Role of Bax and Bak in mitochondrial morphogenesis

Bcl-2 family proteins are potent regulators of programmed cell death. Although their intracellular localization to mitochondria and the endoplasmic reticulum has focused research on these organelles, how they function remains unknown. Two members of the Bcl-2 family, Bax and Bak, change intracellular location early in the promotion of apoptosis to concentrate in focal clusters at sites of mitochondrial division. Here we report that in healthy cells Bax or Bak is required for normal fusion of mitochondria into elongated tubules. Bax seems to induce mitochondrial fusion by activating assembly of the large GTPase Mfn2 and changing its submitochondrial distribution and membrane mobility-properties that correlate with different GTP-bound states of Mfn2. Our results show that Bax and Bak regulate mitochondrial dynamics in healthy cells and indicate that Bcl-2 family members may also regulate apoptosis through organelle morphogenesis machineries.     

线粒体外膜通道蛋白VDAC与靶向肿瘤细胞凋亡

VDAC(Voltage-dependent anion channel)是位于线粒体外膜上的一种主要通道蛋白,参与线粒体内外物质和能量的运输,在线粒体与细胞其它部位的通讯中起重要调节作用.近年来研究发现,VDAC也是线粒体与其它蛋白质相互作用的功能结合位点,可与多种凋亡调节蛋白(如HK-Ⅰ/Ⅱ、Bcl-2家族蛋白、tubulin、MAP2/4等)以及非蛋白调节因子相互作用,参与调控细胞凋亡.因此,VDAC成为线粒体凋亡通路中一种关键的靶蛋白.本文对近年来VDAC在肿瘤细胞凋亡中的作用机制进行简要综述.     

The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore

A sudden increase in permeability of the inner mitochondrial membrane, the so-called mitochondrial permeability transition, is a common feature of apoptosis and is mediated by the mitochondrial permeability transition pore (mtPTP). It is thought that the mtPTP is a protein complex formed by the voltage-dependent anion channel, members of the pro- and anti-apoptotic BAX-BCL2 protein family, cyclophilin D, and the adenine nucleotide (ADP/ATP) translocators (ANTs). The latter exchange mitochondrial ATP for cytosolic ADP and have been implicated in cell death. To investigate the role of the ANTs in the mtPTP, we genetically inactivated the two isoforms of ANT in mouse liver and analysed mtPTP activation in isolated mitochondria and the induction of cell death in hepatocytes. Mitochondria lacking ANT could still be induced to undergo permeability transition, resulting in release of cytochrome c. However, more Ca2+ than usual was required to activate the mtPTP, and the pore could no longer be regulated by ANT ligands. Moreover, hepatocytes without ANT remained competent to respond to various initiators of cell death. Therefore, ANTs are non-essential structural components of the mtPTP, although they do contribute to its regulation.     

Humanin peptide suppresses apoptosis by interfering with Bax activation

Bax (Bcl2-associated X protein) is an apoptosis-inducing protein that participates in cell death during normal development and in various diseases. Bax resides in an inactive state in the cytosol of many cells. In response to death stimuli, Bax protein undergoes conformational changes that expose membrane-targeting domains, resulting in its translocation to mitochondrial membranes, where Bax inserts and causes release of cytochrome c and other apoptogenic proteins. It is unknown what controls conversion of Bax from the inactive to active conformation. Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria. Conversely, reducing HN expression by small interfering RNAs sensitizes cells to Bax and increases Bax translocation to membranes. HN peptides also block Bax association with isolated mitochondria, and suppress cytochrome c release in vitro. Notably, the mitochondrial genome contains an identical open reading frame, and the mitochondrial version of HN can also bind and suppress Bax. We speculate therefore that HN arose from mitochondria and transferred to the nuclear genome, providing a mechanism for protecting these organelles from Bax.     

Calpain的激活与鱼藤酮导致的细胞凋亡

在缺氧或呼吸链抑制剂存在条件下,细胞的呼吸链受到抑制,线粒体的功能受到直接干扰,细胞色素C通过线粒体的外膜特异性通道进入细胞浆内,启动了procaspase-3等一系列凋亡因子,细胞发生与线粒体相关的凋亡。另一方面,因线粒体的功能被抑制,细胞内的钙离子浓度升高,calpain被激活并裂解细胞膜蛋白及细胞内的生物化学分子,促进了细胞凋亡的发生。鱼藤酮作为线粒体呼吸链complexI的抑制剂可导致细胞凋亡,其凋亡途径不仅与caspase介导的机制有关,还有可能与calpain有关。     

Protective paracrine effect of mesenchymal stem cells on cardiomyocytes

Objective: The aim of this study was to test the protective effect of mesenchymal stem cells (MSCs) on cardiomyocytes in vitro and to investigate the anti-apoptotic signaling pathway. Methods: MSCs from Sprague-Dawley (SD) rats were separated and cultured. MSC medium was collected from MSCs cultured in serum-free Dulbecco’s modified eagle medium (DMEM) under hypoxia. Cultured cardiomyocytes from neonatal SD rats were exposed to hypoxia/reoxygenation (H/R) and treated with MSC medium. The apoptotic cardiomyocytes were stained with Annexin-V-fluorescein isothiocyanate (FITC), Hoechst 33342 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). The mitochondrial transmembrane potential of cardiomyocytes was assessed using a fluorescence microscope. The expression of Bcl-2, Bax, cytochrome C, apoptosis-induced factor (AIF), and caspase-3 was tested by Western blot analysis. Results: Our data demonstrated that MSC medium reduced H/R-induced cardiomyocyte apoptosis, increased the Bcl-2/Bax ratio, and reduced the release of cytochrome C and AIF from mitochondria into the cytosol. Conclusion: MSCs protected the cardiomyocytes from H/R-induced apoptosis through a mitochondrial pathway in a paracrine manner.     

Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome

Apoptosis is an evolutionarily conserved cell suicide process executed by cysteine proteases (caspases) and regulated by the opposing factions of the Bcl-2 protein family. Mammalian caspase-9 and its activator Apaf-1 were thought to be essential, because mice lacking either of them display neuronal hyperplasia and their lymphocytes and fibroblasts seem resistant to certain apoptotic stimuli. Because Apaf-1 requires cytochrome c to activate caspase-9, and Bcl-2 prevents mitochondrial cytochrome c release, Bcl-2 is widely believed to inhibit apoptosis by safeguarding mitochondrial membrane integrity. Our results suggest a different, broader role, because Bcl-2 overexpression increased lymphocyte numbers in mice and inhibited many apoptotic stimuli, but the absence of Apaf-1 or caspase-9 did not. Caspase activity was still discernible in cells lacking Apaf-1 or caspase-9, and a potent caspase antagonist both inhibited apoptosis and retarded cytochrome c release. We conclude that Bcl-2 regulates a caspase activation programme independently of the cytochrome c/Apaf-1/caspase-9 'apoptosome', which seems to amplify rather than initiate the caspase cascade.     

Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death

Mitochondria play a critical role in mediating both apoptotic and necrotic cell death. The mitochondrial permeability transition (mPT) leads to mitochondrial swelling, outer membrane rupture and the release of apoptotic mediators. The mPT pore is thought to consist of the adenine nucleotide translocator, a voltage-dependent anion channel, and cyclophilin D (the Ppif gene product), a prolyl isomerase located within the mitochondrial matrix. Here we generated mice lacking Ppif and mice overexpressing cyclophilin D in the heart. Ppif null mice are protected from ischaemia/reperfusion-induced cell death in vivo, whereas cyclophilin D-overexpressing mice show mitochondrial swelling and spontaneous cell death. Mitochondria isolated from the livers, hearts and brains of Ppif null mice are resistant to mitochondrial swelling and permeability transition in vitro. Moreover, primary hepatocytes and fibroblasts isolated from Ppif null mice are largely protected from Ca2+-overload and oxidative stress-induced cell death. However, Bcl-2 family member-induced cell death does not depend on cyclophilin D, and Ppif null fibroblasts are not protected from staurosporine or tumour-necrosis factor-alpha-induced death. Thus, cyclophilin D and the mitochondrial permeability transition are required for mediating Ca2+- and oxidative damage-induced cell death, but not Bcl-2 family member-regulated death.     

Treatment of PC-3 cells with ultrasound combined with microbubbles induces distinct alterations in the expression of Bcl-2 and Bax

The objective of the present study was to investigate whether ultrasound combined with microbubbles induces apoptotic cell death in androgen-independent prostate cancer cells and to identify the probable mechanism. We used ultrasound in continuous wave mode with a frequency of 21 kHz and a spatial-average temporal-average intensity of 46 mW/cm². Ultrasound combined with microbubbles (200 μL SonoVue?) was used to treat androgen-independent human prostate cancer PC-3 cells for 30 s. PC-3 cells were divided into three groups: the control group, the ultrasound group and the ultrasound combined with microbubbles group. Immediately after treatment, trypan blue exclusion was used to assess cell viability. Cell apoptosis at 24 h after treatment was measured using transmission electron microscopy and flow cytometry. Western blotting was used to evaluate the expression of the apoptosis-related proteins, Bcl-2 and Bax. Ultrasound combined with microbubbles had a minimal effect on the viability of PC-3 cells and induced minimal levels of cell lysis. The level of apoptosis in PC-3 cells induced by this modality was significantly higher than in controls (12.77 ± 0.31% vs. 2.56 ± 0.22%, P<0.01). Treatment with ultrasound combined with microbubbles increased the expression of Bax, a pro-apoptotic protein, and decreased the expression of Bcl-2, an anti-apoptotic protein. It was concluded that ultrasound combined with microbubbles induces apoptotic cell death in human prostate cancer PC-3 cells through down-regulation of Bcl-2 and up-regulation of Bax.     

Bcl-2家族蛋白和乙肝病毒x蛋白在肝癌组织中的表达和意义

应用免疫组织化学方法检测了34例肝癌组织及其相对应的癌旁组织,探讨了Bcl-2家族中七种基因(包括促凋亡基因Bak、Bad、Bid、Bax和Bcl-xs及抑凋亡基因Bcl-2、Bcl-w)和乙肝病毒三种抗原(包括HBsAg、HBcAg和HBxAg)在肝癌组织中的表达及意义,结果显示:在肝癌组织中HBsAg、HBcAg和HBxA的阳性率分别为58.8％、26.5％和76.5％、Bcl-2七种蛋白的阳性率分别为58.8％(Bak)、55.9％(Bad)、44.1％(Bid)、41.2％(Bax)、29.4％(Bcl-xs)、35.3％(Bcl-w)和41.2％(Bcl-2)。这七种Bcl-2蛋白的表达均位于肝癌细胞的胞浆,多呈弥漫性分布,少数阳性颗粒呈散在性分布,研究发现,Bcl-2家族中抑凋亡基因Bcl-w和Bcl-2在癌组织中表达的阳性率明显高于癌旁组织(P相似文献   

BAX activation is initiated at a novel interaction site

BAX is a pro-apoptotic protein of the BCL-2 family that is stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed stabilized alpha-helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the human BIM-SAHB-BAX interaction is highlighted by point mutagenesis that disrupts functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis.     

RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels

Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS-RAF-MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)--a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS-RAF-MEK pathway.     

Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis.

The protein Bid is a participant in the pathway that leads to cell death (apoptosis), mediating the release of cytochrome c from mitochondria in response to signals from 'death' receptors known as TNFR1/Fas on the cell surface. It is a member of the proapoptotic Bcd-2 family and is activated as a result of its cleavage by caspase 8, one of a family of proteolytic cell-death proteins. To investigate the role of Bid in vivo, we have generated mice deficient for Bid. We find that when these mice are injected with an antibody directed against Fas, they nearly all survive, whereas wild-type mice die from hepatocellular apoptosis and haemorrhagic necrosis. About half of the Bid-deficient animals had no apparent liver injury and showed no evidence of activation of the effector caspases 3 and 7, although the initiator caspase 8 had been activated. Other Bid-deficient mice survived with only moderate damage: all three caspases (8 and 37) were activated but their cell nuclei were intact and no mitochondrial cytochrome c was released. We also investigated the effects of Bid deficiency in cultured cells treated with anti-Fas antibody (hepatocytes and thymocytes) or with TNFalpha. (fibroblasts). In these Bid-/- cells, mitochondrial dysfunction was delayed, cytochrome c was not released, effector caspase activity was reduced and the cleavage of apoptosis substrates was altered. This loss-of-function model indicates that Bid is a critical substrate in vivo for signalling by death-receptor agonists, which mediates a mitochondrial amplification loop that is essential for the apoptosis of selected cells.     

Bid BH3 peptide, but not its mutant form G94E, induces permeability transition pore opening and cytochrome c release in vitro

It has been shown that Bid and its truncated form tBid could induce cytochrome c (cyt c) release without impacting on PTP. We first show that Bid BH3 peptide, but not its mutant form of Bid BH3 peptide G94E, which is unable to bind to Bcl-xL, induces permeability transition pore (PTP) opening in a dose dependent manner. Bid BH3 peptide also induces the reduction of mitochondria membrane potential (ψm) and cyt c release from mitochondrial in vitro.PTP opening and the loss of ψm were inhibited by Bcl-xL,cyclosporin A and ruthenium red, and the latter was an inhibitor of Ca2+ uniporter in the mitochondrial membrane.These results indicate that Bid BH3 peptide could antagonize Bcl-xL to induced PTP opening and mitochondrial dysfunction.     

Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death

Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosome-a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies-the very first wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.     

Hax1-mediated processing of HtrA2 by Parl allows survival of lymphocytes and neurons

Cytokines affect a variety of cellular functions, including regulation of cell numbers by suppression of programmed cell death. Suppression of apoptosis requires receptor signalling through the activation of Janus kinases and the subsequent regulation of members of the B-cell lymphoma 2 (Bcl-2) family. Here we demonstrate that a Bcl-2-family-related protein, Hax1, is required to suppress apoptosis in lymphocytes and neurons. Suppression requires the interaction of Hax1 with the mitochondrial proteases Parl (presenilin-associated, rhomboid-like) and HtrA2 (high-temperature-regulated A2, also known as Omi). These interactions allow Hax1 to present HtrA2 to Parl, and thereby facilitates the processing of HtrA2 to the active protease localized in the mitochondrial intermembrane space. In mouse lymphocytes, the presence of processed HtrA2 prevents the accumulation of mitochondrial-outer-membrane-associated activated Bax, an event that initiates apoptosis. Together, the results identify a previously unknown sequence of interactions involving a Bcl-2-family-related protein and mitochondrial proteases in the ability to resist the induction of apoptosis when cytokines are limiting.     

DRP-1-mediated mitochondrial fragmentation during EGL-1-induced cell death in C. elegans

Genetic analyses in Caenorhabditis elegans have been instrumental in the elucidation of the central cell-death machinery, which is conserved from C. elegans to mammals. One possible difference that has emerged is the role of mitochondria. By releasing cytochrome c, mitochondria are involved in the activation of caspases in mammals. However, there has previously been no evidence that mitochondria are involved in caspase activation in C. elegans. Here we show that mitochondria fragment in cells that normally undergo programmed cell death during C. elegans development. Mitochondrial fragmentation is induced by the BH3-only protein EGL-1 and can be blocked by mutations in the bcl-2-like gene ced-9, indicating that members of the Bcl-2 family might function in the regulation of mitochondrial fragmentation in apoptotic cells. Mitochondrial fragmentation is independent of CED-4/Apaf-1 and CED-3/caspase, indicating that it occurs before or simultaneously with their activation. Furthermore, DRP-1/dynamin-related protein, a key component of the mitochondrial fission machinery, is required and sufficient to induce mitochondrial fragmentation and programmed cell death during C. elegans development. These results assign an important role to mitochondria in the cell-death pathway in C. elegans.     

An inhibitor of Bcl-2 family proteins induces regression of solid tumours

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.     

Evolutionary conservation of biogenesis of beta-barrel membrane proteins

The outer membranes of mitochondria and chloroplasts are distinguished by the presence of beta-barrel membrane proteins. The outer membrane of Gram-negative bacteria also harbours beta-barrel proteins. In mitochondria these proteins fulfil a variety of functions such as transport of small molecules (porin/VDAC), translocation of proteins (Tom40) and regulation of mitochondrial morphology (Mdm10). These proteins are encoded by the nucleus, synthesized in the cytosol, targeted to mitochondria as chaperone-bound species, recognized by the translocase of the outer membrane, and then inserted into the outer membrane where they assemble into functional oligomers. Whereas some knowledge has been accumulated on the pathways of insertion of proteins that span cellular membranes with alpha-helical segments, very little is known about how beta-barrel proteins are integrated into lipid bilayers and assembled into oligomeric structures. Here we describe a protein complex that is essential for the topogenesis of mitochondrial outer membrane beta-barrel proteins (TOB). We present evidence that important elements of the topogenesis of beta-barrel membrane proteins have been conserved during the evolution of mitochondria from endosymbiotic bacterial ancestors.     

Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis

Apoptotic cells release 'find-me' signals at the earliest stages of death to recruit phagocytes. The nucleotides ATP and UTP represent one class of find-me signals, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 overexpression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the 'selective' plasma membrane permeability of early apoptotic cells to specific dyes. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.