Selective Cytochrome c Displacement by Phosphate and Ca2+ in Brain Mitochondria

In brain mitochondria, phosphate- and Ca²⁺-dependent cytocrome c (cyt c) release reveals pools that interact differently with the inner membrane. Detachment of the phosphate-dependent pool did not influence the pool released by Ca²⁺. Cyt c pools were also detected in a system of cyt c reconstituted in cardiolipin (CL) liposomes. Gradual binding of cyt c (1 nmol) to CL/2–[12-(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino]dodecanoyl-1-hexadecan oyl-sn-glycero-3-phosphocholine (NBDC₁₂-HPC) liposomes (10 nmol) produced NBD fluorescence quenching up to 0.4 nmol of added protein. Additional bound cyt c did not produce quenching, suggesting that cyt c-CL interactions originate distinct cyt c pools. Cyt c was removed from CL/NBDC₁₂-HPC liposomes by either phosphate or Ca²⁺, but only Ca²⁺ produced fluorescence dequenching and leakage of encapsulated 8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis-pyridinium bromide. In mitochondria, complex IV activity and mitochondrial membrane potential (Δψ_m) were not affected by the release of the phosphate-dependent cyt c pool. Conversely, removal of cyt c by Ca²⁺ caused inhibition of complex IV activity and impairment of Δψ_m. In a reconstituted system of mitochondria, nuclei and supernatant, cyt c detached from the inner membrane was released outside mitochondria and triggered events leading to DNA fragmentation. These events were prevented by enriching mitochondria with exogenous CL or by sequestering released cyt c with anti-cyt c antibody.     

Cytochrome c redox state influences the binding and release of cytochrome c in model membranes and in brain mitochondria

Cytochrome c (cyt c), a component of the respiratory chain, promotes apoptosis when released into the cytosol. Cyt c anchorage within mitochondria depends on cardiolipin (CL). Detachment and release have been related to CL loss and peroxidation. We report that NaN₃-dependent complex IV inhibition, accompanied by impairment of respiration, resulted in cyt c release. Contrarily, inhibition of respiration upstream cyt c with complex I and III inhibitors was not accompanied by the release of the protein, despite CL decrease and monolyso-CL increase. No CL changes and H₂O₂ formation were observed by inhibiting complex IV. In cyt c–CL liposomes, breaching cyt c–CL hydrophilic interactions produced a higher release of the reduced, compared to the oxidized form, suggesting that the hydrophobic component of cyt c–CL binding is prevalent in the oxidized form. Free or liposome-reconstituted cyt c was able to form fatty acid–protein complexes (palmitate < linoleate < oleate) only in its reduced form. We hypothesize that reduced cyt c–fatty acid binding favors the dislocation of the protein from anchoring CL. A mechanism for cyt c release independent of CL peroxidation by H₂O₂ is feasible. It could weaken the hydrophobic component of cyt c–CL interactions and might function following complex IV inhibition or in oxygen lack, both conditions producing accumulation of reduced cyt c and free fatty acids.     

Cardiolipin is not required for Bax-mediated cytochrome c release from yeast mitochondria

Cardiolipin (CL) is an inner mitochondrial membrane phospholipid that contributes to optimal mitochondrial function and is gaining widespread attention in studies of mitochondria-mediated apoptosis. Divergent hypotheses describing the role of CL in cytochrome c release and apoptosis have evolved. We addressed this controversy directly by comparing the spontaneous- and Bax-mediated cytochrome c release from mitochondria isolated from two strains of Saccharomyces cerevisiae: one lacking CL-synthase and therefore CL (DeltaCRD1) and the other, its corresponding wild type (WT). We demonstrated by liquid chromatography-mass spectrometry that the main yeast CL species [(16:1)2(18:1)2] differs in fatty acid composition from mammalian CL [(18:2)4], and we verified the absence of the yeast CL species in the DeltaCRD1 strain. We also demonstrated that the mitochondrial association of Bax and the resulting cytochrome c release is not dependent on the CL content of the yeast mitochondrial membranes. Bax inserted equally into both WT and DeltaCRD1 mitochondrial membranes under conditions that lead to the release of cytochrome c from both strains of yeast mitochondria. Furthermore, using models of synthetic liposomes and isolated yeast mitochondria, we found that cytochrome c was bound more "loosely" to the CL-deficient systems compared with when CL is present. These data challenge recent studies implicating that CL is required for Bax-mediated pore formation leading to the release of proteins from the mitochondrial intermembrane space. In contrast, they support our recently proposed two-step mechanism of cytochrome c release, which suggests that CL is required for binding cytochrome c to the inner mitochondrial membrane.     

Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential–sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (ΔΨ_m) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of ΔΨ_m, and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.     

From mitochondrial dynamics to arrhythmias

The reactive oxygen species (ROS)-dependent mitochondrial oscillator described in cardiac cells exhibits at least two modes of function under physiological conditions or in response to metabolic and oxidative stress. Both modes depend upon network behavior of mitochondria. Under physiological conditions cardiac mitochondria behave as a network of coupled oscillators with a broad range of frequencies. ROS weakly couples mitochondria under normal conditions but becomes a strong coupling messenger when, under oxidative stress, the mitochondrial network attains criticality. Mitochondrial criticality is achieved when a threshold of ROS is overcome and a certain density of mitochondria forms a cluster that spans the whole cell. Under these conditions, the slightest perturbation triggers a cell-wide collapse of the mitochondrial membrane potential, Δψ_m, visualized as a depolarization wave throughout the cell which is followed by whole cell synchronized oscillations in Δψ_m, NADH, ROS, and GSH. This dynamic behavior scales from the mitochondrion to the cell by driving cellular excitability and the whole heart into catastrophic arrhythmias. A network collapse of Δψ_m under criticality leads to: (i) energetic failure, (ii) temporal and regional alterations in action potential (AP), (iii) development of zones of impaired conduction in the myocardium, and, ultimately, (iv) a fatal ventricular arrhythmia.     

Exogenous phospholipids specifically affect transmembrane potential of brain mitochondria and cytochrome C release

Release of cytochrome c, a decrease of membrane potential (Deltapsi(m)), and a reduction of cardiolipin (CL) of rat brain mitochondria occurred upon incubation in the absence of respiratory substrates. Since CL is critical for mitochondrial functioning, CL enrichment of mitochondria was achieved by fusion with CL liposomes. Fusion was triggered by potassium phosphate at concentrations producing mitochondrial permeability transition pore opening but not cytochrome c release, which was observed only at >10 mm. Cyclosporin A inhibited phosphate-induced CL fusion, whereas Pronase pretreatment of mitochondria abolished it, suggesting that mitochondrial permeability transition pore and protein(s) are involved in the fusion process. Phosphate-dependent fusion was enhanced in respiratory state 3 and influenced by phospholipid classes in the order CL > phosphatidylglycerol (PG) > phosphatidylserine. The probe 10-nonylacridine orange indicated that fused CL had migrated to the inner mitochondrial membrane. In state 3, CL enrichment of mitochondria resulted in a pH decrease in the intermembrane space. Cytofluorimetric analysis of mitochondria stained with 3,3'-diexyloxacarbocyanine iodide and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzymidazolylcarbocyanine iodide showed Deltapsi(m) increase upon fusion with CL or PG. In contrast, phosphatidylserine fusion required Deltapsi(m) consumption, suggesting that Deltapsi(m) is the driving force in mitochondrial phospholipid importation. Moreover, enrichment with CL and PG brought the low energy mitochondrial population to high Deltapsi(m) values and prevented phosphate-dependent cytochrome c release.     

Loss of cardiolipin in palmitate-treated GL15 glioblastoma cells favors cytochrome c release from mitochondria leading to apoptosis

Unlike oleate and linoleate, palmitate induced mitochondrial apoptosis in GL15 glioblastoma cells. Decrease in membrane potential in a subpopulation of mitochondria of palmitate-treated cells was revealed using the 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide probe. The diminished ability to reduce a tetrazolium salt indicated an impairment of mitochondrial function. Up to 50% cytochrome c (cyt c ) was detached from the inner mitochondrial membrane and released outside mitochondria in palmitate-treated cells, whereas no release was detected after oleate and linoleate treatments. Cyt c release into the cytosol was followed by caspase 3 activation. Released cyt c and caspase 3 activity were not affected by neutral and acid sphingomyelinase inhibitors and by the inhibitor of serine palmitoyltransferase cycloserine, indicating that apoptosis was independent of the ceramide pathway, nor the mitochondrial pro-apoptotic AIF or Bcl-2/Bax factors appeared to be involved in the effect. Utilization of palmitate by GL15 cells altered phospholipid composition. Cardiolipin (CL), the lipid involved in cyt c interaction with the inner mitochondrial membrane, was decreased and highly saturated. This produced an imbalance in hydrophilic/hydrophobic interactions underlying the anchorage of cyt c , by weakening the hydrophobic component and facilitating detachment of the protein and activation of downstream processes. The primary role of CL was explored by supplying GL15 with exogenous CL through a fusion process of CL liposomes with cell plasma membrane. Fused CL moved to mitochondria, as detected by nonylacridine orange probe. Enrichment of mitochondrial membranes with CL prior to palmitate treatment of cells caused decreased cyt c release and caspase 3 activity.     

Mechanistic Issues of the Interaction of the Hairpin-Forming Domain of tBid with Mitochondrial Cardiolipin

Background

The pro-apoptotic effector Bid induces mitochondrial apoptosis in synergy with Bax and Bak. In response to death receptors activation, Bid is cleaved by caspase-8 into its active form, tBid (truncated Bid), which then translocates to the mitochondria to trigger cytochrome c release and subsequent apoptosis. Accumulating evidence now indicate that the binding of tBid initiates an ordered sequences of events that prime mitochondria from the action of Bax and Bak: (1) tBid interacts with mitochondria via a specific binding to cardiolipin (CL) and immediately disturbs mitochondrial structure and function idependently of its BH3 domain; (2) Then, tBid activates through its BH3 domain Bax and/or Bak and induces their subsequent oligomerization in mitochondrial membranes. To date, the underlying mechanism responsible for targeting tBid to mitochondria and disrupting mitochondrial bioenergetics has yet be elucidated.

Principal Findings

The present study investigates the mechanism by which tBid interacts with mitochondria issued from mouse hepatocytes and perturbs mitochondrial function. We show here that the helix αH6 is responsible for targeting tBid to mitochondrial CL and disrupting mitochondrial bioenergetics. In particular, αH6 interacts with mitochondria through electrostatic interactions involving the lysines 157 and 158 and induces an inhibition of state-3 respiration and an uncoupling of state-4 respiration. These changes may represent a key event that primes mitochondria for the action of Bax and Bak. In addition, we also demonstrate that tBid required its helix αH6 to efficiently induce cytochrome c release and apoptosis.

Conclusions

Our findings provide new insights into the mechanism of action of tBid, and particularly emphasize the importance of the interaction of the helix αH6 with CL for both mitochondrial targeting and pro-apoptotic activity of tBid. These support the notion that tBid acts as a bifunctional molecule: first, it binds to mitochondrial CL via its helix αH6 and destabilizes mitochondrial structure and function, and then it promotes through its BH3 domain the activation and oligomerization of Bax and/or Bak, leading to cytochrome c release and execution of apoptosis. Our findings also imply an active role of the membrane in modulating the interactions between Bcl-2 proteins that has so far been underestimated.     

PK11195, a Ligand of the Mitochondrial Benzodiazepine Receptor, Facilitates the Induction of Apoptosis and Reverses Bcl-2-Mediated Cytoprotection

One critical step of the apoptotic process is the opening of the mitochondrial permeability transition (PT) pore leading to the disruption of mitochondrial membrane integrity and to the dissipation of the inner transmembrane proton gradient (ΔΨ_m). The mitochondrial PT pore is a polyprotein structure which is inhibited by the apoptosis-inhibitory oncoprotein Bcl-2 and which is closely associated with the mitochondrial benzodiazepine receptor (mBzR). Here we show that PK11195, a prototypic ligand of the 18-kDa mBzR, facilitates the induction of ΔΨ_mdisruption and subsequent apoptosis by a number of different agents,including agonists of the glucocorticoid receptor,chemotherapeutic agents (etoposide, doxorubicin),gamma irradiation, and the proapoptotic second messenger ceramide. Whereas PK11195 itself has no cytotoxic effect, it enhances apoptosis induction by these agents. This effect is not observed for benzodiazepine diazepam, whose binding site in the mBzR differs from PK11195. PK11195 partially reverses Bcl-2 mediated inhibition of apoptosis in two different cell lines. Thus, transfection-enforced Bcl-2 overexpression confers protection against glucocorticoids and chemotherapeutic agents, and this protection is largely reversed by the addition of PK11195. This effect is observed at the level of ΔΨ_mdissipation as well as at the level of nuclear apoptosis. To gain insights into the site of action of PK11195, we performed experiments on isolated organelles. PK11195 reverses the Bcl-2-mediated mitochondrial retention of apoptogenic factors which cause isolated nuclei to undergo apoptosis in a cell-free system. Mitochondria from control cells, but not mitochondria from Bcl-2-overexpressing cells, readily release such apoptogenic factors in response to atractyloside, a ligand of the adenine nucleotide translocator. However, control and Bcl-2-overexpressing mitochondria respond equally well to a combination of atractyloside and PK11195. Altogether, these findings indicate that PK11195 abolishes apoptosis inhibition by Bcl-2 via a direct effect on mitochondria. Moreover, they suggest a novel strategy for enhancing the susceptibility of cells to apoptosis induction and, concomitantly, for reversing Bcl-2-mediated cytoprotection.     

Loss of molecular interaction between cytochrome c and cardiolipin due to lipid peroxidation.

To explore the molecular mechanism underlying the translocation of cytochrome c from the mitochondrial inner membrane to the cytosol during apoptosis, we analyzed the molecular interaction between cytochrome c and cardiolipin (CL) by (1)H NMR spectroscopy. Bovine heart CL induced a drastic broadening of the linewidth of the downfield signals at 31.4 and 34.2 ppm assigned to the heme methyl group-3 and -8, respectively, of horse heart cytochrome c. In contrast, CL mono- and dihydroperoxides were less active in broadening the signals than CL, and CL trihydroperoxides induced almost no broadening of their linewidth. This finding suggests that the peroxidation of CL induces a release of cytochrome c from mitochondria into the cytosol, which release induces apoptosis in the cells.     

Cardiolipin: Setting the beat of apoptosis

Cardiolipin (CL) is a mitochondria-specific phospholipid which is known to be intimately linked with the mitochondrial bioenergetic machinery. Accumulating evidence now suggests that this unique lipid also has active roles in several of the mitochondria-dependant steps of apoptosis. CL is closely associated with cytochrome c at the outer leaflet of the mitochondrial inner membrane. This interaction makes the process of cytochrome c release from mitochondria more complex than previously assumed, requiring more than pore formation in the mitochondrial outer membrane. While CL peroxidation could be crucial for enabling cytochrome c dissociation from the mitochondrial inner membrane, cytochrome c itself catalyzes CL peroxidation. Moreover, peroxy-CL directly activates the release of cytochrome c and other apoptogenic factors from the mitochondria. CL is also directly involved in mitochondrial outer membrane permeabilization by enabling docking and activation of pro-apoptotic Bcl-2 proteins. It appears therefore that CL has multiple roles in apoptosis and that CL metabolism contributes to the complexity of the apoptotic process.     

Ornithine decarboxylase prevents methotrexate-induced apoptosis by reducing intracellular reactive oxygen species production

Methotrexate (MTX), a folate antagonist, was developed for the treatment of malignancies, and is currently used in rheumatoid arthritis (RA) and other chronic inflammatory disorders. It has been proven in short-term and long-term prospective studies that low doses of MTX (0.75 mg/Kg/week) are effective in controlling the inflammatory manifestations of RA. Low-concentrations of MTX achieve apoptosis and clonal deletion of activated peripheral T cells. One of the mechanisms of the anti-inflammatory and immunosuppressive effects may be the production of reactive oxygen species (ROS). However, the drug resistance of MTX in malignancies remains poorly understood. Ornithine decarboxylase (ODC) plays an important role in diverse biological functions, including cell development, differentiation, transformation, growth and apoptosis. In our previous studies, ODC overexpression was shown to prevent TNFα-induced apoptosis via reducing ROS. Here, we also investigated one mechanism of MTX-induced apoptosis and of drug resistance as to the anti-apoptotic effects of ODC during MTX treatment. We found MTX could induce caspase-dependent apoptosis and promote ROS generation together with disrupting the mitochondrial membrane potential (ΔΨ_m) of HL-60 and Jurkat T cells. Putrescine and ROS scavengers could reduce MTX-induced apoptosis, which leads to the loss of ΔΨ_m, through reducing intracellular ROS. Overexpression of ODC in parental cells had the same effects as putrescine and the ROS scavengers. Moreover, ODC overexpression prevented the decline of Bcl-2 that maintains ΔΨ_m, the cytochrome c release and activations of caspase 9 and 3 following MTX treatment. The results demonstrate that MTX-induced apoptosis is ROS-dependent and occurs along a mitochondria-mediated pathway. Overexpressed ODC cells are resistant to MTX-induced apoptosis by reducing intracellular ROS production.     

Bacterial Porin Disrupts Mitochondrial Membrane Potential and Sensitizes Host Cells to Apoptosis

The bacterial PorB porin, an ATP-binding β-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (ΔΨ_m). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of β-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of ΔΨ_m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce ΔΨ_m loss and apoptosis, demonstrating that dissipation of ΔΨ_m is a requirement for cell death caused by neisserial infection.     

Oxidation-reduction midpoint potentials of mitochondrial flavoproteins and their intramitochondrial localization

Spectrophotometric and fluorimetric substrate couple titrations and potentiometric spectrophotometric titrations were used to determine the oxidation-reduction potentials of components showing absorbance or fluorescence at the wavelengths attributable to the flavoproteins of mitochondria fractionated using digitonin together with sonication. A pure mitoplast fraction devoid of cytochrome b₅ contamination could be obtained using 230 µg digitonin/mg of mitochondrial protein. The digitonin-soluble fraction contained a species havingE _m 7 .4=–123 mV and probably represents the outer membrane flavoproteins. The inner membrane-matrix fraction, treated with ultrasound, provided evidence of a flavoprotein species with low redox potential (E _m 7 .4=–302 mV) in the matrix fraction. The –302 mV component is probably lipoamide dehydrogenase. A high redox potential species withE _m 7 .4=+19 mV in titrations with the succinate fumarate couple was located in the inner membrane vesicles and is probably identical with succinate dehydrogenase. The electron-transferring flavoprotein (ETF) was isolated from bovine heart mitochondria and itsE _m 7 .4=–74 mV determined. The component in the matrix fraction with an apparentE _m 7 .4=–56 mV probably represents ETF, and that in the inner membrane fraction with an apparentE _m 7 .4=–43 mV the NADH dehydrogenase flavoprotein. A component in an apparently low concentration withE _m 7 .4=+30 mV was detected in the inner membrane fraction. This probably represents the ETF-dehydrogenase flavoprotein. The origin of the flavoprotein fluorescence of mitochondria and intact tissues is discussed.     

Fatty acid-promoted mitochondrial permeability transition by membrane depolarization and binding to the ADP/ATP carrier

The mechanism by which non-esterified long-chain fatty acids (FFA) promote mitochondrial permeability transition (MPT) is not clear. We examined with energized rat liver mitochondria the role of two possible actions of FFA in MPT, (i) the reduction of the transmembrane potential (Δψ) and (ii) the increase of the negative surface charge of the inner mitochondrial membrane [Broekemeier, K.M. and Pfeiffer, D.G., Biochemistry 43, (1995) 16440–16449]. It was found that the ability of FFA to stimulate large amplitude swelling is clearly related to their uncoupling activity. Moreover, compared with classical protonophores (FCCP) FFA increase the sensitivity of the pore opening process to Δψ changes. In addition, FFA interact like their thioester derivatives in a structure-dependent manner with the ADP/ATP carrier (measured as inhibition of [³H]atractyloside binding to the AAC protein). It is suggested that not only the protonophoric action of FFA, but also a presumable stabilization of the ‘cytosolic' conformation of AAC contribute to the FFA-promoted MPT.     

Impairment of brain mitochondrial functions by β-hemolytic Group B Streptococcus. Effect of cardiolipin and phosphatidylcholine

Group B Streptococcus (GBS) causes severe infection in the central nervous system. In this study, brain mitochondrial function was investigated by simulating infection of isolated mitochondria with GBS, which resulted in loss of mitochondrial activity. The β-hemolysin expressing strains GBS-III-NEM316 and GBS-III-COH31, but not the gGBS-III-COH31 that does not express β-hemolysin, caused dissipation of preformed mitochondrial membrane potential (Δψ_m). This indicates that β-hemolysin is responsible for decreasing of the reducing power of mitochondria. GBS-III-COH31 interacted with mitochondria causing increase of oxygen consumption, due to uncoupling of respiration, blocking of ATP synthesis, and cytochrome c release outside mitochondria. Moreover, the mitochondrial systems contributing to the control of cellular Ca²⁺ uptake were lost. In spite of these alterations, mitochondrial phospholipid content and composition did not change significantly, as evaluated by MALDI-TOF mass spectrometry. However, exogenous cardiolipin (CL) and dipalmitoylphosphatidylcholine (DPPC) attenuated the uncoupling effect of GBS-III-COH31, although with different mechanisms. CL was effective only when fused to the inner mitochondrial membrane, probably reducing the extent of GBS-induced proton leakage. DPPC, which is not able to fuse with mitochondrial membranes, exerted its effect outside mitochondria, likely by shielding mitochondria against GBS β-hemolysin attack.     

Phosphatidylethanolamine and Cardiolipin Differentially Affect the Stability of Mitochondrial Respiratory Chain Supercomplexes

The mitochondrial inner membrane contains two non-bilayer‐forming phospholipids, phosphatidylethanolamine (PE) and cardiolipin (CL). Lack of CL leads to destabilization of respiratory chain supercomplexes, a reduced activity of cytochrome c oxidase, and a reduced inner membrane potential Δψ. Although PE is more abundant than CL in the mitochondrial inner membrane, its role in biogenesis and assembly of inner membrane complexes is unknown. We report that similar to the lack of CL, PE depletion resulted in a decrease of Δψ and thus in an impaired import of preproteins into and across the inner membrane. The respiratory capacity and in particular the activity of cytochrome c oxidase were impaired in PE-depleted mitochondria, leading to the decrease of Δψ. In contrast to depletion of CL, depletion of PE did not destabilize respiratory chain supercomplexes but favored the formation of larger supercomplexes (megacomplexes) between the cytochrome bc₁ complex and the cytochrome c oxidase. We conclude that both PE and CL are required for a full activity of the mitochondrial respiratory chain and the efficient generation of the inner membrane potential. The mechanisms, however, are different since these non-bilayer‐forming phospholipids exert opposite effects on the stability of respiratory chain supercomplexes.     

Does Oxidation of Mitochondrial Cardiolipin Trigger a Chain of Antiapoptotic Reactions?

Oxidative stress causes selective oxidation of cardiolipin (CL), a fourtail lipid specific for the inner mitochondrial membrane. Interaction with oxidized CL transforms cytochrome c into peroxidase capable of oxidizing even more CL molecules. Ultimately, this chain of events leads to the pore formation in the outer mitochondrial membrane and release of mitochondrial proteins, including cytochrome c, into the cytoplasm. In the cytoplasm, cytochrome c promotes apoptosome assembly that triggers apoptosis (programmed cell death). Because of this amplification cascade, even an occasional oxidation of a single CL molecule by endogenously formed reactive oxygen species (ROS) might cause cell death, unless the same CL oxidation triggers a separate chain of antiapoptotic reactions that would prevent the CL-mediated apoptotic cascade. Here, we argue that the key function of CL in mitochondria and other coupling membranes is to prevent proton leak along the interface of interacting membrane proteins. Therefore, CL oxidation should increase proton permeability through the CL-rich clusters of membrane proteins (CL islands) and cause a drop in the mitochondrial membrane potential (MMP). On one hand, the MMP drop should hinder ROS generation and further CL oxidation in the entire mitochondrion. On the other hand, it is known to cause rapid fission of the mitochondrial network and formation of many small mitochondria, only some of which would contain oxidized CL islands. The fission of mitochondrial network would hinder apoptosome formation by preventing cytochrome c release from healthy mitochondria, so that slowly working protein quality control mechanisms would have enough time to eliminate mitochondria with the oxidized CL. Because of these two oppositely directed regulatory pathways, both triggered by CL oxidation, the fate of the cell appears to be determined by the balance between the CL-mediated proapoptotic and antiapoptotic reactions. Since this balance depends on the extent of CL oxidation, mito-chondria-targeted antioxidants might be able to ensure cell survival in many pathologies by preventing CL oxidation.     

The cardiolipin-cytochrome c interaction and the mitochondrial regulation of apoptosis

While many studies have focused on cytochrome c release from mitochondria, little attention has been given to the specific interaction between cardiolipin (CL) and cytochrome c, the breaching of which likely represents a critical event in the initiation of mitochondrially mediated apoptosis. Mounting evidence suggests that a decrease in the level of CL affects cytochrome c binding to the inner membrane, thus leading to higher levels of soluble cytochrome c in the mitochondrial intermembrane space. Among the factors known to affect CL levels are thyroid status, plasma concentrations of free fatty acids, Ca²⁺ dysregulation, and reactive oxygen species (ROS). These factors, especially Ca²⁺ and ROS, have long been recognized as triggers of cell death and, more recently, as modulators of mitochondrially mediated apoptosis. In this review, we discuss the significance of the disruption of the CL-cytochrome c interaction for cytochrome c release and apoptosis.