Anionic Cardiolipin Research Articles

Anionic cardiolipin stabilizes the transmembrane region of hyaluronan synthase and promotes catalysis-relevant dynamics

Hyaluronic acid (HA) is a glycosaminoglycan essential for cellular processes and finding increasingly applications in medicine, pharmaceuticals, and cosmetics. While membrane-integrated Class I hyaluronan synthase (HAS) catalyzes HA synthesis in most organisms, the molecular mechanisms by which HAS-lipid interactions impact HAS catalysis remain unclear. This study employed coarse-grained molecular dynamics simulation combined with dimensionality reduction to uncover the interplay between lipids and Streptococcus equisimilis HAS (SeHAS). A minimum of 67 % cardiolipin is necessary for HA synthesis, as determined through simulations using gradient-composed membranes. The anionic cardiolipin stabilizes the cationic transmembrane regions of SeHAS and thereby maintains its conformation. Moreover, the highly dynamic cardiolipin is required to modulate the catalysis-relevant motions in HAS and thus facilitate HA synthesis. These findings provide molecular insights essential not only for understanding the physiological functions of HAS, but also for the development of cell factories and enzyme catalysts for HA production.

Lipids uniquely alter the secondary structure and toxicity of amyloid beta 1-42 aggregates.

Abrupt aggregation of amyloid β1-42 (Aβ) peptide is a hallmark of Alzheimer's disease (AD), a severe pathology that affects more than 44million people worldwide. A growing body of evidence suggests that lipids can uniquely alter rates of Aβ1-42 aggregation. However, it remains unclear whether lipids only alter rates of protein aggregation or also uniquely modify the secondary structure and toxicity of Aβ1-42 oligomers and fibrils. In this study, we investigated the effect of phosphatidylcholine (PC), cardiolipin (CL), and cholesterol (Chol) on Aβ1-42 aggregation. We found that PC, CL and Chol strongly accelerated the rate of fibril formation compared to the rate of Aβ1-42 aggregation in the lipid-free environment. Furthermore, anionic CL enabled the strongest acceleration of Aβ1-42 aggregation compared to zwitterionic PC and uncharged Chol. We also found that PC, CL and Chol uniquely altered the secondary structure of early-, middle- and late-stage Aβ1-42 aggregates. Specifically, CL and Chol drastically increased the amount of parallel β-sheet in Aβ1-42 oligomers and fibrils grown in the presence of these lipids. This caused a significant increase in the toxicity of Aβ:CL and Aβ:Chol compared to the toxicity of Aβ:PC and Aβ1-42 aggregates formed in the lipid-free environment. These results demonstrate that toxicity of Aβ aggregates correlates with the amount of their β-sheet content, which, in turn, is determined by the chemical structure of lipids present at the stage of Aβ1-42 aggregation.

Electrostatic Drivers of GPx4 Interactions with Membrane, Lipids, and DNA

Glutathione peroxidase 4 (GPx4) serves as the only enzyme that protects membranes through the reduction of lipid hydroperoxides, preventing membrane oxidative damage and cell death through ferroptosis. Recently, GPx4 has gained attention as a therapeutic target for cancer through inhibition and as a target for inflammatory diseases through activation. In addition, GPx4 isoforms perform several distinct moonlighting functions including cysteine cross-linking of protamines during sperm cell chromatin remodeling, a function for which molecular and structural details are undefined. Despite the importance in biology, disease, and potential for drug development, little is known about GPx4 functional interactions at high resolution. This study presents the first NMR assignments of GPx4, and the electrostatic interaction of GPx4 with the membrane is characterized. Mutagenesis reveals the cationic patch residues that are key to membrane binding and stabilization. The cationic patch is observed to be important in binding headgroups of highly anionic cardiolipin. A novel lipid binding site is observed adjacent to the catalytic site and may enable protection of lipid-headgroups from oxidative damage. Arachidonic acid is also found to engage with GPx4, while cholesterol did not display any interaction. The cationic patch residues were also found to enable DNA binding, the first observation of this interaction. Electrostatic DNA binding explains a mechanism for the nuclear isoform of GPx4 to target DNA-bound protamines and to potentially reduce oxidatively damaged DNA. Together, these results highlight the importance of electrostatics in the function of GPx4 and illuminate how the multifunctional enzyme is able to fill multiple biological roles.

Naja mossambica mossambica Cobra Cardiotoxin Targets Mitochondria to Disrupt Mitochondrial Membrane Structure and Function.

Cobra venom cardiotoxins (CVCs) can translocate to mitochondria to promote apoptosis by eliciting mitochondrial dysfunction. However, the molecular mechanism(s) by which CVCs are selectively targeted to the mitochondrion to disrupt mitochondrial function remains to be elucidated. By studying cardiotoxin from Naja mossambica mossambica cobra (cardiotoxin VII4), a basic three-fingered S-type cardiotoxin, we hypothesized that cardiotoxin VII4 binds to cardiolipin (CL) in mitochondria to alter mitochondrial structure/function and promote neurotoxicity. By performing confocal analysis, we observed that red-fluorescently tagged cardiotoxin rapidly translocates to mitochondria in mouse primary cortical neurons and in human SH-SY5Y neuroblastoma cells to promote aberrant mitochondrial fragmentation, a decline in oxidative phosphorylation, and decreased energy production. In addition, by employing electron paramagnetic resonance (EPR) and protein nuclear magnetic resonance (1H-NMR) spectroscopy and phosphorescence quenching of erythrosine in model membranes, our compiled biophysical data show that cardiotoxin VII4 binds to anionic CL, but not to zwitterionic phosphatidylcholine (PC), to increase the permeability and formation of non-bilayer structures in CL-enriched membranes that biochemically mimic the outer and inner mitochondrial membranes. Finally, molecular dynamics simulations and in silico docking studies identified CL binding sites in cardiotoxin VII4 and revealed a molecular mechanism by which cardiotoxin VII4 interacts with CL and PC to bind and penetrate mitochondrial membranes.

Autoxidation of Reduced Horse Heart Cytochrome c Catalyzed by Cardiolipin-Containing Membranes.

Visible circular dichroism, absorption, and fluorescence spectroscopy were used to probe the binding of horse heart ferrocytochrome c to anionic cardiolipin (CL) head groups on the surface of 1,1',2,2'-tetraoleoyl cardiolipin (TOCL)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (20%:80%) liposomes in an aerobic environment. We found that ferrocytochrome c undergoes a conformational transition upon binding that leads to complete oxidation of the protein at intermediate and high CL concentrations. At low lipid concentrations, the protein maintains a structure that is only slightly different from its native one, whereas an ensemble of misligated predominantly hexacoordinated low-spin states become increasingly populated at high lipid concentrations. A minor fraction of conformations with either high- or quantum-mixed-spin states were detected at a CL to protein ratio of 200 (the largest one investigated). The population of the non-native state is less pronounced than that found for cytochrome c-CL interactions initiated with oxidized cytochrome c. Under anaerobic conditions, the protein maintains its reduced state but still undergoes some conformational change upon binding to CL head groups on the liposome surface. Our data suggest that CL-containing liposomes function as catalysts by reducing the activation barrier for a Fe2+ → O2 electron transfer. Adding NaCl to the existing cytochrome-liposome mixtures under aerobic conditions inhibits protein autoxidation of ferrocytochrome c and stabilizes the reduced state of the membrane-bound protein.

Adsorption of α-Synuclein to Supported Lipid Bilayers: Positioning and Role of Electrostatics

An amyloid form of the protein α-synuclein is the major component of the intraneuronal inclusions called Lewy bodies, which are the neuropathological hallmark of Parkinson's disease (PD). α-Synuclein is known to associate with anionic lipid membranes, and interactions between aggregating α-synuclein and cellular membranes are thought to be important for PD pathology. We have studied the molecular determinants for adsorption of monomeric α-synuclein to planar model lipid membranes composed of zwitterionic phosphatidylcholine alone or in a mixture with anionic phosphatidylserine (relevant for plasma membranes) or anionic cardiolipin (relevant for mitochondrial membranes). We studied the adsorption of the protein to supported bilayers, the position of the protein within and outside the bilayer, and structural changes in the model membranes using two complementary techniques-quartz crystal microbalance with dissipation monitoring, and neutron reflectometry. We found that the interaction and adsorbed conformation depend on membrane charge, protein charge, and electrostatic screening. The results imply that α-synuclein adsorbs in the headgroup region of anionic lipid bilayers with extensions into the bulk but does not penetrate deeply into or across the hydrophobic acyl chain region. The adsorption to anionic bilayers leads to a small perturbation of the acyl chain packing that is independent of anionic headgroup identity. We also explored the effect of changing the area per headgroup in the lipid bilayer by comparing model systems with different degrees of acyl chain saturation. An increase in area per lipid headgroup leads to an increase in the level of α-synuclein adsorption with a reduced water content in the acyl chain layer. In conclusion, the association of α-synuclein to membranes and its adsorbed conformation are of electrostatic origin, combined with van der Waals interactions, but with a very weak correlation to the molecular structure of the anionic lipid headgroup. The perturbation of the acyl chain packing upon monomeric protein adsorption favors association with unsaturated phospholipids preferentially found in the neuronal membrane.

Structure and properties of complexes formed by cationic polymers and anionic cholesterol-containing liposomes

The adsorption of the synthetic polycation poly(N-ethyl-4-vinylpyridinium bromide) on the surface of three-component lipid vesicles (liposomes) formed from a mixture of anionic cardiolipin, electroneutral egg lecithin, and nonionic cholesterol is studied via laser microelectropheresis, dynamic light scattering, conductometry, fluorescence spectroscopy, and UV spectroscopy. The incorporation of cholesterol into the liposomal membrane increases its microviscosity; however, the membrane remains liquid-crystalline. Simultaneously, an increase in the fraction of cholesterol causes the formation of defects in liposome membranes during their binding with poly(N-ethyl-4-vinylpyridium bromide) and makes complexation irreversible. The results of this study are of interest for predicting the behavior of polyelectrolytes and biologically active structures formed on their basis on the surface of cells and the reaction of the cellular membrane to the adsorbed polymer.

Cardiolipin-based respiratory complex activation in bacteria

Anionic lipids play a variety of key roles in membrane function, including functional and structural effects on respiratory complexes. However, little is known about the molecular basis of these lipid-protein interactions. In this study, NarGHI, an anaerobic respiratory complex of Escherichia coli, has been used to investigate the relations in between membrane-bound proteins with phospholipids. Activity of the NarGHI complex is enhanced by anionic phospholipids both in vivo and in vitro. The anionic cardiolipin tightly associates with the NarGHI complex and is the most effective phospholipid to restore functionality of a nearly inactive detergent-solubilized enzyme complex. A specific cardiolipin-binding site is identified on the basis of the available X-ray diffraction data and of site-directed mutagenesis experiment. One acyl chain of cardiolipin is in close proximity to the heme b(D) center and is responsible for structural adjustments of b(D) and of the adjacent quinol substrate binding site. Finally, cardiolipin binding tunes the interaction with the quinol substrate. Together, our results provide a molecular basis for the activation of a bacterial respiratory complex by cardiolipin.

Oxidative lipidomics of hyperoxic acute lung injury: mass spectrometric characterization of cardiolipin and phosphatidylserine peroxidation

Reactive oxygen species have been shown to play a significant role in hyperoxia-induced acute lung injury, in part, by inducing apoptosis of pulmonary endothelium. However, the signaling roles of phospholipid oxidation products in pulmonary endothelial apoptosis have not been studied. Using an oxidative lipidomics approach, we identified individual molecular species of phospholipids involved in the apoptosis-associated peroxidation process in a hyperoxic lung. C57BL/6 mice were killed 72 h after exposure to hyperoxia (100% oxygen). We found that hyperoxia-induced apoptosis (documented by activation of caspase-3 and -7 and histochemical terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining of pulmonary endothelium) was accompanied by nonrandom oxidation of pulmonary lipids. Two anionic phospholipids, mitochondria-specific cardiolipin (CL) and extramitochondrial phosphatidylserine (PS), were the two major oxidized phospholipids in hyperoxic lung. Using electrospray ionization mass spectrometry, we identified several oxygenation products in CL and PS. Quantitative assessments revealed a significant decrease of CL and PS molecular species containing C(18:2), C(20:4), C(22:5), and C(22:6) fatty acids. Similarly, exposure of mouse pulmonary endothelial cells (MLEC) to hyperoxia (95% oxygen; 72 h) resulted in activation of caspase-3 and -7 and significantly decreased the content of CL molecular species containing C(18:2) and C(20:4) as well as PS molecular species containing C(22:5) and C(22:6). Oxygenated molecular species were found in the same two anionic phospholipids, CL and PS, in MLEC exposed to hyperoxia. Treatment of MLEC with a mitochondria-targeted radical scavenger, a conjugate of hemi-gramicidin S with nitroxide, XJB-5-131, resulted in significantly lower oxidation of both CL and PS and a decrease in hyperoxia-induced changes in caspase-3 and -7 activation. We speculate that cytochrome c driven oxidation of CL and PS is associated with the signaling role of these oxygenated species participating in the execution of apoptosis and clearance of pulmonary endothelial cells, thus contributing to hyperoxic lung injury.

Effect of the phase state of the lipid bilayer on the structure and characteristics of the polycation-(anionic liposome) complex

Interaction of the cationic polymer poly-N-ethyl-4-vinylpyridinium bromide with bilayer vesicles (liposomes) composed of zwitterionic dipalmitoylphosphatidylcholine and anionic cardiolipin (the molar fraction of the negatively charged cardiolipin groups is 0.2) is studied. The composition and characteristics of the polycation-liposome complex are shown to be controlled by the phase state of the lipid membrane. Liposomes whose membranes exist in their LC state (“liquid” liposomes) keep their integrity in the complex with polycation. The adsorbed polycation can be completely removed from the liposomal membrane by the addition excess amounts of a competing polyanion. The adsorption of polycation on the surface of liposomes whose membranes exist the gel state (“solid” liposomes) leads to the formation of defects in the membrane, and the polycation’s adsorption with such liposomes becomes irreversible. The defects that form are also preserved when solid liposomes on whose surface the polycation is sorbed are transformed into the liquid state. Moreover, the reversible contact between polycation and liquid liposomes becomes irreversible once the liposomal membranes bound to the polycation transform into the solid state.

Migration of a cationic polymer between lipid vesicles

The adsorption of a synthetic polycation, poly(N-ethyl-4-vinylpyridinium bromide) (PEVP), on the surface of bilayer lipid vesicles (liposomes) and the migration of adsorbed macromolecules between the liposomes are studied. Liposomes of three types are used, including (1) traditional two-component liposomes composed of neutral phosphatidylcholine (PC) and anionic cardiolipin (CL); (2) three-component liposomes consisting of PC, CL, and cationic dicetyldimethylammonium bromide (DCMAB); and (3) anionic PC/CL liposomes with a nonionic surfactant, poly(ethylene oxide)-cetyl alcohol ether (Briij 58), incorporated into their bilayers. The adsorption of PEVP on the surface of PC/CL liposomes is accompanied by their aggregation. Using the fluorescence method, it is shown that the units (segments) of the polycation undergo partial redistribution between the liposomes inside the aggregates formed from PC/CL liposomes (with and without a fluorescent label) and PEVP. On the contrary, three-component PC/CL/DCMAB and PC/CL/Briij liposomes are not aggregated, even with the complete neutralization of their charges by adsorbed PEVP. In both cases, the migration of PEVP molecules between individual (nonaggregated) liposomes is observed. Possible reasons for the aggregative stability of the three-component PC/CL/DCMAB and PC/CL/Briij liposomes and the mechanism of interliposome migration of PEVP in such systems are discussed.

How does the Bax-α1 targeting sequence interact with mitochondrial membranes? The role of cardiolipin

A key event in programmed cell death is the translocation of the apoptotic Bax protein from the cytosol towards mitochondria. The first helix localized at the N-terminus of Bax (Bax-α1) can act here as an addressing sequence, which directs activated Bax towards the mitochondrial surface. Solid state NMR (nuclear magnetic resonance), CD (circular dichroism) and ATR (attenuated total reflection) spectroscopy were used to elucidate this recognition process of a mitochondrial membrane system by Bax-α1. Two potential target membranes were studied, with the outer mitochondrial membrane (OM) mimicked by neutral phospholipids, while mitochondrial contact sites (CS) contained additional anionic cardiolipin. 1H and 31P magic angle spinning (MAS) NMR revealed Bax-α1 induced pronounced perturbations in the lipid headgroup region only in presence of cardiolipin. Bax-α1 could not insert into CS membranes but at elevated concentrations it inserted into the hydrophobic core of cardiolipin-free OM vesicles, thereby adopting β-sheet-like features, as confirmed by ATR. CD studies revealed, that the cardiolipin mediated electrostatic locking of Bax-α1 at the CS membrane surface promotes conformational changes into an α-helical state; a process which seems to be necessary to induce further conformational transition events in activated Bax which finally causes irreversible membrane permeabilization during the mitochondrial apoptosis.

Deficiency in mitochondrial anionic phospholipid synthesis impairs cell wall biogenesis

Cardiolipin (CL) is the signature lipid of the mitochondrial membrane and plays a key role in mitochondrial physiology and cell viability. The importance of CL is underscored by the finding that the severe genetic disorder Barth syndrome results from defective CL composition and acylation. Disruption of PGS1, which encodes the enzyme that catalyses the committed step of CL synthesis, results in loss of the mitochondrial anionic phospholipids phosphatidylglycerol and CL. The pgs1Δ mutant exhibits severe growth defects at 37°C. To understand the essential functions of mitochondrial anionic lipids at elevated temperatures, we isolated suppressors of pgs1Δ that grew at 37°C. The present review summarizes our analysis of suppression of pgs1Δ growth defects by a mutant that has a loss-of-function mutation in KRE5, a gene involved in cell wall biogenesis.

Deficiency in mitochondrial anionic phospholipid synthesis impairs cell wall biogenesis

Cardiolipin (CL) is the signature lipid of the mitochondrial membrane and plays a key role in mitochondrial physiology and cell viability. The importance of CL is underscored by the finding that the severe genetic disorder Barth syndrome results from defective CL composition and acylation. Disruption of PGS1, which encodes the enzyme that catalyses the committed step of CL synthesis, results in loss of the mitochondrial anionic phospholipids phosphatidylglycerol and CL. The pgs1Delta mutant exhibits severe growth defects at 37 degrees C. To understand the essential functions of mitochondrial anionic lipids at elevated temperatures, we isolated suppressors of pgs1Delta that grew at 37 degrees C. The present review summarizes our analysis of suppression of pgs1Delta growth defects by a mutant that has a loss-of-function mutation in KRE5, a gene involved in cell wall biogenesis.

Loss of Function ofKRE5Suppresses Temperature Sensitivity of Mutants Lacking Mitochondrial Anionic Lipids

Disruption of PGS1, which encodes the enzyme that catalyzes the committed step of cardiolipin (CL) synthesis, results in loss of the mitochondrial anionic phospholipids phosphatidylglycerol (PG) and CL. The pgs1Delta mutant exhibits severe growth defects at 37 degrees C. To understand the essential functions of mitochondrial anionic lipids at elevated temperatures, we isolated suppressors of pgs1Delta that grew at 37 degrees C. One of the suppressors has a loss of function mutation in KRE5, which is involved in cell wall biogenesis. The cell wall of pgs1Delta contained markedly reduced beta-1,3-glucan, which was restored in the suppressor. Stabilization of the cell wall with osmotic support alleviated the cell wall defects of pgs1Delta and suppressed the temperature sensitivity of all CL-deficient mutants. Evidence is presented suggesting that the previously reported inability of pgs1Delta to grow in the presence of ethidium bromide was due to defective cell wall integrity, not from "petite lethality." These findings demonstrated that mitochondrial anionic lipids are required for cellular functions that are essential in cell wall biogenesis, the maintenance of cell integrity, and survival at elevated temperature.