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.     

c-Myc functionally cooperates with Bax to induce apoptosis

c-Myc promotes apoptosis by destabilizing mitochondrial integrity, leading to the release of proapoptotic effectors including holocytochrome c. Candidate mediators of c-Myc in this process are the proapoptotic members of the Bcl-2 family. We show here that fibroblasts lacking Bak remain susceptible to c-Myc-induced apoptosis whereas bax-deficient fibroblasts are resistant. However, despite this requirement for Bax, c-Myc activation exerts no detectable effects on Bax expression, localization, or conformation. Moreover, susceptibility to c-Myc-induced apoptosis can be restored in bax-deficient cells by ectopic expression of Bax or by microinjection of a peptide comprising a minimal BH3 domain. Microinjection of BH3 peptide also restores sensitivity to c-Myc-induced apoptosis in p53-deficient primary fibroblasts that are otherwise resistant. By contrast, there is no synergy between BH3 peptide and c-Myc in fibroblasts deficient in both Bax and Bak. We conclude that c-Myc triggers a proapoptotic mitochondrial destabilizing activity that cooperates with proapoptotic members of the Bcl-2 family.     

Structural biology of the Bcl-2 family and its mimicry by viral proteins

Intrinsic apoptosis in mammals is regulated by protein–protein interactions among the B-cell lymphoma-2 (Bcl-2) family. The sequences, structures and binding specificity between pro-survival Bcl-2 proteins and their pro-apoptotic Bcl-2 homology 3 motif only (BH3-only) protein antagonists are now well understood. In contrast, our understanding of the mode of action of Bax and Bak, the two necessary proteins for apoptosis is incomplete. Bax and Bak are isostructural with pro-survival Bcl-2 proteins and also interact with BH3-only proteins, albeit weakly. Two sites have been identified; the in-groove interaction analogous to the pro-survival BH3-only interaction and a site on the opposite molecular face. Interaction of Bax or Bak with activator BH3-only proteins and mitochondrial membranes triggers a series of ill-defined conformational changes initiating their oligomerization and mitochondrial outer membrane permeabilization. Many actions of the mammalian pro-survival Bcl-2 family are mimicked by viruses. By expressing proteins mimicking mammalian pro-survival Bcl-2 family proteins, viruses neutralize death-inducing members of the Bcl-2 family and evade host cell apoptosis during replication. Remarkably, structural elements are preserved in viral Bcl-2 proteins even though there is in many cases little discernible sequence conservation with their mammalian counterparts. Some viral Bcl-2 proteins are dimeric, but they have distinct structures to those observed for mammalian Bcl-2 proteins. Furthermore, viral Bcl-2 proteins modulate innate immune responses regulated by NF-κB through an interface separate from the canonical BH3-binding groove. Our increasing structural understanding of the viral Bcl-2 proteins is leading to new insights in the cellular Bcl-2 network by exploring potential alternate functional modes in the cellular context. We compare the cellular and viral Bcl-2 proteins and discuss how alterations in their structure, sequence and binding specificity lead to differences in behavior, and together with the intrinsic structural plasticity in the Bcl-2 fold enable exquisite control over critical cellular signaling pathways.     

Molecular basis of the regulation of Beclin 1-dependent autophagy by the gamma-herpesvirus 68 Bcl-2 homolog M11

Gamma-herpesviruses (gammaHVs), including important human pathogens such as Epstein Barr virus, Kaposi's sarcoma-associated HV, and the murine gammaHV68, encode homologs of the antiapoptotic, cellular Bcl-2 (cBcl-2) to promote viral replication and pathogenesis. The precise molecular details by which these proteins function in viral infection are poorly understood. Autophagy, a lysosomal degradation pathway, is inhibited by the interaction of cBcl-2s with a key autophagy effector, Beclin 1, and can also be inhibited by gammaHV Bcl-2s. Here we investigate the gammaHV68 M11-Beclin 1 interaction in atomic detail, using biochemical and structural approaches. We show that the Beclin 1 BH3 domain is the primary determinant of binding to M11 and other Bcl-2s, and this domain binds in a hydrophobic groove on M11, reminiscent of the binding of different BH3 domains to other Bcl-2s. Unexpectedly, regions outside of, but contiguous with, the Beclin 1 BH3 domain also contribute to this interaction. We find that M11 binds to Beclin 1 more strongly than do KSHV Bcl-2 or cBcl-2. Further, the differential affinity of M11 for different BH3 domains is caused by subtle, yet significant, variations in the atomic details of each interaction. Consistent with our structural analysis, we find that Beclin 1 residues L116 and F123, and M11 residue pairs G86 + R87 and Y60 + L74, are required for M11 to bind to Beclin 1 and downregulate autophagy. Thus, our results suggest that M11 inhibits autophagy through a mechanism that involves the binding of the Beclin 1 BH3 domain in the M11 hydrophobic surface groove.     

Targeting Bax interaction sites reveals that only homo-oligomerization sites are essential for its activation

Bax is a proapoptotic Bcl-2 family member that has a central role in the initiation of mitochondria-dependent apoptosis. However, the mechanism of Bax activation during apoptosis remains unsettled. It is believed that the activation of Bax is mediated by either dissociation from prosurvival Bcl-2 family members, or direct association with BH3-only members. Several interaction sites on Bax that mediate its interactions with other Bcl-2 family members, as well as its proapoptotic activity, have been identified in previous studies by other groups. To rigorously investigate the functional role of these interaction sites, we knocked in their respective mutants using HCT116 colon cancer cells, in which apoptosis induced by several stimuli is strictly Bax-dependent. Bax-mediated apoptosis was intact upon knock-in (KI) of K21E and D33A, which were shown to block the interaction of Bax with BH3-only activators. Apoptosis was partially reduced by KI of D68R, which impairs the interaction of Bax with prosurvival members, and S184V, a constitutively mitochondria-targeting mutant. In contrast, apoptosis was largely suppressed by KI of L70A/D71A, which blocks homo-oligomerization of Bax and its binding to prosurvival Bcl-2 family proteins. Collectively, our results suggest that the activation of endogenous Bax in HCT116 cells is dependent on its homo-oligomerization sites, but not those previously shown to interact with BH3-only activators or prosurvival proteins only. We therefore postulate that critical interaction sites yet to be identified, or mechanisms other than protein-protein interactions, need to be pursued to delineate the mechanism of Bax activation during apoptosis.     

Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-xL

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the α-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques—yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis—to elucidate specificity determinants for binding to Bcl-x_Lversus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x_L selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x_L, Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x_L-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x_L binders.     

Reconstitution of interactions of Murine gammaherpesvirus 68 M11 with Bcl-2 family proteins in yeast

One of the mechanisms of defense against viral infection is induction of apoptosis in infected cells. To escape this line of protection, genomes of many viruses encode for proteins that inhibit apoptosis. Murid herpesvirus 4 gene M11 encodes for homologue of cellular Bcl-2 proteins that inhibits apoptosis and autophagy in infected cell. To study a role of M11 in regulation of apoptosis we have established a yeast model system in which the action of M11 together with proapoptotic proteins Bax, Bak and Bid can be studied. When expressed in yeast, M11 did not inhibit autophagic pathway, so only effects of expression of M11 on activity of coexpressed proapoptotic proteins could be observed. In this experimental setting M11 potently inhibited both proapoptotic multidomain proteins Bax and Bak. The antiapoptotic activity of M11 was suppressed by coexpression of proapoptotic BH3-only protein tBid, indicating that M11 inhibits apoptosis likely by the same mechanism as cellular antiapoptotic proteins Bcl-2 or Bcl-XL.     

BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and anti-apoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein Bim(S). Regulated expression of Bim(S) in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of Bim(S) showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, Bim(S) enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting.     

Interaction between the antiapoptotic protein Nr-13 and cytochrome c. Antagonistic effect of the BH3 domain of Bax

Mitochondria act as a focal point for upstream apoptosis signals by releasing cytochrome c into the cytosol, leading to the activation of caspases and subsequent cell death. Members of the Bcl-2 protein family regulate this phenomenon by heterodimerization via the BH3 domain of proapoptotic members opposing their pro- and antiapoptotic functions. The mechanism of cytochrome c release from mitochondria and of its regulation remains controversial. In vitro binding studies of purified and biologically active proteins should help in understanding the molecular mechanism of interactions and protein functions. In this work, the Bcl-2-related antiapoptotic chicken protein Nr-13 was overexpressed as a highly soluble recombinant protein which showed correct folding as judged by circular dichroism and fluorescence spectroscopy. Purified Nr-13 inhibits caspase-3 activation in a Xenopus egg-derived cell-free system, and neutralizes the proapoptotic activity of a synthetic peptide containing the BH3 domain of Bax. The latter effect correlates with the high-affinity binding of the BH3 peptide to Nr-13 as monitored by the intrinsic tryptophan fluorescence. On the basis of the structural similarity with Bcl-x(L), putative residues involved in this interaction were identified. Nr-13 exhibits a high-affinity interaction with cytochrome c which is prevented by preincubation with the BH3-Bax peptide. These findings are discussed with respect to a model for the regulation of apoptosis in which a direct interaction between the antiapoptotic protein and cytochrome c may prevent the apoptosis.     

Auto-activation of the apoptosis protein Bax increases mitochondrial membrane permeability and is inhibited by Bcl-2

Interactions among Bcl-2 family proteins mediated by Bcl-2 homology (BH) regions transform apoptosis signals into actions. The interactions between BH3 region-only proteins and multi-BH region proteins such as Bax and Bcl-2 have been proposed to be the dominant interactions required for initiating apoptosis. Experimental evidence also suggests that both homo- and hetero-interactions are mediated primarily by the BH3 regions in all Bcl-2 family proteins and contribute to commitment to or inhibition of apoptosis. We found that a peptide containing the BH3 helix of Bax was not sufficient to activate recombinant Bax to permeabilize mitochondria. However, an extended peptide containing the BH3 helix and additional downstream sequences activated Bax to permeabilize mitochondria and liposomes. Bcl-2 inhibited the membrane-permeabilizing activity of peptide-activated Bax. This activity of Bcl-2 was inhibited by the extended but not the BH3-only peptide despite both peptides binding to Bcl-2 with similar affinity. Further, membrane-bound Bax activation intermediates directly activated soluble Bax further permeabilizing the membrane. Bcl-2 inhibited Bax auto-activation. We therefore propose that Bax auto-activation amplifies the initial death signal produced by BH3-only proteins and that Bcl-2 functions as an inhibitor of Bax auto-activation.     

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.     

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

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

Structural biology of the Bcl-2 family of proteins

The proteins of the Bcl-2 family are important regulators of programmed cell death. Structural studies of Bcl-2 family members have provided many important insights into their molecular mechanism of action and how members of this family interact with one another. To date, structural studies have been performed on six Bcl-2 family members encompassing both anti- (Bcl-x(L), Bcl-2, KSHV-Bcl-2, Bcl-w) and pro-apoptotic (Bax, Bid) members. They all show a remarkably similar fold despite an overall divergence in amino acid sequence and function (pro-apoptotic versus anti-apoptotic). The three-dimensional structures of Bcl-2 family members consist of two central, predominantly hydrophobic alpha-helices surrounded by six or seven amphipathic alpha-helices of varying lengths. A long, unstructured loop is present between the first two alpha-helices. The structures of the Bcl-2 proteins show a striking similarity to the overall fold of the pore-forming domains of bacterial toxins. This finding led to experiments which demonstrated that Bcl-x(L), Bcl-2, and Bax all form pores in artificial membranes. A prominent hydrophobic groove is present on the surface of the anti-apoptotic proteins. This groove is the binding site for peptides that mimic the BH3 region of various pro-apoptotic proteins such as Bak and Bad. Structures of Bcl-x(L) in complex with these BH3 peptides showed that they bind as an amphipathic alpha-helix and make extensive hydrophobic contacts with the protein. These data have not only helped to elucidate the interactions important for hetero-dimerization of Bcl-2 family members but have also been used to guide the discovery of small molecules that block Bcl-x(L) and Bcl-2 function. In the recently determined structure of the anti-apoptotic Bcl-w protein, the protein was also found to have a hydrophobic groove on its surface capable of binding BH3-containing proteins and peptides. However, in the native protein an additional carboxy-terminal alpha-helix interacts with the hydrophobic groove. This is reminiscent of how the carboxy-terminal alpha-helix of the pro-apoptotic protein Bax binds into its hydrophobic groove. This interaction may play a regulatory role and for Bax may explain why it is found predominately in the cytoplasm prior to activation. The hydrophobic groove of the pro-apoptotic protein, Bid protein, is neither as long nor as deep as that found in Bcl-x(L), Bcl-2, or Bax. In addition, Bid contains an extra alpha-helix, which is located between alpha1 and alpha2 with respect to Bcl-x(L), Bcl-2, and Bax. Although there are still many unanswered questions regarding the exact mechanism by which the Bcl-2 family of proteins modulates apoptosis, structural studies of these proteins have deepened our understanding of apoptosis on the molecular level.     

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

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

Antiapoptotic herpesvirus Bcl-2 homologs escape caspase-mediated conversion to proapoptotic proteins

The antiapoptotic Bcl-2 and Bcl-x(L) proteins of mammals are converted into potent proapoptotic factors when they are cleaved by caspases, a family of apoptosis-inducing proteases (E. H.-Y. Cheng, D. G. Kirsch, R. J. Clem, R. Ravi, M. B. Kastan, A. Bedi, K. Ueno, and J. M. Hardwick, Science 278:1966-1968, 1997; R. J. Clem, E. H.-Y. Cheng, C. L. Karp, D. G. Kirsch, K. Ueno, A. Takahashi, M. B. Kastan, D. E. Griffin, W. C. Earnshaw, M. A. Veliuona, and J. M. Hardwick, Proc. Natl. Acad. Sci. USA 95:554-559, 1998). Gamma herpesviruses also encode homologs of the Bcl-2 family. All tested herpesvirus Bcl-2 homologs possess antiapoptotic activity, including the more distantly related homologs encoded by murine gammaherpesvirus 68 (gammaHV68) and bovine herpesvirus 4 (BHV4), as described here. To determine if viral Bcl-2 proteins can be converted into death factors, similar to their cellular counterparts, five herpesvirus Bcl-2 homologs from five different viruses were tested for their susceptibility to caspases. Only the viral Bcl-2 protein encoded by gammaHV68 was susceptible to caspase digestion. However, unlike the caspase cleavage products of cellular Bcl-2, Bcl-x(L), and Bid, which are potent inducers of apoptosis, the cleavage product of gammaHV68 Bcl-2 lacked proapoptotic activity. KSBcl-2, encoded by the Kaposi's sarcoma-associated herpesvirus, was the only viral Bcl-2 homolog that was capable of killing cells when expressed as an N-terminal truncation. However, because KSBcl-2 was not cleavable by caspases, the latent proapoptotic activity of KSBcl-2 apparently cannot be released. The Bcl-2 homologs encoded by herpesvirus saimiri, Epstein-Barr virus, and BHV4 were not cleaved by apoptotic cell extracts and did not possess latent proapoptotic activities. Thus, herpesvirus Bcl-2 homologs escape negative regulation by retaining their antiapoptotic activities and/or failing to be converted into proapoptotic proteins by caspases during programmed cell death.     

Mutation to Bax beyond the BH3 domain disrupts interactions with pro-survival proteins and promotes apoptosis

Pro-survival members of the Bcl-2 family of proteins restrain the pro-apoptotic activity of Bax, either directly through interactions with Bax or indirectly by sequestration of activator BH3-only proteins, or both. Mutations in Bax that promote apoptosis can provide insight into how Bax is regulated. Here, we describe crystal structures of the pro-survival proteins Mcl-1 and Bcl-x(L) in complex with a 34-mer peptide from Bax that encompasses its BH3 domain. These structures reveal canonical interactions between four signature hydrophobic amino acids from the BaxBH3 domain and the BH3-binding groove of the pro-survival proteins. In both structures, Met-74 from the Bax peptide engages with the BH3-binding groove in a fifth hydrophobic interaction. Various Bax Met-74 mutants disrupt interactions between Bax and all pro-survival proteins, but these Bax mutants retain pro-apoptotic activity. Bax/Bak-deficient mouse embryonic fibroblast cells reconstituted with several Bax Met-74 mutants are more sensitive to the BH3 mimetic compound ABT-737 as compared with cells expressing wild-type Bax. Furthermore, the cells expressing Bax Met-74 mutants are less viable in colony assays even in the absence of an external apoptotic stimulus. These results support a model in which direct restraint of Bax by pro-survival Bcl-2 proteins is a barrier to apoptosis.     

Bak but not Bax is essential for Bcl-xS-induced apoptosis

Bcl-x(S), a proapoptotic member of the Bcl-2 protein family, is localized in the mitochondria and induces apoptosis in a caspase- and BH3-dependent manner by a mechanism involving cytochrome c release. The way in which Bcl-x(S) induces caspase activation and cytochrome c release, as well as the relationship between Bcl-x(S) and other proapoptotic members of the Bcl-2 family, is not known. Here we used embryonic fibroblasts derived from mice deficient in the multidomain proapoptotic members of the Bcl-2 family (Bax and Bak) and the apoptotic components of the apoptosome (Apaf-1 and caspase-9) to unravel the cascade of events by which Bcl-x(S) promotes apoptosis. Our results show that Bak but not Bax is essential for Bcl-x(S)-induced apoptosis. Bcl-x(S) induced activation of Bak, which in turn promoted apoptosis by apoptosome-dependent and -independent pathways. These findings provide the first evidence that a proapoptotic Bcl-2 family protein induces apoptosis exclusively via Bak.     

Releasing the spindle assembly checkpoint without tension

Proteins of the Bcl-2 family are critical regulators of apoptosis, but how its BH3-only members activate the essential effectors Bax and Bak remains controversial. The indirect activation model suggests that they simply must neutralize all of the prosurvival Bcl-2 family members, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak directly. As numerous in vitro studies have not resolved this issue, we have investigated Bim''s activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of prosurvival relatives but lost interaction with Bax. Analysis of the mice showed that Bim''s proapoptotic activity is not solely caused by its ability to engage its prosurvival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.     

JAB1 accelerates mitochondrial apoptosis by interaction with proapoptotic BclGs

The Bcl-2 family of proteins is the key regulators of cell apoptosis at the mitochondria level. The BH3-only pro-apoptotic member BclGs was unique among the family due to its highly specific expression in human testis and has been demonstrated to induce apoptosis dependent on the BH3 domain. However, the molecular mechanism of BclGs-induced apoptosis remains unclear. Here we show that overexpression of BclGs could induce Bax expression upregulation and translocation to mitochondria, cytochrome c release and activation of caspase-3. Moreover, we identified JAB1 as a novel BclGs-specific binding protein through a yeast two-hybrid screening in a human testis cDNA library. BclGs interacts with JAB1 both in vitro and in vivo. N-terminal region of BclGs (aa 1-67) was required for the interaction. Importantly, JAB1 and BclGs co-expression synergistically induces apoptosis. JAB1 could compete with Bcl-XL/Bcl-2 to bind to BclGs; thus, promote the apoptosis. RNAi-mediated knock-down of JAB1 results in the reduced proapoptotic activity of BclGs. Taken together, our results provided the first evidence that JAB1 is involved in the regulation of mitochondrial apoptotic pathway through specific interaction with BclGs.     

The putative pore-forming domain of Bax regulates mitochondrial localization and interaction with Bcl-X(L)

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: (i) by disrupting mitochondrial membrane barrier function by formation of ion-permeable pores in mitochondrial membranes and (ii) 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. We mutagenized several charged residues in the alpha5-alpha6 domain of Bax, changing them to alanine. These substitution mutants of Bax constitutively localized to mitochondria and displayed 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 resulted 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: (i) regulation of mitochondrial targeting and (ii) modulation of binding to antiapoptotic Bcl-2 proteins.