Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis

During apoptosis, pro‐apoptotic BAX and BAK are activated, causing mitochondrial outer membrane permeabilisation (MOMP), caspase activation and cell death. However, even in the absence of caspase activity, cells usually die following MOMP. Such caspase‐independent cell death is accompanied by inflammation that requires mitochondrial DNA (mtDNA) activation of cGAS‐STING signalling. Because the mitochondrial inner membrane is thought to remain intact during apoptosis, we sought to address how matrix mtDNA could activate the cytosolic cGAS‐STING signalling pathway. Using super‐resolution imaging, we show that mtDNA is efficiently released from mitochondria following MOMP. In a temporal manner, we find that following MOMP, BAX/BAK‐mediated mitochondrial outer membrane pores gradually widen. This allows extrusion of the mitochondrial inner membrane into the cytosol whereupon it permeablises allowing mtDNA release. Our data demonstrate that mitochondrial inner membrane permeabilisation (MIMP) can occur during cell death following BAX/BAK‐dependent MOMP. Importantly, by enabling the cytosolic release of mtDNA, inner membrane permeabilisation underpins the immunogenic effects of caspase‐independent cell death.     

Fas death receptor signalling: roles of Bid and XIAP

Fas (also called CD95 or APO-1), a member of a subgroup of the tumour necrosis factor receptor superfamily that contain an intracellular death domain, can initiate apoptosis signalling and has a critical role in the regulation of the immune system. Fas-induced apoptosis requires recruitment and activation of the initiator caspase, caspase-8 (in humans also caspase-10), within the death-inducing signalling complex. In so-called type 1 cells, proteolytic activation of effector caspases (-3 and -7) by caspase-8 suffices for efficient apoptosis induction. In so-called type 2 cells, however, killing requires amplification of the caspase cascade. This can be achieved through caspase-8-mediated proteolytic activation of the pro-apoptotic Bcl-2 homology domain (BH)3-only protein BH3-interacting domain death agonist (Bid), which then causes mitochondrial outer membrane permeabilisation. This in turn leads to mitochondrial release of apoptogenic proteins, such as cytochrome c and, pertinent for Fas death receptor (DR)-induced apoptosis, Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding protein with low Pi), an antagonist of X-linked inhibitor of apoptosis (XIAP), which imposes a brake on effector caspases. In this review, written in honour of Juerg Tschopp who contributed so much to research on cell death and immunology, we discuss the functions of Bid and XIAP in the control of Fas DR-induced apoptosis signalling, and we speculate on how this knowledge could be exploited to develop novel regimes for treatment of cancer.     

N‐(3‐Oxo‐acyl)‐homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts

N‐(3‐Oxododecanoyl)‐l ‐homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum‐sensing molecule for bacteria–bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12‐triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in “initiator” caspases or “effector” caspases. Our data indicate that C12 selectively induces the mitochondria‐dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both “initiator” and “effector” caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis.     

Caspase-2 can function upstream of bid cleavage in the TRAIL apoptosis pathway

In many mammalian cell types, engagement of the TRAIL/Apo2L death receptors DR4 and DR5 alters mitochondrial physiology, thereby promoting the release of pro-apoptotic proteins normally contained within this organelle. A contemporary view of this process is that in so-called type II cells death receptor-activated caspase-8 cleaves the Bcl-2 family member Bid, which generates a truncated Bid fragment that collaborates with Bax, another Bcl-2 relative, to promote the release of mitochondrial factors necessary for activation of executioner caspases and apoptosis. Here we show that in some type II cells caspase-2 is necessary for optimal TRAIL-mediated cleavage of Bid. Down-regulation of caspase-2 using RNA interference significantly inhibited TRAIL-induced apoptosis. Analysis of the TRAIL proteolytic cascade following gene silencing of specific pathway components revealed that caspase-2 is necessary for efficient cleavage of Bid; however, caspase-2 proteolytic processing, which occurs downstream of Bax, is not necessary for its role in Bid cleavage.     

Caspase-8 Binding to Cardiolipin in Giant Unilamellar Vesicles Provides a Functional Docking Platform for Bid

Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria.     

Diffusion is capable of translating anisotropic apoptosis initiation into a homogeneous execution of cell death

Background  

Apoptosis is an essential cell death process throughout the entire life span of all metazoans and its deregulation in humans has been implicated in many proliferative and degenerative diseases. Mitochondrial outer membrane permeabilisation (MOMP) and activation of effector caspases are key processes during apoptosis signalling. MOMP can be subject to spatial coordination in human cancer cells, resulting in intracellular waves of cytochrome-c release. To investigate the consequences of these spatial anisotropies in mitochondrial permeabilisation on subsequent effector caspase activation, we devised a mathematical reaction-diffusion model building on a set of partial differential equations.     

Galectin-1 induced activation of the mitochondrial apoptotic pathway: evidence for a connection between death-receptor and mitochondrial pathways in human Jurkat T lymphocytes

Galectin-1 (gal-1) triggers T cell death by several distinct intracellular pathways including the activation of the death-receptor pathway. The aim of this study was to investigate whether gal-1 induced activation of the death-receptor pathway in Jurkat T lymphocytes mediates apoptosis via the mitochondrial pathway linked by truncated Bid (tBid). We demonstrate that gal-1 induced proteolytic cleavage of the death agonist Bid, a member of the Bcl-2/Bcl-xL family and a substrate of activated caspase-8, was inhibited by caspase-8 inhibitor II (Z-IETD-FMK). Downstream of Bid, gal-1 stimulated mitochondrial cytochrome c release as well as the activation and proteolytic processing of initiator procaspase-9 were effectively decreased by caspase-8 inhibitor II. Blocking of gal-1 induced cleavage of effector procaspase-3 by caspase-8 inhibitor II as well as by caspase-9 inhibitors I (Z-LEHD-FMK) and III (Ac-LEHD-CMK) indicates that receptor and mitochondrial pathways converged in procaspase-3 activation and contribute to proteolytic processing of effector procaspase-6 and -7. Western blot analyses and immunofluorescence staining revealed that exposure of Jurkat T cells to gal-1 resulted in the cleavage of the DNA-repair enzyme poly (ADP-ribose) polymerase, cytoskeletal α-fodrin, and nuclear lamin A as substrates of activated caspases. Our data demonstrate that Bid provides a connection between the death receptor and the mitochondrial pathway of gal-1 induced apoptosis in human Jurkat T lymphocytes.     

Cardiolipin-enriched raft-like microdomains are essential activating platforms for apoptotic signals on mitochondria

Cardiolipin (CL) has recently been shown to provide an anchor and an essential activating platform for caspase-8 on mitochondria. We hypothesize that these platforms may correspond to “raft-like” microdomains, which have demonstrated to be detectable on mitochondrial membrane of cells undergoing apoptosis. The role for CL in “raft-like” microdomains could be to anchor caspase-8 at contact sites between inner and outer membranes, facilitating its self-activation, Bid cleavage and apoptosis execution. The role played by “raft-like” microdomains in the apoptotic program could introduce a new task in the pathogenetic studies on human diseases associated with cardiolipin dismetabolism.     

A functional genomics screen identifies PCAF and ADA3 as regulators of human granzyme B-mediated apoptosis and Bid cleavage

The human lymphocyte toxins granzyme B (hGrzB) and perforin cooperatively induce apoptosis of virus-infected or transformed cells: perforin pores enable entry of the serine protease hGrzB into the cytosol, where it processes Bid to selectively activate the intrinsic apoptosis pathway. Truncated Bid (tBid) induces Bax/Bak-dependent mitochondrial outer membrane permeability and the release of cytochrome c and Smac/Diablo. To identify cellular proteins that regulate perforin/hGrzB-mediated Bid cleavage and subsequent apoptosis, we performed a gene-knockdown (KD) screen using a lentiviral pool of short hairpin RNAs embedded within a miR30 backbone (shRNAmiR). We transduced HeLa cells with a lentiviral pool expressing shRNAmiRs that target 1213 genes known to be involved in cell death signaling and selected cells with acquired resistance to perforin/hGrzB-mediated apoptosis. Twenty-two shRNAmiRs were identified in the positive-selection screen including two, PCAF and ADA3, whose gene products are known to reside in the same epigenetic regulatory complexes. Small interfering (si)RNA-mediated gene-KD of PCAF or ADA3 also conferred resistance to perforin/hGrzB-mediated apoptosis providing independent validation of the screen results. Mechanistically, PCAF and ADA3 exerted their pro-apoptotic effect upstream of mitochondrial membrane permeabilization, as indicated by reduced cytochrome c release in PCAF-KD cells exposed to perforin/hGrzB. While overall levels of Bid were unaltered, perforin/hGrzB-mediated cleavage of Bid was reduced in PCAF-KD or ADA3-KD cells. We discovered that PCAF-KD or ADA3-KD resulted in reduced expression of PACS2, a protein implicated in Bid trafficking to mitochondria and importantly, targeted PACS2-KD phenocopied the effect of PCAF-KD or ADA3-KD. We conclude that PCAF and ADA3 regulate Bid processing via PACS2, to modulate the mitochondrial cell death pathway in response to hGrzB.     

Promotion of Fas-mediated apoptosis in Type II cells by high doses of hepatocyte growth factor bypasses the mitochondrial requirement

The death receptor pathway is coupled to the mitochondria apoptosis pathway. However, mitochondrial participation, which is stimulated by Bid but suppressed by Bcl-2/Bcl-x(L), is required in certain cells (Type II), but not in others (Type I). While these differences were originally characterized in the lymphoid cell lines, the typical Type II cells are represented by hepatocytes in vivo. The molecular mechanisms that distinguish Type II from Type I cells and the regulation are not fully understood. Fas can be sequestered by the HGF receptor c-Met and high doses of HGF can promote cell death by freeing Fas from c-Met complex. We thus reasoned that treatment of the Type II cells with high doses of HGF could enhance Fas-mediated apoptosis and spare the mitochondria amplification. Indeed, such treatment led to increased apoptosis in Type II lymphoid cells, which could not be blocked by Bcl-x(L). Moreover, significant hepatocyte apoptosis was induced by this scheme in the absence of Bid with increased dissociation of Fas from c-Met. These findings indicate that high doses of HGF could be used to promote apoptosis in Type II cells bypassing the requirement for mitochondria activation.     

Caspase-9 Activation by the Apoptosome Is Not Required for Fas-mediated Apoptosis in Type II Jurkat Cells

Activation of executioner caspases during receptor-mediated apoptosis in type II cells requires the engagement of the mitochondrial apoptotic pathway. Although it is well established that recruitment of mitochondria in this context involves the cleavage of Bid to truncated Bid (tBid), the precise post-mitochondrial signaling responsible for executioner caspase activation is controversial. Here, we used distinct clones of type II Jurkat T-lymphocytes in which the mitochondrial apoptotic pathway had been inhibited to investigate the molecular requirements necessary for Fas-induced apoptosis. Cells overexpressing either Bcl-2 or Bcl-x_L were protected from apoptosis induced by agonistic anti-Fas antibody. By comparison, Apaf-1-deficient Jurkat cells were sensitive to anti-Fas, exhibiting Bid cleavage, Bak activation, the release of cytochrome c and Smac, and activation of executioner caspase-3. Inhibiting downstream caspase activation with the pharmacological inhibitor Z-DEVD-fmk or by expressing the BIR1/BIR2 domains of X-linked inhibitor of apoptosis protein (XIAP) decreased all anti-Fas-induced apoptotic changes. Additionally, pretreatment of Bcl-x_L-overexpressing cells with a Smac mimetic sensitized these cells to Fas-induced apoptosis. Combined, our findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis do not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells.     

Mitochondrial membrane dynamics, cristae remodelling and apoptosis

Mitochondria form a highly dynamic reticular network in living cells, and undergo continuous fusion/fission events and changes in ultrastructural architecture. Although significant progress has been made in elucidating the molecular events underlying these processes, their relevance to normal cell function remains largely unexplored. Emerging evidence, however, suggests an important role for mitochondrial dynamics in cellular apoptosis. The mitochondria is at the core of the intrinsic apoptosis pathway, and provides a reservoir for protein factors that induce caspase activation and chromosome fragmentation. Additionally, mitochondria modulate Ca2+ homeostasis and are a source of various metabolites, including reactive oxygen species, that have the potential to function as second messengers in response to apoptotic stimuli. One of the mitochondrial factors required for activation of caspases in most intrinsic apoptotic pathways, cytochrome c, is largely sequestered within the intracristae compartment, and must migrate into the boundary intermembrane space in order to allow passage across the outer membrane to the cytosol. Recent evidence argues that inner mitochondrial membrane dynamics regulate this process. Here, we review the contribution of mitochondrial dynamics to the intrinsic apoptosis pathway, with emphasis on the inner membrane.     

B cell receptor cross-linking triggers a caspase-8-dependent apoptotic pathway that is independent of the death effector domain of Fas-associated death domain protein.

We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt's lymphoma cells. Whereas soluble anti-mu Ab triggers caspase-independent mitochondrial activation, cross-linked anti-mu Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt's lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein.     

Apoptosis-inducing factor (AIF) is targeted in IFN-α2a-induced Bid-mediated apoptosis through Bak activation in ovarian cancer cells

Previously we have shown that interferon (IFN)-α induced apoptosis is predominantly mediated by the upregulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) via the caspase-8 pathway. It was also shown that recruitment of mitochondria in IFN-α induced apoptosis involves the cleavage of BH3 interacting domain death agonist (Bid) to truncated Bid (tBid). In the present study, we demonstrate that tBid induced by IFN-α2a activates mitochondrial Bak to trigger the loss of mitochondrial membrane integrity, consequently causing release of apoptosis-inducing factor (AIF) in ovarian cancer cells, OVCAR3. AIF translocates from the mitochondria to the nucleus and induces nuclear fragmentation and cell death. Both a small molecule Bid inhibitor (BI-6C9) or Bid-RNA interference (RNAi) preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated IFN-α2a-induced cell death. Cell death induced by tBid was inhibited by AIF-RNAi, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that BI-6C9 did not prevent the release of cytochrome c from mitochondria to cytosol, while the release of AIF was prevented. In conclusion, IFN-α2a-induced apoptosis is mediated via the mitochondria-associated pathway involving the cleavage of Bid followed by AIF release that involves Bak activation and translocation of AIF from the mitochondria to the nucleus in OVCAR3 cells.     

A central role for Bid in granzyme B-induced apoptosis

Granzyme B, a protease released from cytotoxic lymphocytes, has been proposed to induce target cell death by cleaving and activating the pro-apoptotic Bcl-2 family member Bid. It has also been proposed that granzyme B can induce target cell death by activating caspases directly, by cleaving caspase substrates, and/or by cleaving several non-caspase substrates. The relative importance of Bid in granzyme B-induced cell death has therefore remained unclear. Here we report that cells isolated from various tissues of Bid-deficient mice were resistant to granzyme B-induced cell death. Consistent with the proposed role of Bid in regulating mitochondrial outer membrane permeabilization, cytochrome c remained in the mitochondria of Bid-deficient cells treated with granzyme B. Unlike wild type cells, Bid-deficient cells survived and were then able to proliferate normally, demonstrating the critical role for Bid in mediating granzyme B-induced apoptosis.     

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.     

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.     

Where Killers Meet—Permeabilization of the Outer Mitochondrial Membrane during Apoptosis

Although mitochondria are usually considered as supporters of life, they are also involved in cellular death. Mitochondrial outer membrane permeabilization (MOMP) is a crucial event during apoptosis because it causes the release of proapoptotic factors from the mitochondrial intermembrane space to the cytosol. MOMP is mainly controlled by the Bcl-2 family of proteins, which consists of both proapoptotic and antiapoptotic members. We discuss the current understanding of how activating and inhibitory interactions within this family lead to the activation and oligomerization of MOMP effectors Bax and Bak, which result in membrane permeabilization. The order of events leading to MOMP is then highlighted step by step, emphasizing recent discoveries regarding the formation of Bax/Bak pores on the outer mitochondrial membrane. Besides the Bcl-2 proteins, the mitochondrial organelle contributes to and possibly regulates MOMP, because mitochondrial resident proteins and membrane lipids are prominently involved in the process.Mitochondria are essential for the life of the cell. They produce most of the ATP via oxidative phosphorylation thanks to the respiratory chain that is embedded in the inner mitochondrial membrane. Consequently, mitochondrial dysfunction is implicated in the development of many human diseases, in particular, neurodegenerative disorders (Lin and Beal 2006). Mitochondria are also prominently involved in cell death, because they play a crucial role in many apoptotic responses. Apoptosis is a self-destruction program that is essential during the development of multicellular organisms. Its dysregulation has also been recognized as a main feature of many pathological conditions, especially cancer (Llambi and Green 2011).The executioners of apoptosis are a family of cysteine proteases termed caspases that cleave a variety of cellular targets, resulting in morphological changes, degradation of genomic DNA, and, ultimately, phagocytic removal of the apoptotic cell (Taylor et al. 2008). Caspases are synthesized as inactive zymogens that become activated after regulated limited proteolysis. Two different pathways of apoptotic signaling that result in the activation of executioner caspases 3 and 7 can be distinguished. In the extrinsic pathway, binding of ligands such as FasL or TNFα to a death receptor on the plasma membrane leads to the activation of initiator caspase 8. Active caspase 8 propagates the signal by directly cleaving and thereby activating caspases 3 and 7, which continue a proteolytic cascade ultimately leading to the removal of the cell.The intrinsic pathway, on the other hand, is initiated upon exposure to a number of stress situations, including DNA damage. A subclass of the Bcl-2 protein family termed BH3-only proteins (see below) becomes activated after an internal stress stimulus and translocates to the outer mitochondrial membrane (OMM), where they orchestrate a process called mitochondrial outer membrane permeabilization (MOMP). As an outcome of this process, pores are formed in the OMM, membrane integrity is lost, and contents of the intermembrane space gain access to the cytosol. One of the molecules that is rapidly released to the cytosol is cytochrome c, which is normally a soluble electron carrier between respiratory complexes III and IV. Together with the proapoptotic cytosolic factor APAF1, cytochrome c assembles into a caspase-activating complex termed the “apoptosome.” This complex subsequently activates caspase 9, which is able to cleave caspases 3 and 7, proceeding with the same downstream cascade as in the extrinsic pathway. Other intermembrane space proteins also contribute to cell death after being released into the cytosol (e.g., SMAC/Diablo, which blocks the caspase inhibitor protein XIAP).Remarkably, the two pathways are not completely independent. Cross talk between the extrinsic and intrinsic pathways exists because of caspase 8-dependent cleavage of the BH3-only protein Bid. Upon cleavage, Bid becomes activated, and the truncated version, tBid, translocates to the surface of mitochondria to induce MOMP. In so-called type II cells, this mitochondrial feedback loop is needed to induce apoptosis through the extrinsic pathway, because of the requirement of XIAP antagonism by SMAC.The loss of OMM integrity caused by MOMP is usually considered the point of no return in the whole process, because cells are committed to die once MOMP is initiated. Therefore, this process represents a major checkpoint of apoptosis and must be tightly controlled to ensure that it is initiated at the right time and place. The main molecular players of MOMP belong to the Bcl-2 protein family. Integration of proapoptotic and antiapoptotic signals by the network of Bcl-2 proteins determines whether or not the OMM is permeabilized. In the following sections, we describe in detail the stimulatory and inhibitory protein–protein interactions within this family, discussing various models of how the MOMP effectors, Bax and Bak, become activated. Furthermore, we focus on the actual event of membrane permeabilization, summarizing the current understanding of how pores are formed in the OMM by Bax and Bak oligomers.     

Molecular biology of Bax and Bak activation and action

Bax and Bak are two nuclear-encoded proteins present in higher eukaryotes that are able to pierce the mitochondrial outer membrane to mediate cell death by apoptosis. Thus, organelles recruited by nucleated cells to supply energy can be recruited by Bax and Bak to kill cells. The two proteins lie in wait in healthy cells where they adopt a globular α-helical structure, seemingly as monomers. Following a variety of stress signals, they convert into pore-forming proteins by changing conformation and assembling into oligomeric complexes in the mitochondrial outer membrane. Proteins from the mitochondrial intermembrane space then empty into the cytosol to activate proteases that dismantle the cell. The arrangement of Bax and Bak in membrane-bound complexes, and how the complexes porate the membrane, is far from being understood. However, recent data indicate that they first form symmetric BH3:groove dimers which can be linked via an interface between the α6-helices to form high order oligomers. Here, we review how Bax and Bak change conformation and oligomerize, as well as how oligomers might form a pore. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.