Differential interactions of apo- and holocytochrome c with acidic membrane lipids in model systems and the implications for their import into mitochondria

Monomolecular layers of lipid extracts of microsomal, mitochondrial outer and inner membranes, and pure lipid species have been used to measure their interaction with apo- and holocytochrome c. Large differences were observed both with respect to the nature and the lipid specificity of the interaction. The initial electrostatic interaction of the hemefree precursor apocytochrome c with anionic phospholipids is followed by penetration of the protein in between the acyl chains. Apocytochrome c shows similar interactions for all anionic lipids tested. In strong contrast the holoprotein discriminates enormously between cardiolipin for which it has a high affinity and phosphatidylserine and phosphatidylinositol for which it has a much lower affinity. For these latter lipids the interaction with cytochrome c is primarily electrostatic. The cytochrome c-cardiolipin interaction shows several unique features which suggest the formation of a specific complex between the two molecules. These properties account for the preference in interaction of the apoprotein with the lipid extract of the outer mitochondrial membrane over that of the endoplasmic reticulum and the large preference of cytochrome c for the inner over that of the outer mitochondrial membrane lipid extract. Only apocytochrome c was able to induce close contacts between monolayers of the mitochondrial outer membrane lipids and vesicles of mitochondrial inner membrane lipids. Experiments with fragments of both protein and unfolding experiments with cytochrome c revealed that the differences in interaction between the two proteins are mainly due to differences in their tertiary structure and not the presence of the heme group itself. The initial unfolded structure of apocytochrome c is responsible for the high penetrative power of the protein and its ability to induce close membrane contact, whereas the folded structure of cytochrome c is responsible for the specific interaction with cardiolipin. The results are discussed in the light of the apocytochrome c import process in mitochondria and suggest that lipid-protein interactions contribute to targeting the precursor toward mitochondria and are important for its translocation across the outer mitochondrial membrane and the final localization of cytochrome c toward the outside of the inner mitochondrial membrane.     

Oligomerization of integral membrane proteins under lipid peroxidation

Using polyacrylamide gel electrophoresis in the presence of Na-SDS, the oligomerization of membrane proteins of the retinal rod outer segments of the frog and the wall-eyed pollock and of rabbit skeletal muscle sarcoplasmic reticulum was studied. It was shown that under storage of the retinal rod outer segments the rhodopsin oligomerization is inhibited by the lipid peroxidation inhibitor--ionol. Similar oligomerization was observed under induction of lipid peroxidation in the membranes; the accumulation of the lipid peroxidation product--malonic dialdehyde--was accompanied by disappearance of the rhodopsin monomeric form in the outer segments. The cross-linking agent--glutaric dialdehyde--also causes oligomerization of the rhodopsins. Similar aggregation is also characteristic of the major protein of the sarcoplasmic reticulum fragments, i. e. Ca2+-dependent ATP-ase. Thus, one of the main changes in the protein content of biomembranes under lipid peroxidation is the oligomerization of integral proteins due to their interaction with bifunctional reagents, i. e. lipid peroxidation products.     

Bilayer structure in phospholipid-cytochrome c model membranes.

Lipid protein interactions in biological membranes differ markedly depending on whether the protein is intrinsic or extrinsic. These interactions are studied using lipid spin labels diffused into model systems consisting of phospholipid bilayers and a specific protein. Recently, an intrinsic protein complex, cytochrome oxidase, was examined and the data suggest there is a boundary layer of immobilized lipid between the hydrophobic protein surfaces and adjacent fluid bilayer regions. In the present study, a typical extrinsic protein, cytochrome c, was complexed with a cardiolipin/lecithin (1:4 by weight) mixture. The phospholipids in the presence and absence of cytochrome c exhibit typical bilayer behavior as jedged by four spin-labeling criteria: fluidity gradient, spectral anisotropy of oriented bilayers, response to hydration and the polarity profile. Any effects of cytochrome c on the ESR spectra of lipid spin labels are small, in contrast to the effects of intrinsic proteins. These data are consistent with electrostatic binding of cytochrome c to the charged groups of the phospholipids, and indicate that the presence of extrinsic proteins will not interfere with measurements of boundary lipid in intact biological membranes.     

Destabilization of membranes containing cardiolipin or its precursors by peptides derived from mitogaligin, a cell death protein

Galig, a gene embedded within the galectin-3 gene, induces cell death when transfected in human cells. This death is associated with cell shrinkage, nuclei condensation, and aggregation of mitochondria. Galig contains two different overlapping open reading frames encoding two unrelated proteins. Previous observations have shown that one of these proteins, named mitogaligin, binds to mitochondria and promotes the release of cytochrome c. However, the mechanism of action of this cytotoxic protein remains still obscure. The present study provides evidence that synthetic peptides enclosing the mitochondrial localization signal of mitogaligin bind to anionic biological membranes leading to membrane destabilization, aggregation, and content leakage of mitochondria or liposomes. This binding to anionic phospholipids is the most efficient when cardiolipin, a specific phospholipid of mitochondria, is inserted in the membranes. Thus, cardiolipin may constitute a target of choice for mitogaligin sorting and membrane destabilization activity.     

Two-dimensional infrared correlation spectroscopy study of the interaction of oxidized and reduced cytochrome c with phospholipid model membranes

We have used two-dimensional infrared correlation spectroscopy (2D-IR) to study the interaction and conformation of cytochrome c in the presence of a binary phospholipid mixture composed of a zwitterionic perdeuterated phospholipid and a negatively-charged one. The influence of the main temperature phase transition of the phospholipid model membranes on the conformation of cytochrome c has been evaluated by monitoring both the Amide I' band of the protein and the CH(2) and CD(2) stretching bands of the phospholipids. Synchronous 2D-IR analysis has been used to determine the different secondary structure components of cytochrome c which are involved in the specific interaction with the phospholipids, revealing the existence of a specific interaction between the protein with cardiolipin-containing vesicles but not with phosphatidic acid-containing ones. Interestingly, 2D-IR is capable of showing the existence of significant changes in the protein conformation at the same time that the phospholipid transition occurs. In summary, 2D-IR revealed an important effect of the phospholipid phase transition of cardiolipin on the secondary structure of oxidized cytochrome c but not to either reduced cytochrome c or in the presence of phosphatidic acid, demonstrating the existence of specific intermolecular interactions between cardiolipin and cytochrome c.     

细胞色素C和含心磷脂的人工脂膜相互作用

 本文报告以芘为荧光探剂,研究细胞色素C和含心磷脂的人工脂膜的相互作用。1.由于芘和细胞色素C的血红素团之间的能量转移,细胞色素C与心磷脂结合引起芘的单体荧光发射峰(395nm)强度下降。这种淬灭效应受脂膜的相行为影响,在液晶相时淬灭效应小于凝胶相;2.氧化态细胞色素C与还原态相比,对心磷脂结合的视和度稍高;3.在以芘的激发二聚体荧光峰(475nm)强度与单体荧光峰强度之比做为脂膜流动性的指标,发现还原态细胞色素C与含心磷脂脂膜结合后引起流动性增加的效应高于氧化态的结合。     

The "pro-apoptotic genies" get out of mitochondria: oxidative lipidomics and redox activity of cytochrome c/cardiolipin complexes

One of the prominent consequences of the symbiogenic origin of eukaryotic cells is the unique presence of one particular class of phospholipids, cardiolipin (CL), in mitochondria. As the product originated from the evolution of symbiotic bacteria, CL is predominantly confined to the inner mitochondrial membrane in normally functioning cells. Recent findings identified CL and its oxidation products as important participants and signaling molecules in the apoptotic cell death program. Early in apoptosis, massive membrane translocations of CL take place resulting in its appearance in the outer mitochondrial membrane. Consequently, significant amounts of CL become available for the interactions with cyt c, one of the major proteins of the intermembrane space. Binding of CL with cytochrome c (cyt c) yields the cyt c/CL complex that acts as a potent CL-specific peroxidase and generates CL hydroperoxides. In this review, we discuss the catalytic mechanisms of CL oxidation by the peroxidase activity of cyt c as well as the role of oxidized CL (CLox) in the release of pro-apoptotic factors from mitochondria into the cytosol. Potential implications of cyt c/CL peroxidase intracellular complexes in disease conditions (cancer, neurodegeneration) are also considered. The discovery of the new role of cyt c/CL complexes in early mitochondrial apoptosis offers interesting opportunities for new targets in drug discovery programs. Finally, exit of cyt c from damaged and/or dying (apoptotic) cells into extracellular compartments and its accumulation in biofluids is discussed in lieu of the formation of its peroxidase complexes with negatively charged lipids and their significance in the development of systemic oxidative stress in circulation.     

Mitochondrial kinases and their molecular interaction with cardiolipin

Mitochondrial isoforms of creatine kinase (MtCK) and nucleoside diphosphate kinase (NDPK-D) are not phylogenetically related but share functionally important properties. They both use mitochondrially generated ATP with the ultimate goal of maintaining proper nucleotide pools, are located in the intermembrane/cristae space, have symmetrical oligomeric structures, and show high affinity binding to anionic phospholipids, in particular cardiolipin. The structural basis and functional consequences of the cardiolipin interaction have been studied and are discussed in detail in this review. They mainly result in a functional interaction of MtCK and NDPK-D with inner membrane adenylate translocator, probably by forming proteolipid complexes. These interactions allow for privileged exchange of metabolites (channeling) that ultimately regulate mitochondrial respiration. Further functions of the MtCK/membrane interaction include formation of cardiolipin membrane patches, stabilization of mitochondria and a role in apoptotic signaling, as well as in case of both kinases, a role in facilitating lipid transfer between two membranes. Finally, disturbed cardiolipin interactions of MtCK, NDPK-D and other proteins like cytochrome c and truncated Bid are discussed more generally in the context of apoptosis and necrosis.     

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.     

Cardiolipin content in mitochondria from cultured skin fibroblasts harboring mutations in the mitochondrial <Emphasis Type="Italic">ATP6</Emphasis> gene

The role of phospholipids in normal assembly and organization of the membrane proteins has been well documented. Cardiolipin, a unique tetra-acyl phospholipid localized in the inner mitochondrial membrane, is implicated in the stability of many inner-membrane protein complexes. Loss of cardiolipin content, alterations in its acyl chain composition and/or cardiolipin peroxidation have been associated with dysfunction in multiple tissues in a variety of pathological conditions. The aim of this study was to analyze the phospholipid composition of the mitochondrial membrane in the four most frequent mutations in the ATP6 gene: L156R, L217R, L156P and L217P but, more importantly, to investigate the possible changes in the cardiolipin profile. Mitochondrial membranes from fibroblasts with mutations at codon 217 of the ATP6 gene, showed a different cardiolipin content compared to controls. Conversely, results similar to controls were obtained for mutations at codon 156. These findings may be attributed to differences in the biosynthesis and remodeling of cardiolipin at the level of the inner mitochondrial transmembrane related to some mutations of the ATP6 gene.     

Free radical-induced alterations of myocardial membrane proteins

Rat myocardial membranes exposed to the free radical-generating systems, Fe2+/ascorbate, Cu2+/t-butylhydro-peroxide, linoleic acid hydroperoxide, and soybean lipoxygenase (Type I) undergo lipid peroxidation. This is evidenced by the accumulation of thiobarbituric acid-reactive substances and the loss of both extractable phospholipids and their polyunsaturated acyl groups. Lipid peroxidation is accompanied by alterations of membrane proteins including the general loss of polypeptides and accumulation of high-molecular weight material. The most sensitive protein is a polypeptide with a molecular weight of 28 kDa. At low levels of oxidation, this protein moves incrementally to slightly higher apparent molecular weight. At higher oxidant levels or longer periods of oxidation, the protein disappears completely from the SDS-PAGE gel. The "28K reaction" occurs prior to the massive, oxidant-induced lipid alterations and may thus indicate specific adduct formation between this protein and certain peroxidized membrane phospholipids.     

Cardiolipin is associated with the terminal oxidase of an extremely halophilic archaeon

Membranes having an a high content of cardiolipin were isolated from an extremely halophilic archaeon Halorubrum sp. Absorbance difference spectra of detergent-solubilized plasma membranes reduced by dithionite suggested the presence of b-type cytochromes. Non-denaturing gel electrophoresis revealed only one fraction having TMPD-oxidase activity in which cardiolipin was the major lipid component. The electroeluted fraction showed a cytochrome c oxidase activity characterized by the reduced minus oxidized difference spectra as a terminal heme-copper oxidase. The cytochrome c oxidase activity of the archaeal cardiolipin-rich membranes was inhibited by the cardiolipin-specific fluorescent marker 10-N-nonyl acridine orange (NAO) in a dose-dependent manner. The results indicate that an archaeal analogue of cardiolipin is tightly associated to archaeal terminal oxidases and is required for its optimal functioning.     

Molecular mechanisms of apoptosis. Structure of cytochrome c-cardiolipin complex

One of the functions of cytochrome c in living cells is the initiation of apoptosis by catalyzing lipid peroxidation in the inner mitochondrial membrane, which involves cytochrome c bound with acidic lipids, especially cardiolipin. In this paper the results of studies of cytochrome c-cardiolipin complex structure carried out by different authors mainly on unilamellar cardiolipin-containing phospholipid liposomes are critically analyzed. The principal conclusion from the published papers is that cytochrome c-cardiolipin complex is formed by attachment of a cytochrome c molecule to the membrane surface via electrostatic interactions and the subsequent penetration of one of the fatty-acid cardiolipin chains into the protein globule, this being associated with hydrophobic interactions that break the >Fe…S(Met80) coordinate bond and giving rise to appearance of cytochrome c peroxidase activity. Nevertheless, according to data obtained in our laboratory, cytochrome c and cardiolipin form spherical nanoparticles in which protein is surrounded by a monolayer of cardiolipin molecules. Under the action of cooperative forces, the protein in the globule expands greatly in volume, its conformation is modified, and the protein becomes a peroxidase. In extended membranes, such as giant monolayer liposomes, and very likely in biological membranes, the formation of nanospheres of cytochrome c-cardiolipin complex causes fusion of membrane sections and dramatic chaotization of the whole membrane structure. The subsequent disintegration of the outer mitochondrial membrane is accompanied by cytochrome c release from the mitochondria and triggering of a cascade of programmed cell death reactions.     

Compartmentation of gramicidin 5 in membranes of sensitive bacteria and protein-lipid interactions]

Gramicidin S is sorbed on the isolated membranes of granicidin-sensitive Micrococcus lysodeikticus strain. The antibiotic inhibits the membrane malate dehydrogenase within the temperature range of 9--42 degrees C, i.e. under conditions of gel and liquid-crystalline lipid state; however its effect at 10 degrees C is 10 times as low as is observed at 42 degrees C. The inhibitory effect of gramicidin S on malate dehydrogenase can be eliminated and the antibiotic can be removed from the membrane by an excess of different phospholipids. No transfer of the membrane components on exogenous phospholipids is observed. A prolonged (about 2 hrs, 30 degrees C) incubation of the membranes with gramicidin S results in irreversible inactivation of malate dehydrogenase, although the antibiotic can be still eliminated by an addition of phospholipid emulsions. It is suggested that gramicidin S forms complexes with phospholipids, in which the antibiotic is oriented to water. These complexes disturb the lipid-protein interactions, resulting in relaxation of the binding between the boundary phospholipids and proteins, in the loosening of near-protein lipid zones and simultaneous condensation of acid phospholipids in the whole membrane. Destruction of the lipid zone is accompanied by changes in the enzyme activity, by separation of lipid and protein regions and by transphase enzyme transitions (expulsion or immersion). A slow formation of secondary protein-protein associates may be irreversible.     

Oxidized phospholipids as biomarkers of tissue and cell damage with a focus on cardiolipin

Oxidized phospholipid species are important, biologically relevant, lipid signaling molecules that usually exist in low abundance in biological tissues. Along with their inherent stability issues, these oxidized lipids present themselves as a challenge in their detection and identification. Often times, oxidized lipid species can co-chromatograph with non-oxidized species making the detection of the former extremely difficult, even with the use of mass spectrometry. In this study, a normal-phase and reverse-phase two dimensional high performance liquid chromatography (HPLC)-mass spectrometric system was applied to separate oxidized phospholipids from their non-oxidized counterparts, allowing unambiguous detection in a total lipid extract. We have utilized bovine heart cardiolipin as well as commercially available tetralinoleoyl cardiolipin oxidized with cytochrome c (cyt c) and hydrogen peroxide as well as with lipoxygenase to test the separation power of the system. Our findings indicate that oxidized species of not only cardiolipin, but other phospholipid species, can be effectively separated from their non-oxidized counterparts in this two dimensional system. We utilized three types of biological tissues and oxidative insults, namely rotenone treatment of lymphocytes to induce mitochondrial damage and cell death, pulmonary inhalation exposure to single walled carbon nanotubes, as well as total body irradiation, in order to identify cardiolipin oxidation products, critical to the cell damage/cell death pathways in these tissues following cellular stress/injury. Our results indicate that selective cardiolipin (CL) oxidation is a result of a non-random free radical process. In addition, we assessed the ability of the system to identify CL oxidation products in the brain, a tissue known for its extreme complexity and diversity of CL species. The ability of the two dimensional HPLC-mass spectrometric system to detect and characterize oxidized lipid products will allow new studies to be formulated to probe the answers to biologically important questions with regard to oxidative lipidomics and cellular insult. This article is part of a Special Issue entitled: Oxidized phospholipids - their properties and interactions with proteins.     

Cardiolipin stabilizes respiratory chain supercomplexes

Cardiolipin stabilized supercomplexes of Saccharomyces cerevisiae respiratory chain complexes III and IV (ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase, respectively), but was not essential for their formation in the inner mitochondrial membrane because they were found also in a cardiolipin-deficient strain. Reconstitution with cardiolipin largely restored wild-type stability. The putative interface of complexes III and IV comprises transmembrane helices of cytochromes b and c1 and tightly bound cardiolipin. Subunits Rip1p, Qcr6p, Qcr9p, Qcr10p, Cox8p, Cox12p, and Cox13p and cytochrome c were not essential for the assembly of supercomplexes; and in the absence of Qcr6p, the formation of supercomplexes was even promoted. An additional marked effect of cardiolipin concerns cytochrome c oxidase. We show that a cardiolipin-deficient strain harbored almost inactive resting cytochrome c oxidase in the membrane. Transition to the fully active pulsed state occurred on a minute time scale.     

Reactive oxygen species generated from the mitochondrial electron transport chain induce cytochrome c dissociation from beef-heart submitochondrial particles via cardiolipin peroxidation. Possible role in the apoptosis.

Cytochrome c release from mitochondria is a critical event in the apoptosis induction. Dissociation of cytochrome c from the mitochondrial inner membrane (IMM) is a necessary first step for cytochrome c release. In the present study, the effect of reactive oxygen species (ROS) on the dissociation of cytochrome c from beef-heart submitochondrial particles (SMP) and on the cardiolipin content was investigated. Exposure of SMP to mitochondrial-mediated ROS generation resulted in a large dissociation of cytochrome c from SMP and in a parallel loss of cardiolipin. Both these effects were directly and significantly correlated and also abolished by superoxide dismutase+catalase. These results demonstrate that ROS generation induces the dissociation of cytochrome c from IMM via cardiolipin peroxidation. The data may prove useful in clarifying the molecular mechanism underlying the release of cytochrome c from the mitochondria to the cytosol.     

Effects of lipids on mitochondrial functions

Mitochondria contain two membranes: the outer and inner membrane. Whereas the outer membrane is particularly enriched in phospholipids, the inner membrane has an unusual high protein content and forms large invaginations termed cristae. The proper phospholipid composition of the membranes is crucial for mitochondrial functions. Phospholipids affect activity, biogenesis and stability of protein complexes including protein translocases and respiratory chain supercomplexes. Negatively charged phospholipids such as cardiolipin are important for the architecture of the membranes and recruit soluble factors to the membranes to support mitochondrial dynamics. Thus, phospholipids not only form the hydrophobic core of biological membranes that surround mitochondria, but also create a specific environment to promote functions of various protein machineries. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

Hepatic mitochondrial membrane lipid environment and protein nutrition

Effect of feeding rice diet with and without lysine and threonine supplementation on hepatic mitochondria and its inner and outer membrane proteins, enzymes and phospholipids has been studied. The exchange of phosphatidylcholine and phosphatidylethanolamine between microsomes and mitochondria has also been studied under these conditions. Deficient diet lead to significant decrease in proteins as well as activities of monoamine oxidase, succinate dehydrogenase, cytochrome a + a3 and cytochrome c in mitochondria and its inner and outer membranes. Feeding of the deficient diet also significantly reduced total phospholipids and PC in mitochondria and its outer mitochondrial membrane. In the inner mitochondrial membrane, only PE and cardiolipin were reduced. The incorporation (DPM/microgram PLP) of [methyl-3H]choline and [methyl-14C]methionine into PC of mitochondria and its outer membrane and that of 32Pi into PC and PE of outer mitochondrial membrane but only into PC of inner mitochondrial membrane were significantly reduced in the deficient group. The exchange rates of PC and PE between microsomes and mitochondria were reduced in the deficient group. Supplementation of the deficient diet with lysine and threonine profoundly improved the above biochemical lesions as compared to casein fed rats.