Bax, Bak and beyond mitochondrial performance in apoptosis

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1 STATE-OF-THE-ART REVIEW Bax, Bak and beyond mitochondrial performance in apoptosis Aida Pe~na-Blanco 1 and Ana J. Garcıa-Saez 1,2 1 Interfaculty Institute of Biochemistry, T ubingen University, Germany 2 Max-Planck Institute for Intelligent Systems, Stuttgart, Germany Keywords apoptosis; BAK; BAX; BCL-2 family; DRP1; mitochondria; mitochondrial outer membrane permeabilization Correspondence A. J. Garcıa-Saez, Interfaculty Institute of Biochemistry, T ubingen University, Hoppe- Seyler-Straße 4, Tübingen, Germany Fax: Tel: ana.garcia@uni-tuebingen.de (Received 2 June 2017, revised 12 July 2017, accepted 26 July 2017) doi: /febs Bax and Bak are members of the Bcl-2 family and core regulators of the intrinsic pathway of apoptosis. Upon apoptotic stimuli, they are activated and oligomerize at the mitochondrial outer membrane (MOM) to mediate its permeabilization, which is considered a key step in apoptosis. However, the molecular mechanism underlying Bax and Bak function has remained a key question in the field. Here, we review recent structural and biophysical evidence that has changed our understanding of how Bax and Bak promote MOM permeabilization. We also discuss how the spatial regulation of Bcl-2 family preference for binding partners contributes to regulate Bax and Bak activation. Finally, we consider the contribution of mitochondrial composition, dynamics and interaction with other organelles to apoptosis commitment. A new perspective is emerging, in which the control of apoptosis by Bax and Bak goes beyond them and is highly influenced by additional mitochondrial components. Introduction Apoptosis is an essential programmed cell death pathway, since it is indispensable for tissue homeostasis, embryonic development and immunity. Importantly, deregulated apoptosis has a major role in tumor development, neurodegenerative disorders, and autoimmune diseases [1 3]. Due to its biological relevance and therapeutic applications, apoptosis has been extensively studied during the last three decades. In vertebrates, the Bcl-2 family of proteins controls and regulates the intrinsic or mitochondrial apoptotic pathway. This pathway promotes mitochondrial outer membrane permeabilization (MOMP). MOMP allows the release of proapoptotic factors like cytochrome c and SMAC/DIABLO from the mitochondria into the cytosol to activate the caspase cascade. MOMP is usually considered the point of no return in the apoptotic pathway. After MOMP, caspase activation takes place often within minutes, leading to cell death [4,5]. The Bcl-2 family comprises at least 18 members that are classified into three groups according to their function in apoptosis and the number of Bcl-2 homology (BH) domains they possess [6,7]: (a) the antiapoptotic or prosurvival Bcl-2 proteins (Bcl-2, Bcl-xL, Bcl-w, Mcl1, and A1), which contain four BH domains BH1- BH4 and suppress cell death by binding and inhibiting the proapoptotic Bcl-2 proteins; (b) the proapoptotic effector proteins Bax and Bak, which present BH1- BH4 and directly promote MOMP; and (c) the BH-3 only proteins, which, except Bid, only contain a highly conserved BH3 domain and are very heterogeneous. They have evolved to sense cellular stress and initiate apoptosis. The BH3-only proteins can be further Abbreviations AFM, atomic force microscopy; BH, Bcl-2 homology; Drp1, dynamin-related protein 1; IMS, intermembrane space; Mfn2, mitofusin 2; MOM, mitochondrial outer membrane; MOMP, mitochondrial outer membrane permeabilization; OPA1, optic atrophy The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies

2 A. Pe~na-Blanco and A. J. Garcıa-Saez Bax/Bak and mitochondria in apoptosis classified as direct activators if they directly interact and activate Bax and Bak or sensitizers if they bind antiapoptotic proteins and displace the direct activators from them. [4]. In order to control apoptosis, the Bcl-2 proteins interact with each other and generate a complex interaction network. Thereby, their interplay determines whether a cell lives or dies. The main action of the Bcl-2 proteins occurs at the mitochondria. In healthy cells, Bax and Bak shuttle between cytosol and mitochondrial outer membrane (MOM) with different rates [8 10]. Under apoptotic conditions, Bax and Bak are activated and accumulate at the MOM, where they oligomerize and mediate MOMP, which leads to the release of proapoptotic factors, such as cytochrome c [11,12]. In addition, Bax/Bak regulation by antiapoptotic proteins and BH3-only proteins also takes place at the MOM [12,13]. However, mitochondria are not simple spectators of the function of the Bcl-2 proteins, but they also play an active role in the control of apoptosis. Mitochondrial composition, dynamics, and interactions with other organelles have been shown to impact on the commitment to cell death, although the molecular mechanisms remain poorly defined [14 16]. This suggests that the complexity of the regulation of apoptosis by the Bcl-2 proteins goes beyond the family. Here, we review the recent findings regarding Bax and Bak activation, dimerization and oligomerization to form apoptotic pores at the MOM. We also consider how additional players are involved in the function of Bax and Bak to mediate MOMP. Concretely, we discuss the interplay of Bax and Bak with the rest of the Bcl-2 family and, in addition, we draw attention to mitochondria as the crucial target for apoptosis commitment. Bax and Bak transition into killers How are Bax and Bak activated to promote MOMP? Under normal conditions, Bax is largely cytosolic via constant retrotranslocation from mitochondria to the cytosol mediated by Bcl-xL, which avoids accumulation of toxic Bax levels at the MOM [8]. In this inactive soluble form, Bax has a globular structure comprising nine a helices arranged around a central hydrophobic core composed by helices a2 a5 [17] (Fig. 1A). This structural organization delimits a hydrophobic groove. Importantly, Bax hydrophobic groove accommodates the transmembrane domain of the C-terminal helix a9, maintaining its soluble conformation [17]. Nevertheless, experiments in cells using F oster resonance energy transfer indicated that the C-terminal helix a9 is exposed to the cellular environment, which may facilitate the dynamic equilibrium of Bax between the cytosol and the mitochondria or its binding to other Bcl-2 proteins [18]. It has been long considered that inactive cytosolic Bax exists only as a monomer. However, a recent study has reported that Bax also has an inactive dimer conformation arranged by the N-terminal region of one monomer and the C-terminal region of the other, which includes helix a9 [19]. This autoinhibited dimeric form dissociates to Bax monomers that can be inserted at the MOM upon activation, suggesting a novel Bax activation step and an increased complexity in the regulation of apoptosis. In contrast, in healthy conditions, Bak is mainly mitochondrial with its transmembrane domain (a9) spanning the MOM [20] and presents only a small cytosolic fraction due to retrotranslocation [9]. There is so far no evidence whether inactive Bak could also be found as a dimer in any of the environments. At the membrane, its C-terminal region may not be available for dimerization since it is already inserted in the lipid bilayer. Whether the minor soluble fraction is also able to form autoinhibited dimers remains to be shown. The potential interplay between retrotranslocation and the autoinhibitory mechanism of Bax, and maybe Bak, is another open question. During apoptosis, numerous conformational changes must occur in Bax and Bak to be able to permeabilize the MOM (Fig. 1B). Upon cytotoxic stress, Bax accumulates at the mitochondria [12] and becomes activated by interaction with BH3-only proteins. Two binding sites have been reported for Bax: (a) the rear activation site (helices a1 a6) on the opposite side of the hydrophobic groove, which has been identified by NMR studies using full-length Bax and a stapled Bim- BH3 peptide [21,22], (b) and the canonical hydrophobic groove, which has been solved using crystal structures of truncated versions of Bax in complex with Bid-BH3 peptide and detergents [23]. Interactions of BH3-only proteins with the rear activation site promote the displacement of the loop between helices a1 and a2, which leads to the mobilization of Bax transmembrane domain from the hydrophobic groove and allows Bax insertion into the membrane [11,22] (Fig. 1B). Activator BH3-only proteins can also bind the canonical hydrophobic groove, which leads to the displacement to the cytosol of the N-terminal region preceding helix a1 and the rearrangement of the a2/ BH3 domain. This allows the transient exposure of the BH3 domain, a crucial step for Bax dimerization [23,24]. Therefore, activator BH3-only proteins The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies 417

3 Bax/Bak and mitochondria in apoptosis A. Pe~na-Blanco and A. J. Garcıa-Saez Fig. 1. Bax activation, oligomerization and pore formation at the MOM. (A) Globular Bax is represented using its ribbon visualization (PDB: 1F16): helix a1 (pink), helix a2 (red), helix a3 (orange), helix a4 (yellow), helix a5 (green), helix a6 (blue), helix a7 (cyan), helix a8 (purple), and helix a9 (gray). The transmembrane C-terminal region of the helix a9 is located at the hydrophobic groove, which is composed by helices a2 a5. (B) Proteins are illustrated in their surface representation. Bax (cyan) has a soluble, globular conformation when it is inactive. Upon binding to activator BH3-only proteins (orange), the helix a9 is displaced from the hydrophobic groove, which allows Bax insertion in the membrane. Further rearrangements involve the separation into a dimerization (a2 a5) and a piercing (a6 a9) domain, according to the domain classification in [23,34,35], which allows the exposure of the BH3 domain. The BH3 domain is required for dimerization via BH3-ingroove interaction with another Bax molecule (yellow). Further oligomerization is based on the assembly of dimer units, although the oligomerization interface, in case there is a defined one, still remains unclear. Active Bax dimer topography at the membrane to form a pore is only represented here by the helices a2 a6, where the helix a6 of each monomer lies on the surface of opposite leaflets of the membrane and the dimerization domain (a2 a5) is located at the rim of the pore. It still remains to be elusive whether oligomerization precedes pore formation, if both events occur simultaneously or if only dimerization is sufficient for the opening of the pore. Figure adapted from [48]. (C) Ribbon representation of helices a2 a6 of active Bax dimer at the membrane according to the clamp-like model using the same color code as in A (adapted from [35]). Helices a6 of each monomer are parallel and lay on opposite leaflets of the membrane, whereas helices a2 a5 are placed at the pore edge. promote a stepwise, bimodal activation, in which first Bax is inserted and then the BH3 domain is exposed [24]. According to the kiss-and-run model [25,26], BH3-only proteins only bind transiently to Bax, as they are displaced by the exposed Bax BH3 domain during oligomerization. In the case of Bak, the canonical hydrophobic groove was proposed as the only activation site for activator BH3-only proteins [27,28], but a recent study reported that helix 6 of Bak also participates in the activation by BH3-only proteins [29]. The a1 a2 loop has been identified as an additional activation site in Bak and mitochondrial Bax triggered by generated monoclonal antibodies [30]. This opens exciting possibilities because, although BH3-only proteins do not bind the a1 a2 loop, other Bax/Bak activators may use this novel activation site to promote MOMP. In summary, the molecular mechanism that regulates Bax and Bak initial activation requires the insertion of Bax helix a9 in the membrane and the exposure of the Bax/Bak BH3 domain in order to allow self-assembly with other Bax and Bak molecules. Bax and Bak dimers and higher order oligomers at the mitochondria A number of studies have recently shed new light on the molecular mechanism that regulates Bax and Bak assembly at the membrane. Bax inserts in the bilayer as a monomer [31], but it self-assembles very quickly into higher order oligomers. Interestingly, active Bax exists in the membrane as a mixture of species based on dimer units [31]. Bak also forms dimers before further oligomerization [32] and interdimer interfaces are more labile to detergents than intradimer interactions [33], suggesting an assembly based on dimers units also for Bak. The structural reorganization that drives 418 The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies

4 A. Pe~na-Blanco and A. J. Garcıa-Saez Bax/Bak and mitochondria in apoptosis transition from monomer to dimer requires the separation of the dimerization domain (a2 a5) from piercing domain (a6 a8) in Bax and Bak [23,34,35]. This rearrangement allows the transient exposure of their BH3 domain [23,34]. To form dimers, the BH3 domain of a Bax (or Bak) molecule binds to the canonical groove of another Bax (or Bak) molecule, generating a novel symmetric dimer. This process is known as BH3-in-groove interaction [23,36 39]. Based on this, dimers would be the fundamental unit for the conversion of Bax and Bak into killers. However, the structural mechanism that mediates homodimers association into higher order oligomers remains to be defined. Different studies have identified several sites at helices a1, a3, a5, a6, and a9 where Bax and Bak oligomerize [32,39 43]. Recently, cysteine labeling and linkage analysis of full-length Bak in mitochondria suggested that Bak assembly into higher order oligomers proceeded via dimers association in a disordered and lipid-mediated fashion, with no dominant dimer dimer interface that mediates higher order oligomers formation [33]. This result is attractive, because it is consistent with the identification of several dimer association sites found in the literature. Still, several open questions remain. For example, how is the size of oligomers regulated? Bax and Bak seem to participate in combined oligomers [33,37,44], but how are they arranged in these assemblies? Are there additional proteins or lipids in Bax/Bak oligomers? Pore formation by Bax and Bak: from structural insights to macromolecular organization The main action of the executors Bax and Bak is the disruption of the MOM to allow the release of proapoptotic factors to the cytosol. The nature of the pore formed by Bax and Bak at the MOM has been a long-standing question in the apoptotic field. Now, there is increasing evidence supporting a toroidal pore model. A toroidal pore is characterized by the fusion of the outer and inner bilayer leaflets to form a continuous surface at the edge of the pore, which is formed by both lipids and proteins [45]. Spontaneous pore opening in lipid bilayers is very unlikely, but the probability increases when there is a stress at the membrane that increases the membrane tension, for example the accumulation of proteins in one leaflet of the membrane [46]. After a certain membrane tension threshold, a pore opens. In a toroidal pore, the lipids at the rim of the pore bend to avoid the exposure of their hydrophobic acyl chains to the aqueous environment, which creates a high membrane curvature at the pore edge [47]. This high membrane curvature has an energy cost and causes tension (known as line tension because it depends on the pore diameter) that tends to close the pore. The open pore state can therefore be stabilized by decreasing the line tension, which can be achieved by reducing the energy cost of the rim structure with the presence of proteins [6,48]. A recent study using atomic force microscopy (AFM) of full-length Bax on supported lipid membranes showed for the first time that oligomeric full-length Bax is able to reduce the line tension at the edge of the pore, which promotes pore stabilization [49]. During previous years, indirect evidence using artificial membranes had hinted a toroidal pore for Bax. Helix a5 from Bax had been found to decrease the line tension at the pore edge and to form stable pores [50 53]. In addition, Bax pores are tunable in size and can be regulated by protein density at the membrane [54]. Cryoelectron microscopy of liposomes permeabilized by Bax and Bak resulted in large openings [55]. Importantly, the application of novel biophysical approaches in the characterization of Bax and Bak pores have allowed the visualization of Bax pores in artificial membranes and in the mitochondria of apoptotic cells [56,57]. Superresolution microscopy of Bax tagged with GFP revealed the presence of Bax lines, rings and arcs at the MOM in a wide range of sizes [56,57] (Fig. 2A). In addition, data using AFM on supported bilayers (Fig. 2B), electron microscopy of outer membrane vesicles and lipid nanodiscs containing Bax have also supported the formation of toroidal Bax pores without full protein coverage of the pore rim [56,58,59]. However, there is lack of evidence for such pores in the case of Bak. A study using single molecule localization of Bak clusters in situ has determined that Bak protein density is uniform among clusters, although cluster size at apoptotic mitochondria is highly heterogeneous [60]. In this scenario, visualization of Bak pores and characterization of Bak structures in apoptotic cells will be necessary to complete our understanding of MOMP by Bax and Bak. It has been suggested that Bax and Bak could associate as homodimers or heterodimers at the MOM [33,37,44], but further research is needed to confirm whether they cooperate to form a pore. There are several structural models to explain how Bax and Bak are arranged at the mitochondria to form a toroidal pore. In the long-standing umbrella model, helices a5 and a6 were proposed to insert as a hairpin into the MOM [61]. However, this model seems to be incompatible with current data on Bax and Bak activation, which shows that only helix a6 is The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies 419

5 Bax/Bak and mitochondria in apoptosis A. Pe~na-Blanco and A. J. Garcıa-Saez A C B D has been also used to study Bak disposition at the membrane in relation to a lipidic pore. According to this model, BH3-in-groove homodimers are juxtaposed via the a3/a5 interface, which locates on the curved surface of the pore, although its exact location remains unknown. Helices a6 a8 tether the BH3-in-groove region to the a9 helices, which are located at the flat region of the membrane around the lipidic pore [42]. Interestingly, one feature that arises at all length scales of active Bax and Bak organization in the membrane is flexibility: in its structural arrangement beyond helix 6, in the poorly defined mixture of multimeric species based on dimer units and in the size and shape distribution in the macromolecular assemblies in the membrane. To fully understand how Bax and Bak work, it will be critical to find out how this structural plasticity is relevant for function. Fig. 2. Bax rings and arcs in apoptotic cells and supported lipid bilayers. (A B) Superresolution microscopy images of a GFP-Bax ring (A) and arc (B) in apoptotic cells. Scale bar, 100 nm. Figure obtained from [56]. (C D) Atomic force spectroscopy of fulllength Bax on supported lipid bilayers shows the presence of Bax rings (C) and arcs (D) on the membrane. Scale bar, 50 nm. Reproduced, with permission from [48]. shallowly inserted at the membrane [62]. In a later proposed in-plane model, helices a4 and a5 create an aromatic planar surface on the membrane [62], which might increase membrane tension via local curvature stress, leading to pores at the MOM. In this model, the helices a9 are embedded into the lipid bilayer and helices a5 and a6 are only partially inserted. The disposition of Bax and Bak domains with respect to a membrane pore remains unclear in this model. The clamp model, based on double electron electron resonance of active full-length Bax inserted in liposomes, proposes that Bax dimers embrace the edge of the membrane pore thanks to the opening of the hairpin formed by helices a5 and a6 [35] (Fig. 1C). In this clamp-like conformation, the amphipathic helix a6 of each monomer lies on the surface of opposite leaflets of the membrane and the dimerization or latch domain (helices a2 a5) is located at the rim of the pore (Fig. 1B). However, the arrangement of the dynamic C-terminal region of the protein including the respective orientation of helices a9 in the remains unsolved. The clamp model provides a very attractive explanation for how Bax molecules arrange in the context of the MOM pore and stabilize the highly curved membrane structure at the rim. Double electron electron resonance Control of Bax and Bak activity by the Bcl-2 network Interplay among Bcl-2 proteins: models of interaction Bcl-2 proteins form a complex and intricate interaction network, with multiple, parallel interactions in solution and in membranes, whose outcome establishes a threshold or switch for Bax/Bak activation and apoptosis commitment [6]. Traditionally, several models have been proposed to explain the interplay of Bcl-2 proteins in the regulation of apoptosis. The neutralization model (or derepressor ) [63 65] proposes that in apoptosis BH3-only proteins bind and neutralize antiapoptotic Bcl-2 proteins, which displaces and releases constitutive active Bax and Bak from their inhibitory complex with the antiapoptotic Bcl-2 family proteins. The direct activation model [66 68] suggests that, although the subset of sensitizer BH3-only proteins neutralizes the antiapoptotic Bcl-2 homologs, the direct activator BH3-only s are necessary to directly bind and activate Bax and Bak. Here, the antiapoptotic proteins mainly inhibit the direct activators, whereas the sensitizers restrain the antiapoptotic proteins. The embedded together model [69,70] considers that the antiapoptotic proteins inhibit both direct activators and Bax and Bak, while pointing out the importance of the membrane for Bcl-2 family protein/ protein interactions. Later, the unified model combined previous ideas [71] (Fig. 3A) and ranked the efficiency of the interactions: antiapoptotic proteins inhibit apoptosis by sequestering the direct activators (MODE1) or the 420 The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies

6 A. Pe~na-Blanco and A. J. Garcıa-Saez Bax/Bak and mitochondria in apoptosis executors Bax and Bak (MODE2), with MODE1 being less efficient than MODE2. At low stress levels, direct activators are inhibited by MODE1 inhibition, whereas in high stress levels there is an increase of unbound direct activators and apoptosis can only be prevented by MODE2 inhibition [71]. Beside these, the steady-state retrotranslocation of Bax from the MOM back to the cytosol mediated by Bcl-xL constitutes an additional inhibitory mechanism that is usually incorporated as MODE 0 [8 10]. The fast Bax retrotranslocation to the cytosol mediated by Bcl-xL avoids Bax accumulation at the mitochondria and ensues cell survival. Finally, the recent interconnected hierarchical model [72] (Fig. 3B) established a sequence of events for the execution of apoptosis. It proposes bimodal mechanisms for the stepwise inhibition of antiapoptotic Bcl-2 family proteins and activation of Bax/Bak by BH3-only proteins, as well as for the neutralization of BH3-only proteins and executioners Bax/Bak by antiapoptotic Bcl-2 proteins. Interestingly, Bax and Bak could be autoactivated when antiapoptotic proteins were downregulated in the absence of direct activators [72]. In agreement with this, genetic deletion of all Bcl-2 proteins and reintroduction of Bax alone was sufficient to induce MOMP [73], which suggested that Bax and Bak do not require activation by BH3-only proteins, although unknown activation factors may be involved in this process. Despite their increasing complexity, these models still fail to describe how the Bcl-2 family regulates apoptosis commitment in a general way. The interplay between positive and negative regulators, together with competing back and forward feeding mechanisms yields a highly intricate signaling network with emergent properties that go beyond the sum of the individual components. In this scenario, a systems approach that provides quantitative understanding of the relative affinities between Bcl-2 proteins in solution and in the membrane environment and incorporates them into a mathematical framework is likely necessary to describe appropriately how Bcl-2 proteins regulate MOMP and make accurate predictions upon system perturbations. A number of attempts have been made to build computational models for the mitochondrial pathway of apoptosis [74]. Some of these models provided information about the events that follow MOMP, such as the caspase signaling pathway [75,76]. High-speed livecell imaging was used to measure the spatial onset of MOMP and allowed the mathematical modeling of MOMP dynamics and pore formation kinetics [77]. In addition, single-cell measurements of Bax translocation were used to model Bax localization and oligomerization in relation to MOMP induction [78]. However, a key handicap in these approaches is the lack of quantitative experimental data for the model parameters. Recently, the quantitative analysis of a minimal Bcl-2 family proteins interactome reconstituted in vitro that considered complexes in solution and in membranes has made a step in this direction and is the basis for the integrated model shown in Fig. 3C [79]. The hierarchy of Bcl-2 family of proteins interactions depended on the environment: membrane association redefined the pattern of complexes formed by Bax, cbid, and Bcl-xL. While Bcl-xL and cbid bound both in solution and in membranes, the complex was stabilized in the lipid environment. In contrast, Bcl-xL bound to Bax only in the membrane and only in the absence of cbid. This indicated that in presence of all three proteins, the inhibitory association of Bcl-xL and cbid dominated over the other heterocomplexes. In this chemically controlled system, Bax could spontaneously activate with a positive feedback mechanism at physiological temperature. The rate of activation was significantly increased by presence of cbid, as expected for a catalyst. Interestingly, Bax could also recruit Bcl-xL to membranes, which was sufficient to retrotranslocate Bax back to solution in absence of other cellular components. The advantage of such approach is that it allows increasing the complexity of the network stepwise and systematically by adding additional components or post-translational modifications. On the other hand, comparable quantitative information in the cellular context will be key to improve the construction of a model that better resembles the physiological conditions of the cell. Modulation of apoptosis by the BH3-only proteins With the exception of Bid, BH3-only proteins are intrinsically disordered proteins whose BH3 domain folds into an amphipathic helix upon binding to Bcl-2 homologs [80]. They have evolved to sense cellular stress and to promote apoptosis by binding to the hydrophobic groove of antiapoptotic proteins in order to restrain their activity and, in the case of direct activators, also to that of Bax and Bak to activate them [68,81]. Subtle differences in the BH3 domains of the BH3-only proteins determine the specificity and affinity for their antiapoptotic partners and their regulatory role in apoptosis. The interactions of the BH3-only proteins tbid, Bad and Bim with the antiapoptotic proteins Bcl-2 and Bcl-xL were measured in live cells using fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer [82]. This study showed that tbid and Bad interact through their BH3 domain with both Bcl-2 and Bcl-xL, whereas Bim The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies 421

7 Bax/Bak and mitochondria in apoptosis A. Pe~na-Blanco and A. J. Garcıa-Saez Fig. 3. Models of the interactions of the Bcl-2 family of proteins. (A) The unified model proposes two modes of regulation by the antiapoptotic proteins. One involves the inhibition of the direct activators BH3-only proteins, preventing them from activating Bax and Bak. The other suggests a direct inhibition of the executors Bax and Bak. Although both inhibitory effects happen in the cell, inhibition of MOMP by the antiapoptotic proteins via sequestering Bax and Bak is more efficient. (B) The interconnected hierarchical model considers the order of the events in the regulation of apoptosis by the Bcl-2 family. The antiapoptotic proteins sequester first the direct activators BH3-only proteins and then the executors Bax and Bak in order to prevent apoptosis. Both the activator and the sensitizer BH3-only proteins inhibit the antiapoptotic proteins to promote cell death. In addition, when antiapoptotic proteins are downregulated in the absence of direct activators, Bax and Bak can be autoactivated. (C) The integrated model based on quantitative data of Bax, Bid and Bcl-xL interactions highlights the role of the membrane in the interactions of the Bcl-2 family of proteins. cbid inhibition by Bcl-xL can take place in the cytosol or the membrane, with the latter more stable. Only in the absence of cbid, Bcl-xL can inhibit Bax at the membrane, suggesting that Bcl-xL/ cbid interaction is dominant. In addition, Bax at the membrane recruits Bcl-xL from the cytosol, which promotes Bax retrotranslocation from the mitochondria to the cytosol (dashed lines). In order to promote apoptosis, Bax can spontaneously activate (blue dashed lines), although its activation is significantly enhanced by cbid. binding via its BH3 domain was not so evident. However, some experimental conditions, such as protein overexpression or cell type, among others, could be the reason of the different response of Bim. There is an interest in the mechanism of peptides derived from BH3 domains because they are the basis for the design of BH3 mimetic molecules, which have been recently approved for their use in therapy [2,83]. So far, quantification of affinities between antiapoptotic Bcl-2 members and BH3 peptides had been done in solution [81,84]. However, the membrane environment is a key element in the interaction of Bcl-2 family proteins [11,69,85]. A comparison of the affinities of tbid and Bcl-xL in the membrane and in solution using fluorescence correlation spectroscopy demonstrated the importance of the lipid environment in promoting their association. Two recent studies using isolated mitochondria or artificial membranes have provided quantification of the interactions between BH3-only proteins or peptides and Bcl-2 homologs in the membrane. One of them has compared the ability of BH3 peptides to dissociate the cbid/bcl-xl complex in solution and in membranes [86]. The BH3 peptides from Hrk, Bid, Bim, and Bad were the most efficient to disrupt this complex in the membrane, whereas all of them, except Noxa and Bmf, had a comparable activity in solution. In the second study, Bid chimeras were generated by replacing the BH3 domain of Bid with that of other BH3-only proteins. All Bid chimeras, except those derived of Noxa and Bad, were able to activate both Bax and Bak in isolated mitochondria, suggesting that most of them could act as direct activators under certain conditions [87]. Further studies are thus required to reconsider the actual classification of the BH3-only proteins into direct activators or sensitizers and their specificity for Bax and 422 The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies

8 A. Pe~na-Blanco and A. J. Garcıa-Saez Bax/Bak and mitochondria in apoptosis Bak activation. Although in this study, BH3-only proteins had no preference for Bax or Bak, evidence in cells indicated that Bak is preferentially activated by Bid and Bax by Bim [88]. These differences remain an issue that still needs to be clarified. Bok, an enigmatic member of the Bcl-2 family Bax and Bak are the two accepted executors of the Bcl-2 family, as cells lacking both these proteins are resistant to most apoptotic stimuli [89]. Bok, however, which is a family member with high homology to Bax and Bak, has a more mysterious role. It is predominantly found at the Golgi and endoplasmic reticulum (ER) membranes [90], although a fraction of Bok may also localize at the MOM or the ER-mitochondria contact sites [91]. Recent studies have suggested that Bok induces MOMP in the absence of Bax and Bak [91,92]. Llambi et al. have shown that Bok is a proapoptotic effector that is constitutively active, independently of Bid or Bcl-xL, and whose action is regulated by the ER-associated degradation pathway. This is at odds with biophysical experiments of Bok in liposomes showing that it has pore forming activity unaffected by Bcl-xL but enhanced by cbid [93]. While Bok formed large and stable pores with features of toroidal pores, it was unable to release cytochrome c from Bax / Bak / isolated mitochondria [93]. These discrepancies may be due to differences in the truncated recombinant proteins used in the studies or to the presence in mitochondria of unknown negative regulatory factors. Further studies regarding Bok structure, membrane activity and Bok interaction partners will help to elucidate the biological function of this enigmatic member of the Bcl-2 family. The role of mitochondria in the regulation of Bax and Bak function Mitochondria play an active role in apoptosis. First, most Bcl-2 family protein/protein interactions take place at mitochondria. Second, Bax and Bak form toroidal pores composed by proteins and lipids at the MOM, suggesting that mitochondrial composition participates in pore formation. Third, a crucial step in apoptosis execution is the release of intermembrane space (IMS) proteins to the cytosol, such as SMAC/ DIABLO and cytochrome c. The former requires a proteolytic maturation by the protease PARL at the IMS in order to exert its proapototic function [94], whereas the latter is trapped in the mitochondrial cristae, indicating that mitochondrial reorganization is required before cytochrome c can be released into the cytosol [95]. Four, Bax/Bak-induced MOMP in the absence of caspases triggers the release of mitochondrial DNA, which activates the cgas/sting DNAsensing pathway, leading to the production of type I interferons and causing a proinflammatory type of cell death [96,97]. Therefore, it is likely that mitochondrial architecture, lipid composition and protein constitution are key elements in the complex regulation of apoptosis. It is well known that Bax and Bak colocalize in specific mitochondrial apoptotic foci upon activation [98]. But what are the properties and composition of these foci that make them so attractive for the effectors of the Bcl-2 family? Bax and Bak collaboration with the mitochondria dynamics machinery during apoptosis Besides Bak, so far it is known that Bax colocalizes with the GTPases Dynamin-related protein 1 (Drp1) and mitofusin 2 (Mfn2) [99] at discrete mitochondrial foci during apoptosis. Drp1 is necessary for mitochondrial fission, while Mfn2, together with Mfn1, is a regulator of MOM fusion. In healthy conditions, Drp1 shuttles between cytosol and mitochondria, but upon apoptosis it accumulates in stable foci at the MOM to promote mitochondrial fission, which happens close in time and space to MOMP [100]. To make things more complicated, Drp1-promoted mitochondrial fission occurs at the contact sites between ER and mitochondria [16], via a mechanism in which the ER tubules preconstrict mitochondria, leading to the recruitment and oligomerization of Drp1 at the fission sites. These contact sites also play a key role in other physiological functions, such as lipid and calcium transfer between the two organelles [101], which also play a role in apoptosis. Since mitochondria undergo drastic fragmentation during apoptosis, the interplay between mitochondrial dynamics and apoptosis has been investigated. However, opposite lines of evidence exist regarding the role of Drp1 in the kinetics of apoptosis: in some reports inhibition of mitochondrial fission or enhancement of fusion delay apoptosis [100,102], whereas others indicate that Drp1 and division has no or little impact on the kinetics of MOMP [ ]. Although the role of mitochondrial fission in apoptosis still remains controversial, it is accepted that Drp1 is not required for Bax and Bak activation and foci formation at the mitochondria [99,106] but that the absence of Drp1 delays cytochrome c release [57,103, 104,107]. These observations suggest that Drp1 acts downstream of Bax/Bak activation, but upstream of cytochrome c release. The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies 423

9 Bax/Bak and mitochondria in apoptosis A. Pe~na-Blanco and A. J. Garcıa-Saez While cytochrome c release is delayed in the absence of Drp1, Smac/Diablo release remains unaffected [103,105], which supports a role for Drp1 in the regulation of cytochrome c release. In healthy cells, cytochrome c is located within the IMS, trapped inside the mitochondria cristae [95]. Cristae junctions open and fuse in apoptosis, allowing the complete release of cytochrome c in a process known as cristae remodeling [95,108]. Optic atrophy 1 (OPA1), a GTPase responsible for the fusion of the mitochondrial inner membrane, controls cristae remodeling by preventing the widening of the cristae junctions [109]. OPA1 oligomers form a molecular barrier between adjacent cristae and their disassembly allows the opening of the cristae, which leads to the complete release of cytochrome c. Interestingly, a recent study has revealed that Drp1 affects cristae remodeling and cytochrome c release through its adaptors MiD49/51 and independently of OPA1 disassembly [110]. Previously, Drp1 was also linked to cristae remodeling mediated by an ER-Bik (a BH3-only protein) mechanism [108]. As a result, current evidence suggests a dual role for Drp1 during apoptosis, with participation in both mitochondrial fission and cristae remodeling, specifically promoted via MiD49/51 adaptors. How these processes are coupled and how they relate to Bax and Bak function still remain elusive. In an elegant study, Prudent et al. recently reconciled some open questions about the participation of Drp1, Bax and Bak in MOMP. This study suggested the presence of Drp1 platforms at the ER-mitochondria contact sites during apoptosis [111]. Drp1 is SUMOylated by MAPL during apoptosis, which stabilizes Drp1 oligomers at sites of mitochondrial constriction and fission [111,112]. MAPL function is downstream of Bax and Bak activation and oligomerization, but upstream of cytochrome c release, indicating that the formation of stable platforms is not required for the initiation of Bax/Bak pore structures but still participates in MOMP (Fig. 4). The authors proposed that stabilized ER-mitochondria contact sites in apoptosis provide a platform for calcium and lipid flux from the ER to the mitochondria, which promotes cristae remodeling [15,108,111]. Additional proteins may have similar or regulatory functions in these foci and further research is required to shed light into their composition and structure. We speculate that macromolecular complexes at the ER-mitochondria contact sites may coordinate the activity of Bax, Bak and Drp1 during apoptosis with the manifold mitochondrial alterations in apoptosis [113], including release of proapoptotic factors to the cytosol. But what marks the position of these mitochondrial foci? Are mitochondrial lipids or membrane curvature enough to promote Bax and Bak oligomerization, or are other cellular components required? Impact of mitochondrial lipid composition on Bax/Bak activity It has long been believed that mitochondrial lipids play a role in the activity of Bcl-2 family proteins. Cardiolipin, which mostly localizes at the MIM and is key for MOM/MIM contact sites, promotes tbidmediated Bax activation and formation of large pores in liposomes [ ]. However, Bax is still able to oligomerize and induce cytochrome c release in mitochondria without cardiolipin [117], which brings the role of this lipid in apoptosis into question. Cardiolipin has been also proposed to act as tbid receptor [118]. Yet, due to the low levels of cardiolipin at the MOM, other studies suggested that MOM proteins, such as MTCH2, take over the role of cardiolipin in recruiting tbid in cells [119]. As a result, the role of cardiolipin in apoptosis regulation still remains under debate. Other discussed lipids in apoptosis are ceramides, which accumulate at mitochondria during cell death [120]. Ceramides are synthesized at the ER through the sphingomyelin pathway, consistent with an essential function of the ER-mitochondria contact sites in lipid transfer [101]. Increased levels of ceramides in mitochondria promote Bax activation [ ] and, in turn, Bak activates ceramide synthesis [124,125]. In addition, two metabolites of the ceramide pathway, sphingosine-1-phosphate and hexadecenal, cooperate to promote Bak and Bax mediated MOMP, respectively [126]. Furthermore, a recent study has demonstrated that the inhibition of ceramide synthesis by the antiapoptotic protein Bcl2-L13 (also known as Bclrambo) prevents cell death [127]. Not only the lipid composition but also mitochondrial membrane curvature and size have been proposed as regulators of MOMP. A recent study using as artificial membranes vesicles of different sizes and isolated mitochondria proposed that small vesicles with high curvature prevent Bax helix a9 insertion and execution of MOMP [128]. However, it remains to be clarified how curved membranes with packing defects would restrict protein insertion and whether mitochondrial size has a functional relevance in the cell. Nevertheless, Bax seems to have the ability to stabilize highly curved structures [129] and the formation of hemifusion intermediates by Drp1 has been shown to enhance Bax oligomerization and activity in cell-free systems [130]. This may suggest that the formation of highly curved 424 The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies

10 A. Pe~na-Blanco and A. J. Garcıa-Saez Bax/Bak and mitochondria in apoptosis Fig. 4. Stable platforms at the ER-mitochondria contact sites to mediate cytochrome c release during apoptosis. (A) Upon apoptotic stimuli, Bax and Bak are activated and form foci at the MOM. It is still unclear what marks the localization of active Bax and Bak at the mitochondria. One possibility is that mitochondrial constrictions mediated by the ER tubules may be sufficient to promote Bax/Bak oligomerization, although other still unknown cellular components could also regulate this event. In this situation, OPA1 oligomers prevent cytochrome c release by forming a molecular barrier between adjacent cristae that impedes cristae remodeling. (B) After Bax/Bak activation, Drp1 is recruited to ER-mediated preconstricted mitochondria where it oligomerizes and further constricts the mitochondria. (C) Drp1 is SUMOylated by MAPL during apoptosis, which stabilizes Drp1 oligomers at sites of mitochondrial constriction and fission. This generates Drp1 stable platforms at the ER-mitochondria contact sites during apoptosis (highlighted inside the blue sphere) that are relevant for calcium and lipid transfer between both organelles. As a result of calcium transfer, OPA1 oligomers disassemble and cytochrome c is released. The Drp1 adaptors MiD49/51 are involved in Drp1 cristae remodeling activity. membranes by Drp1, rather than mitochondrial fission, is involved in Bax oligomerization. Given that Bax oligomerization occurs in the absence of Drp1, other mediators of membrane curvature could also be implicated in this process. One possibility could be that local mitochondrial constriction mediated by the ER tubules might promote Bax/Bak oligomerization, although such hypothesis still needs verification. Concluding remarks Our knowledge about Bax and Bak has increased during the last years thanks to the application of novel structural and biophysical techniques. Bax pores at the MOM have been visualized and we understand better the molecular basis for their assembly and structural conversion into killers at mitochondria. Accumulating evidence supports that not only Bax and Bak activity, but also additional mitochondrial components contribute to MOMP execution. In relation to this, the formation of large protein and lipid assemblies at discrete foci that correlate with ER-mitochondria contact sites provides a very attractive explanation for coordination of the multiple alterations that mitochondria undergo during apoptosis. However, the composition and architecture of such macromolecular complexes remains to be defined. Another fascinating question is what marks Bax/Bak oligomerization at specific foci and whether their oligomerization is the only requisite for pore formation or other cellular elements are needed. We envision coming years of exciting research to achieve major progress in our understanding of the role of mitochondria in apoptosis. Acknowledgements The authors thank Dr. Katia Cosentino and Dr. Uris Ros for careful reading of the manuscript and help with figure preparation. The Garcıa-Saez laboratory is funded by the European Research Council (ERC-2012-StG ), the Deutsche Forschungsgemeinschaft (FOR 2036), and the University of T ubingen. Author contributions AP-B and AJG-S conceived and wrote the manuscript. References 1 Czabotar PE, Lessene G, Strasser A & Adams JM (2014) Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol 15, Delbridge AR, Grabow S, Strasser A & Vaux DL (2016) Thirty years of BCL-2: translating cell death The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies 425

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