymatic activity. Thus, E1B 19K blocks TNF-alpha-mediated death signaling by inhibiting a specific form of Bax that interrupts caspase activation downstream of caspase-8 and upstream of caspase-9.     

Induction of apoptosis by human Nbk/Bik, a BH3-containing protein that interacts with E1B 19K.

The E1B 19-kilodalton protein (19K protein) is a potent apoptosis inhibitor and the adenovirus homolog of Bcl-2 (E. White, Genes Dev. 10:1-15, 1996). To obtain a better understanding of the biochemical mechanism by which the E1B 19K protein regulates apoptosis, proteins that interact with 19K have been identified; one of these is Bax (J. Han, P. Sabbatini, D. Perez, L. Rao, D. Mohda, and E. White, Genes Dev. 10:461-477, 1996), and another is Bak (S. N. Farrow, J. H. M. White, I. Martinou, T. Raven, K.-T. Pun, C. J. Grinham, J.-C. Martinou, and R. Brown, Nature (London) 374:731-733, 1995). Bax and Bak are Bcl-2 family members which contain Bcl-2 homology regions 1, 2, and 3 (BH1, BH2, and BH3), which interact with E1B 19K and Bcl-2 and promote apoptosis. Like Bax and Bak, Nbk was cloned from a yeast two-hybrid screen for proteins that interact with E1B 19K. Nbk contained BH3 but not BH1 or BH2. It also interacted with Bcl-2 but not with Bax. Both Bcl-2 and E1B 19K interacted with Nbk in vitro, and this interaction was highly specific. In vivo, the Nbk and E1B 19K proteins may colocalize with cytoplasmic and nuclear membranes. Nbk expression functionally antagonized 19K-mediated inhibition of apoptotic cell death and completely prevented transformation by E1A and E1B 19K. Nbk was sufficient for induction of apoptosis in the presence of mutant p53 and thus low levels of Bax, suggesting that Nbk functions independently of Bax to induce apoptosis. Nbk may therefore represent a novel death regulator which contains only a BH3 that interacts with and antagonizes apoptosis inhibitors such as the E1B 19K protein.     

Identification of a novel antiapoptotic protein, GAM-1, encoded by the CELO adenovirus.

We have developed a simple screening method to identify genes that mimic bcl-2 or adenovirus E1B 19K in enhancing cell survival after transfection and have used this method to identify such a gene in the avian adenovirus CELO. The gene encodes a novel 30-kDa nuclear protein, which we have named GAM-1, that functions comparably to Bcl-2 and adenovirus E1B 19K in blocking apoptosis. However, GAM-1 has no sequence homology to Bcl-2, E1B 19K, or any other known antiapoptotic proteins and thus defines a novel antiapoptotic function.     

Functional complementation of the adenovirus E1B 19-kilodalton protein with Bcl-2 in the inhibition of apoptosis in infected cells.

Expression of the adenovirus E1A oncogene induces apoptosis which impedes both the transformation of primary rodent cells and productive adenovirus infection of human cells. Coexpression of E1A with the E1B 19,000-molecular-weight protein (19K protein) or the Bcl-2 protein, both of which have antiapoptotic activity, is necessary for efficient transformation. Induction of apoptosis by E1A in rodent cells is mediated by the p53 tumor suppressor gene, and both the E1B 19K protein and the Bcl-2 protein can overcome this p53-dependent apoptosis. The functional similarity between Bcl-2 and the E1B 19K protein suggested that they may act by similar mechanisms and that Bcl-2 may complement the requirement for E1B 19K expression during productive infection. Infection of human HeLa cells with E1B 19K loss-of-function mutant adenovirus produces apoptosis characterized by enhanced cytopathic effects (cyt phenotype) and degradation of host cell chromosomal DNA and viral DNA (deg phenotype). Failure to inhibit apoptosis results in premature host cell death, which impairs virus yield. HeLa cells express extremely low levels of p53 because of expression of human papillomavirus E6 protein. Levels of p53 were substantially increased by E1A expression during adenovirus infection. Therefore, E1A may induce apoptosis by overriding the E6-induced degradation of p53 and promoting p53 accumulation. Stable Bcl-2 overexpression in HeLa cells infected with the E1B 19K- mutant adenovirus blocked the induction of the cyt and deg phenotypes. Expression of Bcl-2 in HeLa cells also conferred resistance to apoptosis mediated by tumor necrosis factor alpha and Fas antigen, which is also an established function of the E1B 19K protein. A comparison of the amino acid sequences of Bcl-2 family members and that of the E1B 19K protein indicated that there was limited amino acid sequence homology between the central conserved domains of E1B 19K and Bcl-2. This domain of the E1B 19K protein is important in transformation and regulation of apoptosis, as determined by mutational analysis. The limited sequence homology and functional equivalency provided further evidence that the Bcl-2 and E1B 19K proteins may possess related mechanisms of action and that the E1B 19K protein may be the adenovirus equivalent of the cellular Bcl-2 protein.     

Bak and Bax function to limit adenovirus replication through apoptosis induction

Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B 19K. The mechanism of apoptosis induction and the role that it plays in productive infection are not known. Unlike apoptosis mediated by death receptors, infection with proapoptotic E1B 19K mutant viruses did not induce cleavage of Bid but nonetheless induced changes in Bak and Bax conformation, Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B 19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax conformation. Infection with E1B 19K mutant viruses induced apoptosis in wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically increased E1A expression and virus replication. Thus, Bax- and Bak-mediated apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak function as an antiviral response at the cellular level.     

The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha.

The adenovirus E1A and E1B proteins are required for transformation of primary rodent cells. When expressed in the absence of the 19,000-dalton (19K) E1B protein, however, the E1A proteins are acutely cytotoxic and induce host cell chromosomal DNA fragmentation and cytolysis, analogous to cells undergoing programmed cell death (apoptosis). E1A alone can efficiently initiate the formation of foci which subsequently undergo abortive transformation whereby stimulation of cell growth is counteracted by continual cell death. Cell lines with an immortalized growth potential eventually arise with low frequency. Coexpression of the E1B 19K protein with E1A is sufficient to overcome abortive transformation to produce high-frequency transformation. Like E1A, the tumoricidal cytokine tumor necrosis factor alpha (TNF-alpha) evokes a programmed cell death response in many tumor cell lines by inducing DNA fragmentation and cytolysis. Expression of the E1B 19K protein by viral infection, by transient expression, or in transformed cells completely and specifically blocks this TNF-alpha-induced DNA fragmentation and cell death. Cosegregation of 19K protein transforming activity with protection from TNF-alpha-mediated cytolysis demonstrates that both activities are likely the consequence of the same function of the protein. Therefore, we propose that by suppressing an intrinsic cell death mechanism activated by TNF-alpha or E1A, the E1B 19K protein enhances the transforming activity of E1A and enables adenovirus to evade TNF-alpha-dependent immune surveillance.     

E1B 19K Inhibits Fas-mediated Apoptosis through FADD-dependent Sequestration of FLICE

E1B 19K, the adenovirus Bcl-2 homologue, is a potent inhibitor of apoptosis induced by various stimuli including Fas and tumor necrosis factor-α. Fas and TNFR-1 belong to a family of cytokine-activated receptors that share key components in their signaling pathways, Fas-associating protein with death domain (FADD) and FADD-like interleukin-1β–converting enzyme (FLICE), to induce an apoptotic response. We demonstrate here that E1B 19K and Bcl-x_L are able to inhibit apoptosis induced by FADD, but not FLICE. Surprisingly, apoptosis was abrogated by E1B 19K and Bcl-x_L when FADD and FLICE were coexpressed. Immunofluorescence studies demonstrated that FADD expression produced large insoluble death effector filaments that may represent oligomerized FADD. E1B 19K expression disrupted FADD filament formation causing FADD and FLICE to relocalize to membrane and cytoskeletal structures where E1B 19K is normally localized. E1B 19K, however, does not detectably bind to FADD, nor does it inhibit FADD and FLICE from being recruited to the death-inducing signaling complex (DISC) when Fas is stimulated. Thus, E1B 19K may inhibit Fas-mediated cell death downstream of FADD recruitment of FLICE but upstream of FLICE activation by disrupting FADD oligomerization and sequestering an essential component of the DISC.     

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

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

Oligomerization of membrane-bound Bcl-2 is involved in its pore formation induced by tBid

Both pro-apoptotic Bax and anti-apoptotic Bcl-2 are structurally homologous to the pore-forming domain of bacterial toxins. Bax proteins oligomerize in the mitochondrial outer membranes forming pores that release cytochrome c from the mitochondrial intermembrane space. Bcl-2 proteins also form pores that, however, are much smaller than the Bax pore. It is unknown whether Bcl-2 forms monomeric or oligomeric pores. Here, we characterized the Bcl-2 pore formation in liposomes using biophysical and biochemical techniques. The results show that the Bcl-2 pore enlarges as the concentration of Bcl-2 increases, suggesting that the pore is formed by Bcl-2 oligomers. As expected from oligomerization-mediated pore-formation, the small pores are formed earlier than the large ones. Bcl-2 oligomers form pores faster than the monomer, indicating that the oligomerization constitutes an intermediate step of the pore formation. A Bcl-2 mutant with higher affinity for oligomerization forms pores faster than wild type Bcl-2. Bcl-2 oligomers were detected in the liposomal membranes under conditions that Bcl-2 forms pores, and the extent of oligomerization was positively correlated with the pore-forming activity. Therefore, Bcl-2 oligomerizes in membranes forming pores, but the extent of oligomerization and the size of the resulting pores are much smaller than that of Bax, supporting the model that Bcl-2 is a defective Bax.     

Bax Activates Endophilin B1 Oligomerization and Lipid Membrane Vesiculation

Endophilins participate in membrane scission events that occur during endocytosis and intracellular organelle biogenesis through the combined activity of an N-terminal BAR domain that interacts with membranes and a C-terminal SH3 domain that mediates protein binding. Endophilin B1 (Endo B1) was identified to bind Bax, a Bcl-2 family member that promotes apoptosis, through yeast two-hybrid protein screens. Although Endo B1 does not bind Bax in healthy cells, during apoptosis, Endo B1 interacts transiently with Bax and promotes cytochrome c release from mitochondria. To explore the molecular mechanism of action of Endo B1, we have analyzed its interaction with Bax in cell-free systems. Purified recombinant Endo B1 in solution displays a Stokes radius indicating a tetrameric quarternary structure. However, when incubated with purified Bax, it assembles into oligomers more than 4-fold greater in molecular weight. Although Endo B1 oligomerization is induced by Bax, Bax does not stably associate with the high molecular weight Endo B1 complex. Endo B1 oligomerization requires its C-terminal Src homology 3 domain and is not induced by Bcl-xL. Endo B1 combined with Bax reduces the size and changes the morphology of giant unilamellar vesicles by inducing massive vesiculation of liposomes. This activity of purified Bax protein to induce cell-free assembly of Endo B1 may reflect its activity in cells that regulates apoptosis and/or mitochondrial fusion.     

Vinblastine-induced apoptosis is mediated by discrete alterations in subcellular location, oligomeric structure, and activation status of specific Bcl-2 family members

To gain a broader insight into the role of Bcl-2 proteins in apoptosis induced after mitotic arrest, we investigated the subcellular location, oligomeric structure, and protein interactions of Bax, Bcl-2, and Bcl-xL in vinblastine-treated KB-3 cells. Vinblastine induced the translocation of Bax from the cytosol to the mitochondria, which was accompanied by conformational activation and oligomerization of Bax. Bcl-2 was located in the mitochondria, underwent multisite phosphorylation after vinblastine treatment, and was strictly monomeric under all conditions. In contrast, in control cells, Bcl-xL existed in both monomeric (30 kDa) and oligomeric (150 kDa) forms. Treatment with agents that induced Bcl-xL phosphorylation (microtubule inhibitors) caused loss of the 150-kDa form, but this species was unaffected by apoptotic stimuli that did not stimulate phosphorylation. Vinblastine also promoted Bax activation and Bax oligomerization in HCT116 colon cancer cells. Both wild-type and Bax-deficient HCT116 cells expressed the 150-kDa form of Bcl-xL, which was depleted similarly in both cell lines upon vinblastine treatment. Co-immunoprecipitation studies revealed that in untreated KB-3 cells inactive cytosolic Bax interacted with Bcl-xL, whereas in vinblastine-treated cells, activated mitochondrial Bax did not interact with Bcl-xL. Interaction of Bcl-2 with Bax was not observed under any condition. Overexpression of Bcl-xL inhibited vinblastine-induced Bax activation and Bax dimerization and in parallel inhibited apoptosis. The results indicate that vinblastine-induced apoptosis requires translocation, activation, and oligomerization of Bax and is associated with specific changes in the oligomeric properties of Bcl-xL, which occur independently of Bax.     

Interaction of an adenovirus 14.7-kilodalton protein inhibitor of tumor necrosis factor alpha cytolysis with a new member of the GTPase superfamily of signal transducers.

The adenovirus (Ad) 14.7-kDa E3 protein (E3-14.7K), which can inhibit tumor necrosis factor alpha (TNF-alpha) cytolysis, was used to screen HeLa cell cDNA libraries for interacting proteins in the yeast two-hybrid system. A new member of the low-molecular-weight (LMW) GTP-binding protein family with Ras and ADP-ribosylation factor homology was discovered by this selection and has been named FIP-1 (14.7K-interacting protein). FIP-1 colocalized with Ad E3-14.7K in the cytoplasm especially near the nuclear membrane and in discrete foci on or near the plasma membrane. Its interaction with E3-14.7K was dependent on the FIP-1 GTP-binding domain. The stable expression of FIP-1 antisense message partially protected the cells from TNF-alpha cytolysis. FIP-1 was associated transiently with several unknown phosphorylated cellular proteins within 15 min after treatment with TNF-alpha. FIP-1 mRNA was expressed ubiquitously but at higher levels in human skeletal muscle, heart, and brain. In addition to homology to other LMW GTP-binding proteins, FIP-1 has regions of homology to two prokaryotic metalloproteases. However, there was no homology between FIP-1 and any of the recently isolated death proteins in the TNF-alpha or Fas/APO1 cytolytic pathway and no interaction with several members of the Bcl-2 family of inhibitors of apoptosis. These data suggest that FIP-1, as a cellular target for Ad E3-14.7K, is either a new intermediate on a previously described pathway or part of a novel TNF-alpha-induced cell death pathway. FIP-1 has two consensus sequences for myristoylation which would be expected to facilitate membrane association and also has sequences for Ser/Thr as well as Tyr phosphorylation that could affect its function.     

Viral pro-survival proteins block separate stages in Bax activation but changes in mitochondrial ultrastructure still occur

Mitochondrial dysfunction mediated by Bax and Bak is a critical step in mammalian cell apoptosis. However, the molecular mechanism of Bax activation remains unknown and has been difficult to investigate due to its rapid and stochastic nature. It is currently unclear whether mitochondria play a passive role in the initiation of apoptosis, remaining unaffected by cell stresses until Bax and Bak are active, or whether they actively participate in Bax/Bak activation. Here, two viral proteins, E1B19K and BHRF1, are examined for their ability to block Bax activation at different steps and thereby reveal the timing of mitochondrial changes during apoptosis. We demonstrate that BHRF1 strongly inhibits Bax activation but not upstream apoptotic signaling events, while E1B19K permits initial stages of Bax activation but prevents the subsequent oligomerization of Bax that is required for mitochondrial dysfunction. In this defined system we show that changes in mitochondrial ultrastructure, characteristic of cells undergoing apoptosis, precede Bax activation and are not blocked by E1B19K and BHRF1. We suggest that the ability of mitochondria to respond to apoptotic stress prior to Bax activation indicates that these organelles may play a direct role in activating Bax.     

The E1B19K oncoprotein complexes with Beclin 1 to regulate autophagy in adenovirus-infected cells

The mechanisms underlying adenovirus-mediated autophagy are currently unknown. Recently, members of the Bcl-2 protein family have been associated with autophagy. It was also reported that the Bcl-2 homology-3 (BH3) domain encompassed by both Beclin 1 and Bcl-2-like proteins is essential for their pro-autophagy or anti-autophagy functions. Here, we report for the first time that E1B19K, the adenovirus BH3 domain protein, interacts with Beclin 1 to initiate autophagy. Using immunoprecipitation assays we showed that expression of E1B19K in the host cell disrupted the physical interactions between Beclin 1 and Bcl-2 proteins. The displacement of Bcl-2 was coincident with the recruitment of PI3KC3 to the Beclin 1/E1B19K complexes. As a result of the changes in the components of the Beclin 1 interactome, there was activation of PI3KC3, as showed by the identification of PI3K-mediated lipid phosphorylation, and subsequent formation of autophagosomes. Importantly, the BH3 functional domain of E1B19K protein was required for the heterodimerization with Beclin 1. We also showed that transfer of E1B19K was sufficient to trigger autophagy in cancer cells. Consistent with these data, mutant adenoviruses encompassing a deletion of the E1B19K gene produced a marked deficiency in the capability of the virus to induce autophagy as showed by examining the lipidation and cleavage of LC3-I as well as the subcellular localization of LC3-II, the decrease in the levels of p62, and the formation of autophagosomes. Our work offers new information on the mechanisms of action of the adenoviral E1B19K protein as partner of Beclin 1 and positive regulator of autophagy.     

Molecular dynamics simulation and automated docking of the pro-apoptotic Bax protein and its complex with a peptide designed from the Bax-binding domain of anti-apoptotic Ku70

Bax, a multi-domain protein belonging to the large family of Bcl-2 proteins, has a pivotal role for the initiation of the cytochrome c-mediated apoptosis, a vital physiologic process to eliminate damaged or unwanted cells. In response to specific stimuli Bax translocates from cytosol to mitochondria outer membrane where a process of oligomerization occurs with pore formation through which cytochrome c and other death molecules escape. The pro-death action of Bax is regulated by the interaction with other pro-survival proteins. However, the conformational changes and the structural details necessary for homo and hetero interaction with other regulating proteins are largely unknown. This article reports a combined investigation of molecular dynamics (MD) simulation and automated docking that evidence the molecular regions of Bax involved in the binding with anti-apoptotic exapeptide (Bip) designed from Ku70, a subunit of the protein complex essential for non-homologous DNA repair but that inhibits also the Bax translocation to mitochondria. Since Bip suppresses apoptosis induced by several anti-cancer drugs, it appears relevant to achieve a better understanding of the structural and dynamical aspects that characterize the Bip-Bax complex in view of potential therapeutic implications. The present results show that the Bax region with the highest affinity for Bip is located in proximity of BH3 homology domain of Bax and also involves the alpha-helices 1 and 8. Moreover, the comparison of essential motions of Bax at 300 and 400 K before and after the formation of the complex with Bip evidences how the binding with the exa-peptide affects the collective motions of specific molecular districts of Bax considered to have functional relevance.     

E1B 19,000-Molecular-Weight Protein Interacts with and Inhibits CED-4-Dependent, FLICE-Mediated Apoptosis

Genetic studies of the nematode Caenorhabditis elegans (C. elegans) have identified several important components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homologue CED-9 (or the mammalian Bcl-2 family member Bcl-x_L) and the caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a potent apoptosis inhibitor and the adenovirus homologue of Bcl-2-related apoptosis inhibitors. Since E1B 19K and Bcl-x_L have functional similarity, we determined if E1B 19K interacts with CED-4 and regulates CED-4-dependent caspase activation. Binding analysis indicated that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 was dramatically changed by E1B 19K, supporting the theory of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could not induce cell death, coexpression of CED-4 and FLICE augmented cell death induction by FLICE, which was blocked by expression of E1B 19K. Even though E1B 19K did not prevent FLICE-induced apoptosis, it did inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggested that CED-4 was required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting with CED-4, and presumably a mammalian homologue of CED-4, to inhibit caspase activation and apoptosis.     

Phylogenomics of life-or-death switches in multicellular animals: Bcl-2, BH3-Only, and BNip families of apoptotic regulators

In this report, we conducted a comprehensive survey of Bcl-2 family members, a divergent group of proteins that regulate programmed cell death by an evolutionarily conserved mechanism. Using comparative sequence analysis, we found novel sequences in mammals, nonmammalian vertebrates, and in a number of invertebrates. We then asked what conclusions could be drawn from phyletic distribution, intron/exon structures, sequence/structure relationships, and phylogenetic analyses within the updated Bcl-2 family. First, multidomain members having a sequence pattern consistent with the conservation of the Bcl-X(L)/Bax/Bid topology appear to be restricted to multicellular animals and may share a common ancestry. Next, BNip proteins, which were originally identified based on their ability to bind to E1B 19K/Bcl-2 proteins, form three independent monophyletic branches with different evolutionary history. Lastly, a set of Bcl-2 homology 3-only proteins with unrelated secondary structures seems to have evolved after the origin of Metazoa and exhibits diverse expansion after speciation during vertebrate evolution.