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.     

BcL2蛋白质家族——定位与转位

Bcl-2蛋白质家族的抗凋亡和促凋亡成员,在线粒体水平上决定细胞的存活或死亡.在正常细胞中,这些成员呈现功能适应性的细胞内分布;抗凋亡成员主要定位于细胞内膜系特别是线粒体外膜上：但绝大多数促凋亡成员主要分布于细胞浆中.细胞接受死亡信号后,Bcl-2家族成员本身受到一系列的调节,如磷酸化、裂解、蛋白质-蛋白质相互作用等,结果之一是促凋亡成员发生细胞内定位的改变,从细胞浆转位于线粒体膜上,并引发线粒体功能异常及其内外膜间致凋亡因子的释放,最终导致细胞凋亡.     

凋亡诱导因子是线粒体内介导核凋亡的最主要蛋白质之一

凋亡诱导因子（apoptosis-inducing factor, AIF）基因定位于X染色体上,其编码产物是一种可直接介导细胞核凋亡的效应分子. 鼠AIF前体蛋白在胞浆中合成后,通过其N端的线粒体定位信号（MLS）有效地穿入线粒体膜间隙,然后在102位甘氨酸处水解掉MLS,其余部分再与黄素腺嘌呤二核苷酸（FAD）结合,并重新折叠成为具有促凋亡潜能的成熟AIF分子,分子质量为57 ku.当凋亡信号刺激时,AIF分子从线粒体释放到胞浆,然后转位到细胞核内,引起染色体核周边凝集和DNA呈大片段断裂（～50 kb）. 该作用不受广谱caspases抑制剂z-VAD.fmk的抑制,也不受Bcl-2过量表达的影响.基因剔除实验表明,AIF蛋白的促凋亡活性是胚胎小鼠形态发生过程中类胚体成腔所必需的,而且是AIF独立作用的结果,可以不依赖caspase-3的活性.因此AIF介导的细胞凋亡可能代表了独立于caspase通路之外的另一条更原始、更保守的凋亡途径.     

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。     

关于TRAIL对凋亡的作用

凋亡,即程序性细胞死亡,是允许多细胞动物严格调控细胞生长,防止癌症、免疫缺陷及自身反应性等病理过程的一种关键机制。在哺乳动物细胞中,凋亡可通过两个主要途径起始：一个与死亡受体的参与有关,另一个则与线粒体释放细胞色素c有关。线粒体途径既可由外部信号也可由内部信号如DNA损伤所触发。Bcl-2家族的促凋亡(pro—apoptotic)和抗凋亡(anti—apoptotic)成员可聚集于线粒体表     

线粒体在细胞凋亡中的介导作用

线粒体是细胞内产生能量的重要细胞器,被认为是细胞生存与死亡的调节中心。Bcl-2家族蛋白、内质网和溶酶体能引起线粒体膜通透性的改变,造成线粒体功能损伤,诱导细胞凋亡。本文主要综述线粒体在Bcl-2家族蛋白、内质网和溶酶体诱导细胞凋亡中作用的研究进展。     

线粒体跨膜电位与细胞凋亡

细胞凋亡作为细胞固有的、受机体严密调控的细胞死亡形式,在多细胞生物体清除衰老细胞及无能细胞等方面发挥重要的作用.近年来,细胞凋亡的研究重点已从细胞核转向线粒体.各种死亡信号诱导线粒体膜通透性改变孔（permeability transition pore, PT pore）开放,引起线粒体跨膜电位下降,导致促凋亡物质释放,继而激活caspase,最终使细胞凋亡.Bcl-2和Bcl-X_L通过对线粒体作用而抑制细胞凋亡,而Bax、Bak与Bad通过调节线粒体而诱导细胞凋亡.     

实时监测溶酶体光损伤诱导细胞凋亡过程中Bax定位的时空变化

光敏剂N-aspartyl chlorin e6 (NPe6)能特异性定位于溶酶体,溶酶体光损伤光能触发线粒体凋亡通路从而诱导细胞凋亡.Bax是Bcl-2家族一员,是调控细胞凋亡的关键因子之一.静息态下,Bax定位于细胞质中;而在细胞凋亡过程中,Bax会从细胞质转位到线粒体,损伤线粒体,从而启动细胞凋亡.在本研究中,我们在活细胞内实时监控溶酶体光损伤诱导细胞凋亡过程中Bax亚细胞定位的动态变化.结果表明,溶酶体光损伤后约170 min,Bax开始转位到线粒体,在30 min之内便大量聚集在线粒体上.该研究结果实时动态地展示了细胞凋亡过程中Bax的时空变化过程.     

线粒体在细胞凋亡中的变化与作用

要在各种凋亡信号的诱导下,线粒体会发生显著的结构与功能性的变化,包括各种促凋亡蛋白(如细胞色素c,凋亡诱导因子等）的释放,线粒体膜电位的丢失,电子传递链的变化,以及细胞内氧化还原状态的变化;核转录因子以线粒体为中介也参与了细胞凋亡的调控。线粒体在哺乳动物细胞凋亡中具有核心地位和作用,昆虫细胞凋亡的研究表明,线粒体与昆虫细胞凋亡也有密切的关系。线粒体在细胞凋亡中的作用可能具有普遍意义。     

黄芩甙对肝癌细胞BEL-7402形态学的影响

目的观察黄芩甙对肝癌细胞BEL-7402凋亡的影响,同时观察对肝癌细胞形态及超微结构、线粒体超微结构、线粒体膜电位和细胞内Ca^2＋的影响,探讨线粒体损伤在黄芩甙诱导肝癌细胞凋亡中的作用及可能的机制。方法应用细胞培养技术培养肝癌细胞BEL-7402,光镜、倒置显微镜、扫描电镜、透射电镜观察细胞形态及超微结构的变化尤其是线粒体的变化,应用流式细胞仪检测细胞凋亡百分率及线粒体膜电位、细胞内Ca^2＋的改变,免疫组化法检测细胞Bcl-2、Pax蛋白表达。结果黄芩甙诱导肝癌细胞BEL-7402凋亡呈剂量依赖关系,细胞形态、超微结构及线粒体超微结构出现明显改变,降低肝癌细胞线粒体膜电位,使细胞内Ca^2＋增加,细胞Pax表达增加,广泛分布于胞核和胞质中,Bcl-2表达减少。结论黄芩甙诱导肝癌细胞BEL-7402凋亡,线粒体损伤在黄芩甙诱导肝癌细胞凋亡中起重要作用,其机制可能为抑制肝癌细胞Bcl-2蛋白表达,促进Pax蛋白表达及细胞内Ca^2＋增加,激发线粒体膜通透性转运孔开放,线粒体跨膜电位降低,使肝癌细胞凋亡。     

Co-targeting MCL-1 and ERK1/2 kinase induces mitochondrial apoptosis in rhabdomyosarcoma cells

The RAS/MEK/ERK genetic axis is commonly altered in rhabdomyosarcoma (RMS), indicating high activity of downstream effector ERK1/2 kinase. Previously, we have demonstrated that inhibition of the RAS/MEK/ERK signaling pathway in RMS is insufficient to induce cell death due to residual pro-survival MCL-1 activity. Here, we show that the combination of ERK1/2 inhibitor Ulixertinib and MCL-1 inhibitor S63845 is highly synergistic and induces apoptotic cell death in RMS in vitro and in vivo. Importantly, Ulixertinib/S63845 co-treatment suppresses long-term survival of RMS cells, induces rapid caspase activation and caspase-dependent apoptosis. Mechanistically, Ulixertinib-mediated upregulation of BIM and BMF in combination with MCL-1 inhibition by S63845 shifts the balance of BCL-2 proteins towards a pro-apoptotic state resulting in apoptosis induction. A genetic silencing approach reveals that BIM, BMF, BAK and BAX are all required for Ulixertinib/S63845-induced apoptosis. Overexpression of BCL-2 rescues cell death triggered by Ulixertinib/S63845 co-treatment, confirming that combined inhibition of ERK1/2 and MCL-1 effectively induces cell death of RMS cells via the intrinsic mitochondrial apoptotic pathway. Thus, this study is the first to demonstrate the cytotoxic potency of co-inhibition of ERK1/2 and MCL-1 for RMS treatment.     

Mitochondrial dynamics and apoptosis: a painful separation

Reporting in Molecular Cell, Sheridan et al. (2008) and Breckenridge et al. (2008) show that mitochondrial fragmentation is not required to induce cell death. Meanwhile, Yamaguchi et al. show that proapoptotic Bcl-2 family members promote cytochrome c mobilization through Opa1-mediated cristae remodeling. Therefore, the connection between mitochondrial structure and apoptosis is more complex than previously imagined.     

Activation of Mitochondrial Pathway is Crucial for Tumor Selective Induction of Apoptosis by LAQ824

HDAC inhibitors are promising antitumor drugs with several HDAC inhibitors already in clinical trials. LAQ824, a potent pan-HDAC inhibitor, has been shown to induce cell cycle arrest and cell death. However, the mechanism of its antitumor effects and specially its tumor selectivity are still poorly understood. The focus of this study is to elucidate LAQ824 mediated anti-proliferative effects in lung carcinoma cells and the mechanism underlying the different sensitivity of LAQ824 to cancer and normal cells. In this study, LAQ824 mediated apoptosis was found to occur mainly via activation of the mitochondrial death pathway by inducing Apaf1 and caspase 9 and promoting mitochondrial release of key proapoptotic factors in lung cancer cells, but not in normal fibroblast cells. Using chromatin immunoprecipitation assay, we found that RNA PolII binding and histone H3 acetylation levels at Apaf1 promoter were increased following LAQ824 treatment, explaining LAQ824 induced expression of Apaf1 in lung cancer cells. Furthermore, we showed that LAQ824 only triggered the release of mitochondrial proapoptotic factors such as cytochrome C (Cyto C) and apoptosis inducing factor (AIF) in lung cancer cells but not in normal blast cells. In addition, LAQ824 was found to induce Bax translocation in lung cancer cell, which may play important role in the induction of the release of mitochondrial proapoptotic factors. These data provide insight into the mechanism underlying the selective induction of apoptosis by LAQ824 in cancer cells.     

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.     

Alteration of mitochondrial function and cell sensitization to death

Stimulation of cell death is a powerful instrument in the organism’s struggle with cancer. Apoptosis represents one mode of cell death. However, in a variety of tumor cells proapoptotic mechanisms are downregulated, or not properly activated, whereas antiapoptotic mechanisms are upregulated. Mitochondria are known as key players in the regulation of apoptotic pathways. Specifically, permeabilization of the mitochondrial outer membrane and subsequent release of proapoptotic proteins from the intermembrane space are viewed as decisive events in the initiation and/or execution of apoptosis. Disruption of mitochondrial functions by anticancer drugs, which induce oxidative stress, inhibit mitochondrial respiration, or uncouple oxidative phosphorylation, can sensitize mitochondria in these cells and facilitate outer membrane permeabilization.     

Induction of cancer cell apoptosis by alpha-tocopheryl succinate: molecular pathways and structural requirements.

The vitamin E analog alpha-tocopheryl succinate (alpha-TOS) can induce apoptosis. We show that the proapoptotic activity of alpha-TOS in hematopoietic and cancer cell lines involves inhibition of protein kinase C (PKC), since phorbol myristyl acetate prevented alpha-TOS-triggered apoptosis. More selective effectors indicated that alpha-TOS reduced PKCalpha isotype activity by increasing protein phosphatase 2A (PP2A) activity. The role of PKCalpha inhibition in alpha-TOS-induced apoptosis was confirmed using antisense oligonucleotides or PKCalpha overexpression. Gain- or loss-of-function bcl-2 mutants implied modulation of bcl-2 activity by PKC/PP2A as a mitochondrial target of alpha-TOS-induced proapoptotic signals. Structural analogs revealed that alpha-tocopheryl and succinyl moieties are both required for maximizing these effects. In mice with colon cancer xenografts, alpha-TOS suppressed tumor growth by 80%. This epitomizes cancer cell killing by a pharmacologically relevant compound without known side effects.     

Exposure of any of two proapoptotic domains of presenilin 1-associated protein/mitochondrial carrier homolog 1 on the surface of mitochondria is sufficient for induction of apoptosis in a Bax/Bak-independent manner

Presenilin 1-associated protein/mitochondrial carrier homolog 1 (PSAP/Mtch1) is a proapoptotic outer mitochondrial membrane protein first identified as a presenilin 1-associated protein. The mechanism by which it induces apoptosis upon overexpression in cultured cells is so far unknown. We had previously reported that deletion of two independent regions of PSAP/Mtch1 is required to prevent apoptosis. We now report that mitochondrial targeting of the region containing both proapoptotic domains, or any of them independently, to the outer membrane is sufficient to induce apoptosis. On the other hand, targeting of that region to the surface of the endoplasmic reticulum does not induce apoptosis, indicating that attachment of those domains to the outer mitochondrial membrane, and not just cytosolic exposure, is a requisite for apoptosis. Overexpression of PSAP/Mtch1 in cultured cells causes mitochondrial depolarization and apoptosis that does not depend on Bax or Bak, since apoptosis is induced in mouse embryonic fibroblasts lacking these two proteins. Our results suggest that apoptosis induced by PSAP/Mtch1 likely involves the permeability transition pore.     

The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal transduction by c-Jun NH(2)-terminal kinase

Targeted gene disruption studies have established that the c-Jun NH(2)-terminal kinase (JNK) signaling pathway is required for stress-induced release of mitochondrial cytochrome c and apoptosis. Here we demonstrate that activated JNK is sufficient to induce rapid cytochrome c release and apoptosis. However, activated JNK fails to cause death in cells deficient of members of the Bax subfamily of proapoptotic Bcl2-related proteins. Furthermore, exposure to stress fails to activate Bax, cause cytochrome c release, and induce death in JNK-deficient cells. These data demonstrate that proapoptotic members of the Bax protein subfamily are essential for JNK-dependent apoptosis.     

Adenine nucleotide translocase-1, a component of the permeability transition pore, can dominantly induce apoptosis

Here, we describe the isolation of adenine nucleotide translocase-1 (ANT-1) in a screen for dominant, apoptosis-inducing genes. ANT-1 is a component of the mitochondrial permeability transition complex, a protein aggregate connecting the inner with the outer mitochondrial membrane that has recently been implicated in apoptosis. ANT-1 expression led to all features of apoptosis, such as phenotypic alterations, collapse of the mitochondrial membrane potential, cytochrome c release, caspase activation, and DNA degradation. Both point mutations that impair ANT-1 in its known activity to transport ADP and ATP as well as the NH(2)-terminal half of the protein could still induce apoptosis. Interestingly, ANT-2, a highly homologous protein could not lead to cell death, demonstrating the specificity of the signal for apoptosis induction. In contrast to Bax, a proapoptotic Bcl-2 gene, ANT-1 was unable to elicit a form of cell death in yeast. This and the observed repression of apoptosis by the ANT-1-interacting protein cyclophilin D suggest that the suicidal effect of ANT-1 is mediated by specific protein-protein interactions within the permeability transition pore.