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.     

BMF促细胞凋亡研究进展

BMF(Bcl-2-modifying factor)是一种具有促凋亡作用的Bcl-2家族成员.在正常生理状态下,内源性的BMF持续锚定在胞浆中的细胞骨架上,时刻感受细胞内的变化.一旦损伤刺激作用于细胞,BMF从胞浆转移至线粒体,触发线粒体凋亡途径,造成细胞损伤.BMF的促凋亡作用受到转录、翻译和翻译后修饰的调控,过表达或者转录上调的BMF主要定位于线粒体,诱导细胞凋亡.由此可见,BMF具有强大的促凋亡功能.     

Bcl—2家族蛋白与细胞凋亡

Bcl 2家族蛋白是在细胞凋亡过程中起关键性作用的一类蛋白质。在线粒体上 ,Bcl 2家族蛋白通过与其他凋亡蛋白的协同作用 ,调控线粒体结构与功能的稳定性 ,发挥着细胞凋亡“主开关”的作用。Bcl 2家族包括两类蛋白质 :一类是抗凋亡蛋白 ,另一类是促凋亡蛋白。在细胞凋亡时 ,Bcl 2家族中的促凋亡蛋白成员发生蛋白质的加工修饰 ,易位到线粒体的外膜上 ,引起细胞色素c、凋亡诱导因子等其他促凋亡因子的释放 ,导致细胞凋亡 ;而平时被隔离在线粒体等细胞器内的该家族的抗凋亡蛋白成员则抑制细胞色素c和凋亡诱导因子等促凋亡因子的释放 ,具有抑制细胞凋亡的功能。但一旦这类抗凋亡蛋白成员与激活的促凋亡蛋白发生相互作用后 ,便丧失了对细胞凋亡的抑制作用 ,造成线粒体等细胞器的功能丧失和细胞器内促凋亡因子的释放 ,导致细胞凋亡。现以Bcl 2家族调控细胞凋亡的最新研究进展为基础 ,对Bcl 2家族成员及其蛋白质结构、分布和调控细胞凋亡的分子机制进行综述。     

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。     

p53上调凋亡调制物的促凋亡作用

p53上调凋亡调制物(p53up-regulated modulator of apoptosis,PUMA)是Bcl-2家族中BH3-only(Bcl-2 homology 3-only)蛋白质家族成员,通过其BH3结构域与所有的Bcl-2抗凋亡蛋白质结合,引发线粒体功能障碍和胱天蛋白酶(caspase)级联反应,诱导细胞凋亡。PUMA被证实在多种病理性应激介导的细胞凋亡中发挥着至关重要的作用,因而成为近年研究的热点。     

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     

线粒体通透性增高过程中新的参与者

 应激时,线粒体通透性增高可导致位于线粒体膜间的致凋亡因子释放入细胞质,胱天蛋白酶 (caspase)激活,以及细胞死亡.但线粒体通透性增高的确切机制尚不清楚.许多研究表明,线粒体通透性增高过程需要Bcl-2家族蛋白中促凋亡Bax亚家族蛋白,主要是Bax和Bak的激活;该家族中其它蛋白可对Bax和Bak进行调节.但最近的研究表明,其它非Bcl-2家庭蛋白的蛋白质包括抑制因子和激活因子,也可对线粒体通透性增高过程进行调节.此外,应激时线粒体脂质重新分布,对于线粒体膜通透性增高过程也起重要作用.     

Bax/Bak蛋白在凋亡通路中的调控与激活机制

细胞凋亡是机体维持组织稳态和胚胎发育的重要机制之一,受到多种信号分子的严格调控。促凋亡Bcl-2家族蛋白成员Bax和Bak蛋白在细胞凋亡中扮演着非常重要的角色。在凋亡信号的刺激下,Bax和Bak蛋白被激活并在线粒体上互相凝集成簇,使得线粒体膜的通透性增加,引起凋亡因子的释放,并最终诱导细胞的死亡。本文主要介绍Bax和Bak蛋白在细胞凋亡过程中的调控与激活机制,并详细阐述目前它们在线粒体凋亡通路中的几个激活模型,总结二者在激活线粒体凋亡通路中的作用,为进一步研究线粒体凋亡通路作一铺垫。     

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.     

线粒体与细胞凋亡调控

细胞凋亡是一个受到一系列相关基因严格调控的细胞死亡过程。线粒体是细胞凋亡调控的活动中心。在凋亡因子的刺激下,线粒体释放出不同促凋亡因子如细胞色素C、Smac／Diablo等,激活细胞内凋亡蛋白酶Caspase。我们发现,活化后的Caspase可以反过来作用于线粒体,引发更大量线粒体细胞色素c的释放,构成细胞色素c释放的正反馈调节机制,从而导致电子传递链的中断、膜电势的丧失、胞内ROS的升高以及线粒体产生ATP功能的完全丧失。Bcl-2家族蛋白在细胞色素C释放和细胞凋亡调控中起关键作用。     

Bax and other pro-apoptotic Bcl-2 family "killer-proteins" and their victim the mitochondrion

Two major intracellular apoptosis signaling cascades have been characterized, the mitochondrial pathway and the death receptor pathway. The mitochondrial pathway is regulated by members of the Bcl-2 protein family. The members of this family can be subdivided into anti- and pro-apoptotic proteins. The pro-apoptotic members are further divided into two groups, the multidomain and the 'BH3 domain only' proteins. When cells are exposed to apoptotic stimulation, pro-apoptotic proteins are activated through post-translational modifications or changes in their conformation. The main site of action of the multidomain proteins are the mitochondria, where these proteins induce permeabilization of the outer membrane resulting in the release of proteins, including cytochrome c, from the intermembrane space. In the cytosol cytochrome c activates caspase cascades ultimately leading to cell death. Mounting evidence indicates that apoptosis is involved in a wide range of pathological conditions. Recent studies suggest that the mitochondrial signaling pathway is involved in several diseases. Although, so far, with the exception of C. elegans, most studies on apoptosis have been performed in mammalian systems, recently homologues to the Bcl-2 family members, including pro-apoptotic members, have been identified in Drosophila and zebrafish. Here the structure and function of the various pro-apoptotic Bcl-2 family members, their effects on mitochondria, and their involvement in diseases are discussed.     

Bcl-2 inhibits a Fas-induced conformational change in the Bax N terminus and Bax mitochondrial translocation

Members of the Bcl-2 family of proteins control the cellular commitment to apoptosis, although their role in Fas-induced apoptosis is ill-defined. In this report we demonstrate that activation of the Fas receptor present on a human breast epithelial cell line resulted in a conformational change in the N terminus of the pro-apoptotic protein Bax. This conformational change appeared to occur in the cytosol and precede Bax translocation to the mitochondria. Overexpression of the anti-apoptotic protein Bcl-2 inhibited both the conformational change of Bax as well as its relocalization to the mitochondria. Bcl-2 overexpression did not, however, inhibit Fas-induced cleavage of both procaspase-8 and the pro-apoptotic protein Bid, indicating that Bcl-2 functions downstream of these events. These results suggest that the mechanism by which Bcl-2 inhibits Bax mitochondrial translocation and subsequent amplification of the apoptotic cascade is not by providing a physical barrier to Bax, but rather by inhibiting an upstream event necessary for Bax conformational change.     

Identification of the protein-protein contact site and interaction mode of human VDAC1 with Bcl-2 family proteins

Bcl-2 family of proteins plays differential roles in regulation of mitochondria-mediated apoptosis, by either promoting or inhibiting the release of apoptogenic molecules from mitochondria to cytosol. Bcl-2 family proteins modulate the mitochondrial permeability through interaction with adenine nucleotide translocator (ANT), voltage-dependent anion channel (VDAC), ADP/ATP exchange, or oxidative phosphorylation during apoptosis. Although the mitochondrial homeostasis is affected by the relative ratio of pro- and anti-apoptotic Bcl-2 family members, the molecular mechanism underlying the release of mitochondrial intermembrane proteins remains elusive. Here we reported the biochemical evidence that both pro-apoptotic Bax and anti-apoptotic Bcl-X(L) might simultaneously contact the putative loop regions of human VDAC1, and the existence of VDAC1-Bax-Bcl-X(L) tertiary complex in vitro suggested that VDAC1 channel conformation and mitochondrial permeability could be determined by the delicate balance between Bax and Bcl-X(L).     

Cardiolipin provides specificity for targeting of tBid to mitochondria

Recent evidence supports the theory that mitochondrial homeostasis is the key regulatory step in apoptosis through the actions of members of the Bcl-2 family. Pro-apoptotic members of the family, such as Bax, Bad and Bid, can induce the loss of outer-membrane integrity with subsequent redistribution of pro-apoptotic proteins such as cytochrome c that are normally located in the intermembrane spaces of mitochondria. The anti-apoptotic members of the family, such as Bcl-2 and Bcl-XL, protect the integrity of the mitochondrion and prevent the release of death-inducing factors. Bid normally exists in an inactive state in the cytosol, but after cleavage by caspase 8, the carboxy-terminal portion (tBid) moves from cytosol to mitochondria, where it induces release of cytochrome c. Here we address the question of what mediates specific targeting of tBid to the mitochondria. We provide evidence that cardiolipin, which is present in mitochondrial membranes, mediates the targeting of tBid to mitochondria through a previously unknown three-helix domain in tBid. These findings implicate cardiolipin in the pathway for cytochrome c release.     

Mitochondria and the Bcl-2 family proteins in apoptosis signaling pathways

Two main intracellular apoptosis cascades, the receptor and the mitochondria pathway, have been identified. The mitochondrial pathway is controlled by the Bcl-2 proteins. This protein family contains members with either pro- or anti-apoptotic activity. When activated the pro-apoptotic multidomain proteins permeabilized the outer mitochondrial membrane, resulting in the release of proteins from the intermembrane space. Several proteins, including cytochrome c, Smac/DIABLO, HtrA2/Omi, endonuclease G and AIF, normally sequestered in the mitochondria induce or promote apoptosis once released into the cytosol. Although, apoptosis is an essential physiological process in multicellular organisms it is also involved in a wide range of pathological conditions.     

Mitochondrial Protein Import: A Matter of Death?

Mitochondria play a central role not only in energy generation but also for apoptosis. A key step in mitochondrial apoptosis is the release of mitochondrial proteins, most importantly cytochrome c. This release is orchestrated by the pro- and anti-apoptotic members of the Bcl-2 protein family. The functions of these Bcl-2 family members are clear in terms of order and of principle: the pro-apoptotic BH3-only protein group contains the triggers, which cause the activation of the effectors Bax and Bak, while the anti-apoptotic Bcl-2-like proteins prevent this activation. However, the molecular details are still insufficiently clear and the proposed models have certain gaps and are partly contradictory. We have recently presented evidence that targeting to mitochondria of at least one BH3-only protein is essential for its pro-apoptotic functions. Here we discuss how this mechanism might fit into and expand existing models and speculate about the potential implications of this finding.     

Overexpression of Bcl-2 prevents neomycin-induced hair cell death and caspase-9 activation in the adult mouse utricle in vitro

Mechanosensory hair cells of the inner ear are especially sensitive to death induced by exposure to aminoglycoside antibiotics. This aminoglycoside-induced hair cell death involves activation of an intrinsic program of cellular suicide. Aminoglycoside-induced hair cell death can be prevented by broad-spectrum inhibition of caspases, a family of proteases that mediate apoptotic and programmed cell death in a wide variety of systems. More specifically, aminoglycoside-induced hair cell death requires activation of caspase-9. Caspase-9 activation requires release of mitochondrial cytochrome c into the cytoplasm, indicating that aminoglycoside-induced hair cell death is mediated by the mitochondrial (or "intrinsic") cell death pathway. The Bcl-2 family of pro-apoptotic and anti-apoptotic proteins are important upstream regulators of the mitochondrial apoptotic pathway. Bcl-2 is an anti-apoptotic protein that localizes to the mitochondria and promotes cell survival by preventing cytochrome c release. Here we have utilized transgenic mice that overexpress Bcl-2 to examine the role of Bcl-2 in neomycin-induced hair cell death. Overexpression of Bcl-2 significantly increased hair cell survival following neomycin exposure in organotypic cultures of the adult mouse utricle. Furthermore, Bcl-2 overexpression prevented neomycin-induced activation of caspase-9 in hair cells. These results suggest that the expression level of Bcl-2 has important effects on the pathway(s) important for the regulation of aminoglycoside-induced hair cell death.