Bcl-2 is a better ABT-737 target than Bcl-xL or Bcl-w and only Noxa overcomes resistance mediated by Mcl-1, Bfl-1, or Bcl-B

The novel anticancer drug ABT-737 is a Bcl-2 Homology 3 (BH3)-mimetic that induces apoptosis by inhibiting pro-survival Bcl-2 proteins. ABT-737 binds with equal affinity to Bcl-2, Bcl-xL and Bcl-w in vitro and is expected to overrule apoptosis resistance mediated by these Bcl-2 proteins in equal measure. We have profiled ABT-737 specificity for all six pro-survival Bcl-2 proteins, in p53 wild-type or p53-mutant human T-leukemic cells. Bcl-B was untargeted, like Bfl-1 and Mcl-1, in accord with their low affinity for ABT-737 in vitro. However, Bcl-2 proved a better ABT-737 target than Bcl-xL and Bcl-w. This was reflected in differential apoptosis-sensitivity to ABT-737 alone, or combined with etoposide. ABT-737 was not equally effective in displacing BH3-only proteins or Bax from Bcl-2, as compared with Bcl-xL or Bcl-w, offering an explanation for the differential ABT-737 sensitivity of tumor cells overexpressing these proteins. Inducible expression demonstrated that BH3-only proteins Noxa, but not Bim, Puma or truncated Bid could overrule ABT-737 resistance conferred by Bcl-B, Bfl-1 or Mcl-1. These data identify Bcl-B, Bfl-1 and Mcl-1, but also Bcl-xL and Bcl-w as potential mediators of ABT-737 resistance and indicate that target proteins can be differentially sensitive to BH3-mimetics, depending on the pro-apoptotic Bcl-2 proteins they are complexed with.     

14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation

The Bcl-2 homology 3 (BH3) domain of prodeath Bcl-2 family members mediates their interaction with prosurvival Bcl-2 family members and promotes apoptosis. We report that survival factors trigger the phosphorylation of the proapoptotic Bcl-2 family member BAD at a site (Ser-155) within the BAD BH3 domain. When BAD is bound to prosurvival Bcl-2 family members, BAD Ser-155 phosphorylation requires the prior phosphorylation of Ser-136, which recruits 14-3-3 proteins that then function to increase the accessibility of Ser-155 to survival-promoting kinases. Ser-155 phosphorylation disrupts the binding of BAD to prosurvival Bcl-2 proteins and thereby promotes cell survival. These findings define a mechanism by which survival signals inactivate a proapoptotic Bcl-2 family member, and suggest a role for 14-3-3 proteins as cofactors that regulate sequential protein phosphorylation events.     

C-terminal Residues Regulate Localization and Function of the Antiapoptotic Protein Bfl-1

Unlike other antiapoptotic members of the Bcl-2 family, Bfl-1 does not contain a well defined C-terminal transmembrane domain, and whether the C-terminal tail of Bfl-1 functions as a membrane anchor is not yet clearly established. The molecular modeling study of the full-length Bfl-1 performed within this work suggests that Bfl-1 may co-exist in two distinct conformational states: one in which its C-terminal helix α9 is inserted in the hydrophobic groove formed by the BH1–3 domains of Bfl-1 and one with its C terminus. Parallel analysis of the subcellular localization of Bfl-1 indicates that even if Bfl-1 may co-exist in two distinct conformational states, most of the endogenous protein is tightly associated with the mitochondria by its C terminus in both healthy and apoptotic peripheral blood lymphocytes as well as in malignant B cell lines. However, the helix α9 of Bfl-1, and therefore the binding of Bfl-1 to mitochondria, is not absolutely required for the antiapoptotic activity of Bfl-1. A particular feature of Bfl-1 is the amphipathic character of its C-terminal helix α9. Our data clearly indicate that this property of helix α9 is required for the anchorage of Bfl-1 to the mitochondria but also regulates the antiapoptotic function Bfl-1.Apoptosis is a highly regulated process that plays a key role in maintaining cellular homeostasis, and a delicate balance between proapoptotic and antiapoptotic regulators of apoptosis pathways ensures the proper survival of cells in a variety of tissues. Imbalance between proapoptotic and antiapoptotic proteins occurs in diseases such as cancer, where an overexpression of antiapoptotic proteins endows cells with a selective survival advantage that promotes malignancy. Bcl-2 family members are essential regulators of the intrinsic apoptotic pathway, which act at the level of mitochondria as initiators of cell death (1). This family comprises nearly 20 proteins divided into three main groups. Antiapoptotic members such as Bcl-2, Bcl-x_L, Bcl-w, Bfl-1, and Mcl-1 promote cell survival, whereas proapoptotic members such as Bax and Bak function as death effectors. The life and death balance is displaced in favor of cell death by proapoptotic BH3-only proteins such as Bim, Bad, Bid, Puma, and Noxa, which interact with antiapoptotic proteins and inactivate their function (2) or directly interact with and activate the Bax-like proteins (3).Distinct subcellular localizations of antiapoptotic members have been reported correlating with the accessibility of their C-terminal tail. The C-terminal tail of the antiapoptotic proteins Bcl-2, Bcl-x_L, and Bcl-w possess a hydrophobic region known to be a membrane anchor domain. Thus, Bcl-2 localizes to mitochondria as well as to the endoplasmic reticulum and nuclear membranes (4, 5, 6), and deletion of its C-terminal amino acids abrogates its targeting to the outer mitochondrial membrane (7). In contrast, in healthy cells, Bcl-x_L and Bcl-w localize mainly in the cytosol because their C-terminal tails are sequestered. Bcl-x_L exists as a homodimer through the exchange of the C-terminal tail bound in the hydrophobic groove of the reciprocal dimer partner (8), whereas the C-terminal tail of Bcl-w occupies its own hydrophobic groove in the monomer form (9, 10). It has been proposed that, following apoptotic stimuli, interaction of the BH3 domain from BH3-only proteins with the hydrophobic groove of Bcl-w or Bcl-x_L liberates their C-terminal tail and then the two proteins translocate to the mitochondria (8, 11).Unlike Bcl-2, Bcl-x_L, and Bcl-w, Bfl-1 and its murine homolog, A1, do not contain a well defined C-terminal transmembrane domain (12, 13). C-terminal ends of these two proteins are similar and contain several hydrophilic residues that interrupt their putative transmembrane hydrophobic domain. Whether the C-terminal tail of Bfl-1 functions as a membrane anchor remains to be clarified. Immunofluorescence analyses in an earlier study have shown that overexpressed human Bfl-1 is predominantly localized in the endoplasmic/nuclear envelope regions (14). Then, recent independent studies, with Bfl-1-overexpressing cells, suggested that Bfl-1 localizes to the mitochondria (15, 16, 17) and that the C-terminal end of Bfl-1 is important for anchoring Bfl-1 to the mitochondria due to GFP-Bfl-1 being associated to the mitochondria, whereas GFP-Bfl-1, devoid of its C-terminal tail, also localizes in the cytosol (16, 18). However, localization of endogenous Bfl-1 has never been investigated. In this study, we present a molecular modeling study of full-length Bfl-1 (FL-Bfl-1), based on the crystal structure of a truncated form of Bfl-1 (residues 1–149) in complex with the BIM-BH3 peptide (Protein Data Bank code 2VM6).⁴ Our model suggests that Bfl-1 may co-exist in two distinct conformational states, the first one with its C-terminal helix α9 (residues 155–175) inserted in the hydrophobic groove formed by the BH1–3 domain of Bfl-1, and the second one with its C-terminal tail. Interestingly, helical wheel projection of the C-terminal helix of Bfl-1 highlights its amphipathic character, a feature of transmembrane helices or membrane anchors. These observations incited the reinvestigation of the subcellular localization of Bfl-1 in both malignant B cell lines and peripheral blood lymphocytes (PBLs).⁵ We demonstrate here that endogenous Bfl-1 is preferentially anchored to the mitochondria in malignant B cell lines but also in healthy PBLs. Moreover, we show that both the anchorage of Bfl-1 to the mitochondria and the anti-apoptotic function of the protein are dependent on the amphipathic nature of the C-terminal helix.     

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.     

Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery

Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.     

Fowlpox virus encodes a Bcl-2 homologue that protects cells from apoptotic death through interaction with the proapoptotic protein Bak

Poxviruses are renowned for encoding numerous immunomodulatory proteins capable of undermining potent immune defenses. One effective barrier against infection is apoptosis, a process controlled at the mitochondria by pro- and antiapoptotic members of the highly conserved Bcl-2 family of proteins. Although poxviruses are known to encode an array of effective inhibitors of apoptosis, members of the Avipoxvirus genus, which includes fowlpox virus, encode proteins with Bcl-2 homology. Here, we show that FPV039, a fowlpox virus protein with limited Bcl-2 homology, inhibited apoptosis in response to a variety of cytotoxic stimuli, including virus infection itself. Similar to other antiapoptotic Bcl-2 proteins, FPV039 localized predominantly to the mitochondria in both human and chicken cells and protected human cells from tumor necrosis factor alpha-induced loss of the mitochondrial membrane potential. In addition, coimmunoprecipitation revealed that FPV039 interacted constitutively with the proapoptotic Bcl-2 protein, Bak, in both human and chicken cells. Concordantly, FPV039 also inhibited apoptosis induced by the transient overexpression of Bak. To confirm these results in the context of virus infection, we generated a recombinant vaccinia virus lacking F1L, the endogenous apoptotic inhibitor in vaccinia virus, and expressing FPV039. In the context of vaccinia virus infection, FPV039 retained the ability to localize to the mitochondria and interacted with Bak. Moreover, FPV039 prevented the activation of Bak and protected infected cells from apoptosis induced by staurosporine and virus infection. Together, our data indicate that FPV039 is a functional Bcl-2 homologue that inhibits apoptosis by neutralizing the proapoptotic Bcl-2 family member Bak.     

Estrogen decreases TNF-alpha and oxidized LDL induced apoptosis in endothelial cells

Apoptosis induced by oxidized low-density lipoproteins (oxLDL) and tumor necrosis factor-alpha (TNF-alpha) is believed to contribute to atherosclerosis and vascular dysfunction. Estrogen treatment reduces apoptosis due to TNF-alpha and we hypothesized that it would also reduce apoptosis due to oxLDL. We also explored the anti-apoptotic mechanisms. We used early passage human umbilical vein endothelial cells (HUVEC) grown in steroid-depleted, red phenol-free medium. Cells were synchronized by starvation for 6h and then treated with oxLDL (75microg/ml) or TNF-alpha (20ng/ml) in the presence of 17-beta-estradiol (E2) (20nM). Apoptosis was analyzed by flow cytometry and caspase-3 cleavage. We also assessed expression of Bcl-2 and Bcl-xL and phosphorylation of BAD. At 6h TNF-alpha induced apoptosis but oxLDL did not; E2 did not affect this TNF-alpha induced apoptosis and there was no change in Bcl-2 or Bcl-xL expression. At 24h both TNF-alpha and oxLDL increased apoptosis and E2 reduced the increase. E2 also increased expression of the anti-apoptotic Bcl-2 and Bcl-xL and increased phosphorylation of proapoptotic BAD which reduces its proapoptotic activity at 1h. However at 24h there was also an increase in total BAD so that the proportion of phosphorylation of BAD decreased. oxLDL induced apoptosis occurs later than that of TNF-alpha. E2 decreased this late phase apoptosis and this likely requires the production of anti-apoptotic proteins.     

Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function

Apoptosis is initiated when Bcl-2 and its prosurvival relatives are engaged by proapoptotic BH3-only proteins via interaction of its BH3 domain with a groove on the Bcl-2-like proteins. These interactions have been considered promiscuous, but our analysis of the affinity of eight BH3 peptides for five Bcl-2-like proteins has revealed that the interactions vary over 10,000-fold in affinity, and accordingly, only certain protein pairs associate inside cells. Bim and Puma potently engaged all the prosurvival proteins comparably. Bad, however, bound tightly to Bcl-2, Bcl-xL, and Bcl-w but only weakly to A1 and not to Mcl-1. Strikingly, Noxa bound only Mcl-1 and A1. In accord with their complementary binding, Bad and Noxa cooperated to induce potent killing. The results suggest that apoptosis relies on selective interactions between particular subsets of these proteins and that it should be feasible to discover BH3-mimetic drugs that inactivate specific prosurvival targets.     

JNK suppresses apoptosis via phosphorylation of the proapoptotic Bcl-2 family protein BAD

JNK has been suggested to be proapoptotic, antiapoptotic, or have no role in apoptosis depending on the cell type and stimulus used. The precise mechanism of JNK action, under conditions when it promotes cell survival, is not entirely clear. Here, we report that JNK is required for IL-3-mediated cell survival through phosphorylation and inactivation of the proapoptotic Bcl-2 family protein BAD. IL-3 withdrawal-induced apoptosis is promoted by inhibition of JNK but suppressed by expression of a constitutively active JNK. JNK phosphorylates BAD at threonine 201, thereby inhibiting BAD association with the antiapoptotic molecule BCL-X(L). IL-3 induces BAD phosphorylation at threonine 201, and replacement of threonine 201 by alanine generates a BAD mutant, which promotes IL-3 withdrawal-induced apoptosis. Thus, our results provide a molecular mechanism by which JNK contributes to cell survival.     

How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane?

Bcl-2 family members, like the structurally similar translocation domain of diphtheria toxin, can form ion-selective channels and larger-diameter pores in artificial lipid bilayers. Recent studies show how Bcl-2 family members change topology in membranes during apoptosis and that these different states may either promote or inhibit apoptosis. Binding of BH3-only proteins alters the subcellular localization and/or membrane topology and probably affects the channel formation of Bcl-2, Bcl-xL and Bcl-w. However, it remains unclear how the pore-forming activity functions in cells to regulate mitochondrial membrane permeabilization and cell death. Bcl-2 family members in flies and worms regulate apoptosis by mechanisms seemingly unrelated to membrane permeabilization, leaving a unifying model for the biochemical activity of this protein family unknown. Work linking Bcl-2 family members to mitochondrial morphogenesis in worms and mammals suggests some common functions of Bcl-2 family proteins may exist.     

ABT-737 Induces Bim Expression via JNK Signaling Pathway and Its Effect on the Radiation Sensitivity of HeLa Cells

ABT-737 is a BH3 mimetic small molecule inhibitor that can effectively inhibit the activity of antiapoptotic Bcl-2 family proteins including Bcl2, Bcl-xL and Bcl-w, and further enhances the effect of apoptosis by activating the proapoptotic proteins (t-Bid, Bad, Bim). In this study, we demonstrate that ABT-737 improved the radiation sensitivity of cervical cancer HeLa cells and thereby provoked cell apoptosis. Our results show that ABT-737 inhibited HeLa cell proliferation and activated JNK and its downstream target c-Jun, which caused the up-regulation of Bim expression. Blockade of JNK/c-Jun signaling pathway resulted in significant down-regulation of ABT-737-induced Bim mRNA and protein expression level. Also, ABT-737 could evoke the Bim promoter activity, and enhance the radiation sensitivity of HeLa cells via JNK/c-Jun and Bim signaling pathway. Our data imply that combination of ABT-737 and conventional radiation therapy might represent a highly effective therapeutic approach for future treatment of cervical cancer.     

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.     

Reversible membrane interaction of BAD requires two C-terminal lipid binding domains in conjunction with 14-3-3 protein binding

BAD is a Bcl-2 homology domain 3 (BH3)-only proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Binding of BAD to mitochondria is thought to be exclusively mediated by its BH3 domain. We show here that BAD binds to lipids with high affinities, predominantly to negatively charged phospholipids, such as phosphatidylserine, phosphatidic acid, and cardiolipin, as well as to cholesterol-rich liposomes. Two lipid binding domains (LBD1 and LBD2) with different binding preferences were identified, both located in the C-terminal part of the BAD protein. BAD facilitates membrane translocation of Bcl-XL in a process that requires LBD2. Integrity of LBD1 and LBD2 is also required for proapoptotic activity in vivo. Phosphorylation of BAD does not affect membrane binding but renders BAD susceptible to membrane extraction by 14-3-3 proteins. BAD can be removed efficiently by 14-3-3zeta, -eta, -tau and lesxs efficiently by other 14-3-3 isoforms. The assembled BAD.14-3-3 complex exhibited high affinity for cholesterol-rich liposomes but low affinity for mitochondrial membranes. We conclude that BAD is a membrane-associated protein that has the hallmarks of a receptor rather than a ligand. Lipid binding is essential for the proapoptotic function of BAD in vivo. The data support a model in which BAD shuttles in a phosphorylation-dependent manner between mitochondria and other membranes and where 14-3-3 is a key regulator of this relocation. The dynamic interaction of BAD with membranes is tied to activation and membrane translocation of Bcl-XL.     

Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.     

Mechanism of mitochondrial stress-induced resistance to apoptosis in mitochondrial DNA-depleted C2C12 myocytes

In this study, we show that partial mitochondrial DNA (mtDNA) depletion (mitochondrial stress) induces resistance to staurosporine (STP)-mediated apoptosis in C2C12 myoblasts. MtDNA-depleted cells show a 3-4-fold increased proapoptotic proteins (Bax, BAD and Bid), markedly increased antiapoptotic Bcl-2, and reduced processing of p21 Bid to active tBid. The protein levels and also the ability to undergo STP-mediated apoptosis were restored in reverted cells containing near-normal mtDNA levels and restored mitochondrial transmembrane potential. Inhibition of apoptosis closely correlated with sequestration of Bax, Bid and BAD in the mitochondrial inner membrane, increased Bcl-2 and Bcl-X(L), and inability to process p21 Bid. These factors, together with the reduced activation of caspases 3, 9 and 8 are possible causes of mitochondrial stress-induced resistance to apoptosis. Our results suggest that a highly proliferative and invasive behavior of mtDNA-depleted C2C12 cells is related to their resistance to apoptosis.     

Differential interactions between Beclin 1 and Bcl-2 family members

Autophagy, a cellular degradation system, promotes both cell death and survival. The interaction between Bcl-2 family proteins and Beclin 1, a Bcl-2 interacting protein that promotes autophagy, can mediate crosstalk between autophagy and apoptosis. We investigated the interaction between anti-and pro-apoptotic Bcl-2 proteins with Beclin 1. Our results show that Beclin 1 directly interacts with Bcl-2, Bcl-x(L), Bcl-w and to a lesser extent with Mcl-1. Beclin 1 does not bind the pro-apoptotic Bcl-2 proteins. The interaction between Beclin 1 and the anti-apoptotic protein Bcl-x(L) was inhibited by BH3-only proteins, but not by multi-domain proteins. Sequence alignment and structural modeling suggest that Beclin 1 contains a putative BH3-like domain which may interact with the hydrophobic grove of Bcl-x(L). Mutation of the Beclin 1 amino acids predicted to mediate this interaction inhibited the association of Beclin 1 with Bcl-x(L). Our results suggest that BH3 only proapoptotic Bcl-2 proteins may modulate the interactions between Bcl-x(L) and Beclin 1.     

Bcl-2-related protein family gene expression during oligodendroglial differentiation

Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All 'multidomain' anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for 'multidomain' pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among 'BH3 domain-only' members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.     

Conversion of Bfl-1, an anti-apoptotic Bcl-2 family protein,to a potent pro-apoptotic protein by fusion with green fluorescent protein (GFP)

Human Bfl-1 is an anti-apoptotic Bcl-2 family member. Here, we found that Bfl-1 was converted into a potent death-promoting protein by green fluorescent protein (GFP) fusion with its N-terminus. The transient expression of GFP-Bfl-1 induced cytochrome c release and triggered apoptosis in 293T cells, which depended on the mitochondrial localization of GFP-Bfl-1. Apoptosis induced by GFP-Bfl-1 was significantly blocked by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone, but was not blocked by either Bcl-xL or Bfl-1. Our findings provide a useful model for understanding the structural basis of Bcl-2 family proteins that act in an opposite way despite sharing structural similarity between anti-apoptotic and pro-apoptotic proteins.     

Dynamics of the BH3-Only Protein Binding Interface of Bcl-xL

The balance and interplay between pro-death and pro-survival members of the B-cell lymphoma-2 (Bcl-2) family proteins play key roles in regulation of the mitochondrial pathway of programmed cell death. Recent NMR and biochemical studies have revealed that binding of the proapoptotic BH3-only protein PUMA induces significant unfolding of antiapoptotic Bcl-xL at the interface, which in turn disrupts the Bcl-xL/p53 interaction to activate apoptosis. However, the molecular mechanism of such regulated unfolding of Bcl-xL is not fully understood. Analysis of the existing Protein Data Bank structures of Bcl-xL in both bound and unbound states reveal substantial intrinsic heterogeneity at its BH3-only protein binding interface. Large-scale atomistic simulations were performed in explicit solvent for six representative structures to further investigate the intrinsic conformational dynamics of Bcl-xL. The results support that the BH3-only protein binding interface of Bcl-xL is much more dynamic compared to the rest of the protein, both unbound and when bound to various BH3-only proteins. Such intrinsic interfacial conformational dynamics likely provides a physical basis that allows Bcl-xL to respond sensitively to detailed biophysical properties of the ligand. The ability of Bcl-xL to retain or even enhance dynamics at the interface in bound states could further facilitate the regulation of its interactions with various BH3-only proteins such as through posttranslational modifications.