A yeast BH3-only protein mediates the mitochondrial pathway of apoptosis

Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-X(L) and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.     

Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site

The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation.     

Lipidic pore formation by the concerted action of proapoptotic BAX and tBID

BCL-2 homology 3 (BH3)-only proteins of the BCL-2 family such as tBID and BIM(EL) assist BAX-type proteins to breach the permeability barrier of the outer mitochondrial membrane, thereby allowing cytoplasmic release of cytochrome c and other active inducers of cell death normally confined to the mitochondrial inter-membrane space. However, the exact mechanism by which tBID and BIM(EL) aid BAX and its close homologues in this mitochondrial protein release remains enigmatic. Here, using pure lipid vesicles, we provide evidence that tBID acts in concert with BAX to 1) form large membrane openings through both BH3-dependent and BH3-independent mechanisms, 2) cause lipid transbilayer movement concomitant with membrane permeabilization, and 3) disrupt the lipid bilayer structure of the membrane by promoting positive monolayer curvature stress. None of these effects were observed with BAX when BIM(EL) was substituted for tBID. Based on these data, we propose a novel model in which tBID assists BAX not only via protein-protein but also via protein-lipid interactions to form lipidic pore-type non-bilayer structures in the outer mitochondrial membrane through which intermembrane prodeath molecules exit mitochondria during apoptosis.     

Neurons exclusively express N-Bak, a BH3 domain-only Bak isoform that promotes neuronal apoptosis

Bak is generally recognized as a multidomain, pro-apoptotic member of the Bcl-2 family. Bak and Bax are functionally redundant in non-neuronal cells and represent a mitochondrial convergence point for cell death signaling pathways. This functional redundancy, however, may not exist in neurons in which the single deletion of Bax is sufficient to confer protection against a variety of cytotoxic insults. In the present study, we demonstrate that postnatal cortical and cerebellar granule neurons exclusively express an alternatively spliced, BH3 domain-only form of Bak (N-Bak), whereas astrocytes express only the full-length, multidomain form. Overexpression of N-Bak promotes Bax translocation in HeLa cells and induces neuronal cell death in cortical, hippocampal, and cerebellar granule neurons in a Bax-dependent manner. N-Bak interacts with Bcl-XL but not BAX, suggesting an indirect mechanism for promoting Bax translocation to the mitochondria. N-Bak message and protein levels are elevated in cortical neurons in response to DNA damage, and subsequent induction of neuronal death is significantly delayed by expressing a full-length Bak antisense plasmid. These results demonstrate that postnatal neurons solely express a BH3 domain-only form of Bak, which contributes to DNA damage-induced neuronal apoptosis. The absence of full-length Bak expression explains the near exclusive requirement for Bax in neuronal apoptosis.     

MCL-1ES Induces MCL-1L-Dependent BAX- and BAK-Independent Mitochondrial Apoptosis

MCL-1 (myeloid cell leukemia-1), a member of the BCL-2 family, has three splicing variants, antiapoptotic MCL-1L, proapoptotic MCL-1S, and MCL-1ES. We previously reported cloning MCL-1ES and characterizing it as an apoptotic molecule. Here, we investigated the molecular mechanism by which MCL-1ES promotes cell death. MCL-1ES was distinct from other proapoptotic BCL-2 members that induce apoptosis by promoting BAX or BAK oligomerization, leading to mitochondrial outer membrane permeabilization (MOMP), in that MCL-1ES promoted mitochondrial apoptosis independently of both BAX and BAK. Instead, MCL-1L was crucial for the apoptotic activity of MCL-1ES by facilitating its proper localization to the mitochondria. MCL-1ES did not interact with any BCL-2 family proteins except for MCL-1L, and antiapoptotic BCL-2 members failed to inhibit apoptosis induced by MCL-1ES. The BCL-2 homology 3 (BH3) domain of MCL-1ES was critical for both MCL-1ES association with MCL-1L and apoptotic activity. MCL-1ES formed mitochondrial oligomers, and this process was followed by MOMP and cytochrome c release in a MCL-1L-dependent manner. These findings indicate that MCL-1ES, as a distinct proapoptotic BCL-2 family protein, may be useful for intervening in diseases that involve uncontrolled MCL-1L.     

A stapled BID BH3 helix directly binds and activates BAX

BAX is a multidomain proapoptotic BCL-2 family protein that resides in the cytosol until activated by an incompletely understood trigger mechanism, which facilitates BAX translocation to mitochondria and downstream death events. Whether BAX is activated by direct contact with select BH3-only members of the BCL-2 family is highly debated. Here we detect and quantify a direct binding interaction between BAX and a hydrocarbon-stapled BID BH3 domain, which triggers the functional activation of BAX at nanomolar doses in vitro. Chemical reinforcement of BID BH3 alpha helicity was required to reveal the direct BID BH3-BAX association. We confirm the specificity of this BH3 interaction by characterizing a stapled BAD BH3 peptide that interacts with antiapoptotic BCL-X(L) but does not bind or activate BAX. We further demonstrate that membrane targeting of stapled BID BH3 optimizes its ability to activate BAX, supporting a model in which BID directly engages BAX to trigger mitochondrial apoptosis.     

Upregulation of Bcl2 inhibits apoptosis-driven BAX insertion but favors BAX relocalization in mitochondria

Protein-protein interactions between the Bcl2 family proteins regulate apoptosis. An imbalance of this interaction network due to the upregulation of the proto-oncogene Bcl2 leads to a resistance to apoptosis associated with tumor formation. Bcl2 overexpression inhibits BAX oligomerization and mitochondrial outer membrane (MOM) permeabilization. However, Bcl2 effects on earlier steps of BAX-mediated apoptosis are not fully understood. Bcl2 overexpression inhibits BAX insertion into the MOM but spontaneously increases BAX relocalization to the mitochondria. Also, a physical interaction between BAX and Bcl2 is necessary for these two effects to occur. Taken together, these results suggest upregulated Bcl2 stabilizes BAX loose binding to mitochondrial membranes, inhibiting its insertion into the MOM and consequently cytochrome c release.

Structured summary

MINT-7945271: BAX (uniprotkb:Q07813) physically interacts (MI:0915) with Bcl-2 (uniprotkb:P10417) by anti bait coimmunoprecipitation (MI:0006)     

Src Regulates the Activity of the ING1 Tumor Suppressor

The INhibitor of Growth 1 (ING1) is stoichiometric member of histone deacetylase (HDAC) complexes and functions as an epigenetic regulator and a type II tumor suppressor. It impacts cell growth, aging, apoptosis, and DNA repair, by affecting chromatin conformation and gene expression. Down regulation and mislocalization of ING1 have been reported in diverse tumor types and Ser/Thr phosphorylation has been implicated in both of these processes. Here we demonstrate that both in vitro and in vivo, the tyrosine kinase Src is able to physically associate with, and phosphorylate ING1, which results in a nuclear to cytoplasmic relocalization of ING1 in cells and a decrease of ING1 stability. Functionally, Src antagonizes the ability of ING1 to induce apoptosis, most likely through relocalization of ING1 and down regulation of ING1 levels. These effects were due to both kinase-dependent and kinase-independent properties of Src, and were most apparent at elevated levels of Src expression. These findings suggest that Src may play a major role in regulating ING1 levels during tumorigenesis in those cancers in which high levels of Src expression or activity are present. These data represent the first report of tyrosine kinase-mediated regulation of ING1 levels and suggest that kinase activation can impact chromatin structure through the ING1 epigenetic regulator.     

Selenium Compounds Induced ROS-Dependent Apoptosis in Myelodysplasia Cells

Several authors have demonstrated the chemoprotective and anti-carcinogenic role of selenium. However, the therapeutic potential of selenium in myelodysplastic syndrome (MDS) as single agent and as co-adjuvant of the current therapies has not been previously studied. Sodium selenite and selenomethionine, alone and in combination with cytarabine, induce a decrease in cell viability in a time-, dose- and administration-dependent manner inducing cell death by apoptosis in F36P cells (MDS cell line). These compounds increased superoxide production and induced mitochondrial membrane depolarization. The increase in BAX/BCL-2 ratio and in the activated caspase 3 expression levels, the decrease in mitochondria membrane potential, as well as the increase in superoxide production, supports the mitochondria contribution on selenium-induced apoptosis. These findings suggest that selenium may offer a new therapeutic approach in myelodysplastic syndrome in monotherapy and/or as co-adjuvant therapy to conventional anti-carcinogenic.     

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.     

Platinum resistant cancer cells conserve sensitivity to BH3 domains and obatoclax induced mitochondrial apoptosis

Resistance to cisplatin chemotherapy remains a major hurdle preventing effective treatment of many solid cancers. BAX and BAK are pivotal regulators of the mitochondrial apoptosis pathway, however little is known regarding their regulation in cisplatin resistant cells. Cisplatin induces DNA damage in both sensitive and resistant cells, however the latter exhibits a failure to initiate N-terminal exposure of mitochondrial BAK or mitochondrial SMAC release. Both phenotypes are highly sensitive to mitochondrial permeabilisation induced by exogenous BH3 domain peptides derived from BID, BIM, NOXA (which targets MCL-1 and A1), and there is no significant change in their prosurvival BCL2 protein expression profiles. Obatoclax, a small molecule inhibitor of pro-survival BCL-2 family proteins including MCL-1, decreases cell viability irrespective of platinum resistance status across a panel of cell lines selected for oxaliplatin resistance. In summary, selection for platinum resistance is associated with a block of mitochondrial death signalling upstream of BAX/BAK activation. Conservation of sensitivity to BH3 domain induced apoptosis can be exploited by agents such as obatoclax, which directly target the mitochondria and BCL-2 family.     

The MUC1-C oncoprotein binds to the BH3 domain of the pro-apoptotic BAX protein and blocks BAX function

The pro-apoptotic BAX protein contains a BH3 domain that is necessary for its dimerization and for activation of the intrinsic apoptotic pathway. The MUC1 (mucin 1) heterodimeric protein is overexpressed in diverse human carcinomas and blocks apoptosis in the response to stress. In this study, we demonstrate that the oncogenic MUC1-C subunit associates with BAX in human cancer cells. MUC1-C·BAX complexes are detectable in the cytoplasm and mitochondria and are induced by genotoxic and oxidative stress. The association between MUC1-C and BAX is supported by the demonstration that the MUC1-C cytoplasmic domain is sufficient for the interaction with BAX. The results further show that the MUC1-C cytoplasmic domain CQC motif binds directly to the BAX BH3 domain at Cys-62. Consistent with binding to the BAX BH3 domain, MUC1-C blocked BAX dimerization in response to (i) truncated BID in vitro and (ii) treatment of cancer cells with DNA-damaging agents. In concert with these results, MUC1-C attenuated localization of BAX to mitochondria and the release of cytochrome c. These findings indicate that the MUC1-C oncoprotein binds directly to the BAX BH3 domain and thereby blocks BAX function in activating the mitochondrial death pathway.     

Biochemical and genetic analysis of the mitochondrial response of yeast to BAX and BCL-X(L)

The BCL-2 family includes both proapoptotic (e.g., BAX and BAK) and antiapoptotic (e.g., BCL-2 and BCL-X(L)) molecules. The cell death-regulating activity of BCL-2 members appears to depend on their ability to modulate mitochondrial function, which may include regulation of the mitochondrial permeability transition pore (PTP). We examined the function of BAX and BCL-X(L) using genetic and biochemical approaches in budding yeast because studies with yeast suggest that BCL-2 family members act upon highly conserved mitochondrial components. In this study we found that in wild-type yeast, BAX induced hyperpolarization of mitochondria, production of reactive oxygen species, growth arrest, and cell death; however, cytochrome c was not released detectably despite the induction of mitochondrial dysfunction. Coexpression of BCL-X(L) prevented all BAX-mediated responses. We also assessed the function of BCL-X(L) and BAX in the same strain of Saccharomyces cerevisiae with deletions of selected mitochondrial proteins that have been implicated in the function of BCL-2 family members. BAX-induced growth arrest was independent of the tested mitochondrial components, including voltage-dependent anion channel (VDAC), the catalytic beta subunit or the delta subunit of the F(0)F(1)-ATP synthase, mitochondrial cyclophilin, cytochrome c, and proteins encoded by the mitochondrial genome as revealed by [rho(0)] cells. In contrast, actual cell killing was dependent upon select mitochondrial components including the beta subunit of ATP synthase and mitochondrial genome-encoded proteins but not VDAC. The BCL-X(L) protection from either BAX-induced growth arrest or cell killing proved to be independent of mitochondrial components. Thus, BAX induces two cellular processes in yeast which can each be abrogated by BCL-X(L): cell arrest, which does not require aspects of mitochondrial biochemistry, and cell killing, which does.     

BH3 Peptides Induce Mitochondrial Fission and Cell Death Independent of BAX/BAK

BH3 only proteins trigger cell death by interacting with pro- and anti-apoptotic members of the BCL-2 family of proteins. Here we report that BH3 peptides corresponding to the death domain of BH3-only proteins, which bind all the pro-survival BCL-2 family proteins, induce cell death in the absence of BAX and BAK. The BH3 peptides did not cause the release of cytochrome c from isolated mitochondria or from mitochondria in cells. However, the BH3 peptides did cause a decrease in mitochondrial membrane potential but did not induce the opening of the mitochondrial permeability transition pore. Interestingly, the BH3 peptides induced mitochondria to undergo fission in the absence of BAX and BAK. The binding of BCL-X_L with dynamin-related protein 1 (DRP1), a GTPase known to regulate mitochondrial fission, increased in the presence of BH3 peptides. These results suggest that pro-survival BCL-2 proteins regulate mitochondrial fission and cell death in the absence of BAX and BAK.     

MCL-1 inhibits BAX in the absence of MCL-1/BAX Interaction

The BCL-2 family of proteins plays a major role in the control of apoptosis as the primary regulator of mitochondrial permeability. The pro-apoptotic BCL-2 homologues BAX and BAK are activated following the induction of apoptosis and induce cytochrome c release from mitochondria. A second class of BCL-2 homologues, the BH3-only proteins, is required for the activation of BAX and BAK. The activity of both BAX/BAK and BH3-only proteins is opposed by anti-apoptotic BCL-2 homologues such as BCL-2 and MCL-1. Here we show that anti-apoptotic MCL-1 inhibits the function of BAX downstream of its initial activation and translocation to mitochondria. Although MCL-1 interacted with BAK and inhibited its activation, the activity of MCL-1 against BAX was independent of an interaction between the two proteins. However, the anti-apoptotic function of MCL-1 required the presence of BAX. These results suggest that the pro-survival activity of MCL-1 proceeds via inhibition of BAX function at mitochondria, downstream of its activation and translocation to this organelle.     

Caffeine sensitizes human H358 cell line to p53-mediated apoptosis by inducing mitochondrial translocation and conformational change of BAX protein

The mechanisms involved in p53-mediated cell death remain controversial. In the present study, we investigated this cell death pathway by stably transfecting the p53-null H358 cell line with a tetracycline-dependent wild type p53-expressing vector. Restoration of p53 triggered a G(2)/M cell cycle arrest and enhanced BAX protein expression, without inducing apoptosis or potentiating the cytotoxic effect of etoposide, vincristine, and cis-platinum. Accordingly, overexpression of BAX in H358 cells, through stable transfection of a tetracycline-regulated expression vector, did not induce cell death. Interestingly, the methylxanthine caffeine (4 mm) promoted the translocation of BAX from the cytosol to the mitochondria. In the setting of an overexpression of BAX, caffeine induced a conformational change of the protein and apoptosis. The consequences of caffeine were independent of its cell cycle-related activities. All together, caffeine synergizes with p53 for inducing cell death through a cell cycle-independent mechanism, involving mitochondrial translocation and conformational change of BAX protein.