The third model of Bax/Bak activation: a Bcl-2 family feud finally resolved?

Bax and Bak, two functionally similar, pro-apoptotic proteins of the Bcl-2 family, are known as the gateway to apoptosis because of their requisite roles as effectors of mitochondrial outer membrane permeabilization (MOMP), a major step during mitochondria-dependent apoptosis. The mechanism of how cells turn Bax/Bak from inert molecules into fully active and lethal effectors had long been the focal point of a major debate centered around two competing, but not mutually exclusive, models: direct activation and indirect activation. After intensive research efforts for over two decades, it is now widely accepted that to initiate apoptosis, some of the BH3-only proteins, a subclass of the Bcl-2 family, directly engage Bax/Bak to trigger their conformational transformation and activation. However, a series of recent discoveries, using previously unavailable CRISPR-engineered cell systems, challenge the basic premise that undergirds the consensus and provide evidence for a novel and surprisingly simple model of Bax/Bak activation: the membrane (lipids)-mediated spontaneous model. This review will discuss the evidence, rationale, significance, and implications of this new model.

Structural insight into tanapoxvirus-mediated inhibition of apoptosis.

Premature programmed cell death or apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Tanapoxvirus (TANV) is a large double-stranded DNA virus belonging to the poxviridae that causes mild monkeypox-like infections in humans and primates. TANV encodes for a putative apoptosis inhibitory protein 16L. We show that TANV16L is able to bind to a range of peptides spanning the BH3 motif of human proapoptotic Bcl-2 proteins and is able to counter growth arrest of yeast induced by human Bak and Bax. We then determined the crystal structures of TANV16L bound to three identified interactors, Bax, Bim and Puma BH3. TANV16L adopts a globular Bcl-2 fold comprising 7 α-helices and utilizes the canonical Bcl-2 binding groove to engage proapoptotic host cell Bcl-2 proteins. Unexpectedly, TANV16L is able to adopt both a monomeric and a domain-swapped dimeric topology where the α1 helix from one protomer is swapped into a neighbouring unit. Despite adopting two different oligomeric forms, the canonical ligand binding groove in TANV16L remains unchanged from monomer to domain-swapped dimer. Our results provide a structural and mechanistic basis for tanapoxvirus-mediated inhibition of host cell apoptosis and reveal the capacity of Bcl-2 proteins to adopt differential oligomeric states whilst maintaining the canonical ligand binding groove in an unchanged state. DATABASE: Structural data are available in the Protein Data Bank (PDB) under the accession numbers 6TPQ, 6TQQ and 6TRR.

A small molecule interacts with VDAC2 to block mouse BAK-driven apoptosis.

Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.

A non-death function of the mitochondrial apoptosis apparatus in immunity.

Apoptosis is a frequent form of programmed cell death, but the apoptotic signaling pathway can also be engaged at a low level, in the absence of cell death. We here report that such sub-lethal engagement of mitochondrial apoptosis signaling causes the secretion of cytokines from human epithelial cells in a process controlled by the Bcl-2 family of proteins. We further show that sub-lethal signaling of the mitochondrial apoptosis pathway is initiated by infections with all tested viral, bacterial, and protozoan pathogens and causes damage to the genomic DNA. Epithelial cells infected with these pathogens secreted cytokines, and this cytokine secretion upon microbial infection was substantially reduced if mitochondrial sub-lethal apoptosis signaling was blocked. In the absence of mitochondrial pro-apoptotic signaling, the ability of epithelial cells to restrict intracellular bacterial growth was impaired. Triggering of the mitochondrial apoptosis apparatus thus not only causes apoptosis but also has an independent role in immune defense.

Increased Ripk1-mediated bone marrow necroptosis leads to myelodysplasia and bone marrow failure in mice.

Hematopoiesis is a dynamic system that requires balanced cell division, differentiation, and death. The 2 major modes of programmed cell death, apoptosis and necroptosis, share molecular machinery but diverge in outcome with important implications for the microenvironment; apoptotic cells are removed in an immune silent process, whereas necroptotic cells leak cellular contents that incite inflammation. Given the importance of cytokine-directed cues for hematopoietic cell survival and differentiation, the impact on hematopoietic homeostasis of biasing cell death fate to necroptosis is substantial and poorly understood. Here, we present a mouse model with increased bone marrow necroptosis. Deletion of the proapoptotic Bcl-2 family members Bax and Bak inhibits bone marrow apoptosis. Further deletion of the BH3-only member Bid (to generate Vav CreBaxBakBid triple-knockout [TKO] mice) leads to unrestrained bone marrow necroptosis driven by increased Rip1 kinase (Ripk1). TKO mice display loss of progenitor cells, leading to increased cytokine production and increased stem cell proliferation and exhaustion and culminating in bone marrow failure. Genetically restoring Ripk1 to wild-type levels restores peripheral red cell counts as well as normal cytokine production. TKO bone marrow is hypercellular with abnormal differentiation, resembling the human disorder myelodysplastic syndrome (MDS), and we demonstrate increased necroptosis in MDS bone marrow. Finally, we show that Bid impacts necroptotic signaling through modulation of caspase-8-mediated Ripk1 degradation. Thus, we demonstrate that dysregulated necroptosis in hematopoiesis promotes bone marrow progenitor cell death that incites inflammation, impairs hematopoietic stem cells, and recapitulates the salient features of the bone marrow failure disorder MDS.

Differential role of FL-BID and t-BID during verotoxin-1-induced apoptosis in Burkitt's lymphoma cells.

The globotriaosylceramide Gb3 is a glycosphingolipid expressed on a subpopulation of germinal center B lymphocytes which has been recognized as the B cell differentiation antigen CD77. Among tumoral cell types, Gb3/CD77 is strongly expressed in Burkitt's lymphoma (BL) cells as well as other solid tumors including breast, testicular and ovarian carcinomas. One known ligand of Gb3/CD77 is Verotoxin-1 (VT-1), a Shiga toxin produced in specific E. coli strains. Previously, we have reported that in BL cells, VT-1 induces apoptosis via a caspase-dependent and mitochondria-dependent pathway. Yet, the respective roles of various apoptogenic factors remained to be deciphered. Here, this apoptotic pathway was found to require cleavage of the BID protein by caspase-8 as well as activation of two other apoptogenic proteins, BAK and BAX. Surprisingly however, t-BID, the truncated form of BID resulting from caspase-8 cleavage, played no role in the conformational changes of BAK and BAX. Rather, their activation occurred under the control of full length BID (FL-BID). Indeed, introducing a non-cleavable form of BID (BID-D59A) into BID-deficient BL cells restored BAK and BAX activation following VT-1 treatment. Still, t-BID was involved along with FL-BID in the BAK-dependent and BAX-dependent cytosolic release of CYT C and SMAC/DIABLO from the mitochondrial intermembrane space: FL-BID was found to control the homo-oligomerization of both BAK and BAX, likely contributing to the initial release of CYT C and SMAC/DIABLO, while t-BID was needed for their hetero-oligomerization and ensuing release amplification. Together, our results reveal a functional cooperation between BAK and BAX during VT-1-induced apoptosis and, unexpectedly, that activation of caspase-8 and production of t-BID were not mandatory for initiation of the cell death process.

Ectopic overexpression of LAPTM5 results in lysosomal targeting and induces Mcl-1 down-regulation, Bak activation, and mitochondria-dependent apoptosis in human HeLa cells.

Human lysosomal-associated protein multispanning membrane 5 (LAPTM5) was identified by an ordered differential display-polymerase chain reaction (ODD-PCR) as an up-regulated cDNA fragment during 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced differentiation of U937 cells into monocytes/macrophages. After TPA-treatment, the levels of LAPTM5 mRNA and protein increased and reached a maximum at 18-36 h. In healthy human tissues, LAPTM5 mRNA was expressed at high levels in hematopoietic cells and tissues, at low levels in the lung and fetal liver, and was not detected in other non-hematopoietic tissues. LAPTM5 mRNA was detected in immature malignant cells of myeloid lineage, such as K562, HL-60, U937, and THP-1 cells, and in unstimulated peripheral T cells, but was absent or barely detectable in lymphoid malignant or non-hematopoietic malignant cells. The LAPTM5 level in HL-60 cells increased more significantly during TPA-induced monocyte/macrophage differentiation than during DMSO-induced granulocyte differentiation. Ectopic expression of GFP-LAPTM5 or LAPTM5 in HeLa cells exhibited the localization of LAPTM5 to the lysosome. In HeLa cells overexpressing LAPTM5, the Mcl-1 and Bid levels declined markedly and apoptosis was induced via Bak activation, Δψm loss, activation of caspase-9, -8 and -3, and PARP degradation without accompanying necrosis. However, these LAPTM5-induced apoptotic events except for the decline of Bid level were completely abrogated by concomitant overexpression of Mcl-1. The pan-caspase inhibitor (z-VAD-fmk) could suppress the LAPTM5-induced apoptotic sub-G1 peak by ~40% but failed to block the induced Δψm loss, whereas the broad-range inhibitor of cathepsins (Cathepsin Inhibitor I) could suppress the LAPTM5-induced apoptotic sub-G1 peak and Δψm loss, by ~22% and ~23%, respectively, suggesting that the LAPTM5-mediated Δψm loss was exerted at least in part in a cathepsin-dependent manner. Together, these results demonstrate that ectopic overexpression of LAPTM5 in HeLa cells induced apoptosis via cleavage of Mcl-1 and Bid by a LAPTM5-associated lysosomal pathway, and subsequent activation of the mitochondria-dependent caspase cascade.

Developmental Regulation of Mitochondrial Apoptosis by c-Myc Governs Age- and Tissue-Specific Sensitivity to Cancer Therapeutics.

It is not understood why healthy tissues can exhibit varying levels of sensitivity to the same toxic stimuli. Using BH3 profiling, we find that mitochondria of many adult somatic tissues, including brain, heart, and kidneys, are profoundly refractory to pro-apoptotic signaling, leading to cellular resistance to cytotoxic chemotherapies and ionizing radiation. In contrast, mitochondria from these tissues in young mice and humans are primed for apoptosis, predisposing them to undergo cell death in response to genotoxic damage. While expression of the apoptotic protein machinery is nearly absent by adulthood, in young tissues its expression is driven by c-Myc, linking developmental growth to cell death. These differences may explain why pediatric cancer patients have a higher risk of developing treatment-associated toxicities.

The Roles of the Interaction of BCL2-Antagonist/Killer 1, Apoptotic Peptidase Activating Factor 1 and Selenium in the Pathogenesis of Kashin-Beck Disease.

BCL2-antagonist/killer 1 (BAK1) and apoptotic peptidase activating factor 1 (APAF1) are significant genes in apoptosis signalling pathway of Kashin-Beck disease (KBD). We aimed to verify the protein expression levels of BAK1 and APAF1 in the cartilage and chondrocytes of patients with KBD. Additionally, we explored the relationship between the levels of these proteins and selenium concentration. Chondrocytes was cultured and treated with sodium selenite in vitro. Immunohistochemistry and Western blotting were used to verify the expression levels of BAK1 and APAF1. Compared with the control samples, APAF1 was upregulated and BAK1 was downregulated in the cartilage and chondrocytes of KBD patients. APAF1 expression was higher in the middle and deep zone in the KBD cartilage. APAF1 levels decreased gradually with the increasing selenium concentration (0.05, 0.10 and 0.25 mg/L). BAK1 expression in the 0.25 mg/L selenium group was lower than that of the control group. Different selenium concentrations had varying effects on BAK1 and APAF1 levels. APAF1 may play an important role in the pathogenesis of KBD. APAF1-related apoptosis was more pronounced in the middle and deep zones of the KBD cartilage. APAF may represent a potentially novel molecular target, which may be a biomarker of the role of selenium on the prevention and treatment of KBD. The role of BAK1 in the pathogenesis of KBD requires further study.

Impact of the combined loss of BOK, BAX and BAK on the hematopoietic system is slightly more severe than compound loss of BAX and BAK.

It is well established that BAX and BAK play crucial, overlapping roles in the intrinsic pathway of apoptosis. Gene targeted mice lacking both BAX and BAK have previously been generated, but the majority of these animals died perinatally. BOK is a poorly studied relative of BAX and BAK that shares extensive amino acid sequence homology to both proteins, but its function remains largely unclear to date. To determine whether BOK plays an overlapping role with BAX and BAK, we utilized a hematopoietic reconstitution model where lethally irradiated wild type mice were transplanted with Bok(-/-)Bax(-/-)Bak(-/-) triple knockout (TKO) fetal liver cells, and compared alongside mice reconstituted with a Bax(-/-)Bak(-/-) double knockout (DKO) hematopoietic compartment. We report here that mice with a TKO and DKO hematopoietic system died at a similar rate and much earlier than control animals, mostly due to severe autoimmune pathology. Both TKO and DKO reconstituted mice also had altered frequencies of various leukocyte subsets in the thymus, bone marrow and spleen, displayed leukocyte infiltrates and autoimmune pathology in multiple tissues, as well as elevated levels of anti-nuclear autoantibodies. Interestingly, the additional deletion of BOK (on top of BAX and BAK loss) led to a further increase in peripheral blood lymphocytes, as well as enhanced lymphoid infiltration in some organs. These findings suggest that BOK may have some functions that are redundant with BAX and BAK in the hematopoietic system.

Proapoptotic activities of protein disulfide isomerase (PDI) and PDIA3 protein, a role of the Bcl-2 protein Bak.

Protein disulfide isomerase (PDI) family proteins are classified as enzymatic chaperones for reconstructing misfolded proteins. Previous studies have shown that several PDI members possess potential proapoptotic functions. However, the detailed molecular mechanisms of PDI-mediated apoptosis are not completely known. In this study, we investigated how two members of PDI family, PDI and PDIA3, modulate apoptotic signaling. Inhibiting PDI and PDIA3 activities pharmacologically alleviates apoptosis induced by various apoptotic stimuli. Although a decrease of PDIA3 expression alleviates apoptotic responses, overexpression of PDIA3 exacerbates apoptotic signaling. Importantly, Bak, but not Bax, is essential for PDIA3-induced proapoptotic signaling. Furthermore, both purified PDI and PDIA3 proteins induce Bak-dependent, but not Bax-dependent, mitochondrial outer membrane permeabilization in vitro, probably through triggering Bak oligomerization on mitochondria. Our results suggest that both of PDI and PDIA3 possess Bak-dependent proapoptotic function through inducing mitochondrial outer membrane permeabilization, which provides a new mechanism linking ER chaperone proteins and apoptotic signaling.

Role of Bax/Bcl-2 family members in green tea polyphenol induced necroptosis of p53-deficient Hep3B cells.

Green tea polyphenol (GTP) is one of the most promising chemopreventive agent for cancer; it can inhibit cancer cell proliferation and induce apoptosis through p53-dependent cell signaling pathways. Unfortunately, many tumor cells lack the functional p53, and little is known about the effect of GTP on the p53-deficient/mutant cancer cells. To understand the p53-independent mechanisms in GTP-treated p53-dificient/mutant cancer cells, we have now examined GTP-induced cytotoxicity in human hepatoma Hep3B cells (p53-deficient). The results showed that GTP could induce Bax and Bak activation, cytochrome c release, caspase activation, and necroptosis of Hep3B cells. Bax and Bak, two key molecules of mitochondrial permeability transition pore (MPTP), were interdependently activated by GTP, with translocation and homo-oligomerization on the mitochondria. Bax and Bak induce cytochrome c release. Importantly, cytochrome c release and necroptosis were diminished in Hep3B cells (Bax(-/-)) and Hep3B cells (Bak(-/-)). Furthermore, overexpression of Bcl-2 could ameliorate GTP-induced cytochrome c release and necroptosis. Together, the findings suggested that GTP-induced necroptosis was modulated by the p53-independent pathway, which was related to the translocation of Bax and Bak to mitochondria, release of cytochrome c, and activation of caspases.

Bak and Mcl-1 are essential for Temozolomide induced cell death in human glioma.

Temozolomide (TMZ) is an alkylating agent used for the treatment of glioblastoma multiforme (GBM), the main form of human brain tumours in adults. It has been reported that TMZ induced DNA lesions that subsequently trigger cell death but the actual mechanisms involved in the process are still unclear. We investigated the implication of major proteins of the Bcl-2 family in TMZ-induced cell death in GBM cell lines at concentrations closed to that reached in the brain during the treatments. We did not observe modulation of autophagy at these concentrations but we found an induction of apoptosis. Using RNA interference, we showed that TMZ induced apoptosis is dependent on the pro-apoptotic protein Bak but independent of the pro-apoptotic protein Bax. Apoptosis was not enhanced by ABT-737, an inhibitor of Bcl-2/Bcl-Xl/Bcl-W but not Mcl-1. The knock-down of Mcl-1 expression increased TMZ induced apoptosis. Our results identify a Mcl-1/Bak axis for TMZ induced apoptosis in GBM and thus unravel a target to overcome therapeutic resistance toward TMZ.

3-Substituted-N-(4-hydroxynaphthalen-1-yl)arylsulfonamides as a novel class of selective Mcl-1 inhibitors: structure-based design, synthesis, SAR, and biological evaluation.

Mcl-1, an antiapoptotic member of the Bcl-2 family of proteins, is a validated and attractive target for cancer therapy. Overexpression of Mcl-1 in many cancers results in disease progression and resistance to current chemotherapeutics. Utilizing high-throughput screening, compound 1 was identified as a selective Mcl-1 inhibitor and its binding to the BH3 binding groove of Mcl-1 was confirmed by several different, but complementary, biochemical and biophysical assays. Guided by structure-based drug design and supported by NMR experiments, comprehensive SAR studies were undertaken and a potent and selective inhibitor, compound 21, was designed which binds to Mcl-1 with a Ki of 180 nM. Biological characterization of 21 showed that it disrupts the interaction of endogenous Mcl-1 and biotinylated Noxa-BH3 peptide, causes cell death through a Bak/Bax-dependent mechanism, and selectively sensitizes Eµ-myc lymphomas overexpressing Mcl-1, but not Eµ-myc lymphoma cells overexpressing Bcl-2. Treatment of human leukemic cell lines with compound 21 resulted in cell death through activation of caspase-3 and induction of apoptosis.

Both leukaemic and normal peripheral B lymphoid cells are highly sensitive to the selective pharmacological inhibition of prosurvival Bcl-2 with ABT-199.

Overexpression of the prosurvival protein Bcl-2 marks many B-lymphoid malignancies and contributes to resistance to many commonly used chemotherapeutic agents. The first effective BH3 mimetic inhibitors of Bcl-2, ABT-737 and navitoclax, also target Bcl-xL, causing dose-limiting thrombocytopenia. This prompted the development of the Bcl-2-selective antagonist, ABT-199. Here we show that in lymphoid cells, ABT-199 specifically causes Bax/Bak-mediated apoptosis that is triggered principally by the initiator BH3-only protein Bim. As expected, malignant cells isolated from patients with chronic lymphocytic leukaemia are highly sensitive to ABT-199. However, we found that normal, untransformed mature B cells are also highly sensitive to ABT-199, both in vitro and in vivo. By contrast, the B-cell precursors are largely spared, as are cells of myeloid origin. These results pinpoint the probable impact of the pharmacological inhibition of Bcl-2 by ABT-199 on the normal mature haemopoietic cell lineages in patients, and have implications for monitoring during ABT-199 therapy as well as for the clinical utility of this very promising targeted agent.

Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice.

A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death. DOI:http://dx.doi.org/10.7554/eLife.00772.001.

OCT4 promotes tumorigenesis and inhibits apoptosis of cervical cancer cells by miR-125b/BAK1 pathway.

Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene that maintains the pluripotency and self-renewal properties of embryonic stem cells. Although there is emerging evidence that it can function as oncogene in several cancers, the role in mediating cervical cancer remains unexplored. Here we found that OCT4 protein expression showed a pattern of gradual increase from normal cervix to cervical carcinoma in situ and then to invasive cervical cancer. Overexpression of OCT4 in two types of cervical cancer cells promotes the carcinogenesis, and inhibits cancer cell apoptosis. OCT4 induces upregulation of miR-125b through directly binding to the promoter of miR-125b-1 confirmed by chromatin immunoprecipitation analysis. MiRNA-125b overexpression suppressed apoptosis and expression of BAK1 protein. In contrast, miR-125b sponge impaired the anti-apoptotic effect of OCT4, along with the upregulated expression of BAK1. Significantly, Luciferase assay showed that the activity of the wild-type BAK1 3'-untranslated region reporter was suppressed and this suppression was diminished when the miR-125b response element was mutated or deleted. In addition, we observed negative correlation between levels of BAK1 and OCT4, and positive between OCT4 and miR-125b in primary cervical cancers. These findings suggest an undescribed regulatory pathway in cervical cancer, by which OCT4 directly induces expression of miR-125b, which inhibits its direct target BAK1, leading to suppression of cervical cancer cell apoptosis.

Propofol protects against nitrosative stress-induced apoptotic insults to cerebrovascular endothelial cells via an intrinsic mitochondrial mechanism.

BACKGROUND: Cerebrovascular endothelial cells (CECs), major component cells of the blood-brain barrier, can be injured by oxidative stress. Propofol can protect cells from oxidative injury. The aim of this study was to evaluate the effects of propofol on nitrosative stress-induced insults to CECs and its possible mechanisms. METHODS: Primary CECs isolated from mouse cerebral capillaries were exposed to2 nitric oxide (NO) donors: sodium nitroprusside (SNP) or S-nitrosoglutathione (GSNO). Cellular NO levels, cell morphologies, and cell viabilities were analyzed. DNA fragmentation and apoptotic cells were quantified using flow cytometry. Proapoptotic Bcl2-antagonist-killer (Bak) and cytochrome c were immunodetected. Bak translocation was analyzed using confocal microscopy. Caspases-9 and -3 activities were measured fluorometrically. Permeability of the CEC monolayer was assayed by measuring the transendothelial electrical resistance. RESULTS: Exposure of CECs to SNP increased cellular NO levels and simultaneously decreased cell viability (P < .01). Meanwhile, treatment of CECs with propofol at a therapeutic concentration (50 µM) decreased SNP-induced cell death (P < .01). SNP induced DNA fragmentation and cell apoptosis, but propofol decreased the cell injury (P < .01). Sequentially, propofol decreased SNP-enhanced Bak levels and translocation from the cytoplasm to mitochondria (P < .05). Exposure of CECs to propofol attenuated GSNO-induced cell death, apoptosis, and caspase-3 activation (P < .01). Additionally, propofol protected CECs against SNP-induced disruption of the CEC monolayer (P < .05). Consequently, SNP-enhanced cascade activation of caspases-9 and -3 was decreased by propofol (P < .01). CONCLUSION: This study suggested that propofol at a therapeutic concentration can protect against nitrosative stress-induced apoptosis of CECs due to downregulation of the intrinsic Bak-mitochondrion-cytochrome c-caspase protease pathway.

Mcl-1 antagonizes Bax/Bak to promote effector CD4(+) and CD8(+) T-cell responses.

Members of the Bcl-2 family have critical roles in regulating tissue homeostasis by modulating apoptosis. Anti-apoptotic molecules physically interact and restrain pro-apoptotic family members preventing the induction of cell death. However, the specificity of the functional interactions between pro- and anti-apoptotic Bcl-2 family members remains unclear. The pro-apoptotic Bcl-2 family member Bcl-2 interacting mediator of death (Bim) has a critical role in promoting the death of activated, effector T cells following viral infections. Although Bcl-2 is an important Bim antagonist in effector T cells, and Bcl-xL is not required for effector T-cell survival, the roles of other anti-apoptotic Bcl-2 family members remain unclear. Here, we investigated the role of myeloid cell leukemia sequence 1 (Mcl-1) in regulating effector T-cell responses in vivo. We found, at the peak of the response to lymphocytic choriomeningitis virus (LCMV) infection, that Mcl-1 expression was increased in activated CD4(+) and CD8(+) T cells. Retroviral overexpression of Mcl-1-protected activated T cells from death, whereas deletion of Mcl-1 during the course of infection led to a massive loss of LCMV-specific CD4(+) and CD8(+) T cells. Interestingly, the co-deletion of Bim failed to prevent the loss of Mcl-1-deficient T cells. Furthermore, lck-driven overexpression of a Bcl-xL transgene only partially rescued Mcl-1-deficient effector T cells suggesting a lack of redundancy between the family members. In contrast, additional loss of Bax and Bak completely rescued Mcl-1-deficient effector T-cell number and function, without enhancing T-cell proliferation. These data suggest that Mcl-1 is critical for promoting effector T-cell responses, but does so by combating pro-apoptotic molecules beyond Bim.

Bcl-2 and Bcl-xL suppress glucose signaling in pancreatic ß-cells.

B-cell lymphoma 2 (Bcl-2) family proteins are established regulators of cell survival, but their involvement in the normal function of primary cells has only recently begun to receive attention. In this study, we demonstrate that chemical and genetic loss-of-function of antiapoptotic Bcl-2 and Bcl-x(L) significantly augments glucose-dependent metabolic and Ca(2+) signals in primary pancreatic ß-cells. Antagonism of Bcl-2/Bcl-x(L) by two distinct small-molecule compounds rapidly hyperpolarized ß-cell mitochondria, increased cytosolic Ca(2+), and stimulated insulin release via the ATP-dependent pathway in ß-cell under substimulatory glucose conditions. Experiments with single and double Bax-Bak knockout ß-cells established that this occurred independently of these proapoptotic binding partners. Pancreatic ß-cells from Bcl-2(-/-) mice responded to glucose with significantly increased NAD(P)H levels and cytosolic Ca(2+) signals, as well as significantly augmented insulin secretion. Inducible deletion of Bcl-x(L) in adult mouse ß-cells also increased glucose-stimulated NAD(P)H and Ca(2+) responses and resulted in an improvement of in vivo glucose tolerance in the conditional Bcl-x(L) knockout animals. Our work suggests that prosurvival Bcl proteins normally dampen the ß-cell response to glucose and thus reveals these core apoptosis proteins as integrators of cell death and physiology in pancreatic ß-cells.