Oligomeric BAX induces mitochondrial permeability transition and complete cytochrome c release without oxidative stress

In the present study, we investigated the mechanism of cytochrome c release from isolated brain mitochondria induced by recombinant oligomeric BAX (BAX_oligo). We found that BAX_oligo caused a complete release of cytochrome c in a concentration- and time-dependent manner. The release was similar to those induced by alamethicin, which causes maximal mitochondrial swelling and eliminates barrier properties of the OMM. BAX_oligo also produced large amplitude mitochondrial swelling as judged by light scattering assay and transmission electron microscopy. In addition, BAX_oligo resulted in a strong mitochondrial depolarization. ATP or a combination of cyclosporin A and ADP, inhibitors of the mPT, suppressed BAX_oligo-induced mitochondrial swelling and depolarization as well as cytochrome c release but did not influence BAX_oligo insertion into the OMM. Both BAX_oligo- and alamethicin-induced cytochrome c releases were accompanied by inhibition of ROS generation, which was assessed by measuring mitochondrial H₂O₂ release with an Amplex Red assay. The mPT inhibitors antagonized suppression of ROS generation caused by BAX_oligo but not by alamethicin. Thus, BAX_oligo resulted in a complete cytochrome c release from isolated brain mitochondria in the mPT-dependent manner without involvement of oxidative stress by the mechanism requiring mitochondrial remodeling and permeabilization of the OMM.     

VDAC2 is required for truncated BID‐induced mitochondrial apoptosis by recruiting BAK to the mitochondria

Truncated BID (tBID), a proapoptotic BCL2 family protein, induces BAK/BAX‐dependent release of cytochrome c and other mitochondrial intermembrane proteins to the cytosol to induce apoptosis. The voltage‐dependent anion channels (VDACs) are the primary gates for solutes across the outer mitochondrial membrane (OMM); however, their role in apoptotic OMM permeabilization remains controversial. Here, we report that VDAC2^?/? (V2^?/?) mouse embryonic fibroblasts (MEFs) are virtually insensitive to tBID‐induced OMM permeabilization and apoptosis, whereas VDAC1^?/?, VDAC3^?/? and VDAC1^?/?/VDAC3^?/? MEFs respond normally to tBID. V2^?/? MEFs regain tBID sensitivity after VDAC2 expression. Furthermore, V2^?/? MEFs are deficient in mitochondrial BAK despite normal tBID–mitochondrial binding and BAX/BAK expression. tBID sensitivity of BAK^?/? MEFs is also reduced, although not to the same extent as V2^?/? MEFs, which might result from their strong overexpression of BAX. Indeed, addition of recombinant BAX also sensitized V2^?/? MEFs to tBID. Thus, VDAC2 acts as a crucial component in mitochondrial apoptosis by allowing the mitochondrial recruitment of BAK, thereby controlling tBID‐induced OMM permeabilization and cell death.     

Oligomeric BAX induces mitochondrial permeability transition and complete cytochrome c release without oxidative stress

In the present study, we investigated the mechanism of cytochrome c release from isolated brain mitochondria induced by recombinant oligomeric BAX (BAX(oligo)). We found that BAX(oligo) caused a complete release of cytochrome c in a concentration- and time-dependent manner. The release was similar to those induced by alamethicin, which causes maximal mitochondrial swelling and eliminates barrier properties of the OMM. BAX(oligo) also produced large amplitude mitochondrial swelling as judged by light scattering assay and transmission electron microscopy. In addition, BAX(oligo) resulted in a strong mitochondrial depolarization. ATP or a combination of cyclosporin A and ADP, inhibitors of the mPT, suppressed BAX(oligo)-induced mitochondrial swelling and depolarization as well as cytochrome c release but did not influence BAX(oligo) insertion into the OMM. Both BAX(oligo)- and alamethicin-induced cytochrome c releases were accompanied by inhibition of ROS generation, which was assessed by measuring mitochondrial H(2)O(2) release with an Amplex Red assay. The mPT inhibitors antagonized suppression of ROS generation caused by BAX(oligo) but not by alamethicin. Thus, BAX(oligo) resulted in a complete cytochrome c release from isolated brain mitochondria in the mPT-dependent manner without involvement of oxidative stress by the mechanism requiring mitochondrial remodeling and permeabilization of the OMM.     

S-palmitoylation represents a novel mechanism regulating the mitochondrial targeting of BAX and initiation of apoptosis

The intrinsic pathway of apoptotic cell death is mainly mediated by the BCL-2-associated X (BAX) protein through permeabilization of the mitochondrial outer membrane (MOM) and the concomitant release of cytochrome c into the cytosol. In healthy, non-apoptotic cells, BAX is predominantly localized in the cytosol and exhibits a dynamic shuttle cycle between the cytosol and the mitochondria. Thus, the initial association with mitochondria represents a critical regulatory step enabling BAX to insert into MOMs, promoting the release of cytochrome c and ultimately resulting in apoptosis. However, the molecular mode of how BAX associates with MOMs and whether a cellular regulatory mechanism governs this process is poorly understood. Here we show that in both primary tissues and cultured cells, the association with MOMs and the proapoptotic action of BAX is controlled by its S-palmitoylation at Cys-126. A lack of BAX palmitoylation reduced BAX mitochondrial translocation, BAX oligomerization, caspase activity and apoptosis. Furthermore, ectopic expression of specific palmitoyl transferases in cultured healthy cells increases BAX S-palmitoylation and accelerates apoptosis, whereas malignant tumor cells show reduced BAX S-palmitoylation consistent with their reduced BAX-mediated proapoptotic activity. Our findings suggest that S-palmitoylation of BAX at Cys126 is a key regulatory process of BAX-mediated apoptosis.     

Dissimilar mechanisms of cytochrome c release induced by octyl glucoside-activated BAX and by BAX activated with truncated BID

In the present study, we compared alkali-resistant BAX insertion into the outer mitochondrial membrane, mitochondrial remodeling, mitochondrial membrane potential changes, and cytochrome c (Cyt c) release from isolated brain mitochondria triggered by recombinant BAX oligomerized with 1% octyl glucoside (BAX_oligo) and by a combination of monomeric BAX (BAX_mono) and caspase 8-cleaved C-terminal fragment of recombinant BID (truncated BID, t^cBID). We also examined whether the effects induced by BAX_oligo or by BAX_mono activated with t^cBID depended on induction of the mitochondrial permeability transition. The results obtained in this study revealed that t^cBID plus BAX_mono produced BAX insertion and Cyt c release without overt changes in mitochondrial morphology. On the contrary, treatment of mitochondria with BAX_oligo resulted in BAX insertion and Cyt c release, which were accompanied by gross distortion of mitochondrial morphology. The effects of BAX_oligo could be at least partially suppressed by mitochondrial depolarization. The effects of t^cBID plus BAX_mono were insensitive to depolarization. BAX_oligo produced similar BAX insertion, mitochondrial remodeling, and Cyt c release in KCl- and in N-methyl-d-glucamine-based incubation media indicating a non-essential role for K⁺ influx into mitochondria in these processes. A combination of cyclosporin A and ADP, inhibitors of the mitochondrial permeability transition, attenuated Cyt c release, mitochondrial remodeling, and depolarization induced by BAX_oligo, but failed to influence the effects produced by t^cBID plus BAX_mono. Thus, our results suggest a significant difference in the mechanisms of the outer mitochondrial membrane permeabilization and Cyt c release induced by detergent-oligomerized BAX_oligo and by BAX activated with t^cBID.     

Upregulation of Bcl2 inhibits apoptosis-driven BAX insertion but favors BAX relocalization in mitochondria

Protein-protein interactions between the Bcl2 family proteins regulate apoptosis. An imbalance of this interaction network due to the upregulation of the proto-oncogene Bcl2 leads to a resistance to apoptosis associated with tumor formation. Bcl2 overexpression inhibits BAX oligomerization and mitochondrial outer membrane (MOM) permeabilization. However, Bcl2 effects on earlier steps of BAX-mediated apoptosis are not fully understood. Bcl2 overexpression inhibits BAX insertion into the MOM but spontaneously increases BAX relocalization to the mitochondria. Also, a physical interaction between BAX and Bcl2 is necessary for these two effects to occur. Taken together, these results suggest upregulated Bcl2 stabilizes BAX loose binding to mitochondrial membranes, inhibiting its insertion into the MOM and consequently cytochrome c release.

Structured summary

MINT-7945271: BAX (uniprotkb:Q07813) physically interacts (MI:0915) with Bcl-2 (uniprotkb:P10417) by anti bait coimmunoprecipitation (MI:0006)     

Ca-mediated regulation of VDAC1 expression levels is associated with cell death induction

VDAC1, an outer mitochondrial membrane (OMM) protein, is crucial for regulating mitochondrial metabolic and energetic functions and acts as a convergence point for various cell survival and death signals. VDAC1 is also a key player in apoptosis, involved in cytochrome c (Cyto c) release and interactions with anti-apoptotic proteins. Recently, we demonstrated that various pro-apoptotic agents induce VDAC1 oligomerization and proposed that a channel formed by VDAC1 oligomers mediates cytochrome c release. As VDAC1 transports Ca^2 + across the OMM and because Ca^2 + has been implicated in apoptosis induction, we addressed the relationship between cytosolic Ca^2 + levels ([Ca^2 +]i), VDAC1 oligomerization and apoptosis induction. We demonstrate that different apoptosis inducers elevate cytosolic Ca^2 + and induce VDAC1 over-expression. Direct elevation of [Ca^2 +]i by the Ca^2 +-mobilizing agents A23187, ionomycin and thapsigargin also resulted in VDAC1 over-expression, VDAC1 oligomerization and apoptosis. In contrast, decreasing [Ca^2 +]i using the cell-permeable Ca^2 +-chelating reagent BAPTA-AM inhibited VDAC1 over-expression, VDAC1 oligomerization and apoptosis. Correlation between the increase in VDAC1 levels and oligomerization, [Ca^2 +]i levels and apoptosis induction, as induced by H₂O₂ or As₂O₃, was also obtained. On the other hand, cells transfected to overexpress VDAC1 presented Ca^2 +-independent VDAC1 oligomerization, cytochrome c release and apoptosis, suggesting that [Ca^2 +]i elevation is not a pre-requisite for apoptosis induction when VDAC1 is over-expressed. The results suggest that Ca^2 + promotes VDAC1 over-expression by an as yet unknown signaling pathway, leading to VDAC1 oligomerization, ultimately resulting in apoptosis. These findings provide a new insight into the mechanism of action of existing anti-cancer drugs involving induction of VDAC1 over-expression as a mechanism for inducing apoptosis. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau     

Two pathways for tBID-induced cytochrome c release from rat brain mitochondria: BAK- versus BAX-dependence

The mechanisms of truncated BID (tBID)-induced Cyt c release from non-synaptosomal brain mitochondria were examined. Addition of tBID to mitochondria induced partial Cyt c release which was inhibited by anti-BAK antibodies, implicating BAK. Immunoblotting showed the presence of BAK, but not BAX, in brain mitochondria. tBID did not release Cyt c from rat liver mitochondria, which lacked both BAX and BAK. This indicated that tBID did not act independently of BAX and BAK. tBID plus monomeric BAX produced twice as much Cyt c release as did tBID or oligomeric BAX alone. Neither tBID alone nor in combination with BAX induced mitochondrial swelling. In both cases Cyt c release was insensitive to cyclosporin A plus ADP, inhibitors of the mitochondrial permeability transition (mPT). Recombinant Bcl-xL inhibited Cyt c release induced by tBID alone or in combination with monomeric BAX. Koenig's polyanion, an inhibitor of VDAC, suppressed tBID-induced Cyt c release from brain mitochondria mediated by BAK but not by BAX. Thus, tBID can induce mPT-independent Cyt c release from brain mitochondria by interacting with exogenous BAX and/or with endogenous BAK that may involve VDAC. In contrast, neither adenylate kinase nor Smac/DIABLO was released from isolated rat brain mitochondria via BAK or BAX.     

Activation of calcium-independent phospholipase A (iPLA) in brain mitochondria and release of apoptogenic factors by BAX and truncated BID

Cleaved or truncated BID (tBID) is known to oligomerize both BAK and BAX. Previously, BAK and BAX lacing the C-terminal fragment (BAXDeltaC) were shown to induce modest cytochrome c (Cyt c) release from rat brain mitochondria when activated by tBID. We now show that tBID plus monomeric full-length BAX induce extensive release of Cyt c, Smac/DIABLO, and Omi/HtrA2 (but not endonuclease G and the apoptosis inducing factor) comparable to the release induced by alamethicin. This occurs independently of the permeability transition without overt changes in mitochondrial morphology. The mechanism of the release may involve formation of reactive oxygen species (ROS) and activation of calcium-independent phospholipase A(2) (iPLA(2)). Indeed, increased ROS production and activated iPLA(2) were observed prior to massive Cyt c release. Furthermore, the extent of inhibition of Cyt c release correlated with the degree of suppression of iPLA(2) by the inhibitors propranolol, dibucaine, 4-bromophenacyl bromide, and bromenol lactone. Consistent with a requirement for iPLA(2) in Cyt c release from brain mitochondria, synthetic liposomes composed of lipids mimicking the outer mitochondrial membrane (OMM) but lacing iPLA(2) failed to release 10 kDa fluorescent dextran (FD-10) in response to tBID plus BAX. We propose that tBID plus BAX activate ROS generation, which subsequently augments iPLA(2) activity leading to changes in the OMM that allow translocation of certain mitochondrial proteins from the intermembrane space.     

Mitochondria as the target of the pro-apoptotic protein Bax

During apoptosis, engagement of the mitochondrial pathway involves the permeabilization of the outer mitochondrial membrane (OMM), which leads to the release of cytochrome c and other apoptogenic proteins such as Smac/DIABLO, AIF, EndoG, Omi/HtraA2 and DDP/TIMM8a. OMM permeabilization depends on activation, translocation and oligomerization of multidomain Bcl-2 family proteins such as Bax or Bak. Factors involved in Bax conformational change and the function(s) of the distinct domains controlling the addressing and the insertion of Bax into mitochondria are described in this review. We also discuss our current knowledge on Bax oligomerization and on the molecular mechanisms underlying the different models accounting for OMM permeabilization during apoptosis.     

tBID Homooligomerizes in the mitochondrial membrane to induce apoptosis.

Activation of the tumor necrosis factor R1/Fas receptor results in the cleavage of cytosolic BID to truncated tBID. tBID translocates to the mitochondria to induce the oligomerization of BAX or BAK, resulting in the release of cytochrome c (Cyt c). Here we demonstrate that in tumor necrosis factor alpha-activated FL5.12 cells, tBID becomes part of a 45-kDa cross-linkable mitochondrial complex that does not include BAX or BAK. Using fluorescence resonance energy transfer analysis and co-immunoprecipitation, we demonstrate that tBID-tBID interactions occur in the mitochondria of living cells. Cross-linking experiments using a tBID-GST chimera indicated that tBID forms homotrimers in the mitochondrial membrane. To test the functional consequence of tBID oligomerization, we expressed a chimeric FKBP-tBID molecule. Enforced dimerization of FKBP-tBID by the bivalent ligand FK1012 resulted in Cyt c release, caspase activation, and apoptosis. Surprisingly, enforced dimerization of tBID did not result in the dimerization of either BAX or BAK. Moreover, a tBID BH3 mutant (G94E), which does not interact with or induce the dimerization of either BAX or BAK, formed the 45-kDa complex and induced both Cyt c release and apoptosis. Thus, tBID oligomerization may represent an alternative mechanism for inducing mitochondrial dysfunction and apoptosis.     

Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site

The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation.     

BID-dependent and BID-independent pathways for BAX insertion into mitochondria

In the absence of an apoptotic signal, BAX adopts a conformation that constrains the protein from integrating into mitochondrial membranes. Here, we show that caspases, including caspase-8, can initiate BAX insertion into mitochondria in vivo and in vitro. The cleavage product of caspase-8, tBID, induced insertion of BAX into mitochondria in vivo, and reconstitution in vitro showed that tBID, either directly or indirectly, relieved inhibition of the BAX transmembrane signal-anchor by the NH2-terminal domain, resulting in integration of BAX into mitochondrial membrane. In contrast to these findings, however, Bid-null mouse embryo fibroblasts supported Bax insertion into mitochondria in response to death signaling by either TNFalpha or E1A, despite the fact that cytochrome c release from the organelle was inhibited. We conclude, therefore, that a parallel Bid-independent pathway exists in these cells for mitochondrial insertion of Bax and that, in the absence of Bid, cytochrome c release can be uncoupled from Bax membrane insertion.     

Membranotropic effects of ω-hydroxypalmitic acid and Ca<Superscript>2+</Superscript> on rat liver mitochondria and lecithin liposomes. Aggregation and membrane permeabilization

The paper examines membranotropic Ca²⁺-dependent effects of ω-hydroxypalmitic acid (HPA), a product of ω-oxidation of fatty acids, on the isolated rat liver mitochondria and artificial membrane systems (liposomes). It was established that in the presence of Ca²⁺, HPA induced aggregation of liver mitochondria, which was accompanied by the release of cytochrome c from the organelles. It was further demonstrated that the addition of Ca²⁺ to HPA-containing liposomes induced their aggregation and/or fusion. Ca²⁺ also caused the release of the fluorescent dye sulforhodamine B from liposomes, indicating their permeabilization. HPA was shown to induce a high-amplitude swelling of Ca²⁺-loaded mitochondria, to decrease their membrane potential, to induce the release of Ca²⁺ from the organelles and to result in the oxidation of the mitochondrial NAD(P)H pool. Those effects of HPA were not blocked by the MPT pore inhibitor CsA, but were suppressed by the mitochondrial calcium uniporter inhibitor ruthenium red. The effects of HPA were also observed when Ca²⁺ was replaced with Sr²⁺ (but not with Ba²⁺ or Mg²⁺). A supposition is made that HPA can induce a Ca²⁺-dependent aggregation of mitochondria, as well as Ca²⁺dependent CsA-insensitive permeabilization of the inner mitochondrial membrane – with the subsequent lysis of the organelles.     

Pro-apoptotic complexes of BAX and BAK on the outer mitochondrial membrane

In multicellular organisms the regulated cell death apoptosis is critically important for both ontogeny and homeostasis. Mitochondria are indispensable for stress-induced apoptosis. The BCL-2 protein family controls mitochondrial apoptosis and initiates cell death through the pro-apoptotic activities of BAX and BAK at the outer mitochondrial membrane (OMM). Cellular survival is ensured by the retrotranslocation of mitochondrial BAX and BAK into the cytosol by anti-apoptotic BCL-2 proteins. BAX/BAK-dependent OMM permeabilization releases the mitochondrial cytochrome c (cyt c), which initiates activation of caspase-9. The caspase cascade leads to cell shrinkage, plasma membrane blebbing, chromatin condensation, and apoptotic body formation. Although it is clear that ultimately complexes of active BAX and BAK commit the cell to apoptosis, the nature of these complexes is still enigmatic. Excessive research has described a range of complexes, varying from a few molecules to several 10,000, in different systems. BAX/BAK complexes potentially form ring-like structures that could expose the inner mitochondrial membrane. It has been suggested that these pores allow the efflux of small proteins and even mitochondrial DNA. Here we summarize the current state of knowledge for mitochondrial BAX/BAK complexes and the interactions between these proteins and the membrane.     

Induction of Ca2+-dependent cyclosporin a-insensitive nonspecific permeability of the inner membrane of liver mitochondria and cytochrome c release by α,ω-hexadecanedioic acid in media of varying ionic strength

In liver mitochondria loaded with Ca²⁺ or Sr²⁺, α,ω-hexadecanedioic acid (HDA) can induce nonspecific permeability of the inner membrane (mitochondrial pore) by the mechanism insensitive to cyclosporin A (CsA). In this work we studied the effect of ionic strength of the incubation medium on the kinetics of the processes that accompany Ca²⁺-dependent induction of the mitochondrial pore by fatty acid: organelle swelling, Ca²⁺ release from the matrix, changes in transmembrane potential (Δψ) and rate of oxygen consumption, and the release of cytochrome c from the intermembrane space. Two basic incubation media were used: sucrose medium and isotonic ionic medium containing KCl without sucrose. We found that 200 μM Ca²⁺ and 20 μM HDA in the presence of CsA effectively induce high-amplitude swelling of mitochondria both in the case of sucrose and in the ionic incubation medium. In the presence of CsA, mitochondria can rapidly absorb Ca²⁺ and retain it in the matrix for a while without reducing Δψ. Upon incubation in the ionic medium, mitochondria retain most of the added Ca²⁺ in the matrix for a short time without reducing the Δψ. In both cases the addition of HDA to the mitochondria 2 min after the introduction of Ca²⁺ leads to the rapid release of these ions from the matrix and total drop in Δψ. The mitochondrial swelling induced by Ca²⁺ and HDA in non-ionic medium is accompanied by almost maximal stimulation of respiration. Under the same conditions, but during incubation of mitochondria in the ionic medium, it is necessary to add cytochrome c for significant stimulation of respiration. The mitochondrial swelling induced by Ca²⁺ and HDA leads to the release of cytochrome c in a larger amount in the case of ionic medium than for the sucrose medium. We conclude that high ionic strength of the incubation medium determines the massive release of cytochrome c from mitochondria and liberates it from the respiratory chain, which leads to blockade of electron transport along the respiratory chain and consequently to disruption of the energy functions of the organelles.     

Lipidic pore formation by the concerted action of proapoptotic BAX and tBID

BCL-2 homology 3 (BH3)-only proteins of the BCL-2 family such as tBID and BIM(EL) assist BAX-type proteins to breach the permeability barrier of the outer mitochondrial membrane, thereby allowing cytoplasmic release of cytochrome c and other active inducers of cell death normally confined to the mitochondrial inter-membrane space. However, the exact mechanism by which tBID and BIM(EL) aid BAX and its close homologues in this mitochondrial protein release remains enigmatic. Here, using pure lipid vesicles, we provide evidence that tBID acts in concert with BAX to 1) form large membrane openings through both BH3-dependent and BH3-independent mechanisms, 2) cause lipid transbilayer movement concomitant with membrane permeabilization, and 3) disrupt the lipid bilayer structure of the membrane by promoting positive monolayer curvature stress. None of these effects were observed with BAX when BIM(EL) was substituted for tBID. Based on these data, we propose a novel model in which tBID assists BAX not only via protein-protein but also via protein-lipid interactions to form lipidic pore-type non-bilayer structures in the outer mitochondrial membrane through which intermembrane prodeath molecules exit mitochondria during apoptosis.     

Mitochondrial outer membrane permeabilization during apoptosis: The role of mitochondrial fission

Mitochondria continually fuse and divide to yield a dynamic interconnected network throughout the cell. During apoptosis, concomitantly with permeabilization of the mitochondrial outer membrane (MOMP) and cytochrome c release, mitochondria undergo massive fission. This results in the formation of small, round organelles that tend to aggregate around the nucleus. Under some circumstances, preceding their fission, mitochondria tend to elongate and to hyperfuse, a process that is interpreted as a cell defense mechanism. Since many years, there is a controversy surrounding the physiological relevance of mitochondrial fragmentation in apoptosis. In this review, we present recent advances in this field, describe the mechanisms that underlie this process, and discuss how they could cooperate with Bax to trigger MOMP and cytochrome c release. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Melatonin inhibits cardiolipin peroxidation in mitochondria and prevents the mitochondrial permeability transition and cytochrome c release

Cardiolipin oxidation is emerging as an important factor in mitochondrial dysfunction as well as in the initial phase of the apoptotic process. We have previously shown that exogenously added peroxidized cardiolipin sensitizes mitochondria to Ca²⁺-induced mitochondrial permeability transition (MPT) pore opening and promotes the release of cytochrome c. In this work, the effects of intramitochondrial cardiolipin peroxidation on Ca²⁺-induced MPT and on the cytochrome c release from mitochondria were studied. The effects of melatonin, a compound known to protect the mitochondria from oxidative damage, on both of these processes were also tested. tert-Butylhydroperoxide (t-BuOOH), a lipid-soluble peroxide that promotes lipid peroxidation, was used to induce intramitochondrial cardiolipin peroxidation. Exposure of heart mitochondria to t-BuOOH resulted in the oxidation of cardiolipin, associated with an increased sensitivity of mitochondria to Ca²⁺-induced MPT and with the release of cytochrome c from the mitochondria. All these processes were inhibited by micromolar concentrations of melatonin. It is proposed that melatonin inhibits cardiolipin peroxidation in mitochondria, and this effect seems to be responsible for the protection afforded by this agent against the MPT induction and cytochrome c release. Thus, manipulating the oxidation sensitivity of cardiolipin with melatonin may help to control MPT and cytochrome c release, events associated with cell death, and thus, be used for treatment of those disorders characterized by mitochondrial cardiolipin oxidation and Ca²⁺ overload.     

A novel paradigm for rapid ABT-737-induced apoptosis involving outer mitochondrial membrane rupture in primary leukemia and lymphoma cells

Primary chronic lymphocytic leukemia (CLL) cells are exquisitely sensitive to ABT-737, a small molecule BCL2-antagonist, which induces many of the classical biochemical and ultrastructural features of apoptosis, including BAX/BAK oligomerization, cytochrome c release, caspase activation and chromatin condensation. Surprisingly, ABT-737 also induces mitochondrial inner membrane permeabilization (MIMP) resulting in mitochondrial matrix swelling and rupture of the outer mitochondrial membrane (OMM), so permitting the rapid efflux of cytochrome c from mitochondrial cristae and facilitating rapid caspase activation and apoptosis. BAX and BAK appear to be involved in the OMM discontinuities as they localize to the OMM break points. Notably, ABT-737 induced mitochondrial matrix swelling and OMM discontinuities in other primary B-cell malignancies, including mantle cell, follicular and marginal zone lymphoma cells but not in several cell lines studied. Thus, we describe a new paradigm of apoptosis in primary B-cell malignancies, whereby targeting of BCL2 results in all the classical features of apoptosis together with OMM rupture independent of caspase activation. This mechanism may be far more prevalent than hitherto recognized due to the failure of most methods, used to measure apoptosis, to recognize such a mechanism.