CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13

The pathway to cell death in Caenorhabditis elegans is well established. In cells undergoing apoptosis, the Bcl-2 homology domain 3 (BH3)-only protein EGL-1 binds to CED-9 at the mitochondrial membrane to cause the release of CED-4, which oligomerises and facilitates the activation of the caspase CED-3. However, despite many studies, the biophysical features of the CED-4/CED-9 complex have not been fully characterised. Here, we report the purification of a soluble and stable 2 : 2 heterotetrameric complex formed by recombinant CED-4 and CED-9 coexpressed in bacteria. Consistent with previous studies, synthetic peptides corresponding to the BH3 domains of worm BH3-only proteins (EGL-1, CED-13) dissociate CED-4 from CED-9, but not from the gain-of-function CED-9 (G169E) mutant. Surprisingly, the ability of worm BH3 domains to dissociate CED-4 was specific since mammalian BH3-only proteins could not do so.     

Evidence that CED-9/Bcl2 and CED-4/Apaf-1 localization is not consistent with the current model for C. elegans apoptosis induction

In C. elegans, the BH3-only domain protein EGL-1, the Apaf-1 homolog CED-4 and the CED-3 caspase are required for apoptosis induction, whereas the Bcl-2 homolog CED-9 prevents apoptosis. Mammalian B-cell lymphoma 2 (Bcl-2) inhibits apoptosis by preventing the release of the Apaf-1 (apoptotic protease-activating factor 1) activator cytochrome c from mitochondria. In contrast, C. elegans CED-9 is thought to inhibit CED-4 by sequestering it at the outer mitochondrial membrane by direct binding. We show that CED-9 associates with the outer mitochondrial membrane within distinct foci that do not overlap with CED-4, which is predominantly perinuclear and does not localize to mitochondria. CED-4 further accumulates in the perinuclear space in response to proapoptotic stimuli such as ionizing radiation. This increased accumulation depends on EGL-1 and is abrogated in ced-9 gain-of-function mutants. CED-4 accumulation is not sufficient to trigger apoptosis execution, even though it may prime cells for apoptosis. Our results suggest that the cell death protection conferred by CED-9 cannot be solely explained by a direct interaction with CED-4.     

RNA aptamers targeting the cell death inhibitor CED-9 induce cell killing in Caenorhabditis elegans

Bcl-2 family proteins include anti- and proapoptotic factors that play important roles in regulating apoptosis in diverse species. Identification of compounds that can modulate the activities of Bcl-2 family proteins will facilitate development of drugs for treatment of apoptosis-related human diseases. We used an in vitro selection method named systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers that bind the Caenorhabditis elegans Bcl-2 homolog CED-9 with high affinity and specificity and tested whether these aptamers modulate programmed cell death in C. elegans. Five CED-9 aptamers were isolated and classified into three groups based on their predicted secondary structures. Biochemical analyses indicated that two of these aptamers, R9-2 and R9-7, and EGL-1, an endogenous CED-9-binding proapoptotic protein, bound to distinct regions of CED-9. However, these two aptamers shared overlapping CED-9 binding sites with CED-4, another CED-9-binding proapoptotic factor. Importantly ectopic expression of these two aptamers in touch receptor neurons induced efficient killing of these neurons largely in a CED-3 caspase-dependent manner. These findings suggest that RNA aptamers can be used to modulate programmed cell death in vivo and can potentially be used to develop drugs to treat human diseases caused by abnormal apoptosis.     

Disruption of the CED-9.CED-4 complex by EGL-1 is a critical step for programmed cell death in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the apoptotic machinery is composed of four basic elements: the caspase CED-3, the Apaf-1 homologue CED-4, and the Bcl-2 family members CED-9 and EGL-1. The ced-9(n1950) gain-of-function mutation prevents most, if not all, somatic cell deaths in C. elegans. It encodes a CED-9 protein with a glycine-to-glutamate substitution at position 169, which is located within the highly conserved Bcl-2 homology 1 domain. We performed biochemical analyses with the CED-9G169E protein to gain insight into the mechanism of programmed cell death. We find that CED-9G169E retains the ability to bind both EGL-1 and CED-4, although its affinity for EGL-1 is reduced. In contrast to the behavior of wild-type CED-9, the interaction between CED-9G169E and CED-4 is not disrupted by expression of EGL-1. Furthermore, CED-4 and CED-9G169E co-localizes with EGL-1 to the mitochondria in mammalian cells, and expression of EGL-1 does not induce translocation of CED-4 to the cytosol. Finally, the ability of EGL-1 to promote apoptosis is impaired by the replacement of wild-type CED-9 with CED-9G169E, and this effect is correlated with the inability of EGL-1 to induce the displacement of CED-4 from the CED-9.CED-4 complex. These studies suggest that the release of CED-4 from the CED-9.CED-4 complex is a necessary step for induction of programmed cell death in C. elegans.     

Structural, biochemical, and functional analyses of CED-9 recognition by the proapoptotic proteins EGL-1 and CED-4

Programmed cell death in Caenorhabditis elegans is initiated by the binding of EGL-1 to CED-9, which disrupts the CED-4/CED-9 complex and allows CED-4 to activate the cell-killing caspase CED-3. Here we demonstrate that the C-terminal half of EGL-1 is necessary and sufficient for binding to CED-9 and for killing cells. Structure of the EGL-1/CED-9 complex revealed that EGL-1 adopts an extended alpha-helical conformation and induces substantial structural rearrangements in CED-9 upon binding. EGL-1 interface mutants failed to bind to CED-9 or to release CED-4 from the CED-4/CED-9 complex, and were unable to induce cell death in vivo. A surface patch on CED-9, different from that required for binding to EGL-1, was identified to be responsible for binding to CED-4. These data suggest a working mechanism for the release of CED-4 from the CED-4/CED-9 complex upon EGL-1 binding and provide a mechanistic framework for understanding apoptosis activation in C. elegans.     

CED-9及其在细胞凋亡中的调控机制

细胞凋亡与生物体发育、疾病等息息相关,已成为近年来生命科学领域的研究热点之一。线虫作为一种模式生物是动物细胞凋亡研究的极好材料。CED-9是Bcl-2抗凋亡蛋白家族中的成员,是调控线虫细胞凋亡的关键蛋白质。与Bcl-2家族中的其他抗凋亡蛋白相比,它具有自身独特的结构特点。通过与促凋亡蛋白CED-4结合,对CED-4在细胞内的定位产生影响,从而改变下游的CED-3的活性,最终调控细胞凋亡。同时,CED-9自身的活性受到另一种促凋亡蛋白EGL-1的调控,EGL—1诱导CED-9的构象发生变化,两者形成稳定的复合物,同时CED-4从CED-9上脱离并释放到细胞质中,进而激活CED-3,使细胞发生凋亡。目前,从线虫中克隆出来的ced-9基因转入其他生物比如动物细胞、高等植物和酵母中进行研究。在论述CED-9的独特结构的基础上,进一步阐述了CED-9对CED-4在细胞内定位的影响,与EGL-1、CED-4相互作用机制,以及近年来它在高等植物和酵母中的研究进展。     

Direct and selective small-molecule activation of proapoptotic BAX

BCL-2 family proteins are key regulators of the apoptotic pathway. Antiapoptotic members sequester the BCL-2 homology 3 (BH3) death domains of proapoptotic members such as BAX to maintain cell survival. The antiapoptotic BH3-binding groove has been successfully targeted to reactivate apoptosis in cancer. We recently identified a geographically distinct BH3-binding groove that mediates direct BAX activation, suggesting a new strategy for inducing apoptosis by flipping BAX's 'on switch'. Here we applied computational screening to identify a BAX activator molecule that directly and selectively activates BAX. We demonstrate by NMR and biochemical analyses that the molecule engages the BAX trigger site and promotes the functional oligomerization of BAX. The molecule does not interact with the BH3-binding pocket of antiapoptotic proteins or proapoptotic BAK and induces cell death in a BAX-dependent fashion. To our knowledge, we report the first gain-of-function molecular modulator of a BCL-2 family protein and demonstrate a new paradigm for pharmacologic induction of apoptosis.     

Structural and biochemical bases for the inhibition of autophagy and apoptosis by viral BCL-2 of murine gamma-herpesvirus 68

All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2) to counter the clearance of infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2 proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine gamma-herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3) domain-containing peptides derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the 1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the virus.     

Structural and Biochemical Bases for the Inhibition of Autophagy and Apoptosis by Viral BCL-2 of Murine ��-Herpesvirus 68

All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2) to counter the clearance of infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2 proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine γ-herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3) domain-containing peptides derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the 1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the virus.     

MCL-1ES Induces MCL-1L-Dependent BAX- and BAK-Independent Mitochondrial Apoptosis

MCL-1 (myeloid cell leukemia-1), a member of the BCL-2 family, has three splicing variants, antiapoptotic MCL-1L, proapoptotic MCL-1S, and MCL-1ES. We previously reported cloning MCL-1ES and characterizing it as an apoptotic molecule. Here, we investigated the molecular mechanism by which MCL-1ES promotes cell death. MCL-1ES was distinct from other proapoptotic BCL-2 members that induce apoptosis by promoting BAX or BAK oligomerization, leading to mitochondrial outer membrane permeabilization (MOMP), in that MCL-1ES promoted mitochondrial apoptosis independently of both BAX and BAK. Instead, MCL-1L was crucial for the apoptotic activity of MCL-1ES by facilitating its proper localization to the mitochondria. MCL-1ES did not interact with any BCL-2 family proteins except for MCL-1L, and antiapoptotic BCL-2 members failed to inhibit apoptosis induced by MCL-1ES. The BCL-2 homology 3 (BH3) domain of MCL-1ES was critical for both MCL-1ES association with MCL-1L and apoptotic activity. MCL-1ES formed mitochondrial oligomers, and this process was followed by MOMP and cytochrome c release in a MCL-1L-dependent manner. These findings indicate that MCL-1ES, as a distinct proapoptotic BCL-2 family protein, may be useful for intervening in diseases that involve uncontrolled MCL-1L.     

Mutational analysis of the interacting cell death regulators CED-9 and CED-4

The genes ced-3, ced-4 and ced-9 are central components in the cell death pathway of the nematode C. elegans. Ced-9, which functions to inhibit cell death, is homologous to the Bcl-2 family of mammalian anti-apoptotic genes. The ced-3 gene encodes a protein homologous to the caspases, a family of cysteine proteases involved in the execution of programmed cell death. It has recently been demonstrated that CED-4, an inducer of apoptosis for which no mammalian equivalent has been reported, can interact with CED-9 and Bcl-x(L). Here we confirm that CED-9 and CED-4 interact and using a series of deletion mutants, demonstrate that only short N-terminal deletions are tolerated in each molecule without loss-of-interaction. Two loss-of-function point mutations in different regions of CED-4 also lead to a significant loss of interaction suggesting further that the relevant interaction domains are not short linear sequences, but rather, are formed by more complex structural determinants in each molecule. Furthermore, we demonstrate that CED-4 not only interacts with Bcl-x(L) but also with its homologue, Bcl-2, and that the unstructured loop region present in Bcl-x(L) and Bcl-2 can regulate the CED-4 interaction. Lastly, we show that a BH3 peptide that can inhibit Bcl-2 family interactions also inhibits the interaction between Bcl-x(L) and CED-4.     

Role for CED-9 and Egl-1 as regulators of mitochondrial fission and fusion dynamics

Bcl-2 family proteins play central roles in apoptosis by regulating the release of mitochondrial intermembrane space proteins such as cytochrome c. Death-promoting Bcl-2 family members, such as Bax, can promote cytochrome c release and fragmentation of the mitochondrial network, whereas apoptosis-inhibitory members, such as Bcl-2 and Bcl-xL, can antagonize these events. It remains unclear whether CED-9, the worm Bcl-2 relative, can regulate mitochondrial fission/fusion dynamics or the release of proteins from the mitochondrial intermembrane space. Here, we show that CED-9 interacts with Mitofusin-2/fuzzy onions and can promote mitochondrial clustering and dramatic reorganization of mitochondrial networks. Consistent with its ability to neutralize CED-9 function, EGL-1 antagonized CED-9-dependent remodeling of the mitochondrial network. However, CED-9 failed to inhibit mitochondrial cytochrome c release or apoptosis induced by diverse triggers in mammalian cells. These data suggest that the ability to regulate mitochondrial fission/fusion dynamics is an evolutionarily conserved property of the Bcl-2 family.     

A stapled BID BH3 helix directly binds and activates BAX

BAX is a multidomain proapoptotic BCL-2 family protein that resides in the cytosol until activated by an incompletely understood trigger mechanism, which facilitates BAX translocation to mitochondria and downstream death events. Whether BAX is activated by direct contact with select BH3-only members of the BCL-2 family is highly debated. Here we detect and quantify a direct binding interaction between BAX and a hydrocarbon-stapled BID BH3 domain, which triggers the functional activation of BAX at nanomolar doses in vitro. Chemical reinforcement of BID BH3 alpha helicity was required to reveal the direct BID BH3-BAX association. We confirm the specificity of this BH3 interaction by characterizing a stapled BAD BH3 peptide that interacts with antiapoptotic BCL-X(L) but does not bind or activate BAX. We further demonstrate that membrane targeting of stapled BID BH3 optimizes its ability to activate BAX, supporting a model in which BID directly engages BAX to trigger mitochondrial apoptosis.     

A surface groove essential for viral Bcl-2 function during chronic infection in vivo

Antiapoptotic Bcl-2 family proteins inhibit apoptosis in cultured cells by binding BH3 domains of proapoptotic Bcl-2 family members via a hydrophobic BH3 binding groove on the protein surface. We investigated the physiological importance of the BH3 binding groove of an antiapoptotic Bcl-2 protein in mammals in vivo by analyzing a viral Bcl-2 family protein. We show that the gamma-herpesvirus 68 (gammaHV68) Bcl-2 family protein (gammaHV68 v-Bcl-2), which is known to inhibit apoptosis in cultured cells, inhibits both apoptosis in primary lymphocytes and Bax toxicity in yeast. Nuclear magnetic resonance determination of the gammaHV68 v-Bcl-2 structure revealed a BH3 binding groove that binds BH3 domain peptides from proapoptotic Bcl-2 family members Bax and Bak via a molecular mechanism shared with host Bcl-2 family proteins, involving a conserved arginine in the BH3 peptide binding groove. Mutations of this conserved arginine and two adjacent amino acids to alanine (SGR to AAA) within the BH3 binding groove resulted in a properly folded protein that lacked the capacity of the wild-type gammaHV68 v-Bcl-2 to bind Bax BH3 peptide and to block Bax toxicity in yeast. We tested the physiological importance of this v-Bcl-2 domain during viral infection by engineering viral mutants encoding a v-Bcl-2 containing the SGR to AAA mutation. This mutation resulted in a virus defective for both efficient reactivation of gammaHV68 from latency and efficient persistent gammaHV68 replication. These studies demonstrate an essential functional role for amino acids in the BH3 peptide binding groove of a viral Bcl-2 family member during chronic infection.     

B Cell Lymphoma-2 (BCL-2) Homology Domain 3 (BH3) Mimetics Demonstrate Differential Activities Dependent upon the Functional Repertoire of Pro- and Anti-apoptotic BCL-2 Family Proteins

The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these “BH3 mimetics” in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.     

Antiapoptotic Bcl‐2 homolog CED‐9 in Caenorhabditis elegans: Dynamics of BH3 and CED‐4 binding regions and comparison with mammalian antiapoptotic Bcl‐2 proteins

Proteins belonging to Bcl‐2 family regulate intrinsic cell death pathway. Although mammalian antiapoptotic Bcl‐2 members interact with multiple proapoptotic proteins, the Caenorhabditis elegans Bcl‐2 homolog CED‐9 is known to have only two proapoptotic partners. The BH3‐motif of proapoptotic proteins bind to the hydrophobic groove of prosurvival proteins formed by the Bcl‐2 helical fold. CED‐9 is also known to interact with CED‐4, a homolog of the human cell death activator Apaf1. We have performed molecular dynamics simulations of CED‐9 in two forms and compared the results with those of mammalian counterparts Bcl‐X_L, Bcl‐w, and Bcl‐2. Our studies demonstrate that the region forming the hydrophobic cleft is more flexible compared with the CED‐4‐binding region, and this is generally true for all antiapoptotic Bcl‐2 proteins studied. CED‐9 is the most stable protein during simulations and its hydrophobic pocket is relatively rigid explaining the absence of functional redundancy in CED‐9. The BH3‐binding region of Bcl‐2 is less flexible among the mammalian proteins and this lends support to the studies that Bcl‐2 binds to less number of BH3 peptides with high affinity. The C‐terminal helix of CED‐9 lost its helical character because of a large number of charged residues. We speculate that this region probably plays a role in intracellular localization of CED‐9. The BH4‐motif accessibility in CED‐9 and Bcl‐w is controlled by the loop connecting the first two helices. Although CED‐9 adopts the same Bcl‐2 fold, our studies highlight important differences in the dynamic behavior of CED‐9 and mammalian antiapoptotic homologs. Proteins 2014; 82:1035–1047. © 2013 Wiley Periodicals, Inc.     

The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CED-4.

Bcl-2 and close homologues such as Bcl-xL promote cell survival, while other relatives such as Bax antagonize this function. Since only the pro-survival family members possess a conserved N-terminal region denoted BH4, we have explored the role of this amphipathic helix for their survival function and for interactions with several agonists of apoptosis, including Bax and CED-4, an essential regulator in the nematode Caenorhabditis elegans. BH4 of Bcl-2 could be replaced by that of Bcl-x without perturbing function but not by a somewhat similar region near the N-terminus of Bax. Bcl-2 cell survival activity was reduced by substitutions in two of ten conserved BH4 residues. Deletion of BH4 rendered Bcl-2 (and Bcl-xL) inactive but did not impair either Bcl-2 homodimerization or ability to bind to Bax or five other pro-apoptotic relatives (Bak, Bad, Bik, Bid or Bim). Hence, association with these death agonists is not sufficient to promote cell survival. Significantly, however, Bcl-xL lacking BH4 lost the ability both to bind CED-4 and antagonize its pro-apoptotic activity. These results favour the hypothesis that the BH4 domain of pro-survival Bcl-2 family members allows them to sequester CED-4 relatives and thereby prevent apoptosis.     

MCL-1S, a splicing variant of the antiapoptotic BCL-2 family member MCL-1, encodes a proapoptotic protein possessing only the BH3 domain

MCL-1 (myeloid cell leukemia-1) is an antiapoptotic BCL-2 family protein discovered as an early induction gene during myeloblastic leukemia cell differentiation. This survival protein has the BCL-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region. We identified a short splicing variant of the MCL-1 mRNA in the human placenta encoding a protein, termed MCL-1 short (MCL-1S), with an altered C terminus as compared with the full-length MCL-1 long (MCL-1L), leading to the loss of BH1, BH2, and the transmembrane domains. Analysis of the human MCL-1 gene indicated that MCL-1S results from the splicing out of exon 2 during mRNA processing. MCL-1S, unlike MCL-1L, does not interact with diverse proapoptotic BCL-2-related proteins in the yeast two-hybrid system. In contrast, MCL-1S dimerizes with MCL-1L in the yeast assay and coprecipitates with MCL-1L in transfected mammalian cells. Overexpression of MCL-1S induces apoptosis in transfected Chinese hamster ovary cells, and the MCL-1S action was antagonized by the antiapoptotic MCL-1L. Thus, the naturally occurring MCL-1S variant represents a new proapoptotic BH3 domain-only protein capable of dimerizing with the antiapoptotic MCL-1L. The fate of MCL-1-expressing cells could be regulated through alternative splicing mechanisms and interactions of the resulting anti- and proapoptotic gene products.     

A unified model of mammalian BCL-2 protein family interactions at the mitochondria

During apoptosis, the BCL-2 protein family controls mitochondrial outer membrane permeabilization (MOMP), but the dynamics of this regulation remain controversial. We employed chimeric proteins composed of exogenous BH3 domains inserted into a tBID backbone that can activate the proapoptotic effectors BAX and BAK to permeabilize membranes without being universally sequestered by all antiapoptotic BCL-2 proteins. We thus identified two "modes" whereby prosurvival BCL-2 proteins can block MOMP, by sequestering direct-activator BH3-only proteins ("MODE 1") or by binding active BAX?and BAK ("MODE 2"). Notably, we found that MODE 1 sequestration is less efficient and more easily derepressed to promote MOMP than MODE 2. Further, MODE 2 sequestration prevents mitochondrial fusion. We provide a unified model of BCL-2 family function that helps to explain otherwise paradoxical observations relating to MOMP, apoptosis, and mitochondrial dynamics.