Portal de Periódicos da CAPES

Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases

2011; Springer Nature; Volume: 30; Issue: 18 Linguagem: Inglês

10.1038/emboj.2011.307

1460-2075

Andreas Strasser, Suzanne Cory, Jerry M. Adams,

Autophagy in Disease and Therapy

EMBO Member's Review23 August 2011free access Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases Andreas Strasser Corresponding Author Andreas Strasser The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia Department of Medical Biology, Melbourne University, Melbourne, Australia Search for more papers by this author Suzanne Cory Corresponding Author Suzanne Cory The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia Department of Medical Biology, Melbourne University, Melbourne, Australia Search for more papers by this author Jerry M Adams Corresponding Author Jerry M Adams The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia Department of Medical Biology, Melbourne University, Melbourne, Australia Search for more papers by this author Andreas Strasser Corresponding Author Andreas Strasser The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia Department of Medical Biology, Melbourne University, Melbourne, Australia Search for more papers by this author Suzanne Cory Corresponding Author Suzanne Cory The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia Department of Medical Biology, Melbourne University, Melbourne, Australia Search for more papers by this author Jerry M Adams Corresponding Author Jerry M Adams The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia Department of Medical Biology, Melbourne University, Melbourne, Australia Search for more papers by this author Author Information Andreas Strasser 1,2, Suzanne Cory 1,2 and Jerry M Adams 1,2 1The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia 2Department of Medical Biology, Melbourne University, Melbourne, Australia *Corresponding authors: The Walter and Eliza Hall, Institute of Medical Research, 1G Royal Parade, Parkville Victoria 3050, Australia. Tel.: +61 3 9345 2624; Fax: +61 3 9347 0852; E-mail: [email protected] or Tel.: +61 3 9345 2555; Fax: +61 3 9347 0852; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2011)30:3667-3683https://doi.org/10.1038/emboj.2011.307 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Apoptosis, the major form of programmed cell death in metazoan organisms, plays critical roles in normal development, tissue homeostasis and immunity, and its disturbed regulation contributes to many pathological states, including cancer, autoimmunity, infection and degenerative disorders. In vertebrates, it can be triggered either by engagement of ‘death receptors’ of the tumour necrosis factor receptor family on the cell surface or by diverse intracellular signals that act upon the Bcl-2 protein family, which controls the integrity of the mitochondrial outer membrane through the complex interactions of family members. Both pathways lead to cellular demolition by dedicated proteases termed caspases. This review discusses the groundbreaking experiments from many laboratories that have clarified cell death regulation and galvanised efforts to translate this knowledge into novel therapeutic strategies for the treatment of malignant and perhaps certain autoimmune and infectious diseases. Introduction Cell death programmes have evolved to meet many needs. While certain unicellular organisms can invoke cell death to match cell numbers to the nutrient supply, multicellular organisms utilise ‘altruistic’ cell suicide for diverse essential purposes (Golstein, 1998). During embryogenesis, cell death sculpts the tissues by removing superfluous cells—hollowing out ducts, for example, or deleting interdigital cells during limb formation. During neuronal development, cell death is critical for matching neuron numbers with their targets (e.g. other neurons or muscle cells). In adults, cell death counterbalances cell proliferation to maintain homeostasis in rapidly renewing tissues (e.g. the intestinal epithelium or haemopoietic tissues), and drives mammary gland involution at weaning and thymus atrophy on ageing (Kerr et al, 1972). Cell death also eliminates irreparably damaged or potentially dangerous cells, such as those undergoing neoplastic transformation or lymphocytes that target self-tissues (Strasser et al, 2000). Finally, to limit pathogen spread, both the innate and adaptive immune systems target infected cells (Trambas and Griffiths, 2003)—provoking an arms race in which pathogens evolve new ways to prevent host cell suicide while their hosts refine their weaponry for killing infected cells (Vaux et al, 1994). The major mode of programmed cell death, apoptosis, is prominent in diverse animal species and has been intensely investigated in mammals, the fruit fly Drosophila, and the worm Caenorhabditis elegans. Although evidence for additional types of programmed cell death processes (e.g. necroptosis) is growing, they have been reviewed recently (Hotchkiss et al, 2009; Yuan and Kroemer, 2010) and will only be briefly addressed here. Morphologically, apoptosis is characterised by chromatin condensation and shrinkage of the nucleus and cytoplasm, followed by fragmentation of the cell into plasma membrane-bound ‘apoptotic bodies’, which are quickly engulfed by nearby phagocytic cells and ultimately digested in lysosomes (Kerr et al, 1972). Removal of the cellular corpse, triggered by ‘eat me’ signals on the dying cells, is so rapid that apoptosis is not readily visible histologically, even in tissues with massive cell turnover, such as the thymus (Egerton et al, 1990). In contrast to necrosis, apoptosis does not rupture the plasma membrane, thereby minimising release of inflammatory cellular contents and the risk of inducing autoimmunity (Nagata et al, 2010). The foundation stones for current understanding of apoptosis were laid by a remarkable confluence of pioneering genetic studies in C. elegans with discoveries emerging from mammalian cancer genetics and biochemistry. The first molecularly defined cell death regulator emerged after the breakpoint of a recurrent chromosome translocation in human follicular lymphoma revealed the previously unknown gene BCL-2 (Tsujimoto et al, 1984). In a seminal discovery, enforced Bcl-2 expression was found to render cultured haemopoietic cells refractory to cell death upon cytokine deprivation (Vaux et al, 1988) and to promote B-cell accumulation in vivo (McDonnell et al, 1989; Strasser et al, 1991b), often culminating in autoimmune disease or cancer (see below). Strikingly, CED-9, which prevents developmentally programmed cell death in the worm (Hengartner et al, 1992), proved to be the functional homologue of BCL-2 (Vaux et al, 1992; Hengartner and Horvitz, 1994), heralding a partially conserved genetic programme (Figure 1). The exciting finding that the essential worm killer protein CED-3 resembled a new type of mammalian protease (Yuan et al, 1993) revealed that cell demolition relied on dedicated aspartate-specific cysteine proteases, soon termed caspases. Subsequently, elegant biochemical studies disclosed that the proteolytic cascade often initiates on the scaffold protein APAF-1 (Zou et al, 1997), which proved to be the homologue of worm CED-4 (Yuan and Horvitz, 1992). Collectively, these and other discoveries discussed below delineated the genetic programme for controlling apoptotic cell death, with immense ramifications for biology and human health. Figure 1.Comparison of the pathway of programmed cell death in C. elegans with the major one in vertebrates. The worm has a homologue of Bcl-2 (CED-9), of its BH3-only apoptosis inducers (EGL-1), of the caspase-activator Apaf-1 (CED-4) and of the proteolytic caspases (CED-3). However, a striking difference is that CED-9 directly binds and inhibits CED-4, until CED-4 is displaced by EGL-1, whereas vertebrate Bcl-2 does not bind to Apaf-1 but instead prevents the activation of its pro-apoptotic siblings Bax and Bak, thereby preventing their permeabilisation of the MOM and release of cytochrome c (cyt c), an essential cofactor for Apaf-1. Download figure Download PowerPoint This review will concentrate upon apoptosis in mammalian cells, because defects in its control contribute to many human diseases (Hotchkiss et al, 2009), particularly cancer (Cory and Adams, 2002). We first briefly describe the effectors of apoptosis, the caspases, and then focus on its major regulators, particularly the Bcl-2 protein family. We discuss the physiological function of Bcl-2 family members and the remarkable ways, as yet incompletely understood, in which their interactions flip the apoptotic switch. We briefly describe the proposed link from Bcl-2 via Beclin-1 to autophagy, another ancient cell death/survival mechanism. Finally, we discuss how apoptosis is impaired in cancer, restricting current treatment modalities, and how directly targeting the apoptotic machinery is offering new hope for improved therapy for cancer and perhaps also for certain autoimmune and infectious diseases. Caspases: the cellular demolition crew Regardless of the initiating death stimulus or cell type, apoptosis culminates in the fragmentation of several hundred proteins and the DNA. Aspartate-specific cysteinyl proteases termed caspases mediate the proteolysis (Timmer and Salvesen, 2007) and also activate the responsible DNAse, CAD (caspase-activated DNAse), by cleaving its chaperone/inhibitor ICAD (inhibitor of CAD) (Liu et al, 1997; Enari et al, 1998), allowing CAD to chop the chromatin into a characteristic ‘ladder’ of nucleosomes. By structure and function, caspases fall into two groups: initiator (or apical) caspases and effector (or executioner) caspases (Riedl and Salvesen, 2007; Timmer and Salvesen, 2007). The executioners, caspases-3, -6 and -7, which perform nearly all the cellular proteolysis and activate CAD, are synthesised as single-chain zymogens (catalytically inactive pro-forms) with short pro-domains. The proteolytic cascade is launched when an initiator caspase cleaves them into fragments of ∼20 (p20) and ∼10 (p10) kDa that assemble into the active tetrameric (p202p102) proteases. The initiator caspases, such as caspase-8 or -9, have long pro-domains that, following an apoptotic signal, target them to specific scaffold proteins (FADD/Mort1 for caspase-8 and Apaf-1 for caspase-9; Figure 2), where conformational changes provoke their activation (Riedl and Salvesen, 2007). Figure 2.The two major pathways to caspase activation in vertebrates: the death receptor or extrinsic pathway, engaged by the indicated members of the TNF receptor family on the cell surface, and the Bcl-2-regulated mitochondrial or intrinsic pathway. The death receptors lead, via the adaptor FADD (with help by TRADD in certain death receptors), to activation of caspase-8, which then activates the effector caspases-3, -6 and -7. Caspase-8 also processes the BH3-only protein Bid, and the truncated Bid (tBid) can then activate the Bcl-2-regulated pathway. Upon MOMP, that pathway leads to effector caspase activation via Apaf-1 and caspase-9. The cytosolic E3 ubiquitin ligase XIAP can inhibit caspases-3 and -7 (and perhaps caspase-9), but that inhibition is blocked by SMAC/DIABLO when it is released from mitochondria. E3 ubiquitin ligases cIAP1 and cIAP2 work instead in part by preventing formation of the pro-apoptotic signalling complex from TNF-R1 and by regulating pro-survival NF-κB survival pathways. Download figure Download PowerPoint Some caspases have non-apoptotic functions. Caspase-1 and its adaptors, which process pro-IL-1β and IL-18, are critical for inflammatory responses (Martinon et al, 2002). Perplexingly, caspase-8 and its adaptor FADD are essential not only for the apoptosis induced by ‘death receptors’ (see below) but also for blood vessel development, macrophage differentiation and the proliferation of certain cell types (Newton et al, 1998; Varfolomeev et al, 1998; Zhang et al, 1998). To elicit apoptosis, caspase-8 must be processed to the p202p102 tetramer, but the non-apoptotic functions require only its non-processed activated form (Kang et al, 2008). That form can prevent the RIP1 and RIP3 kinases from provoking necroptosis (programmed necrosis) (Kaiser et al, 2011; Oberst et al, 2011; Zhang et al, 2011). Two distinct but convergent pathways to caspase activation Vertebrates have two distinct apoptosis signalling pathways (Strasser et al, 1995) that ultimately converge upon effector caspase activation (Figure 2). The death receptor (or extrinsic) pathway is engaged on the plasma membrane by ligation of members of the tumour necrosis factor (TNF) receptor super-family containing intracellular ‘death domains’, such as Fas and TNF-R1. As reviewed elsewhere (Strasser et al, 2009), these receptors trigger apoptosis by forming a ‘death-inducing signalling complex’ (Kischkel et al, 1995), within which the FADD adaptor protein, assisted in certain death receptors (e.g. TNF-R1) by the adaptor TRADD, recruits and activates caspase-8 (and caspase-10 in humans and certain other species but not mouse). In the Bcl-2-regulated mitochondrial (or intrinsic) pathway, developmental cues and diverse cytotoxic insults, including growth factor deprivation and exposure to DNA damage or cancer therapeutics, initiate apoptosis by activating pro-apoptotic members of the Bcl-2 protein family (see below). Their action leads to the release from the mitochondrial intermembrane space not only of cytochrome c, which triggers APAF-1-mediated activation of caspase-9, but also of other apoptogenic proteins, such as SMAC/DIABLO, which prevents the Inhibitor of Apoptosis protein XIAP from inhibiting its caspase targets (Green and Kroemer, 2004; Jost et al, 2009). Experiments with genetically modified mice revealed that FADD and caspase-8 are essential for death receptor-induced apoptosis but dispensable for apoptosis initiated by the mitochondrial pathway (Newton et al, 1998; Varfolomeev et al, 1998; Zhang et al, 1998). Conversely, cells lacking caspase-9 or its activator APAF-1 have defects in Bcl-2-regulated apoptosis but not death receptor-induced killing (Hakem et al, 1998; Kuida et al, 1998). Interestingly, however, although loss or inhibition of caspase-8 allows long-term (clonogenic) survival of cells stimulated through a death receptor (Smith et al, 1996; Longthorne and Williams, 1997; Varfolomeev et al, 1998; Salmena et al, 2003), loss of caspase-9 or APAF-1 does not allow long-term cell survival if the mitochondrial pathway is engaged (Marsden et al, 2002, 2004, 2006; Ekert et al, 2004; van Delft et al, 2009). This is because mitochondrial outer membrane permeabilisation (MOMP) by activated Bax and Bak (see below) commits the cell to die (Green and Kroemer, 2004). Even in the absence of caspase activity, the reduced respiration following cytochrome c release soon triggers a backup cell death and clearance programme (Goldstein et al, 2000). The Bcl-2 family: the cellular life/death switch The vertebrate Bcl-2 family contains three functional subgroups (Figure 3A). Bcl-2 and its closest homologues (Bcl-xL, Bcl-w, Mcl-1, A1/Bfl1 and, in humans, Bcl-B), which contain four conserved sequence motifs (called Bcl-2 homology (BH) domains), all promote cell survival. The pro-apoptotic effectors Bax, Bak and the much less studied Bok share extensive similarity with their pro-survival relatives, including structural features of all four BH regions (Kvansakul et al, 2008). Despite this similarity, once activated, Bax and Bak damage rather than protect mitochondria, and either protein suffices for MOMP, indicative of functional redundancy (Lindsten et al, 2000). Lastly, the apoptosis initiators, the BH3-only proteins (which include Bad, Bik, Hrk, Bid, Bim, Bmf, Noxa and Puma) share with each other and the Bcl-2 family at large only the ∼26-residue BH3 domain. This amphipathic α-helix allows them to engage and inactivate their pro-survival relatives (Sattler et al, 1997) and perhaps also to transiently bind and activate Bax and Bak (Letai et al, 2002; Gavathiotis et al, 2008) (see below). The BH3-only proteins are activated by distinct cytotoxic stimuli in various ways, including enhanced transcription and post-translational modifications (Puthalakath and Strasser, 2002). Figure 3.(A) The three functional subgroups of the Bcl-2 family. Sequences most homologous to Bcl-2 (BH domains) and α-helical regions are indicated. The BH3-only proteins share sequence homology only within the BH3 domain, which mediates association between family members. Bid has a defined 3D structure but the others are relatively unstructured. The pro-survival group, which shares four regions of sequence homology, includes Bcl-B in humans but its mouse homologue (Boo) appears to be inactive due to a mutation of essential residues in BH1. The pro-apoptotic Bax/Bak group, which includes the little studied Bok, is remarkably similar in sequence and structure to the pro-survival group, including an α-helix resembling BH4 near the N-terminus (Kvansakul et al, 2008). Most family members contain a C-terminal hydrophobic trans-membrane (TM) region, which mediates their targeting and anchoring to the MOM and/or the ER, either constitutively (e.g. Bcl-2 or Bak) or after an apoptotic stimulus (e.g. Bcl-xL or Bax). (B,C) Predominant interactions within the Bcl-2 family, including those of BH3-only proteins with their pro-survival relatives (B) and the major interactions of the latter with Bax and Bak (C). Download figure Download PowerPoint While most BH3-only proteins are unstructured prior to engaging pro-survival proteins (Hinds et al, 2007), Bid forms an α-helical bundle resembling Bax or Bcl-2 (Chou et al, 1999; McDonnell et al, 1999), despite the lack of sequence homology except for the BH3 domain (Youle and Strasser, 2008). Bid can link the death receptor and Bcl-2-regulated pathways because its cleavage by caspase-8 generates an active C-terminal segment termed tBid, which promotes Bax/Bak-mediated MOMP (Figure 2). This tBid-activated amplification mechanism is essential for death receptor-induced killing in so-called type 2 cells, such as hepatocytes, but dispensable in type 1 cells, such as thymocytes (Scaffidi et al, 1998; Yin et al, 1999; Kaufmann et al, 2007). The Bcl-2 family can be regarded as a tripartite switch that sets the threshold for commitment to cell death, primarily by interactions within the family. The pro-survival members can bind with high affinity to members of both the Bax/Bak-like and the BH3-only subgroups, via association of the BH3 domain of the pro-apoptotic proteins with a hydrophobic groove on the surface of the pro-survival proteins (Sattler et al, 1997). These interactions, however, exhibit specificity (Figure 3B, C). The affinities of BH3-only proteins for the pro-survival proteins differ markedly (Chen et al, 2005; Kuwana et al, 2005): Bim, Puma and perhaps tBid bind all with high affinity, whereas other BH3-only proteins show more selectivity (Figure 3B). Most strikingly, Bad binds Bcl-2, Bcl-xL and Bcl-w but not Mcl-1 or A1, whereas Noxa interacts strongly only with Mcl-1 and A1. Accordingly, enforced expression of either Bim or Puma potently kills cells, whereas Bad and Noxa can efficiently induce cell death only if coexpressed (Chen et al, 2005). Bax and Bak also differ in their interaction with the pro-survival proteins (Figure 3C). Bak can be bound tightly by Bcl-xL and Mcl-1 but only poorly by Bcl-2 (Willis et al, 2005), whereas all the pro-survival proteins probably can constrain Bax activity (Willis et al, 2007). How the Bcl-2 apoptotic switch is flipped Two distinct, albeit not mutually exclusive, models have been proposed to describe how the interplay between the three Bcl-2 factions activates Bax and Bak and hence produces MOMP (Adams and Cory, 2007; Chipuk and Green, 2008). The direct activation model (Figure 4A) posits that certain BH3-only proteins (termed ‘activators’), namely Bim, tBid and probably Puma (Letai et al, 2002; Kuwana et al, 2005; Kim et al, 2009), must transiently bind and activate Bax and Bak, whereas the other BH3-only proteins, termed ‘sensitisers’ (e.g. Bad, Noxa), can only bind their pro-survival relatives (‘Bcl-2 et al’ in Figure 4A). In this model, the pro-survival Bcl-2 proteins function by sequestering the ‘activators’, and apoptosis proceeds when ‘sensitisers’ displace the activators from the pro-survival proteins, allowing them to bind and activate Bax and Bak. The activator BH3-only proteins were widely assumed to target a hydrophobic surface groove on Bax and Bak resembling that on the pro-survival proteins, but they may instead (or in addition) bind to a proposed distal site involving Bax α-helices 1 and 6 (Gavathiotis et al, 2008, 2010; Kim et al, 2009). The binding of certain BH3 domains to Bax or Bak has been described as ‘hit and run’ (transient and low affinity) and this so-called ‘rear site’ is not yet well defined. A very recent study provides strong support for direct activation of Bak by Bim, tBid and, surprisingly, Noxa, but they were clearly shown to bind to the ‘front site’ of Bak (Dai et al, 2011). Perhaps both sites can be used, one for promoting initial activation of Bax/Bak and the other for recruiting additional Bax (Bak) molecules (see below). Figure 4.Models for how the BH3-only proteins activate Bax and Bak. (A) In the direct activation model, the activator BH3-only proteins (Bim, tBid and probably Puma), via their BH3 domain (red triangle), can directly engage and activate Bax (or Bak), but in healthy cells the pro-survival family members (‘Bcl-2 et al’) prevent this by sequestering the BH3-only activators. After an apoptotic signal, the sensitiser BH3-only proteins (e.g. Bad, Noxa, Bik) free the activators to target Bax or Bak. Inactive cytosolic Bax has its BH3 domain buried and its C-terminal hydrophobic helix (α9) lies in its surface groove, but during activation that helix is freed and can target Bax to the membrane. (B) In the indirect activation model, the BH3-only proteins need only target their pro-survival relatives, which primarily prevent activation of Bax and Bak by sequestering any Bax or Bak that becomes active (‘primed’) by exposure of its BH3 domain (red triangle). (C) The priming–capture–displacement model proposed here incorporates features of both direct and indirect activation. In it, any Bax or Bak that becomes primed, either spontaneously or by BH3-only proteins or other potential signals (e.g. phosphorylation), is immediately captured by a pro-survival relative, until the primed Bax or Bak is displaced by further activation of BH3-only proteins (e.g. Bad or Bim). The displaced Bax or Bak can then form dimers and higher oligomers (see Figure 5). Download figure Download PowerPoint The indirect activation model (Willis et al, 2007) (Figure 4B) posits that, probably even in healthy cells, some Bax and Bak molecules (perhaps spontaneously) assume a ‘primed conformation’, that is, one in which their BH3 domain is exposed, and that pro-survival family members prevent apoptosis by binding to this domain, thereby preventing Bax and Bak from oligomerising (see further below). In this model, the primary role of all BH3-only proteins is to bind to the pro-survival proteins, and apoptosis can only proceed when all the relevant pro-survival proteins have been neutralised, thereby liberating the ‘primed’ Bax or Bak to oligomerise and cause MOMP. Consistent with this model, Bax molecules with BH3 mutations that prevent their sequestration by pro-survival relatives provoke unrestrained apoptosis (Fletcher et al, 2008; Czabotar et al, 2011). The direct and indirect models were recently interrogated in vivo using gene-targeted mice in which the Bim BH3 domain (binding all pro-survival Bcl-2 proteins and possibly Bax) was replaced in situ with that of Bad, Noxa or Puma, creating alleles encoding, respectively, BimBad (binding only Bcl-2, Bcl-xL, Bcl-w), BimNoxa (binding only Mcl-1, A1) and BimPuma (binding all pro-survival Bcl-2-like proteins but not Bax). The results showed that, for optimal cell death, Bim must be able to bind all anti-apoptotic Bcl-2 family members, as in the indirect model, but that its interaction with Bax may contribute to cell killing, suggesting that physiological cell death may follow both models (Merino et al, 2009). The two models could represent alternative paths to cell death or different stages of a single pathway. For example, we propose in the priming–capture–displacement model (Figure 4C) that direct activation by BH3-only proteins is at least one of the ways that ‘primed’ Bax and Bak are generated, but that pro-survival relatives immediately capture this ‘primed form’ of Bax and Bak, until BH3-only proteins displace it, as in the indirect activation model. Interestingly, the pro-survival family members may regulate Bax and Bak in multiple ways. According to a recent report, Bax in healthy cells spontaneously translocates from the cytosol to associate peripherally with mitochondria, but Bcl-xL then binds and ‘retrotranslocates’ Bax back into the cytosol, whereupon this heterodimer disassociates (Edlich et al, 2011). Also, the ‘embedded together model’ posits that Bax and Bcl-2 interact only after both have rearranged to bury their α5 and α6 helices as well as their terminal α9 helix within the MOM (Leber et al, 2007) (see below). Furthermore, in elegant simplified systems using recombinant proteins with liposomes or isolated mitochondria, tBid rapidly bound to the membrane and recruited Bax, but its recruitment was opposed by Bcl-xL, which could sequester both tBid and Bax, until addition of Bad displaced Bax from Bcl-xL (Lovell et al, 2008). One of the great mysteries of cell death is how Bax and Bak homo-oligomerise and disrupt the MOM. Although no 3D structure of any active form of Bax or Bak is yet available, some veils have been lifted. Antibody and biochemical probes indicate that the globular structures of inactive Bax (Suzuki et al, 2000) and Bak (Moldoveanu et al, 2006) are substantially altered during their activation by several rearrangements (Kim et al, 2009; Gavathiotis et al, 2010). Notably, in an early step, Bak exposes its BH3 domain, which can then intercalate into the partial surface groove of another equivalently activated Bak molecule, forming a dimer that appears to be symmetric (Dewson et al, 2008) (Figure 5A). These novel dimers can then multimerise into larger oligomers through a separate interface involving the Bak α6 helix (Dewson et al, 2009). Bax oligomerisation is thought to proceed similarly (Westphal et al, 2011). Nevertheless, the proposed ‘rear’ site on Bax raises the possibility that Bax could instead insert an exposed BH3 domain into that site on another Bax molecule, forming ‘asymmetric’ dimers that would produce a ‘daisy chain’ of monomers (Figure 5B). This is consistent with some evidence (Shamas-Din et al, 2011) but not another study (Dewson et al, 2009). In any case, a key step in Bax and Bak oligomerisation and membrane permeabilisation may well be insertion of their α5 and α6 helices as a hairpin across the bi-layer of the MOM (Leber et al, 2007) (Figure 5B). Figure 5.Models for the oligomerisation of Bax and Bak. (A) In this dimer multimerisation model, upon activation by an apoptotic signal, Bak (or Bax) first extrudes its BH3 domain, which allows it to engage another ‘primed’ Bak (or Bax) molecule to form a ‘symmetric’ (face-to-face) dimer (Dewson et al, 2008). These dimers then multimerise by a different interface, such as one between α6 helices, to form the large oligomers that provoke MOMP, allowing cytochrome c (cyt c) to escape to the cytosol (Dewson et al, 2009). (B) In an alternative model (Shamas-Din et al, 2011), prompted by the proposal that certain BH3 domains can engage a novel ‘rear’ site on Bax, ‘primed’ Bak (or Bax) can engage the proposed rear site of another activated molecule to form an ‘asymmetric (face-to-back) dimer’, which could be extended by monomer recruitment. In this model, insertion of the α5–α6 hairpin as well as α9 is depicted. That insertion might also feature in the model shown in (A). Download figure Download PowerPoint Whether Bax and Bak produce homo-oligomers of a defined size is unclear, but proteinaceous ‘pores’ large enough to allow egress of all the intermembrane proteins probably would require 8–12 mers (Green and Kroemer, 2004). However, the oligomers may instead simply create lipidic pores of undefined size by disrupting the lipid bi-layer (Green and Kroemer, 2004). To clarify these pivotal steps, there is a pressing need for the determination of 3D structures of activated forms of Bax and Bak and of their full-length heterodimers with pro-survival relatives, preferably anchored within the membrane. An evolutionary conundrum Given the central role of Bax/Bak-driven MOMP in vertebrate apoptosis, it is paradoxical that mitochondrial disruption plays little if any role in worms and flies. C. elegans lacks any pro-apoptotic Bax/Bak equivalent, and the worm APAF-1 homologue CED-4 does not require cytochrome c to activate the caspase CED-3 (Hengartner, 2000) (Figure 1). Instead, CED-9 directly sequesters CED-4, until EGL-1 binds to CED-9, liberating CED-4 to activate CED-3 (Hengartner, 2000). Furthermore, whereas Bcl-2 homologues have the crucial cell survival role in the worm and vertebrates, that role is dominated in Drosophila by the direct control of caspases by IAPs rather than by the two fly Bcl-2-related proteins, and mitochondrial disruption is not implicated (Dorstyn et al,