BNIP3 heterodimerizes with Bcl-2/Bcl-X(L) and induces cell death independent of a Bcl-2 homology 3 (BH3) domain at both mitochondrial and nonmitochondrial sites

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 was sufficient for Bcl-X(L) interaction. Additionally, the removal of the BH3-like domain in BNIP3 did 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) directed protein expression to nonmitochondrial sites and still promoted apoptosis and heterodimerization with Bcl-2 and Bcl-X(L). We propose 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.     

The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis in a Bax- and Bim-dependent manner

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.     

MAP-1, a novel proapoptotic protein containing a BH3-like motif that associates with Bax through its Bcl-2 homology domains

A novel Bax-associating protein, named MAP-1 (Modulator of Apoptosis), has been identified in a yeast two-hybrid screen. MAP-1 contains a BH3-like (BH: Bcl-2 homology) motif and mediates caspase-dependent apoptosis in mammalian cells when overexpressed. MAP-1 homodimerizes and associates with the proapoptotic Bax and the prosurvival Bcl-2 and Bcl-X(L) of the Bcl-2 family in vitro and in vivo in mammalian cells. Mutagenesis analyses revealed that the BH3-like domain in MAP-1 is not required for its association with Bcl-X(L) but is required for association with Bax and for mediating apoptosis. Interestingly, in contrast to other Bax-associating proteins such as Bcl-X(L) and Bid, which require the BH3 and BH1 domains of Bax, respectively, for binding, the binding of MAP-1 to Bax appears to require all three BH domains (BH1, BH2, and BH3) of Bax, because point mutation of the critical amino acid in any one of these domains is sufficient to abolish its binding to MAP-1. These data suggest that MAP-1 mediates apoptosis through a mechanism that involves binding to Bax.     

Herpesvirus saimiri encodes a functional homolog of the human bcl-2 oncogene.

Here we demonstrate that open reading frame 16 (ORF16) of the oncogenic herpesvirus saimiri protects cells from heterologous virus-induced apoptosis. The BH1 and BH2 homology domains are highly conserved in ORF16, and ORF16 heterodimerizes with Bcl-2 family members Bax and Bak. However, ORF16 lacks the core sequence of the conserved BH3 homology domain, suggesting that this region is not essential for anti-apoptotic activity. Conservation of a functional bcl-2 homolog among gammaherpesviruses suggests that inhibition of programmed cell death is important in the biology of these viruses.     

Crystal structure of the Bcl-XL-Beclin 1 peptide complex: Beclin 1 is a novel BH3-only protein

Bcl-2 family proteins are key regulators of apoptosis and have recently been shown to modulate autophagy. The tumor suppressor Beclin 1 has been proposed to coordinate both apoptosis and autophagy through direct interaction with anti-apoptotic family members Bcl-2 and/or Bcl-X(L). However, the molecular basis for this interaction remains enigmatic. Here we report that Beclin 1 contains a conserved BH3 domain, which is both necessary and sufficient for its interaction with Bcl-X(L). We also report the crystal structure of a Beclin BH3 peptide in complex with Bcl-X(L) at 2.5A resolution. Reminiscent of previously determined Bcl-X(L)-BH3 structures, the amphipathic BH3 helix of Beclin 1 bound to a conserved hydrophobic groove of Bcl-X(L). These results define Beclin 1 as a novel BH3-only protein, implying that Beclin 1 may have a direct role in initiating apoptotic signaling. We propose that this putative apoptotic function may be linked to the ability of Beclin 1 to suppress tumor formation in mammals.     

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.     

Bcl-B, a novel Bcl-2 family member that differentially binds and regulates Bax and Bak

A novel human member of the Bcl-2 family was identified, Bcl-B, which is closest in amino acid sequence homology to the Boo (Diva) protein. The Bcl-B protein contains four Bcl-2 homology (BH) domains (BH1, BH2, BH3, BH4) and a predicted carboxyl-terminal transmembrane (TM) domain. The BCL-B mRNA is widely expressed in adult human tissues. The Bcl-B protein binds Bcl-2, Bcl-X(L), and Bax but not Bak. In transient transfection assays, Bcl-B suppresses apoptosis induced by Bax but not Bak. Deletion of the TM domain of Bcl-B impairs its association with intracellular organelles and diminishes its anti-apoptotic function. Bcl-B thus displays a unique pattern of selectivity for binding and regulating the function of other members of the Bcl-2 family.     

Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1

The anti-apoptotic proteins Bcl-2 and Bcl-X(L) bind and inhibit Beclin-1, an essential mediator of autophagy. Here, we demonstrate that this interaction involves a BH3 domain within Beclin-1 (residues 114-123). The physical interaction between Beclin-1 and Bcl-X(L) is lost when the BH3 domain of Beclin-1 or the BH3 receptor domain of Bcl-X(L) is mutated. Mutation of the BH3 domain of Beclin-1 or of the BH3 receptor domain of Bcl-X(L) abolishes the Bcl-X(L)-mediated inhibition of autophagy triggered by Beclin-1. The pharmacological BH3 mimetic ABT737 competitively inhibits the interaction between Beclin-1 and Bcl-2/Bcl-X(L), antagonizes autophagy inhibition by Bcl-2/Bcl-X(L) and hence stimulates autophagy. Knockout or knockdown of the BH3-only protein Bad reduces starvation-induced autophagy, whereas Bad overexpression induces autophagy in human cells. Gain-of-function mutation of the sole BH3-only protein from Caenorhabditis elegans, EGL-1, induces autophagy, while deletion of EGL-1 compromises starvation-induced autophagy. These results reveal a novel autophagy-stimulatory function of BH3-only proteins beyond their established role as apoptosis inducers. BH3-only proteins and pharmacological BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin-1 and Bcl-2 or Bcl-X(L).     

Bcl-2 family members: dual regulators of apoptosis and autophagy

The essential autophagy protein and haplo-insufficient tumor suppressor, Beclin 1, interacts with several cofactors (Ambra1, Bif-1, UVRAG) to activate the lipid kinase Vps34, thereby inducing autophagy. In normal conditions, Beclin 1 is bound to and inhibited by Bcl-2 or the Bcl-2 homolog Bcl-X(L). This interaction involves a Bcl-2 homology 3 (BH3) domain in Beclin 1 and the BH3 binding groove of Bcl-2/Bcl-X(L). Other proteins containing BH3 domains, called BH3-only proteins, can competitively disrupt the interaction between Beclin 1 and Bcl-2/Bcl-X(L) to induce autophagy. Nutrient starvation, which is a potent physiologic inducer of autophagy, can stimulate the dissociation of Beclin 1 from its inhibitors, either by activating BH3-only proteins (such as Bad) or by posttranslational modifications of Bcl-2 (such as phosphorylation) that may reduce its affinity for Beclin 1 and BH3-only proteins. Thus, anti-apoptotic Bcl-2 family members and pro-apoptotic BH3-only proteins may participate in the inhibition and induction of autophagy, respectively. This hitherto neglected crosstalk between the core machineries regulating autophagy and apoptosis may redefine the role of Bcl-2 family proteins in oncogenesis and tumor progression.     

Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32

Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74-282)) and the conserved C-terminal region (p32 (1-221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.     

The anti-apoptotic Bcl-B protein inhibits BECN1-dependent autophagic cell death

Bcl-2 family members are key modulators of apoptosis that have recently been shown to also regulate autophagy. It has been previously reported that Bcl-2 and Bcl-X(L) bind and inhibit BECN1, an essential mediator of autophagy. Bcl-B is an anti-apoptotic member of the Bcl-2 family that possesses the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal trans-membrane domain. Although the anti-apoptotic properties of Bcl-B are well characterized, its physiological function remains to be established. In the present study, we first established that Bcl-B interacts with the BH3 domain of BECN1. We also showed that Bcl-B overexpression reduces autophagy triggered by a variety of pro-autophagic stimuli. This impairment of autophagy was closely related to the capacity of Bcl-B to bind to BECN1. Importantly, we have demonstrated that Bcl-B knockdown triggers autophagic cell death and sensitizes cells to amino acid starvation. The cell death induced by Bcl-B knockdown was partially dependent on components of the autophagy machinery (LC3; BECN1; ATG5). These findings reveal a new role of Bcl-B in the regulation of autophagy.     

Bcl-G, a novel pro-apoptotic member of the Bcl-2 family

A new member of the Bcl-2 family was identified, Bcl-G. The human BCL-G gene consists of 6 exons, resides on chromosome 12p12, and encodes two proteins through alternative mRNA splicing, Bcl-G(L) (long) and Bcl-G(S) (short) consisting of 327 and 252 amino acids in length, respectively. Bcl-G(L) and Bcl-G(S) have identical sequences for the first 226 amino acids but diverge thereafter. Among the Bcl-2 homology (BH) domains previously recognized in Bcl-2 family proteins, the BH3 domain is found in both Bcl-G(L) and Bcl-G(S), but only the longer Bcl-G(L) protein possesses a BH2 domain. Bcl-G(L) mRNA is expressed widely in adult human tissues, whereas Bcl-G(S) mRNA was found only in testis. Overexpression of Bcl-G(L) or Bcl-G(S) in cells induced apoptosis although Bcl-G(S) was far more potent than Bcl-G(L). Apoptosis induction by Bcl-G(S) depended on the BH3 domain and was suppressed by coexpression of anti-apoptotic Bcl-X(L) protein. Bcl-X(L) also coimmunoprecipitated with Bcl-G(S) but not with mutants of Bcl-G(S) in which the BH3 domain was deleted or mutated or with Bcl-G(L). Bcl-G(S) was predominantly localized to cytosolic organelles, whereas Bcl-G(L) was diffusely distributed throughout the cytosol. A mutant of Bcl-G(L) in which the BH2 domain was deleted displayed increased apoptotic activity and coimmunoprecipitated with Bcl-X(L), suggesting that the BH2 domain autorepresses Bcl-G(L).     

BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L)

Beclin 1 has recently been identified as novel BH3-only protein, meaning that it carries one Bcl-2-homology-3 (BH3) domain. As other BH3-only proteins, Beclin 1 interacts with anti-apoptotic multidomain proteins of the Bcl-2 family (in particular Bcl-2 and its homologue Bcl-X(L)) by virtue of its BH3 domain, an amphipathic alpha-helix that binds to the hydrophobic cleft of Bcl-2/Bcl-X(L). The BH3 domains of other BH3-only proteins such as Bad, as well as BH3-mimetic compounds such as ABT737, competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-X(L). This causes autophagy of mitochondria (mitophagy) but not of the endoplasmic reticulum (reticulophagy). Only ER-targeted (not mitochondrion-targeted) Bcl-2/Bcl-X(L) can inhibit autophagy induced by Beclin 1, and only Beclin 1-Bcl-2/Bcl-X(L) complexes present in the ER (but not those present on heavy membrane fractions enriched in mitochondria) are disrupted by ABT737. These findings suggest that the Beclin 1-Bcl-2/Bcl-X(L) complexes that normally inhibit autophagy are specifically located in the ER and point to an organelle-specific regulation of autophagy. Furthermore, these data suggest a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrion-permeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-X(L) at the level of the endoplasmic reticulum.     

Apoptosis-based dual molecular targeting by INNO-406, a second-generation Bcr-Abl inhibitor, and ABT-737, an inhibitor of antiapoptotic Bcl-2 proteins, against Bcr-Abl-positive leukemia

Bcr-Abl is the cause of Philadelphia-positive (Ph(+)) leukemias and also constitutes their principal therapeutic target, as exemplified by dramatic effects of imatinib mesylate. However, mono-targeting of Bcr-Abl does not always achieve complete leukemia eradication, and additional strategies those enable complete elimination of leukemic cells are desired to develop. Here we demonstrate that INNO-406, a much more active Bcr-Abl tyrosine kinase inhibitor than imatinib, augments the activities of several proapoptotic Bcl-2 homology (BH)3-only proteins (Bim, Bad, Bmf and Bik) and induces apoptosis in Ph(+) leukemia cells via Bcl-2 family-regulated intrinsic apoptosis pathway. ABT-737, an inhibitor of antiapoptotic Bcl-2 and Bcl-X(L), greatly enhanced the apoptosis by INNO-406, even in INNO-406-less sensitive cells with Bcr-Abl point mutations except T315I mutation. In contrast, co-treatment with INNO-406 and other pharmacologic inducers of those BH3-only proteins, such as 17-allylaminogeldanamycin, an heat shock protein-90 inhibitor, or PS-341, a proteasome inhibitor, did not further increase the BH3-only protein levels or sensitize leukemic cells to INNO-406-induced apoptosis, suggesting a limit to how much expression levels of BH3-only proteins can be increased by anticancer agents. Thus, double-barrelled molecular targeting for Bcr-Abl-driven oncogenic signaling and the cell protection by antiapoptotic Bcl-2 family proteins may be the rational therapeutic approach for eradicating Ph(+) leukemic cells.     

Identification of a novel regulatory domain in Bcl-X(L) and Bcl-2.

Bcl-X(L), a member of the Bcl-2 family, can inhibit many forms of programed cell death. The three-dimensional structure of Bcl-X(L) identified a 60 amino acid loop lacking defined structure. Although amino acid sequence within this region is not conserved among Bcl-2 family members, structural modeling suggested that Bcl-2 also contains a large unstructured region. Compared with the full-length protein, loop deletion mutants of Bcl-X(L) and Bcl-2 displayed an enhanced ability to inhibit apoptosis. Despite enhanced function, the deletion mutants did not have significant alterations in the ability to bind pro-apoptotic proteins such as Bax. The loop deletion mutant of Bcl-2 also displayed a qualitative difference in its ability to inhibit apoptosis. Full-length Bcl-2 was unable to prevent anti-IgM-induced cell death of the immature B cell line WEHI-231. In contrast, the Bcl-2 deletion mutant protected WEHI-231 cells from death. Substantial differences were observed in the ability of WEHI-231 cells to phosphorylate the deletion mutant of Bcl-2 compared with full-length Bcl-2. Bcl-2 phosphorylation was found to be dependent on the presence of an intact loop domain. These results suggest that the loop domain in Bcl-X(L) and Bcl-2 can suppress the anti-apoptotic function of these genes and may be a target for regulatory post-translational modifications.     

The proapoptotic BH3-only protein BAD transduces cell death signals independently of its interaction with Bcl-2

The BH3-only protein BAD binds to Bcl-2 family proteins through its BH3 domain. Recent studies suggest that BAD binds to both Bcl-2 and Bcl-X(L), however mediates its pro-apoptotic functions through inhibition of Bcl-X(L), but not Bcl-2. In this paper we addressed this issue using a BAD mutant within the BH3 domain, by substitution of Asp 119 with Gly (BAD(D119G)), which selectively abrogates an ability to interact with Bcl-2. Confocal microscopy revealed that mutation of BAD at D119 does not affect BAD targeting to the mitochondrial membrane in serum-starved COS-7 cells. However, co-precipitation assays indicated that, whereas wild-type BAD (BADwt) directly interacts with Bcl-2 and Bcl-X(L), BAD(D119G) interacts only with Bcl-X(L). Nevertheless both BADwt and BAD(D119G) could introduce apoptosis and diminish the anti-apoptotic effect of Bcl-2 and Bcl-X(L) in a similar manner in a co-transfection assay. These data thus suggest that Asp119 is a crucial site within the BH3 domain of BAD for interaction of BAD with Bcl-2, but is dispensable for the interaction of BAD with Bcl-X(L), for its targeting to mitochondria, and most importantly, for its pro-apoptotic functions. Thus, we confirm that neutralization of Bcl-2 function is marginal for BAD-mediated apoptosis.     

BH3-only protein Bim inhibits activity of antiapoptotic members of Bcl-2 family when expressed in yeast

Proteins of the Bcl-2 family regulate programmed cell death in mammals by promoting the release of cytochrome c from mitochondria in response to various proapoptotic stimuli. The mechanism by which BH3-only members of the family activate multidomain proapoptotic proteins Bax and Bak to form a pore in mitochondrial membranes remains under dispute. We report that cell death promoting activity of BH3-only protein Bim can be reconstituted in yeast when both Bax and antiapoptotic protein Bcl-X(L) are present, suggesting that Bim likely activates Bax indirectly by inhibiting antiapoptotic proteins.     

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

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