The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability

Mitochondria are well known as sites of electron transport and generators of cellular ATP. Mitochondria also appear to be sites of cell survival regulation. In the process of programmed cell death, mediators of apoptosis can be released from mitochondria through disruptions in the outer mitochondrial membrane; these mediators then participate in the activation of caspases and of DNA degradation. Thus the regulation of outer mitochondrial membrane integrity is an important control point for apoptosis. The Bcl-2 family is made up of outer mitochondrial membrane proteins that can regulate cell survival, but the mechanisms by which Bcl-2 family proteins act remain controversial. Most metabolites are permeant to the outer membrane through the voltage dependent anion channel (VDAC), and Bcl-2 family proteins appear to be able to regulate VDAC function. In addition, many Bcl-2 family proteins can form channels in vitro, and some pro-apoptotic members may form multimeric channels large enough to release apoptosis promoting proteins from the intermembrane space. Alternatively, Bcl-2 family proteins have been hypothesized to coordinate the permeability of both the outer and inner mitochondrial membranes through the permeability transition (PT) pore. Increasing evidence suggests that alterations in cellular metabolism can lead to pro-apoptotic changes, including changes in intracellular pH, redox potential and ion transport. By regulating mitochondrial membrane physiology, Bcl-2 proteins also affect mitochondrial energy generation, and thus influence cellular bioenergetics. Cell Death and Differentiation (2000) 7, 1182 - 1191     

Mitochondrial Ca(2+) and apoptosis

Mitochondria are key decoding stations of the apoptotic process. In support of this view, a large body of experimental evidence has unambiguously revealed that, in addition to the well-established function of producing most of the cellular ATP, mitochondria play a fundamental role in triggering apoptotic cell death. Various apoptotic stimuli cause the release of specific mitochondrial pro-apoptotic factors into the cytosol. The molecular mechanism of this release is still controversial, but there is no doubt that mitochondrial calcium (Ca(2+)) overload is one of the pro-apoptotic ways to induce the swelling of mitochondria, with perturbation or rupture of the outer membrane, and in turn the release of mitochondrial apoptotic factors into the cytosol. Here, we review as different proteins that participate in mitochondrial Ca(2+) homeostasis and in turn modulate the effectiveness of Ca(2+)-dependent apoptotic stimuli. Strikingly, the final outcome at the cellular level is similar, albeit through completely different molecular mechanisms: a reduced mitochondrial Ca(2+) overload upon pro-apoptotic stimuli that dramatically blunts the apoptotic response.     

The mitochondrial gateway to cell death

Mitochondria play a key role in death signaling. The intermembrane space of these organelles contains a number of proteins which promote cell death once they are redistributed to the cytosol. The formation of pores in the outer membrane of mitochondria defines a gateway through which the apoptogenic proteins pass during death signaling. Interactions between pro-apoptotic and pro-survival members of the Bcl-2 family of proteins are decisive in the initiation of pore opening. While the specific composition of the pore in molecular terms is still subject to debate and continuing investigation, it is recognized functionally as a passive channel which not only allows egress of proteins to cytosol but also entry in the reverse direction. A variety of constraints may restrict the release of proteins from the intermembrane space to the cytosol. These include trapping in the intercristal spaces formed by the convoluted invaginations of the inner membrane, binding of proteins to the inner membrane or to other soluble proteins of the intermembrane space, or insertion of proteins into the inner membrane. There is a corresponding variety of mechanisms that facilitate release of apoptogenic proteins from such entrapment. Morphological changes that expand the inner membrane enable proteins to be released from enclosure in intercristal spaces, allowing these proteins access to the mitochondrial gateway. Specific cases include cytochrome c molecules bound to inner membrane cardiolipin and released upon oxidation of that lipid component. Further, AIF that is embedded in the inner membrane is released by proteases (caspases or calpains), which enter from the cytosol once the outer membrane pore has opened. The facilitation (or restriction) of apoptogenic protein release through the mitochondrial gateway may provide new opportunities for regulating cell death.     

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-x(L) blocks a mitochondrial inner membrane channel and prevents Ca2+ overload-mediated cell death

Apoptosis is an active process that plays a key role in many physiological and pathological conditions. One of the most important organelles involved in apoptosis regulation is the mitochondrion. An increase in intracellular Ca(2+) is a general mechanism of toxicity in neurons which occurs in response to different noxious stimuli like excitotoxicity and ischemia producing apoptotic and necrotic cell death through mitochondria-dependent mechanisms. The Bcl-2 family of proteins modulate the release of pro-apoptotic factors from the mitochondrial intermembrane space during cell death induction by different stimuli. In this work, we have studied, using single-cell imaging and patch-clamp single channel recording, the mitochondrial mechanisms involved in the neuroprotective effect of Bcl-x(L) on Ca(2+) overload-mediated cell death in human neuroblastoma SH-SY5Y cells. We have found that Bcl-x(L) neuroprotective actions take place at mitochondria where this antiapoptotic protein delays both mitochondrial potential collapse and opening of the permeability transition pore by preventing Ca(2+)-mediated mitochondrial multiple conductance channel opening. Bcl-x(L) neuroprotective actions were antagonized by the Bcl-x(L) inhibitor ABT-737 and potentiated by the Ca(2+) chelator BAPTA-AM. As a consequence, this would prevent free radical production, mitochondrial membrane permeabilization, release from mitochondria of pro-apoptotic molecules, caspase activation and cellular death.     

Mitochondrial regulation of apoptotic cell death

Mitochondria play a decisive role in the regulation of both apoptotic and necrotic cell death. Permeabilization of the outer mitochondrial membrane and subsequent release of intermembrane space proteins are important features of both models of cell death. The mechanisms by which these proteins are released depend presumably on cell type and the nature of stimuli. Of the mechanisms involved, mitochondrial permeability transition appears to be associated mainly with necrosis, whereas the release of caspase activating proteins during early apoptosis is regulated primarily by the Bcl-2 family of proteins. However, there is increasing evidence for interaction and co-operation between these two mechanisms. The multiple mechanisms of mitochondrial permeabilization may explain diversities in the response of mitochondria to numerous apoptotic stimuli in different types of cells.     

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.     

Regulation of mitochondrial membrane permeabilization by BCL-2 family proteins and caspases

Mitochondria play an important role in the integration and transmission of cell death signals, activating caspases and other cell death execution events by releasing apoptogenic proteins from the intermembrane space. The BCL-2 family of proteins localize (or can be targeted) to mitochondria and regulate the permeability of the mitochondrial outer membrane to these apoptotic factors. Recent evidence suggests that multiple mechanisms may regulate the release of mitochondrial factors, some of which depend on the action of caspases.     

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.     

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.     

Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death

Mitochondria are critically involved in necrotic cell death induced by Ca(2+) overload, hypoxia and oxidative damage. The mitochondrial permeability transition (MPT) pore - a protein complex that spans both the outer and inner mitochondrial membranes - is considered the mediator of this event and has been hypothesized to minimally consist of the voltage-dependent anion channel (Vdac) in the outer membrane, the adenine-nucleotide translocase (Ant) in the inner membrane and cyclophilin-D in the matrix. Here, we report the effects of deletion of the three mammalian Vdac genes on mitochondrial-dependent cell death. Mitochondria from Vdac1-, Vdac3-, and Vdac1-Vdac3-null mice exhibited a Ca(2+)- and oxidative stress-induced MPT that was indistinguishable from wild-type mitochondria. Similarly, Ca(2+)- and oxidative-stress-induced MPT and cell death was unaltered, or even exacerbated, in fibroblasts lacking Vdac1, Vdac2, Vdac3, Vdac1-Vdac3 and Vdac1-Vdac2-Vdac3. Wild-type and Vdac-deficient mitochondria and cells also exhibited equivalent cytochrome c release, caspase cleavage and cell death in response to the pro-death Bcl-2 family members Bax and Bid. These results indicate that Vdacs are dispensable for both MPT and Bcl-2 family member-driven cell death.     

Mitochondria frozen with trehalose retain a number of biological functions and preserve outer membrane integrity

In apoptosis, Bcl-2-family proteins regulate the barrier function of the mitochondrial outer membrane (MOM), controlling the release of proapoptotic proteins from the intermembrane space into the cytoplasm. This process can be studied in vitro with freshly isolated mouse liver mitochondria. Unfortunately, mitochondria frozen/thawed in standard sucrose-mannitol buffers become leaky and useless for apoptosis research. However, here we show that mitochondria frozen in buffer containing the sugar, trehalose, maintained MOM integrity and responsiveness to Bcl-2-family proteins, much like fresh mitochondria. Trehalose also preserved ultrastructure, as well as biological functions such as ATP synthesis, calcium-induced swelling, transmembrane potential, and the import and processing of protein precursors. However, bioenergetic function was somewhat reduced. Thus, trehalose-frozen mitochondria retained most of the biological features of mitochondria including MOM integrity. Although not ideal for studies involving bioenergetics, this method will facilitate research on apoptosis and other mitochondrial functions that rely on an intact MOM.     

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 membrane permeabilization: the sine qua non for cell death

Mitochondria are essential for maintaining cell life but they also play a role in regulating cell death, which occurs when their membranes become permeabilized. Mitochondria possess two distinct membrane systems including an outer membrane in close communication with the cytosol and an inner membrane involved in energy transduction. Outer membrane permeabilization is regulated by Bcl-2 family proteins, which control the release of proteins from the mitochondrial intermembrane space; these proteins then activate apoptosis. Inner membrane permeabilization is regulated by the mitochondrial permeability transition (MPT), which is activated by calcium and oxidative stress and leads to bioenergetic failure and necrosis. The purpose of this review is to discuss the biochemical mechanisms regulating mitochondrial membrane permeabilization; this is crucial to our understanding of the role of cell death in diseases such as cancer and the neurodegenerative diseases.     

Modulation of mitochondrial apoptosis by PI3K inhibitors

Most anticancer therapies exert their action by triggering programmed cell death (apoptosis) in cancer cells. The mitochondrial pathway of apoptosis is initiated by mitochondrial outer membrane permeabilization, leading to the release of apoptogenic factors such as cytochrome c or Smac from the mitochondrial intermembrane space into the cytosol. Mitochondrial outer membrane permeabilization is tightly controlled, for example by pro- and anti-apoptotic proteins of the Bcl-2 family. Recent evidence indicates that inhibition of the PI3K/Akt/mTOR pathway by small-molecule PI3K inhibitors primes cancer cells to mitochondrial apoptosis by tipping the balance towards pro-apoptotic Bcl-2 proteins, resulting in increased mitochondrial outer membrane permeabilization. Thus, mitochondrial apoptotic events play an important role in PI3K inhibitor-mediated sensitization for apoptosis.     

The endoplasmic reticulum gateway to apoptosis by Bcl-X(L) modulation of the InsP3R

Members of the Bcl-2 protein family modulate outer mitochondrial membrane permeability to control apoptosis. However, these proteins also localize to the endoplasmic reticulum (ER), the functional significance of which is controversial. Here we provide evidence that anti-apoptotic Bcl-2 proteins regulate the inositol 1,4,5-trisphosphate receptor (InsP(3)R) ER Ca(2+) release channel resulting in increased cellular apoptotic resistance and enhanced mitochondrial bioenergetics. Anti-apoptotic Bcl-X(L) interacts with the carboxyl terminus of the InsP(3)R and sensitizes single InsP(3)R channels in ER membranes to low [InsP(3)], enhancing Ca(2+) and InsP(3)-dependent regulation of channel activity in vitro and in vivo, reducing ER Ca(2+) content and stimulating mitochondrial energetics. The pro-apoptotic proteins Bax and tBid antagonize this effect by blocking the biochemical interaction of Bcl-X(L) with the InsP(3)R. These data support a novel model in which Bcl-X(L) is a direct effector of the InsP(3)R, increasing its sensitivity to InsP(3) and enabling ER Ca(2+) release to be more sensitively coupled to extracellular signals. As a consequence, cells are protected against apoptosis by a more sensitive and dynamic coupling of ER to mitochondria through Ca(2+)-dependent signal transduction that enhances cellular bioenergetics and preserves survival.     

MAC and Bcl-2 family proteins conspire in a deadly plot

Apoptosis is an elemental form of programmed cell death; it is fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Apoptosis is also involved in many pathologies including cancer, neurodegenerative diseases, aging, and infarcts. This cell death program is tightly regulated by Bcl-2 family proteins by controlling the formation of the mitochondrial apoptosis-induced channel or MAC. Assembly of MAC corresponds to permeabilization of the mitochondrial outer membrane, which is the so called commitment step of apoptosis. MAC provides the pathway through the mitochondrial outer membrane for the release of cytochrome c and other pro-apoptotic factors from the intermembrane space. While overexpression of anti-apoptotic Bcl-2 eliminates MAC activity, oligomers of the pro-apoptotic members Bax and/or Bak are essential structural component(s) of MAC. Assembly of MAC from Bax or Bak was monitored in real time by directly patch-clamping mitochondria with micropipettes containing the sentinel tBid, a direct activator of Bax and Bak. Herein, a variety of high affinity inhibitors of MAC (iMAC) that may prove to be crucial tools in mechanistic studies have recently been identified. This review focuses on characterization of MAC activity, its regulation by Bcl-2 family proteins, and a discussion of how MAC can be pharmacologically turned on or off depending on the pathology to be treated.     

The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet

Mitochondria are 'life-essential' organelles for the production of metabolic energy in the form of ATP. Paradoxically mitochondria also play a key role in controlling the pathways that lead to cell death. This latter role of mitochondria is more than just a 'loss of function' resulting in an energy deficit but is an active process involving different mitochondrial proteins. Cytochrome c was the first characterised mitochondrial factor shown to be released from the mitochondrial intermembrane space and to be actively implicated in apoptotic cell death. Since then, other mitochondrial proteins, such as AIF, Smac/DIABLO, endonuclease G and Omi/HtrA2, were found to undergo release during apoptosis and have been implicated in various aspects of the cell death process. Members of the Bcl-2 protein family control the integrity and response of mitochondria to apoptotic signals. The molecular mechanism by which mitochondrial intermembrane space proteins are released and the regulation of mitochondrial homeostasis by Bcl-2 proteins is still elusive. This review summarises and evaluates the current knowledge concerning the complex role of released mitochondrial proteins in the apoptotic process.     

Changes in astrocyte mitochondrial function with stress: effects of Bcl-2 family proteins

Mitochondria are central to both apoptotic and necrotic cell death, as well as to normal physiological function. Astrocytes are crucial for neuronal metabolic, antioxidant, and trophic support, as well as normal synaptic function. In the setting of stress, such as during cerebral ischemia, astrocyte dysfunction may compromise the ability of neurons to survive. Despite their central importance, the response of astrocyte mitochondria to stress has not been extensively studied. Limited data already suggest clear differences in the response of neuronal and astrocytic mitochondria to oxygen-glucose deprivation (GD). Prominent mitochondrial alterations during stress that can contribute to cell death include changes in production of reactive oxygen species (ROS) and release of death regulatory and signaling molecules from the intermembrane space. In response to stress mitochondrial respiratory function and membrane potential also change, and these changes appear to depend on cell type. Bcl-2 family proteins are the best studied regulators of cell death, especially apoptosis, and mitochondria are a major site of action for these proteins. Although much data supports the role of Bcl-2 family proteins in the regulation of some of these mitochondrial alterations, this remains an area of active investigation. This mini-review summarizes current knowledge regarding mitochondrial control of cell survival and death in astrocytes and the effects of anti-apoptotic Bcl-2 proteins on astrocyte mitochondrial function.     

BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation of Bax

During mitochondrial apoptosis, pro-apoptotic BH3-only proteins cause the translocation of cytosolic Bcl-2-associated X protein (Bax) to the outer mitochondrial membrane (OMM) where it is activated to release cytochrome c from the mitochondrial intermembrane space, but the mechanism is under dispute. We show that most BH3-only proteins are mitochondrial proteins that are imported into the OMM via a C-terminal tail-anchor domain in isolated yeast mitochondria, independently of binding to anti-apoptotic Bcl-2 proteins. This C-terminal domain acted as a classical mitochondrial targeting signal and was sufficient to direct green fluorescent protein to mitochondria in human cells. When expressed in mouse fibroblasts, these BH3-only proteins localised to mitochondria and were inserted in the OMM. The BH3-only proteins Bcl-2-interacting mediator of cell death (Bim), tBid and p53-upregulated modulator of apoptosis sensitised isolated mitochondria from Bax/Bcl-2 homologous antagonist/killer-deficient fibroblasts to cytochrome c-release by recombinant, extramitochondrial Bax. For Bim, this activity is shown to require the C-terminal-targeting signal and to be independent of binding capacity to and presence of anti-apoptotic Bcl-2 proteins. Bim further enhanced Bax-dependent killing in yeast. A model is proposed where OMM-tail-anchored BH3-only proteins permit passive 'recruitment' and catalysis-like activation of extra-mitochondrial Bax. The recognition of C-terminal membrane-insertion of BH3-only proteins will permit the development of a more detailed concept of the initiation of mitochondrial apoptosis.