Bcl-2 family of proteins: life-or-death switch in mitochondria

An increase in the permeability of outer mitochondrial membrane is central to apoptotic cell death, and results in the release of several apoptogenic factors such as cytochrome c into the cytoplasm to activate downstream destructive programs. The voltage-dependent anion channel (VDAC or mitochondrial porin) plays an essential role in disrupting the mitochondrial membrane barrier and is regulated directly by members of the Bcl-2 family proteins. Anti-apoptotic Bcl-2 family members interact with and close the VDAC, whereas some, but not all, proapoptotic members interact with VDAC to open protein-conducting pore through which apoptogenic factors pass. Although the VDAC is involved directly in breaking the mitochondrial membrane barrier and is a known component of the permeability transition pore complex, VDAC-dependent increase in outer membrane permeability can be independent of the permeability transition event such as mitochondrial swelling followed by rupture of the outer mitochondrial membrane. VDAC interacts not only with Bcl-2 family members but also with proteins such as gelsolin, an actin regulatory protein, and appears to be a convergence point for a variety of cell survival and cell death signals.     

VDAC regulation by the Bcl-2 family of proteins

The Bcl-2 family of proteins consists of anti-apoptotic and pro-apoptotic members, which determine the life or death of cells by altering mitochondrial membrane permeability. Pro-apoptotic Bcl-2 family members increase mitochondrial membrane permeability, resulting in the release of mitochondrial apoptogenic factors such as cytochrome c that activates death proteases called caspases, whereas anti-apoptotic family members prevent this increase of mitochondrial membrane permeability. The release of cytochrome c is central to apoptotic signal transduction in mammals, and has been studied extensively, leading to the development of several models for cytochrome c release including rupture of the mitochondrial outer membrane and involvement of specific channels. This article describes the important role of a mitochondrial outer membrane channel, the voltage-dependent anion channel (VDAC), in apoptogenic cytochrome c release and its regulation by Bcl-2 family members, and also discusses the molecular architecture of the life - death switch in mammalian cells. Cell Death and Differentiation (2000) 7, 1174 - 1181     

Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c

The Bcl-2 family of proteins, consisting of anti-apoptotic and pro-apoptotic members, regulates cell death by controlling mitochondrial membrane permeability that is crucial for apoptotic signal transduction. We have recently shown that some of these proteins, such as Bcl-x(L), Bax, and Bak, directly modulate the mitochondrial voltage-dependent anion channel (VDAC) and thus regulate apoptogenic cytochrome c release and potential loss. To elucidate the molecular mechanisms of VDAC regulation by Bcl-2 family proteins, an electrophysiological study was carried out. It was found that VDAC and pro-apoptotic Bax created a large pore, with conductance levels 4- and 10-fold greater than those of the VDAC and Bax channels, respectively. Although the VDAC and Bax channels both show ion selectivity and voltage-dependent modulation of their activity, the VDAC-Bax channel had neither of their properties. Anti-apoptotic Bcl-x(L) and its BH4 oligopeptide completely closed the VDAC, in contrast to the Bax. Cytochrome c passed through a single VDAC-Bax channel but not through the VDAC or Bax channel in a planar lipid bilayer. These data provide direct evidence that VDAC forms a novel large pore together with Bax.     

VDAC activation by the 18 kDa translocator protein (TSPO), implications for apoptosis

The voltage dependent anion channel (VDAC), located in the outer mitochondrial membrane, functions as a major channel allowing passage of small molecules and ions between the mitochondrial inter-membrane space and cytoplasm. Together with the adenine nucleotide translocator (ANT), which is located in the inner mitochondrial membrane, the VDAC is considered to form the core of a mitochondrial multiprotein complex, named the mitochondrial permeability transition pore (MPTP). Both VDAC and ANT appear to take part in activation of the mitochondrial apoptosis pathway. Other proteins also appear to be associated with the MPTP, for example, the 18 kDa mitochondrial Translocator Protein (TSPO), Bcl-2, hexokinase, cyclophylin D, and others. Interactions between VDAC and TSPO are considered to play a role in apoptotic cell death. As a consequence, due to its apoptotic functions, the TSPO has become a target for drug development directed to find treatments for neurodegenerative diseases and cancer. In this context, TSPO appears to be involved in the generation of reactive oxygen species (ROS). This generation of ROS may provide a link between activation of TSPO and of VDAC, to induce activation of the mitochondrial apoptosis pathway. ROS are known to be able to release cytochrome c from cardiolipins located at the inner mitochondrial membrane. In addition, ROS appear to be able to activate VDAC and allow VDAC mediated release of cytochrome c into the cytosol. Release of cytochrome c from the mitochondria forms the initiating step for activation of the mitochondrial apoptosis pathway. These data provide an understanding regarding the mechanisms whereby VDAC and TSPO may serve as targets to modulate apoptotic rates. This has implications for drug design to treat diseases such as neurodegeneration and cancer.     

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     

Cell death regulation by the Bcl-2 protein family in the mitochondria

An increase in the permeability of the outer mitochondrial membrane is central to apoptotic cell death, since it leads to the release of several apoptogenic factors, such as cytochrome c and Smac/Diablo, into the cytoplasm that activate downstream death programs. During apoptosis, the mitochondria also release AIF and endonuclease G, both of which are translocated to the nucleus and are implicated in apoptotic nuclear changes that occur in a caspase-independent manner. Mitochondrial membrane permeability is directly controlled by the major apoptosis regulator, i.e., the Bcl-2 family of proteins, mainly through regulation of the formation of apoptotic protein-conducting pores in the outer mitochondrial membrane, although the precise molecular mechanisms are still not completely understood. Here, I focus on the mechanisms by which Bcl-2 family members control the permeability of mitochondrial membrane during apoptosis.     

Mitochondrial membrane permeability transition and cell death

Mitochondria are important organelles for energy production, Ca2+ homeostasis, and cell death. In recent years, the role of the mitochondria in both apoptotic and necrotic cell death has received much attention. In apoptotic and necrotic death, an increase of mitochondrial membrane permeability is considered to be one of the key events, although the detailed mechanism remains to be elucidated. The mitochondrial membrane permeability transition (MPT) is a Ca2+-dependent increase in the permeability of the mitochondrial membrane that leads to loss of Deltapsi, mitochondrial swelling, and rupture of the outer mitochondrial membrane. The MPT is thought to occur after the opening of a channel, which is termed the permeability transition pore (PTP) and putatively consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), cyclophilin D (Cyp D: a mitochondrial peptidyl prolyl-cis, trans-isomerase), and other molecule(s). Our studies of mice lacking Cyp D have revealed that it is essential for occurrence of the MPT and that the Cyp D-dependent MPT regulates some forms of necrotic cell death, but not apoptotic death. We have also shown that two anti-apoptotic proteins, Bcl-2 and Bcl-x(L), block the MPT by directly inhibition of VDAC activity. Here we summarize a role of the MPT in cell death.     

NMR structural investigation of the mitochondrial outer membrane protein VDAC and its interaction with antiapoptotic Bcl-xL

Bcl-2 family proteins are essential regulators of cell death and exert their primary pro- or antiapoptotic roles at the mitochondrial outer membrane. Previously, pro- and antiapoptotic Bcl-2 proteins have been shown to interact with the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. VDAC is a 283-residue integral membrane protein that forms an aqueous pore in the outer mitochondrial membrane, through which metabolites and other small molecules pass between the cytosol and intermembrane space. The essential life-sustaining function of VDAC in metabolite trafficking is believed to be regulated by proteins of the Bcl-2 family. The protective role of antiapoptotic Bcl-xL may be through its interaction with VDAC. Here, VDAC has been expressed, purified, and refolded into a functional form amenable to NMR studies. Various biophysical experiments indicate that micelle-bound VDAC is in intermediate exchange between monomer and trimer. Using NMR spectroscopy, gel filtration, and chemical cross-linking, we obtained direct evidence for binding of Bcl-xL to VDAC in a detergent micelle system. The VDAC-interacting region of Bcl-xL was characterized by NMR with chemical shift perturbation and transferred cross-saturation. The interaction region was mapped to a putative helical hairpin motif of Bcl-xL that was found to insert into detergent micelles. Our results suggest that Bcl-xL can bind to one or two VDAC molecules forming heterodimers and heterotrimers. Our characterization of the VDAC/Bcl-xL complex offers initial structural insight into the role of antiapoptotic Bcl-xL in regulating apoptotic events in the mitochondrial outer membrane.     

Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.     

Phosphorothioate oligonucleotides reduce mitochondrial outer membrane permeability to ADP

G3139, an antisense Bcl-2 phosphorothioate oligodeoxyribonucleotide, induces apoptosis in melanoma and other cancer cells. This apoptosis happens before and in the absence of the downregulation of Bcl-2 and thus seems to be Bcl-2-independent. Binding of G3139 to mitochondria and its ability to close voltage-dependent anion-selective channel (VDAC) have led to the hypothesis that G3139 acts, in part, by interacting with VDAC channels in the mitochondrial outer membrane (21). In this study, we demonstrate that G3139 is able to reduce the mitochondrial outer membrane permeability to ADP by a factor of 6 or 7 with a K_i between 0.2 and 0.5 µM. Because VDAC is responsible for this permeability, this result strengthens the aforesaid hypothesis. Other mitochondrial respiration components are not affected by [G3139] up to 1 µM. Higher levels begin to inhibit respiration rates, decrease light scattering and increase uncoupled respiration. These results agree with accumulating evidence that VDAC closure favors cytochrome c release. The speed of this effect (within 10 min) places it early in the apoptotic cascade with cytochrome c release occurring at later times. Other phosphorothioate oligonucleotides are also able to induce VDAC closure, and there is some length dependence. The phosphorothioate linkages are required to induce the reduction of outer membrane permeability. At levels below 1 µM, phosphorothioate oligonucleotides are the first specific tools to restrict mitochondrial outer membrane permeability. respiration; voltage-dependent anion-selective channel; apoptosis; cell death     

VDAC blockage by phosphorothioate oligonucleotides and its implication in apoptosis

Apoptosis is a crucial process that regulates the homeostasis of multicellular organisms. Impaired apoptosis contributes to cancer development, while enhanced apoptosis is detrimental in neurodegenerative diseases. The intrinsic apoptotic pathway is initiated by cytochrome c release from mitochondria. Research published in the recent decade has suggested that cytochrome c release can be influenced by the conducting states of VDAC, the channel in the mitochondrial outer membrane (MOM) responsible for metabolite flux. This review will describe the evidence that VDAC gating or blockage and subsequent changes in MOM permeability influence cytochrome c release and the onset of apoptosis. The blockage of VDAC by G3139, a proapoptotic phosphorothioate oligonucleotide, provides strong evidence for the role of VDAC in the initiation of apoptosis. The proapoptotic activity and VDAC blockage are linked in that both require the PS (phosphorothioate) modification, both are enhanced by an increase in oligonucleotide length, and both are insensitive to the nucleotide sequence. Thus, the mitochondrial outer membrane permeability regulated by VDAC gating may play an important role in mitochondrial function and in the control of apoptosis. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

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.     

Mouse uterine epithelial apoptosis is associated with expression of mitochondrial voltage-dependent anion channels,release of cytochrome C from mitochondria,and the ratio of Bax to Bcl-2 or Bcl-X

The release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members and is considered to take place through voltage-dependent anion channels (VDACs) on the outer membranes of mitochondria, results in activation of effector caspases, such as caspase-3, which induce apoptosis. We studied the involvement of the mitochondrial apoptosis pathway in uterine epithelial apoptosis. Estradiol-17beta pellets were implanted into ovariectomized mice and removed 4 days later (Day 0). The apoptotic index (percentage of apoptotic cells) of the luminal epithelium increased markedly, peaking on Day 2, whereas that of the glandular epithelium increased much less. Expression of VDAC1, 2, and 3 mRNAs increased in the luminal epithelium in correlation with the apoptotic index of the luminal epithelium. No increases in VDAC1, 2, and 3 mRNA levels were observed in the stroma or muscle, where no apoptosis occurs. VDAC1 protein levels in the uterus also correlated well with the apoptotic index of the luminal epithelium. In addition, the apoptotic index showed good correlation with the release of cytochrome c from mitochondria, activation of caspase-3, which was immunohistochemically detected only in the epithelium, and the mRNA and protein ratios of Bax:Bcl-2 and Bax:Bcl-X in the uterus. The present results suggest that the release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members, plays a role in uterine epithelial apoptosis after estrogen deprivation. The increase in VDAC expression may facilitate the release of cytochrome c during apoptosis.     

Voltage-dependent Anion Channel 1-based Peptides Interact with Bcl-2 to Prevent Antiapoptotic Activity

The antiapoptotic proteins of the Bcl-2 family are expressed at high levels in many types of cancer. However, the mechanism by which Bcl-2 family proteins regulate apoptosis is not fully understood. Here, we demonstrate the interaction of Bcl-2 with the outer mitochondrial membrane protein, voltage-dependent anion channel 1 (VDAC1). A direct interaction of Bcl-2 with bilayer-reconstituted purified VDAC was demonstrated, with Bcl-2 decreasing channel conductance. Expression of Bcl-2-GFP prevented apoptosis in cells expressing native but not certain VDAC1 mutants. VDAC1 sequences and amino acid residues important for interaction with Bcl-2 were defined through site-directed mutagenesis. Synthetic peptides corresponding to the VDAC1 N-terminal region and selected sequences bound specifically, in a concentration- and time-dependent manner, to immobilized Bcl-2, as revealed by the real-time surface plasmon resonance. Moreover, expression of the VDAC1-based peptides in cells over-expressing Bcl-2 prevented Bcl-2-mediated protection against staurosporine-induced apoptotic cell death. Similarly, a cell-permeable VDAC1-based synthetic peptide was also found to prevent Bcl-2-GFP-mediated protection against apoptosis. These results point to Bcl-2 as promoting tumor cell survival through binding to VDAC1, thereby inhibiting cytochrome c release and apoptotic cell death. Moreover, these findings suggest that interfering with the binding of Bcl-2 to mitochondria by VDAC1-based peptides may serve to potentiate the efficacy of conventional chemotherapeutic agents.     

In vitro interactions between the two mitochondrial membrane proteins VDAC and cytochrome c oxidase

VDAC, a mitochondrial outer membrane channel, is involved in the control of aerobic metabolism and in apoptotic processes via numerous protein-protein interactions. To unveil those interactions, we screened a human liver cDNA library with the phage display methodology optimized to target VDAC reconstituted into a membrane environment. One positively selected clone yielded a sequence matching a part of the subunit I of human cytochrome c oxidase (COX), a mitochondrial inner membrane enzyme. Such putative interaction was never reported before. This interaction proved to be functional as evidenced by the effect of the human and yeast isoforms of VDAC on the oxidation of cytochrome c by the pure holoenzyme and by the effect of the COX epitope on VDAC permeability. Our results providing four independently obtained evidences of VDAC-COX interaction in vitro, would support a novel and potentially important level of mitochondrial regulation given the respective locations and functions of both proteins.     

Role of the mitochondrial membrane permeability transition in cell death

In recent years, the role of the mitochondria in both apoptotic and necrotic cell death has received considerable attention. An increase of mitochondrial membrane permeability is one of the key events in apoptotic or necrotic death, although the details of the mechanism involved remain to be elucidated. The mitochondrial membrane permeability transition (MPT) is a Ca²⁺-dependent increase of mitochondrial membrane permeability that leads to loss of Δψ, mitochondrial swelling, and rupture of the outer mitochondrial membrane. The MPT is thought to occur after the opening of a channel that is known as the permeability transition pore (PTP), which putatively consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), cyclophilin D (Cyp D: a mitochondrial peptidyl prolyl-cis, trans-isomerase), and other molecule(s). Recently, significant progress has been made by studies performed with mice lacking Cyp D at several laboratories, which have convincingly demonstrated that Cyp D is essential for the MPT to occur and that the Cyp D-dependent MPT regulates some forms of necrotic, but not apoptotic, cell death. Cyp D-deficient mice have also been used to show that the Cyp D-dependent MPT plays a crucial role in ischemia/reperfusion injury. The anti-apoptotic proteins Bcl-2 and Bcl-x_L have the ability to block the MPT, and can therefore block MPT-dependent necrosis in addition to their well-established ability to inhibit apoptosis.     

Mitochondria as the central control point of apoptosis

Mitochondria play a major role in apoptosis triggered by many stimuli. They integrate death signals through Bcl-2 family members and coordinate caspase activation through the release of cytochrome c as a result of the outer mitochondrial membrane becoming permeable. The mechanisms that lead to this permeability are not yet completely understood. Here, we attempt to summarize our current view of the mechanisms that lead to the efflux of many proteins from mitochondria during apoptosis and the role played by Bcl-2 family proteins in the control of this event.     

Regulation of the mitochondrial apoptosis-induced channel, MAC, by BCL-2 family proteins

Programmed cell death or apoptosis is central to many physiological processes and pathological conditions such as organogenesis, tissue homeostasis, cancer, and neurodegenerative diseases. Bcl-2 family proteins tightly control this cell death program by regulating the permeabilization of the mitochondrial outer membrane and, hence, the release of cytochrome c and other pro-apoptotic factors. Control of the formation of the mitochondrial apoptosis-induced channel, or MAC, is central to the regulation of apoptosis by Bcl-2 family proteins. MAC is detected early in apoptosis by patch clamping the mitochondrial outer membrane. The focus of this review is on the regulation of MAC activity by Bcl-2 family proteins. The role of MAC as the putative cytochrome c release channel during early apoptosis and insights concerning its molecular composition are also discussed.     

Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis?

Apoptosis is a phenomenon fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Bcl-2 family proteins tightly control this cell death program by regulating the permeabilization of the mitochondrial outer membrane and, hence, the release of cytochrome c and other proapoptotic factors. Mitochondrial apoptosis-induced channel (MAC) is the mitochondrial apoptosis-induced channel and is responsible for cytochrome c release early in apoptosis. MAC activity is detected by patch clamping mitochondria at the time of cytochrome c release. The Bcl-2 family proteins regulate apoptosis by controlling the formation of MAC. Depending on cell type and apoptotic inducer, Bax and/or Bak are structural component(s) of MAC. Overexpression of the antiapoptotic protein Bcl-2 eliminates MAC activity. The focus of this review is a biophysical characterization of MAC activity and its regulation by Bcl-2 family proteins, and ends with some discussion of therapeutic targets.     

Human Bop is a novel BH3-only member of the Bcl-2 protein family

One group of Bcl-2 protein family, which shares only the BH3 domain (BH3-only), is critically involved in the regulation of programmed cell death. Herein we demonstrated a novel human BH3-only protein (designated as Bop) which could induce apoptosis in a BH3 domain-dependent manner. Further analysis indicated that Bop mainly localized to mitochondria and used its BH3 domain to contact the loop regions of voltage dependent anion channel 1 (VDAC1) in the outer mitochondrial membrane. In addition, purified Bop protein induced the loss of mitochondrial transmembrane potential (ΔΨm) and the release of cytochrome c. Furthermore, Bop used its BH3 domain to contact pro-survival Bcl-2 family members (Bcl-2, Bcl-X_L, Mcl-1, A1 and Bcl-w), which could inhibit Bop-induced apoptosis. Bop would be constrained by pro-survival Bcl-2 proteins in resting cells, because Bop became released from phosphorylated Bcl-2 induced by microtubule-interfering agent like vincristine (VCR). Indeed, knockdown experiments indicated that Bop was partially required for VCR induced cell death. Finally, Bop might need to function through Bak and Bax, likely by releasing Bak from Bcl-X_L sequestration. In conclusion, Bop may be a novel BH3-only factor that can engage with the regulatory network of Bcl-2 family members to process intrinsic apoptotic signaling.