Bcl-2家族蛋白调控线粒体膜通透性和细胞色素C释放的新机制

Bcl-2家族蛋白在调控线粒体功能和细胞色素C释放中起重要作用。最近发现Bcl-2分子通过与其他促凋亡分子相互作用调控线粒体外膜通透性,其具体分子机制尚不完全清楚。本课题组采用化学生物学方法,在研究Bax/Bak非依赖的细胞凋亡途径中,发现了一些小分子化合物能够诱导Bim表达量急剧升高,Bim能转位到线粒体上,与Bcl-2相互作用增强,并直接促进Bcl-2构象变化。有意义的是,Bim可以诱导Bcl-2功能发生转换并能够形成大的复合体通道来介导细胞色素C释放。研究结果提示Bcl-2分子可变成促凋亡分子,参与Bax/Bak非依赖的细胞色素C释放和细胞凋亡。     

Underphosphorylated BAD interacts with diverse antiapoptotic Bcl-2 family proteins to regulate apoptosis

Survival factors activate kinases which, in turn, phosphorylate the proapoptotic Bcl-xl/Bcl-2-associated death promoter homolog (BAD) protein at key serine residues. Phosphorylated BAD interacts with 14-3-3 proteins, and overexpression of 14-3-3 attenuates BAD-mediated apoptosis. Although BAD is known to interact with Bcl-2, Bcl-w, and Bcl-xL, the exact relationship between BAD and anti- or proapoptotic Bcl-2 proteins has not been analyzed systematically. Using the yeast two-hybrid protein interaction assay, we found that BAD interacted negligibly with proapoptotic Bcl-2 proteins. Even though wild type BAD only interacted with selected numbers of antiapoptotic proteins, underphosphorylated mutant BAD interacted with all antiapoptotic Bcl-2 proteins tested (Bcl-2, Bcl-w, Bcl-xL, Bfl-1/A1, Mcl-1, Ced-9, and BHRF-1). Using nonphosphorylated recombinant BAD expressed in bacteria, direct interactions between BAD and diverse antiapoptotic Bcl-2 members were also observed. Furthermore, apoptosis induced by BAD was blocked by coexpression with Bcl-2, Bcl-w, and Bfl-1. Comparison of BAD orthologs from zebrafish to human indicated the conservation of a 14-3-3 binding site and the BH3 domain during evolution. Thus, highly conserved BAD interacts with diverse antiapoptotic Bcl-2 members to regulate apoptosis.     

Dopamine-induced programmed cell death is associated with cytochrome c release and caspase-3 activation in snail salivary gland cells

Background information. PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). Results. SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling)‐positive cells both in inactive and feeding animals. The DA‐induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto‐c (cytochrome c) translocation into the cytosol and methyl‐β‐cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto‐c was under the control of Bcl‐2 and Bad. DA also increased the active caspase‐3 in gland cells while D2 receptor antagonists and TEA attenuated it. Conclusion. Our results provide evidence for a type of transmitter‐mediated pathway that regulates the PCD of secretory cells in a mitochondrial‐caspase‐dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto‐c, ceramide, Bcl‐2 proteins and caspase‐3, but not caspase‐8, was demonstrated in cells involved in the DA‐induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.     

4-HPR-mediated leukemia cell cytotoxicity is triggered by ceramide-induced mitochondrial oxidative stress and is regulated downstream by Bcl-2

We have previously reported that, in leukemia cells, the cytotoxicity of the anticancer agent N-(4-hydroxyphenyl)retinamide (4-HPR) is mediated by mitochondria-derived reactive oxygen species (ROS) and cardiolipin peroxidation. Here, we have analyzed at greater depth the 4-HPR-triggered molecular events, demonstrating that 4-HPR induces an early (15 min) increase in ceramide levels by sphingomyelin hydrolysis and later (from 1 h) by de novo synthesis. Using specific inhibitors of both pathways, we demonstrate that ceramide accumulation is responsible for early ROS generation, which act as apoptotic signalling intermediates leading to conformational activation of Bak and Bax, loss of mitochondrial membrane potential (ΔΨm), mitochondrial membrane permeabilization (MMP) and cell death. Enforced expression of Bcl-2 has no effect on 4-HPR-induced oxidative stress, but notably prevents mitochondrial alterations and apoptosis, indicating that Bcl-2 functions by regulating events downstream of ROS generation. In conclusion, our study delineates for the fist time the sequence and timing of the principal events induced by 4-HPR in leukemia cells and points to the potential use of modulators of ceramide metabolism as enhancers in 4-HPR-based therapies.     

4-HPR-mediated leukemia cell cytotoxicity is triggered by ceramide-induced mitochondrial oxidative stress and is regulated downstream by Bcl-2

We have previously reported that, in leukemia cells, the cytotoxicity of the anticancer agent N-(4-hydroxyphenyl)retinamide (4-HPR) is mediated by mitochondria-derived reactive oxygen species (ROS) and cardiolipin peroxidation. Here, we have analyzed at greater depth the 4-HPR-triggered molecular events, demonstrating that 4-HPR induces an early (15 min) increase in ceramide levels by sphingomyelin hydrolysis and later (from 1 h) by de novo synthesis. Using specific inhibitors of both pathways, we demonstrate that ceramide accumulation is responsible for early ROS generation, which act as apoptotic signalling intermediates leading to conformational activation of Bak and Bax, loss of mitochondrial membrane potential (ΔΨm), mitochondrial membrane permeabilization (MMP) and cell death. Enforced expression of Bcl-2 has no effect on 4-HPR-induced oxidative stress, but notably prevents mitochondrial alterations and apoptosis, indicating that Bcl-2 functions by regulating events downstream of ROS generation. In conclusion, our study delineates for the fist time the sequence and timing of the principal events induced by 4-HPR in leukemia cells and points to the potential use of modulators of ceramide metabolism as enhancers in 4-HPR-based therapies.     

Bcl-2 overexpression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome c translocation and caspase-3 activity

Bcl-2 protects against both apoptotic and necrotic death induced by several cerebral insults. We and others have previously demonstrated that defective herpes simplex virus vectors expressing Bcl-2 protect against various insults in vitro and in vivo, including cerebral ischemia. Because the infarct margin may be a region that is most amenable to treatment, we first determined whether gene transfer to the infarct margin is possible using a focal ischemia model. Since ischemic injury with and without reperfusion may occur by different mechanisms, we also determined whether Bcl-2 protects against focal cerebral ischemic injury either with or without reperfusion in rats. Bax expression, cytochrome c translocation and activated caspase-3 expression were also assessed. Viral vectors overexpressing Bcl-2 were delivered to the infarct margin. Reperfusion resulted in larger infarcts than permanent occlusion. Bcl-2 overexpression significantly improved neuron survival in both ischemia models. Bcl-2 overexpression did not alter overall Bax expression, but inhibited cytosolic accumulation of cytochrome c and caspase-3 activation. Thus, we provide the first evidence that gene transfer to the infarct margin is feasible, that overexpression of Bcl-2 protects against damage to the infarct margin induced by ischemia with and without reperfusion, and that Bcl-2 overexpression using gene therapy attenuates apoptosis-related proteins. This suggests a potential therapeutic strategy for stroke.     

HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53

HAMLET (Human α-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.     

Drosophila Bcl-2 proteins participate in stress-induced apoptosis, but are not required for normal development

Many developing tissues require programmed cell death (PCD) for proper formation. In mice and C. elegans, developmental PCD is regulated by the Bcl-2 family of proteins. Two bcl-2 genes are encoded in the Drosophila genome (debcl/dBorg1/Drob-1/dBok and buffy/dBorg2) and previous RNAi-based studies suggested a requirement for these in embryonic development. However, we report here that, despite the fact that many tissues in fruit flies are shaped by PCD, deletion of the bcl-2 genes does not perturb normal development. We investigated whether the fly bcl-2 genes regulate non-apoptotic processes that require caspases, but found these to be bcl-2 gene-independent. However, irradiation of the mutants demonstrates that DNA damage-induced apoptosis, mediated by Reaper, is blocked by buffy and that debcl is required to inhibit buffy. Our results demonstrate that developmental PCD regulation in the fly does not rely upon the Bcl-2 proteins, but that they provide an added layer of protection in the apoptotic response to stress.     

Bcl-2 attenuates anticancer agents-induced apoptosis by sustained activation of Akt/protein kinase B in U937 cells

Aberrant overexpression of antiapoptotic members of the Bcl-2 protein family contributes to resistance to anticancer therapeutic drugs. Thus, this protein represent attractive target for novel anticancer agents. In the present study, we determined the effect of the anti-apoptosis protein Bcl-2 on caspase-3 activation, PLC-γ1 degradation and Akt activation during the various anticancer agents-induced apoptosis. Treatment with chrysin for 12 h produced morphological features of apoptosis in U937 cells, which was associated with caspase-3 activation and PLC-γ1 degradation. Induction of apoptosis was also accompanied by down-regulation of XIAP and inactivation of Akt. Chrysin-induced caspase-3 activation, PLC-γ1 degradation and apoptosis were significantly attenuated in Bcl-2 overexpressing U937/Bcl-2 cells. Ectopic expression of Bcl-2 appeared to inhibit ceramide-, and Akt specific inhibitor (SH-6)-induced apoptosis by sustained Akt activation. Thus, our findings imply that some of the biological functions of Bcl-2 may be attributed to their ability to inhibit anticancer agents-induced apoptosis through the sustained Akt activation.     

Demonstration of exogenous nuclear histone H3 binding to mitochondria and subsequent cytochrome c release in cauliflower

The life cycle of a cell is partly regulated by the programmed cell death (PCD) process. From development to demise, a cell's PCD process must respond to external signals and internal factors mediated by mitochondria. Previous studies show that the release of histones into the cytosol caused by DNA damage or loss of nuclear integrity is correlated with apoptosis in mammalian cells. These released histones bind to mitochondria and permeabilize its inner and outer membranes, which causes the release of cytochrome c into the cytosol that leads to caspase activation and the demise of the cell. Owing to the high conservation of histones, we hypothesize that histone‐mediated cytochrome c release from mitochondria may be conserved across a wide range of eukaryotes. We investigated this histone–mitochondrial interaction in cauliflower using density‐gradient purified mitochondria and exogenous histones from a crude histone fraction, then added the exogenous histone fractions to the purified cauliflower mitochondria and analyzed the mitochondrial pellets and supernatants by immunoblotting against cytochrome c and H3. Our data clearly shows that histone‐enriched fractions elicited cytochrome c release from mitochondria, and that mitochondria bind exogenous histone H3.     

Bcl-2 family member Bcl-G is not a proapoptotic protein

The three major subgroups of the Bcl-2 family, including the prosurvival Bcl-2-like proteins, the proapoptotic Bcl-2 homology (BH)3-only proteins and Bax/Bak proteins, regulate the mitochondrial apoptotic pathway. In addition, some outliers within the Bcl-2 family do not fit into these subgroups. One of them, Bcl-G, has a BH2 and a BH3 region, and was proposed to trigger apoptosis. To investigate the physiological role of Bcl-G, we have inactivated the gene in the mouse and generated monoclonal antibodies to determine its expression. Although two isoforms of Bcl-G exist in human, only one is found in mice. mBcl-G is expressed in a range of epithelial as well as in dendritic cells. Loss of Bcl-G did not appear to affect any of these cell types. mBcl-G only binds weakly to prosurvival members of the Bcl-2 family, and in a manner that is independent of its BH3 domain. To understand what the physiological role of Bcl-G might be, we searched for Bcl-G-binding partners through immunoprecipitation/mass spectroscopy and yeast-two-hybrid screening. Although we did not uncover any Bcl-2 family member in these screens, we found that Bcl-G interacts specifically with proteins of the transport particle protein complex. We conclude that Bcl-G most probably does not function in the classical stress-induced apoptosis pathway, but rather has a role in protein trafficking inside the cell.     

Cyclosporin A inhibits caspase-independent death of NGF-deprived sympathetic neurons: a potential role for mitochondrial permeability transition

Opening of the permeability transition pore (PTP) has been implicated as an important mitochondrial event that occurs during apoptosis. We examined the role of the PTP in the well-characterized cell death of rat sympathetic neurons deprived of nerve growth factor (NGF) in vitro. Removal of NGF causes these neurons to undergo either a classic apoptotic cell death or, when treated with a broad-spectrum caspase inhibitor such as boc-aspartyl(OMe)-fluoromethylketone (BAF), a delayed, nonapoptotic cell death. The PTP inhibitor, cyclosporin A (CsA), blocked commitment-to-die in the presence of BAF, as defined by the ability of NGF readdition to rescue cells, but had little effect on commitment-to-die in the absence of BAF. CsA did not have trophic effects on BAF-saved cells, but did block the decrease in mitochondrial membrane potential. These data suggest that PTP opening is a critical event in caspase-independent, nonapoptotic (but not caspase-dependent, apoptotic) death of NGF-deprived rat sympathetic neurons.     

Heat-induced programmed cell death in Leishmania infantum is reverted by Bcl-XL expression

An increasing number of reports indicate that single-celled organisms are able to die following what seems to be an ordered program of cell death with strong similarities to apoptosis from higher eukaryotes. DNA degradation and several other apoptotic-like processes have also been described in the parasitic protozoa Leishmania. However, the existence of an apoptotic death in this parasite is still a matter of controversy. Our results indicate that most of the processes of macromolecular degradation and organelle dysfunction observed in mammalian cells during apoptosis can also be reproduced in promastigotes of the genus Leishmania when incubated at temperatures above 38°C. These processes can be partially reversed by the expression of the anti-apoptotic mammalian gene Bcl-X_L, which suggests that this family of apoptosis-regulating proteins was present very early in the evolution of eukaryotic cells.     

Abiotic stress induces apoptotic-like features in tobacco that is inhibited by expression of human Bcl-2

The shared features between plant and animal programmed cell death are becoming increasingly apparent. In this study, human Bcl-2, an anti-apoptotic member of the Bcl-2 family of cell death regulators, was stably expressed in tobacco. Previously, we have shown that such plants were resistant/tolerant to several necrotrophic fungal pathogens. In this study, we show that transgenic plants are protected by several lethal abiotic stresses including heat, cold, menadione and hydrogen peroxide. Importantly, wild type tobacco, exposed to these treatments, not only died but during the death process exhibited features associated with mammalian apoptosis including DNA laddering, fragmentation, and the development of apoptotic bodies. These features were not observed in viable transgenic tobacco. Thus, abiotic stress induced cell death in plants can be accompanied by apoptotic-like features that are inhibited by expression of Bcl-2. These observations add to the growing body of evidence indicating trans-kingdom conservation of programmed cell death mechanisms.     

How the Bcl-2 family of proteins interact to regulate apoptosis

Commitment of cells to apoptosis is governed largely by protein-protein interactions between members of the Bcl-2 protein family. Its three sub-families have distinct roles： the BH3-only proteins trigger apoptosis by binding via their BH3 domain to pro-survival relatives, while the pro-apoptotic Bax and Bak have an essential downstream role involving disruption of organellar membranes and induction of caspase activation. The BH3-only proteins act as damage sensors, held inert until their activation by stress signals. Once activated, they were thought to bind promiscuously to pro-survival protein targets but unexpected selectivity has recently emerged from analysis of their interactions. Some BH3-only proteins also bind to Bax and Bak. Whether Bax and Bak are activated directly by these BH3-only proteins, or indirectly as a consequence of BH3-only proteins neutralizing their pro-survival targets is the subject of intense debate. Regardless of this, a detailed understanding of the interactions between family members, which are often selective, has notable implications for designing anti-cancer drugs to target the Bcl-2 family.     

定点整合人Bcl-2基因的CHO/dhfr-细胞株的建立

构建重组载体质粒pMCEfrt—Bcl-2,利用FIp—In^TM定点重组系统,在CHO—dhfr^-细胞内定点整合人Bcl-2基因,通过Western印迹检测重组细胞Bcl-2蛋白的表达。通过流式细胞仪和DNA Ladder检测在高NH4C1条件下细胞的凋亡情况;用台盼蓝染色检测在无血清IMDM培养基中细胞的活细胞数目和活细胞比例。结果获得了稳定表达Bcl-2基因的细胞株CHO—Bcl-2,该细胞株能高水平表达Bcl-2蛋白。在无血清培养过程中,CHO—Bcl-2细胞比对照细胞保持高约15％的活细胞比例,细胞总数高25％。CHO-Bcl-2在高NH4^＋（50mmol／L）培养条件下具有较低的凋亡水平。建立了能够高表达Bcl-2基因并具有一定的抗凋亡能力的重组CHO／dhfr^-细胞株。     

细胞凋亡中的Bcl-2家族蛋白及其BH3结构域的功能研究

凋亡相关蛋白中的Bcl-2家族是细胞凋亡的关键调节分子,由抗凋亡和促凋亡成员组成,这些成员之间通过相互协同作用调节了线粒体结构与功能的稳定性,从而在线粒体水平发挥着细胞凋亡的“开关”作用.抗凋亡成员大都分布于线粒体的外膜,与促凋亡成员的BH3结构域相互作用对细胞凋亡发挥抵抗作用.促凋亡成员则主要分布于细胞浆中,细胞接受死亡信号刺激后,促凋亡成员自身受到一系列的调节,如典型的Bax构象改变、BAD和Bik的磷酸化调节以及Bid和Bim的蛋白裂解效应等,使得促凋亡成员在凋亡信号的刺激下整合于线粒体外膜,最终导致线粒体通透转换孔的开放,进而释放包括细胞色素c、凋亡诱导因子、Smac等重要的凋亡因子,随后caspase被激活进而断裂重要的细胞内结构蛋白与功能分子,执行细胞凋亡.     

Down-regulation of Bcl-2 enhances estrogen apoptotic action in long-term estradiol-depleted ER<Superscript>+</Superscript> breast cancer cells

Postmenopausal women with estrogen receptor positive (ER⁺) breast cancer frequently respond paradoxically to estrogen administration with tumor regression. Using both LTED and E8CASS cells derived from MCF-7 breast cancer cells by long-term estrogen-deprivation, we previously reported that 17 -estradiol (estradiol) is a powerful, pro-apoptotic hormone which kills the cancer cells through activation of the Fas/FasL death receptor pathway. We postulated that the mitochondrial interactive protein Bcl-2 might play a role in the regulation of estradiol-induced apoptosis in both LTED and E8CASS cells. In this study, we assessed estradiol effects on cell growth, proliferation and apoptosis. Additionally we investigated the effect of estradiol on caspase activation, NF-KB and Bcl-2 expression. The functional role of Bcl-2 in estradiol-induced apoptosis was further studied by knockdown or decrease of Bcl-2 with siRNA. Our results show that estradiol significantly inhibited cell growth primarily through a pro-apoptotic action involving caspase-7 and 9 activations (p < 0.01). Basal Bcl-2 and NF-KB levels were greatly elevated and estradiol decreased NF-KB, but not Bcl-2 expression. Knockdown of Bcl-2 expression with siRNA decreased the levels of this protein by 9 fold (p < 0.01). This reduction markedly sensitized both LTED and E8CASS cells to the pro-apoptotic action of estradiol, leading to a synergistic induction of apoptosis and a concomitant reduction in cell number (p < 0.01). Therefore, down-regulation of Bcl-2 synergistically enhanced estradiol-induced apoptosis in ER⁺ postmenopausal breast cancer cells.     

Proposed mechanism for regulation of H2O2-induced programmed cell death in plants by binding of cytochrome c to 14-3-3 proteins

Programmed cell death (PCD) is crucial for development and homeostasis of all multicellular organisms. In human cells, the double role of extra-mitochondrial cytochrome c in triggering apoptosis and inhibiting survival pathways is well reported. In plants, however, the specific role of cytochrome c upon release from the mitochondria remains in part veiled yet death stimuli do trigger cytochrome c translocation as well. Here, we identify an Arabidopsis thaliana 14-3-3ι isoform as a cytosolic cytochrome c target and inhibitor of caspase-like activity. This finding establishes the 14-3-3ι protein as a relevant factor at the onset of plant H₂O₂-induced PCD. The in vivo and in vitro studies herein reported reveal that the interaction between cytochrome c and 14-3-3ι exhibits noticeable similarities with the complex formed by their human orthologues. Further analysis of the heterologous complexes between human and plant cytochrome c with plant 14-3-3ι and human 14-3-3ε isoforms corroborated common features. These results suggest that cytochrome c blocks p14-3-3ι so as to inhibit caspase-like proteases, which in turn promote cell death upon H₂O₂ treatment. Besides establishing common biochemical features between human and plant PCD, this work sheds light onto the signaling networks of plant cell death.