Mitochondrial regulation of apoptotic cell death

Chemico-Biological Interactions 163 (2006) 4–14

Mitochondrial regulation of apoptotic cell death Vladimir Gogvadze, Sten Orrenius ∗ Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden Available online 30 April 2006

Abstract Mitochondria play a decisive role in the regulation of both apoptotic and necrotic cell death. Permeabilization of the outer mitochondrial membrane and subsequent release of intermembrane space proteins are important features of both models of cell death. The mechanisms by which these proteins are released depend presumably on cell type and the nature of stimuli. Of the mechanisms involved, mitochondrial permeability transition appears to be associated mainly with necrosis, whereas the release of caspase activating proteins during early apoptosis is regulated primarily by the Bcl-2 family of proteins. However, there is increasing evidence for interaction and co-operation between these two mechanisms. The multiple mechanisms of mitochondrial permeabilization may explain diversities in the response of mitochondria to numerous apoptotic stimuli in different types of cells. © 2006 Published by Elsevier Ireland Ltd. Keywords: Mitochondria; Apoptosis; Cytochrome c; Cardiolipin

1. Introduction Apoptosis and necrosis are two modes of cell death with distinct morphological and biochemical features. Apoptosis is an active process characterized by cell shrinkage, nuclear and cytoplasmic condensation, chromatin fragmentation and phagocytosis of dying cells. In contrast, necrosis is a passive form of cell death associated with inflammation and certain forms of cell injury. One of the characteristic features of necrosis is cellular and organelle swelling, rupture of the plasma membrane and spilling of cellular contents into the extracellular milieu. Different toxicants may trigger either apoptotic or necrotic cell death, depending on the cell type and severity of insult. Further, completion of the apoptotic death program requires maintenance of a sufficient intracellular energy level and of a redox

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Corresponding author at: Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden. Tel.: +46 8 33 58 74; fax: +46 8 32 90 41. E-mail address: [email protected] (S. Orrenius). 0009-2797/$ – see front matter © 2006 Published by Elsevier Ireland Ltd. doi:10.1016/j.cbi.2006.04.010

state compatible with caspase activation. Thus, ATP depletion or severe oxidative stress may re-direct otherwise apoptotic cell death to necrosis. Mitochondria play a key role in the regulation of apoptotic cell death [1]. Specifically, different pro-apoptotic proteins, such as Cytochrome c and Smac/Diablo, which are normally present in the intermembrane space of these organelles are released during the early stages of apoptosis [2,3]. Once in the cytosol, Cytochrome c participates in the formation of the apoptosome complex together with its adaptor molecule, Apaf-1, resulting in the recruitment, processing and activation of procaspase-9 in the presence of dATP or ATP [4]. Subsequently, caspase-9 cleaves and activates pro-caspase-3 and -7; these effector caspases are responsible for the cleavage of various proteins leading to biochemical and morphological features characteristic of apoptosis [5]. The release of Cytochrome c is therefore considered a key initiative step in the apoptotic process, although the precise mechanisms regulating this event remain unclear. Several mechanisms have been proposed to explain the mitochondrial outer membrane permeabilization. The first pathway, which may be engaged during both

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necrotic and apoptotic cell death, involves the induction of mitochondrial permeability transition (MPT), which for a long time was regarded as the prime mechanism responsible for the permeabilization of the mitochondrial outer membrane. 1.1. Induction of mitochondrial permeability transition This phenomenon was described some 30 years ago by Haworth and Hunter in a series of seminal papers [6–8], in which they showed that Ca2+ uptake stimulates drastic changes in mitochondrial morphology and functional activity due to the opening of a non-specific pore in the mitochondrial inner membrane, commonly known as the MPT pore. The existence of a non-specific channel in the inner mitochondrial membrane was confirmed by electrophysiological experiments. A multiple conductance channel (MCC) has been identified by Kinnally et al. [9]. Zoratti and co-workers found a similar high-conductance channel in the membrane of rat liver mitoplasts (mitochondrial megachannel, MMC) [10]. Different characteristics of MMC, such as size, voltage dependence, activation by Ca2+ and inhibition by cyclosporin A (CSA), Mg2+ , Mn2+ , Sr2+ , H+ , and ADP, resembled those of the MPT pore [11]. Both channels, MCC and MMC, revealed similar conductance ranges and multiple substrates, including the “half-conductance” one. Interestingly, overexpression of Bcl-2 suppressed activation of MCC by calcium, indicating a possible involvement of this channel in apoptosis [12]. Measurements of single-channel current of excised patches with reconstituted purified mitochondrial adenine nucleotide translocase revealed the presence of a large cation-selective channel [13]. The properties of the channel were similar to the MPT pore, and resembled both MCC [9] and MMC channels [10]. The authors concluded that the adenine nucleotide translocase, when converted into a large unselective channel, is a key component of the MPT pore. The channel opening was proposed to be caused by binding of Ca2+ to cardiolipin, which is tightly bound to the adenine nucleotide translocase, thus releasing positive charges within the adenine nucleotide translocase to open the gate. MPT results in osmotic swelling of the mitochondrial matrix, mitochondrial uncoupling, rupture of the mitochondrial outer membrane, and the release of intermembrane space proteins, including Cytochrome c, into the cytosol [14,15]. However, recent observations have questioned the importance of MPT as prime mechanism for the release of Cytochrome c from the mito-

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chondria under apoptotic conditions. Thus, overexpression of cyclophilin-D, a component of the MPT pore complex, had opposite effects on apoptosis and necrosis; while NO-induced necrosis was promoted, NOand staurosporine-induced apoptosis was inhibited [16]. These findings suggest that MPT leads to cell necrosis, but argue against its involvement in apoptosis. Furthermore, recent genetic studies demonstrated that cyclophilin-D-deficient cells died normally in response to various apoptotic stimuli, but were resistant to necrotic cell death induced by reactive oxygen species and Ca2+ overload [17,18]. In addition, cyclophilin-D-deficient mice showed resistance to ischemia/reperfusion-induced cardiac injury. These results also support the assumption that the cyclophilin-D-dependent MPT mediates some forms of necrotic cell death. Hence, this model of mitochondrial outer membrane permeabilization may be most relevant during ischemia/reperfusion injury, or in response to cytotoxic stimuli resulting in localized mitochondrial Ca2+ overload (for recent review, see [19]). On the other hand, transient pore opening in a sub-fraction of mitochondria could result in the release mitochondrial proteins without observable large-amplitude swelling, or drop in membrane potential, of the entire organelle population [20]. This could occur in close proximity to calcium “hot spots” — microdomains, in which the local concentration of ionized calcium far exceeds the average cytosolic concentration [21]. This local Ca2+ elevation might be high enough to induce Ca2+ overload and subsequent pore opening in a sub-fraction of mitochondria. The frequency of such spontaneous pore opening and closure might increase under the influence of apoptotic stimuli, contributing to translocation of intermembrane space proteins into the cytosol. 1.2. Bcl-2 family proteins and mitochondrial outer membrane permeabilization The most important mechanism of outer membrane permeabilization under apoptotic conditions involves members of the Bcl-2 family of proteins. The Bcl-2 family consists of more than 30 proteins, which can be divided into three subgroups: Bcl-2-like survival factors, Bax-like death factors, and BH3-only death factors. Residues from BH1–3 form a hydrophobic groove, with which BH3-only death factors interact through their BH3 domain, whereas the N-terminal BH4 domain stabilizes this pocket (for recent review, see [22]). Early indications of the importance of these proteins for the release of Cytochrome c were obtained in 1997, when two groups independently showed that overexpres-

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sion of Bcl-2 prevented the efflux of Cytochrome c from the mitochondria as well as the initiation of apoptosis [23,24]. The same year, the ability of a pro-apoptotic protein, Bax, to stimulate Cytochrome c release was demonstrated in yeast overexpressing Bax [25]. Other components of the mitochondrial inner membrane (the bc1 complex and ATPase) were unaffected. Co-expression of Bcl-XL almost fully prevented the effect of Bax. The importance of Bax interaction with mitochondria for the apoptotic process was shown by Jurgensmeier et al. [26]. Addition of submicromolar amounts of recombinant Bax protein to isolated mitochondria induced Cytochrome c release, whereas a peptide representing the Bax BH3 domain was inactive. When added to purified cytosol, neither mitochondria nor Bax alone induced proteolytic processing and activation of caspases. In contrast, addition of a combination of Bax and mitochondria triggered release of Cytochrome c from the mitochondria and induced caspase activation in the cytosol. Supernatants from Bax-treated mitochondria also triggered caspase processing and activation, while recombinant Bcl-XL protein abrogated Bax-induced release of Cytochrome c from isolated mitochondria and prevented caspase activation. Several assumptions were made to explain the ability of Bax to release Cytochrome c from mitochondria. In experiments with lipid membranes it was shown that Bax forms pores in the lipid bilayer and triggers the release of liposome-encapsulated carboxyfluorescein that was blocked by Bcl-2 [27]. Further, using isolated mitochondria Narita et al. [28] reported that pro-apoptotic Bcl-2 family proteins, Bax or Bak, can release Cytochrome c by interacting with MPT pore components, in particular the voltage-dependent anion channel (VDAC). In addition to Cytochrome c release, these proteins caused mitochondrial alterations typical of MPT, such as loss of ψ and swelling of the organelles. All of these changes were Ca2+ -dependent and were prevented by MPT inhibitors — cyclosporin A (CsA) and bongkrekic acid [28]. Furthermore, antibodies that inhibited VDAC activity prevented Bax-induced Cytochrome c release and loss of ψ [29]. Contrary to this data, it was demonstrated that Cytochrome c release was facilitated by Mg2+ , an inhibitor of pore opening [30]. These results strongly suggest the existence of two distinct mechanisms leading to Cytochrome c release, one of which is stimulated by calcium and inhibited by CsA, while the other is Baxdependent and Mg2+ -sensitive but CsA-insensitive. Hence, the involvement of VDAC in Bax-induced, CsA-insensitive Cytochrome c release was suggested by the experiments with artificial lipid membranes. VDAC is responsible for most of the metabolite flux across the

mitochondrial outer membrane [31]. However, even in the open state it is not large enough (3 nm) to allow penetration of Cytochrome c (14 kDa). In spite of this, it was suggested that Bax or Bak stimulate the opening of VDAC incorporated into liposomes and allow encapsulated Cytochrome c to pass, and the passage can be prevented by Bcl-XL [32]. In contrast to this report, Rostovtseva et al. [33] found no electrophysiologically detectable interaction between VDAC channels isolated from mammalian mitochondria and either monomeric or oligomeric forms of Bax. In contrast, another pro-apoptotic protein, tBid, proteolytically cleaved by caspase-8, affected the voltage gating of VDAC by inducing channel closure [33]. Therefore, although it appears that VDAC might play an important role in the permeabilization of the outer membrane, the precise mechanism(s) of its engagement in the cell death process is still unclear. Permeabilization of the outer mitochondrial membrane was shown to be a property of the oligomeric form of Bax, whereas monomeric Bax was ineffective [34]. Oligomerization of Bax is a result of binding to the truncated form of the BH3 domain-only pro-apoptotic protein Bid [35]. Hence, it was shown that tBid triggers the homo-oligomerization of Bax (or Bak) [36], resulting in the release of Cytochrome c from mitochondria. Cells lacking both Bax and Bak, but not cells lacking only one of these proteins, have been found to be resistant to tBid-induced Cytochrome c release and apoptosis [37]. Moreover, Bax- and Bak-deficient cells were also resistant to a variety of apoptotic stimuli that act through the mitochondrial pathway, such as staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimulus thapsigargin. Thus, activation of a “multidomain” pro-apoptotic Bcl-2 family member Bax or Bak, appears to be the predominant gateway to the mitochondrial release of proteins required for cell death in response to diverse stimuli. A different mechanism of tBid-induced release of Cytochrome c was proposed by Scorrano et al. [38], who found that tBid added to mouse liver mitochondria stimulated Cytochrome c release via mechanisms that did not require the BH3 domain of tBid, or the presence of Bak (Bax), but were sensitive to CsA. According to the authors, tBid induces structural re-arrangement of mitochondria; individual cristae become fused, and the junctions between the cristae and the intermembrane space open. The ability of CsA to block tBid-induced release of Cytochrome c suggested that the MPT pore complex was involved in this remodeling process. However, the physiological importance of this pathway is doubtful, since the amount of tBid required for the Cytochrome c

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release via remodeling of cristae was almost two orders of magnitude higher than that required for activation of the Bax/Bak-mediated pathway [39]. The permeability of the outer mitochondrial membrane is regulated by the concerted operation of pro- and anti-apoptotic proteins. Anti-apoptotic proteins, such as Bcl-2, Bcl-XL , Mcl-1, and Bcl-w, interact with the proapoptotic proteins, Bax and Bak, thereby preventing their oligomerization. For instance, Mcl-1 suppresses the pore forming activity of Bak by the formation of complexes with this pro-apoptotic protein [40]. This type of regulation is quite specific, for example, Bak can be sequestered by both Mcl-1 and Bcl-XL but not by Bcl-2 or Bclw [41]. Suppression of Bax pro-apoptotic activity was also shown to occur via formation of complexes with the DNA repair factor Ku70. After induction of apoptosis Ku70 becomes acetylated at particular lysine residues, causing dissociation of Bax [42]. Disturbance of the balance between anti- and proapoptotic Bcl-2 family members in favor of the latter can proceed by mechanisms involving BH3-only proteins that bind to and occupy the anti-apoptotic proteins, thereby liberating Bax and Bak. For example, the BH3only proteins PUMA and NOXA, which are expressed in a p53-dependent manner upon DNA damage, were shown to cause outer membrane permeabilization [43]. Co-immunoprecipitation studies showed that NOXA binds to Bcl-2 and Bcl-XL , depending on a functional BH3 motif of NOXA, but not to Bax [44]. Interestingly, cytosolic p53 can not only induce PUMA and NOXA, but can also directly activate Bax and thereby cause permeabilization of the mitochondrial outer membrane [45], although the precise mechanism of this activation is still obscure. Different mechanisms of Cytochrome c release can co-exist within one model of cell death. Thus, arsenicinduced Cytochrome c release triggered by low (up to 20 ␮M) doses of As2 O3 was found to be Bax/Bakdependent and, hence, blocked in Bax/Bak double knockout mouse embryonic fibroblasts [46]. However, at higher arsenic concentrations (above 200 ␮M) Cytochrome c release was caused by a direct effect of the toxicant on mitochondria resulting in MPT induction, which occurred to a similar extent in both wild type cells and cells lacking Bax and Bak [46,47]. Bcl-2 family proteins do not only regulate the permeabilization of the mitochondrial outer membrane via formation of pores, but can also modulate MPT induction. Thus, the presence of a higher level of Bcl-2 protein in mitochondria of Zajdela hepatoma cells was reported to cause a delay in MPT induction as compared to liver mitochondria [48]. Conversely, it has been demonstrated

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that cell death resulting from Bax overexpression can occur via induction of MPT, since it was prevented by inhibition of the MPT with CsA in combination with the phospholipase A2 inhibitor aristolochic acid [49]. In another study, inclusion of recombinant oligomeric Bax in the incubation buffer markedly stimulated Ca2+ mediated MPT induction [50]. In contrast to MPT-induced Cytochrome c release, the release mediated by Bcl-2 family proteins occurs without apparent alterations of ultrastructure and main mitochondrial functions, even when the loss of Cytochrome c was almost complete [51]. Apparently, under these circumstances mitochondrial ψ might be maintained via hydrolysis of glycolytically generated ATP, since the severe loss of Cytochrome c should have blocked mitochondrial respiration and ATP formation by oxidative phosphorylation. 2. The two-step process of Cytochrome c release Cytochrome c is normally bound to the inner mitochondrial membrane by an association with the anionic phospholipid cardiolipin, where it can reversibly interact with complexes III and IV of the respiratory chain. Cardiolipin is unique to mitochondria and is present predominantly, if not exclusively, in the inner mitochondrial membrane [52,53]. Due to its intracellular distribution, cardiolipin has been postulated, and later demonstrated, to be an essential component in many mitochondrial processes such as electron transport, ADP/ATP translocation, ion permeability, membrane integrity, and protein function and transport [53–55]. The molecular interaction between cardiolipin and Cytochrome c has been extensively studied using multiple biochemical and analytical approaches. It is well established that Cytochrome c specifically and stoichiometrically binds to cardiolipin [55], which anchors the hemoprotein to the inner mitochondrial membrane. Binding to cardiolipin involves at least two conformations: (1) a loosely bound conformation provided by electrostatic interaction with positively charged lysine residues of Cytochrome c and negatively charged phosphate groups of cardiolipin [56] and (2) a tightly bound conformation wherein hydrophobic interactions accompany a loosening of the tertiary structure, resulting in partial embedding of the protein into the membrane [57,58]. An alternate model for the tightly bound conformation has been proposed, such that the hydrophobic interaction between an expanded acyl chain of cardiolipin and a hydrophobic inlet of Cytochrome c anchors the protein to the membrane [59]. In either case, because of its association with cardiolipin, it seems that permeabilization of the outer membrane, alone, would

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be insufficient to stimulate the release of Cytochrome c. In other words, a disruption of the Cytochrome ccardiolipin interaction would seem to have to occur before, or concomitantly with, permeabilization of the outer membrane in order for Cytochrome c to be released from mitochondria. Evidence suggests that dissociation of Cytochrome c from cardiolipin might be a critical first step for Cytochrome c release into the cytosol and the induction of apoptosis [60–64]. In particular, it was demonstrated that exposing submitochondrial particles to reactive oxygen species (ROS) produced by the mitochondrial electron transport chain stimulates a mobilization of Cytochrome c and a concomitant loss of cardiolipin [60]. Similarly, other studies showed that lowering mitochondrial cardiolipin content correlates not only with a decrease in respiration [61], but also with a stoichiometric increase in Cytochrome c release [62]. Combined, these findings indicate that cardiolipin plays an important role not only in the function of the respiratory chain, but also in the retention of Cytochrome c within the intermembrane space. Data from our laboratory and others demonstrated that changes in the levels [62,63], or chemical structure through oxidation of cardiolipin [60,64–67], result in the formation of a soluble pool of Cytochrome c that can be released into the cytosol upon permeabilization of the outer mitochondrial membrane. Based on these results we hypothesized that Cytochrome c release occurs by a two-step process, involving the initial detachment of this protein from the inner membrane followed by permeabilization of the outer membrane and the release of the hemoprotein c into the extramitochondrial milieu [64]. In vitro, in experiments with isolated mitochondria, depending on the detachment stimulus, two distinct pools of Cytochrome c can be mobilized. The first pool is sensitive to electrostatic alterations that can be elicited by changes in ionic strength, surface-charge density, or pH [59,68] and thus most likely reflects Cytochrome c present in the loosely bound conformation. The second pool can be mobilized by oxidative modification of mitochondrial lipids, specifically cardiolipin, and therefore, likely represents tightly bound Cytochrome c that is detached because of disturbances in membrane structure. The two-step concept of Cytochrome c release from mitochondria has been confirmed in several subsequent studies. For example, recent observations demonstrated that in the absence of complex I inhibitors, recombinant oligomeric Bax protein elicited only a minimal Cytochrome c release (∼18%) from brain mitochondria. However, when the mitochondria were incubated with both recombinant Bax and complex I inhibitors, (which were shown to stimulate ROS production and,

hence, cardiolipin oxidation) up to 65% of the mitochondrial Cytochrome c was released. Thus, according to the two-step concept, neither ROS production via complex I inhibition, nor permeabilization of the outer membrane with Bax, alone, triggered overt release of Cytochrome c, whereas their combination resulted in a marked release (>60%) of this pro-apoptotic molecule [69]. In another study, deficits of complex I stimulated intramitochondrial oxidative stress, which, in turn, increased the releasable soluble pool of Cytochrome c within the mitochondrial intermembrane space [70]. Upon mitochondrial permeabilization by Bax, more Cytochrome c was released into the cytosol from brain mitochondria with impaired complex I activity. Based on these results, the authors proposed a model in which defects of complex I lower the threshold for activation of mitochondria-dependent apoptosis by Bax, thereby rendering compromised neurons more prone to degenerate. 3. Possible mechanisms of cardiolipin oxidation Mitochondria are known as one of the most powerful sources of ROS. The respiratory chain of the mitochondria is the major site of superoxide formation. The extent of ROS production depends on many factors, such as respiratory substrates, uncoupling proteins, the presence or absence of Ca2+ , etc. Further, production of ROS was stimulated in the presence of Bax plus a BH3 domain peptide [71], apparently due to increased auto-oxidation following Cytochrome c release [72]. Increased ROS production may further contribute to Cytochrome c release by activating lipid peroxidation [67] and facilitating Cytochrome c dissociation from cardiolipin [73]. Another mechanism potentially involved in the disruption of the outer mitochondrial membrane is activation of mitochondrial phospholipases, either calciumdependent or -independent. Mitochondria from rat liver and rabbit heart have been shown to contain a Ca2+ independent phospholipase A2 (iPLA2) [74]. iPLA2 activity was found to be localized to the contact sites between the outer and the inner membranes in liver mitochondria [75]. iPLA2 can be activated via uncoupling mitochondria through the action of CCCP, opening of the permeability transition pore, or in the presence of alamethicin [74,76]. Analysis of iPLA2-mediated mitochondrial deterioration and Cytochrome c release was performed by Pfeiffer et al. [77]. They demonstrated that accumulation of free fatty acids after activation of iPLA2 does not require permeability transition, because it can occur in the absence of apparent swelling and in

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the presence of the inhibitor of permeability transition CsA. Furthermore, it is unrelated to the Ca2+ ion per se, because Sr2+ accumulation was similarly effective, both in terms of depressing membrane potential and promoting the accumulation of FFA. The authors conclude that, in this model, the release of Cytochrome c occurs via induction of permeability transition, which might happen after several hours, even in the presence of CsA and EGTA. Although CsA is very potent as an inhibitor of pore opening, its action still is transient considering the time scale of apoptosis [78]. Furthermore, it was demonstrated that tBid plus BAX activate ROS generation, which subsequently augments iPLA2 activity leading to changes in the outer mitochondrial membrane that allow translocation of certain mitochondrial proteins from the intermembrane space into the cytosol [79]. An inhibitor of iPLA2, propranolol, inhibited all three processes: activation of ROS generation, iPLA2 activity, and permeabilization of the outer mitochondrial membrane. Earlier, propranolol was shown to inhibit the release of entrapped 10 kDa dextran from protein-free liposomes treated with Bax and tBid. Since membrane insertion of Bax was not inhibited by propranolol, it was concluded that dibucaine and propranolol inhibit Bax-induced permeability changes through a direct interaction with the lipid membrane [80]. Selective peroxidation of the unique mitochondrial phospholipid cardiolipin was recently shown to precede Cytochrome c release by Kagan et al. [81]. The authors demonstrated that a pool of cardiolipin-bound mitochondrial Cytochrome c can catalyze cardiolipin peroxidation, which facilitates the detachment of Cytochrome c from the outer surface of the inner mitochondrial membrane and its subsequent release into the cytoplasm through pores in the outer membrane. The peroxidase function of the cardiolipin–Cytochrome c complex is compatible with the proposed two-step mechanism of Cytochrome c release and provides a plausible explanation for the protective effects against apoptosis reported for multiple mitochondrial antioxidant enzymes. One possible source of ROS production in mitochondria is the p66Shs protein, a redox enzyme that utilizes reducing equivalents of the mitochondrial electron transfer chain through the oxidation of Cytochrome c [82]. Redox-defective mutants of p66Shc are unable to induce mitochondrial ROS generation and swelling in vitro and are resistant to mitochondrially mediated apoptosis in vivo. Interestingly, a fraction of this cytosolic enzyme localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Upon stimulation of apop-

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tosis, dissociation of this complex occurs, followed by the release of monomeric p66Src and its interaction with Cytochrome c to generate hydrogen peroxide. An additional aspect of the role of cardiolipin in Cytochrome c release from mitochondria, that has surfaced recently, is the hypothesis that cardiolipin is required for permeabilization of the outer mitochondrial membrane. Although, cardiolipin is present almost exclusively in the inner mitochondrial membrane, it also can be found in the outer membrane; however, its content in the outer membrane depends on the source of mitochondria. In contrast to outer membrane vesicles from rat liver mitochondria which are virtually free of cardiolipin, outer membrane vesicles from yeast mitochondria, as well as those from Neurospora crassa, contain more cardiolipin than can be accounted for by contamination with inner membrane fragments [83]. Thus, although cardiolipin is primarily an inner membrane phospholipid, it has some access to the outer membrane [84]. Experiments with liposomes composed of lipids mimicking the mitochondrial outer membrane, or the contact sites between the outer and inner membranes, revealed that tBid binds poorly to liposomes resembling the mitochondrial outer membrane, but binds effectively to liposomes resembling the contact sites. Analysis of the role of individual phospholipids demonstrated that the most efficient binding was observed in the presence of 20% cardiolipin. Although the cardiolipin content used in the liposome experiment far exceeds its physiological content in the outer mitochondrial membrane, the authors postulated that cardiolipin seems to be required for the recruitment of tBid to mitochondria, in particular at contact sites where it would be enriched [85]. The requirement of cardiolipin for Cytochrome c release was supported by a study showing that cardiolipin is obligatory for Bax-mediated pore formation in liposomes [86]. Specifically, the authors used reconstituted membrane and/or synthetic liposomes encapsulating fluorescently labeled dextran molecules, to demonstrate that Bax-mediated dextran release required the presence of cardiolipin in the liposomes [86]. Bid, or its BH3 domain peptide, activated monomeric Bax to produce membrane openings that allowed the passage of very large (2 MDa) dextran molecules, mimicking the translocation of large mitochondrial proteins during apoptosis. This process required cardiolipin and was inhibited by anti-apoptotic Bcl-XL . Thus, they concluded that mitochondrial protein release in apoptosis can be mediated by supramolecular openings in the outer mitochondrial membrane, promoted by BH3/Bax/lipid interaction and directly inhibited by Bcl-XL . However, how protein–lipid interaction might lead to formation

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of the huge pores in the outer mitochondrial membrane is uncertain. Besides, as highlighted [79], it is difficult to understand why such pores would not be formed in the inner membrane in which the content of cardiolipin is incomparably higher than in the outer membrane. In order to study the role of cardiolipin in outer membrane permeabilization, we used a model of cardiolipindeficient and wild type yeast mitochondria [87]. We demonstrated that neither the mitochondrial association of exogenous recombinant Bax, nor the resulting Cytochrome c release, was dependent on the cardiolipin content of the yeast mitochondrial membranes. In our experiments, Bax associated equally with both wild type and cardiolipin-deficient mitochondrial membranes under conditions that lead to the release of Cytochrome c from both strains. Furthermore, we found that Cytochrome c was bound more “loosely” to the cardiolipin-deficient inner mitochondrial membrane compared to the wild type control. These data support the two-step mechanism of Cytochrome c release described above, implicating that cardiolipin is required for binding Cytochrome c to the inner mitochondrial membrane, thereby limiting its release in case of pore formation in the outer membrane [64]. 4. Effect of caspases on mitochondria Since the discovery of interleukin-1-converting enzyme (ICE or caspase-1), a number of additional caspases have been identified [88]. All caspases are present constitutively in precursor forms (30–50 kDa), which must be proteolytically cleaved for activation. Each procaspase consists of a pro-domain, a large (∼20 kDa) and a small (∼10 kDa) subunit. Caspases are commonly divided into two groups: initiator caspases with long pro-domain (caspase-2, -8, -9 and -10) and effector (caspase-3, -6 and -7) caspases with short pro-domain. Initiator caspases are activated with the help of adaptor molecules, e.g., Apaf-1 and FADD/MORT1, which bring these zymogens into close proximity, permitting auto-processing. Once activated, initiator caspases are responsible for cleaving and activating effector caspases. Effector caspases, in turn, cleave other pro-caspases as well as a range of other cellular proteins, leading to morphological and biochemical characteristic features of apoptosis. Permeabilization of the outer mitochondrial membrane and subsequent release of Cytochrome c result in activation of downstream caspases, in particular, caspase-9 and, subsequently, caspase-3. On the other hand, ligation of cell surface death receptors and acti-

vation of caspase-8 can result in cleavage of the proapoptotic protein Bid and tBid-mediated release of Cytochrome c from the mitochondria. Caspases other than caspase-8, in particular caspase-3, -6, and -7, were also shown to stimulate the release of Cytochrome c when added to isolated mitochondria in vitro [89]. This effect was dependent on the presence of cytosol, and none of these caspases had a direct effect on intact mitochondria at physiological concentrations. Hence, the cleavage of cytosolic Bid was tested in the same experiment. Bid was only cleaved by caspase-3 and 8. In addition, Bid was less effectively processed by caspase-3 than by caspase-8, although both caspases released Cytochrome c with similar, rapid kinetics. Neither caspase-6, nor caspase-7, cleaved Bid despite their ability to induce Cytochrome c release. Hence the authors concluded that cytosol may contain a caspase substrate, which differs from Bid and which, in response to cleavage by effector caspases, releases Cytochrome c from mitochondria [89]. Recently obtained data further demonstrate that caspases do not only stimulate permeabilization of the outer mitochondrial membrane, but also can affect mitochondrial functional activity. In UV-irradiated or staurosporine-treated cells inhibition of caspases protected mitochondrial protein import despite the loss of Cytochrome c. Thus, it appears that Bid and Bax act only on the outer membrane, and that lesions in the mitochondrial inner membrane occurring during apoptosis are secondary, caspase-dependent events [51]. Involvement of caspases in mitochondrial dysfunction during apoptosis was further analyzed by Green and co-workers [90]. Addition of recombinant caspase-3 following limited permeabilization of the mitochondrial outer membrane by Bax/Bak resulted in inhibition of complexes I and II, loss of ψ, increased production of ROS, and disruption of mitochondrial morphology. It was concluded that after outer membrane permeabilization and Cytochrome c release, activated caspases might target the permeabilized mitochondria. This alters mitochondrial functional activity (loss of ψ) and generates ROS through effects of caspases on complexes I and II in the electron transport chain [90]. All these events were attenuated in cells expressing a caspase-resistant p75 mutant. The authors suggested that the target for caspase-3 is the 75-kDa subunit of complex I of the electron transport chain, although the expression of noncleavable p75D255A produced only partial protection from loss of ψ and alteration of mitochondrial morphology in cells undergoing apoptosis [91]. It is also unclear how the presence of noncleavable p75D255A in complex I would protect Complex II

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of the mitochondrial respiratory chain from caspase proteolysis. 5. Caspase-2 permeabilization of mitochondria Recent evidence indicates that caspase-2 can be directly involved in the release of Cytochrome c from mitochondria in apoptotic cells. Caspase-2 is the best conserved caspase across species and was the first human apoptotic caspase to be cloned [92]. It is activated early in response to genotoxic stress and can function as an upstream modulator of the mitochondrial apoptotic pathway. Recent studies of caspase-2 activation have shown that this seems to occur within a multiprotein complex, containing the p53-inducible protein, PIDD, the adaptor protein, RAIDD, and additional proteins [93,94]. DNA damage induced by the chemotherapeutic drug etoposide triggers the onset of a series of intracellular events characteristic of apoptosis. Among the early changes observed is the release of Cytochrome c from mitochondria. Cells treated with the caspase-2 inhibitor, benzyloxycarbonyl–Val–Asp–Val–Ala–Asp–fluoromethyl ketone (z-VDVAD-fmk), or stably transfected with pro-caspase-2 antisense, are refractory to Cytochrome c release stimulated by etoposide. Apart from inhibiting Cytochrome c release, undermining caspase-2 processing results in the attenuation of downstream apoptotic events in etoposide-treated cells, including caspase-9 and -3 activation, phosphatidylserine exposure on the cell surface, and DNA fragmentation. Taken together, these data suggest that caspase-2 provides an important link between etoposide-induced DNA damage and the engagement of the mitochondrial apoptotic pathway [95]. In model experiments with isolated rat liver mitochondria fully processed caspase-2 stimulated the release of Cytochrome c and Smac/DIABLO, but not AIF, from mitochondria. Importantly, these events occurred independently of several Bcl-2 family proteins, including Bax, Bak and Bcl-2 [96], although the presence of Bcl-2 family proteins was reported to be necessary for caspase-2-mediated apoptosis in another study [97]. It should be noted that no other caspase tested (caspase-3, -6, -7, and -8) had a similar permeabilizing effect on the mitochondria [97]. Inactivation experiments revealed that the proteolytic activity of caspase-2 was not required for Cytochrome c release. Combined, these data suggest that caspase-2 retains a unique ability to engage directly the mitochondrial apoptotic pathway, an effect that requires processing of the zymogen but not the associated catalytic activity [96]. Recent experiments using permeabilized

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cells, isolated mitochondria, and protein-free liposomes, revealed that this effect is direct and depends neither on the presence or cleavage of other proteins nor on a specific phospholipid composition of the liposomal membrane [98]. Interestingly, caspase-2 was also shown to disrupt the interaction of Cytochrome c with anionic phospholipids, notably cardiolipin, and thereby enhance the release of the hemoprotein caused by treatment of mitochondria with digitonin or the pro-apoptotic protein Bax [98]. 6. Concluding remarks It is now clear that the mitochondria play a critical role in the regulation of both apoptotic and necrotic cell death. Mitochondrial permeabilization and release of intermembrane space proteins are important features of both models of cell death. Of the mechanisms involved, MPT appears to be associated mainly with necrosis, whereas the release of caspase activating proteins during early apoptosis is regulated primarily by the Bcl-2 family of proteins. However, there is increasing evidence for interaction and co-operation between these two mechanisms. Finally, mitochondrial permeabilization by processed caspase-2 represents a novel, previously unknown mechanism of apoptosis regulation, whose physiological significance under different conditions requires further study. Acknowledgments This work in the authors laboratory was supported by grants from the Swedish Research Council (31X-0247137A), and the EC-RTD grant (QLK3-CT-2002-01956). References [1] S. Orrenius, Mitochondrial regulation of apoptotic cell death, Toxicol. Lett. 149 (2004) 19–23. [2] J. Cai, J. Yang, D.P. Jones, Mitochondrial control of apoptosis: the role of Cytochrome c, Biochim. Biophys. Acta 1366 (1998) 139–149. [3] D.R. Green, J.C. Reed, Mitochondria and apoptosis, Science 281 (1998) 1309–1312. [4] H. Zou, Y. Li, X. Liu, X. Wang, An APAF-1 Cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9, J. Biol. Chem. 274 (1999) 11549–11556. [5] J.D. Robertson, S. Orrenius, B. Zhivotovsky, Review: nuclear events in apoptosis, J. Struct. Biol. 129 (2000) 346–358. [6] D.R. Hunter, R.A. Haworth, The Ca2+ -induced membrane transition in mitochondria. Part I. The protective mechanisms, Arch. Biochem. Biophys. 195 (1979) 453–459. [7] R.A. Haworth, D.R. Hunter, The Ca2+ -induced membrane transition in mitochondria. Part II. Nature of the Ca2+ trigger site, Arch. Biochem. Biophys. 195 (1979) 460–467.

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