Interaction of Adriamycin and N-Trifluoroacetyladriamycin-14-Valerate with Cardiolipin-Containing Lipid Bilayers

Abstract

The interaction of adriamycin (ADR) and N-Trifluoroacetyladriamycin-14-valerate (AD 32) with cardiolipin (CL)-containing multilamellar vesicles was studied by high-sensitivity differential scanning calorimetry, using liposomes formed from either dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) containing small amounts of CL. the drugs partitioned into cardiolipin-containing neutral and acidic bilayers in a manner similar to that observed earlier with CL-free bilayers. the partition coefficient of adriamycin in bilayers of vesicles prepared from DPPG or DPPG in admixture with CL was much higher than that obtained with neutral DPPC vesicles or the DPPC together with CL. Under all conditions, AD 32 was essentially completely partitioned into the lipid phase of the investigated phospholipid membranes. As expected, addition of adriamycin to CL-containing vesicles did not significantly change the thermotropic behavior of these bilayers, whereas the fluidizing effect of AD 32 was directly related to the CL content of the vesicles. the multipeak transitions produced by the anthracyclines in pure DPPG bilayers were preserved in the presence of CL, but the endotherms were broader and slightly shifted to lower temperatures, a finding indicative of stronger interactions. Chlorpromazine (CPZ), which was used as a reference compound to compare the effects produced by the anthracyclines, was found to behave similarly to AD 32 and to be more effective than quinidine (QND), in agreement with their behavior in CL-free liposomes.     

Membrane Permeabilization by Trypanosome Lytic Factor, a Cytolytic Human High Density Lipoprotein

Trypanosome lytic factor (TLF) is a subclass of human high density lipoprotein (HDL) that mediates an innate immune killing of certain mammalian trypanosomes, most notably Trypanosoma brucei brucei, the causative agent of a wasting disease in cattle. Mechanistically, killing is initiated in the lysosome of the target trypanosome where the acidic pH facilitates a membrane-disrupting activity by TLF. Here we utilize a model liposome system to characterize the membrane binding and permeabilizing activity of TLF and its protein constituents, haptoglobin-related protein (Hpr), apolipoprotein L-1 (apoL-1), and apolipoprotein A-1 (apoA-1). We show that TLF efficiently binds and permeabilizes unilamellar liposomes at lysosomal pH, whereas non-lytic human HDL exhibits inefficient permeabilizing activity. Purified, delipidated Hpr and apoL-1 both efficiently permeabilize lipid bilayers at low pH. Trypanosome lytic factor, apoL-1, and apoA-1 exhibit specificity for anionic membranes, whereas Hpr permeabilizes both anionic and zwitterionic membranes. Analysis of the relative particle sizes of susceptible liposomes reveals distinctly different membrane-active behavior for native TLF and the delipidated protein components. We propose that lysosomal membrane damage in TLF-susceptible trypanosomes is initiated by the stable association of the TLF particle with the lysosomal membrane and that this is a property unique to this subclass of human HDL.High density lipoproteins (HDL)² are complex yet ordered macromolecules consisting of characteristic proteins embedded in a phospholipid monolayer that surrounds a hydrophobic core of esterified cholesterol and triglycerides. A subclass of HDL is responsible for an innate immune killing of the African blood stream parasite Trypanosoma brucei brucei (1–3), and very recently, has been shown to be cytotoxic to intracellular Leishmania promastigotes (4). The trypanolytic HDL particle, termed trypanosome lytic factor (TLF), is characterized by the presence of two proteins, apolipoprotein L-1 (apoL-1) and haptoglobin-related protein (Hpr), as well as the HDL ubiquitous apolipoprotein A-1 (apoA-1) (1, 5–7). Killing of the susceptible parasite involves high affinity binding to a cell-surface receptor, endocytosis, and trafficking of the TLF particle to the lysosome (8–12). The acidic lysosomal environment facilitates a membrane-disrupting activity by the TLF particle and subsequent cell death (9, 13). It has been shown that purified, delipidated apoL-1 or Hpr are sufficient for trypanosome killing. When these proteins are incorporated into the same lipoprotein particle, a several hundredfold increase in killing activity is exhibited (5). In addition, Molina-Portela et al. (14) show that maximal protection against T. b. brucei in a transgenic mouse model requires the expression of human Hpr, apoL-1, and apoA-1, supporting a synergistic mode of action.Haptoglobin-related protein evolved during primate evolution and is restricted to apes, old world monkeys, and humans (15). Haptoglobin-related protein is highly similar (92%) to the acute phase serum protein haptoglobin (Hp) (16). All mammals use Hp as a scavenger of hemoglobin (Hb) released during hemolysis associated with infection or trauma. Haptoglobin binds cell-free Hb with high affinity and facilitates its removal from the circulation through a receptor-mediated process in the liver (17). Like Hp, Hpr binds free Hb, yet this Hpr·Hb complex is not recognized by the requisite receptors in mammals and is thus not removed from the circulation (18). TLF uptake by susceptible trypanosomes requires specific binding to an Hpr·Hb complex that facilitates trafficking of the TLF particle to the lysosome (10). It has been proposed that once inside the lysosomal compartment, Hpr·Hb contributes directly to membrane disruption through the generation of oxygen radicals with the bound Hb providing the iron necessary for Fenton chemistry (7, 10, 19).Apolipoprotein L-1 is a unique member of the apolipoprotein L protein family in that, unlike the remaining apoL proteins, it possesses an N-terminal signal sequence and is thus secreted from cells. As is the case for Hpr, apoL-1 appeared during primate evolution (20–22). Within the circulation of primates, apoL-1 is exclusively associated with HDL, and the majority of the protein is in the TLF subclass (5). The apoL family members are all predicted to adopt amphipathic α-helical conformations, suggesting that their physiological role involves membrane interaction (20). Apolipoprotein L-1 shares limited homology with channel-forming colicins and, consistent with this observation, has been shown to function as an ion channel when incorporated into lipid bilayers (23).The ultimate fate of TLF-targeted lysosomal membranes is not firmly established. Several studies employing both in vivo cellular analysis and artificial membrane systems address this point with conflicting results. Electron microscopy studies with gold-conjugated TLF revealed accumulation of TLF in intracellular vesicles and subsequent vesicle membrane breakdown and appearance of gold particles in the cytoplasm (9). Widener et al. (10) observed efflux of lysosomally localized large molecular mass dextrans (500 kDa) in TLF-treated T. b. brucei. These data suggest that the lysosomal membrane experiences large scale disruption. In contrast, Perez-Morga et al. (23) and Vanhollebeke et al. (24) report uncontrollable lysosomal swelling in susceptible trypanosomes treated with normal human serum, suggesting stability of the lamellar structure of the lysosomal membrane after TLF attack. Swelling is attributed to apoL-1-mediated influx of Cl^– ions and concomitant osmotic flow of water into the lysosome. However, Molina-Portela et al. (25) observed the formation of cation-selective pores in TLF-treated planar lipid bilayers composed of trypanosome lipids. The diversity of activities reported for TLF and normal human serum may reflect the packaging of multiple toxins within the same complex that can act synergistically to provide optimal killing activity (5, 14).Here we utilize model liposomes to monitor the membrane activity of TLF and its protein constituents. We describe the effects of TLF, delipidated Hpr, apoL-1, and apoA-1 on the permeability of unilamellar liposomes. Additionally, we show that TLF, apoL-1, and apoA-1 exhibit lipid specificity and that Hpr, apoL-1, and apoA-1 induce large scale changes in the geometry of liposomes. These results provide a molecular basis for the recognition of lysosomal membranes by this toxic HDL and support a multicomponent mechanism for trypanosome killing.     

Interspecies Variation in the Functional Consequences of Mutation of Cytochrome c

The naturally occurring human cytochrome c variant (G41S) is associated with a mild autosomal dominant thrombocytopenia (Thrombocytopenia Cargeeg) caused by dysregulation of platelet production. The molecular basis of the platelet production defect is unknown. Despite high conservation of cytochrome c between human and mouse (91.4% identity), introducing the G41S mutation into mouse cytochrome c in a knockin mouse (Cycs ^G41S/G41S) did not recapitulate the low platelet phenotype of Thrombocytopenia Cargeeg. While investigating the cause of this disparity we found a lack of conservation of the functional impact of cytochrome c mutations on caspase activation across species. Mutation of cytochrome c at residue 41 has distinct effects on the ability of cytochrome c to activate caspases depending on the species of both the cytochrome c and its binding partner Apaf-1. In contrast to our previous results showing the G41S mutation increases the ability of human cytochrome c to activate caspases, here we find this activity is decreased in mouse G41S cytochrome c. Additionally unlike wildtype human cytochrome c, G41S cytochrome c is unable to activate caspases in Xenopus embryo extracts. Taken together these results demonstrate a previously unreported species-specific component to the interaction of cytochrome c with Apaf-1. This suggests that the electrostatic interaction between cytochrome c and Apaf-1 is not the sole determinant of binding, with additional factors controlling binding specificity and affinity. These results have important implications for studies of the effects of cytochrome c mutations on the intrinsic apoptosis pathway.     

Regulation of Lytic and Non-Lytic Functions of Gasdermin Pores

Pyroptosis is a necrotic form of cell death that was initially found to be induced upon activation of inflammatory caspases by inflammasome complexes. Mechanistically, pyroptosis induction requires cleavage of the caspase substrate gasdermin D (GSDMD), and the release of the GSDMD N-terminal fragment, which targets the plasma membrane to form large β-barrel pores. GSDMD shares this pore-forming ability with other gasdermin family members, which induce pyroptosis during infection or upon treatment with chemotherapy drugs. While induction of cell death has been assumed to be the main function of the gasdermin pores, increasing evidence suggests that these pores have non-lytic functions, such as in releasing cytokines or alarmins and in regulating intracellular signaling via ionic fluxes. Here we discuss how gasdermin pore formation is regulated to induce membrane permeabilization or lysis, how gasdermin pores achieve specificity for cargo-release and how cells repair gasdermin-induced damage to the plasma membrane.     

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。     

Domain-Swapped Dimer of Pseudomonas aeruginosa Cytochrome c 551: Structural Insights into Domain Swapping of Cytochrome c Family Proteins

Cytochrome c (cyt c) family proteins, such as horse cyt c, Pseudomonas aeruginosa cytochrome c ₅₅₁ (PA cyt c ₅₅₁), and Hydrogenobacter thermophilus cytochrome c ₅₅₂ (HT cyt c ₅₅₂), have been used as model proteins to study the relationship between the protein structure and folding process. We have shown in the past that horse cyt c forms oligomers by domain swapping its C-terminal helix, perturbing the Met–heme coordination significantly compared to the monomer. HT cyt c ₅₅₂ forms dimers by domain swapping the region containing the N-terminal α-helix and heme, where the heme axial His and Met ligands belong to different protomers. Herein, we show that PA cyt c ₅₅₁ also forms domain-swapped dimers by swapping the region containing the N-terminal α-helix and heme. The secondary structures of the M61A mutant of PA cyt c ₅₅₁ were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping. The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins. These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins.     

SERR Spectroelectrochemical Study of Cytochrome cd 1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c 552 on Biocompatible Metal Electrodes

Cytochrome cd1 nitrite reductases (cd ₁NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd ₁NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd ₁NiR from Marinobacter hydrocarbonoclasticus (Mhcd ₁) depends on the presence of its physiological redox partner, cytochrome c ₅₅₂ (cyt c ₅₅₂), we show here a detailed surface enhanced resonance Raman characterization of Mhcd ₁ and cyt c ₅₅₂ attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd ₁ and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd ₁ // cyt c ₅₅₂ and Ag // SAM // cyt c ₅₅₂ // Mhcd ₁ constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c ₅₅₂ retains its native properties, while the redox potential of apparently intact Mhcd ₁ undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd ₁, reinforcing the idea that subtle and very specific interactions between Mhcd ₁ and cyt c ₅₅₂ govern efficient intermolecular electron transfer and catalytic activity of Mhcd ₁.     

Structural Changes of Surfactant Protein A Induced by Cations Reorient the Protein on Lipid Bilayers

Surfactant protein A (SP-A) is an octadecameric hydrophilic glycoprotein and is the major protein component of pulmonary surfactant. This protein complex plays several roles in the body, such as regulation of surfactant secretion, recycling and adsorption of surfactant lipids, and non-serum-induced immune response. Many of SP-A's activities are dependent upon the presence of cations, especially calcium. Here, we have studiedin vitrothe effect of cations on the interaction of purified bovine SP-A with phospholipid vesicles made of dipalmitoylphosphatidylcholine and unsaturated phosphatidylcholine. We have found that SP-A octadecamers exist in an “opened-bouquet” conformation in the absence of cations and interact with lipid membranes via one or two globular headgroups. Calcium-induced structural changes in SP-A lead to the formation of a clearly identifiable stem in a “closed-bouquet” conformation. This change, in turn, seemingly results in all of SP-A's globular headgroups interacting with the lipid membrane surface and with the stem pointing away from the membrane surface. These results represent direct evidence that the headgroups of SP-A (comprising carbohydrate recognition domains), and not the stem (comprising the amino-terminus and collagen-like region), interact with lipid bilayers. Our data support models of tubular myelin in which the headgroups, not the tails, interact with the lipid walls of the lattice.     

Structure of Oriented Lipid Bilayers

X-ray diffraction studies show that lipid hydrocarbon chains are uniformly packed in bilayers and oriented so that their free ends are near the centre. This provides a model for biological membranes.     

Curcumin Mitigates the Intracellular Lipid Deposit Induced by Antipsychotics In Vitro

Scope

First- and second-generation antipsychotics (FGAs and SGAs, respectively), both inhibit cholesterol biosynthesis and impair the intracellular cholesterol trafficking, leading to lipid accumulation in the late endosome/lysosome compartment. In this study we examined if curcumin, a plant polyphenol that stimulates exosome release, can alleviate antipsychotic-induced intracellular lipid accumulation.

Methods

HepG2 hepatocarcinoma cells were treated with antipsychotics or placebo and DiI-labelled LDL for 18 h and then exposed to curcumin for the last 2 h. Cells and media were collected separately and used for biochemical analyses, electron microscopy and immunocytochemistry. Exosomes were isolated from the incubation medium by ultracentrifugation.

Results

Curcumin treatment reduced the number of heterolysosomes and shifted their subcellular localization to the periphery, as revealed by electron microscopy, and stimulated the release of lysosomal β-hexosaminidase and exosome markers flotillin-2 and CD63 into the media. The presence of DiI in exosomes released by cells preloaded with DiI-LDL demonstrated the endolysosomal origin of the microvesicles. Furthermore, curcumin increased the secretion of cholesterol as well as LDL-derived DiI and [³H]-cholesterol, in association with a decrease of intracellular lipids. Thus, the disruption of lipid trafficking induced by FGAs or SGAs can be relieved by curcumin treatment. This polyphenol, however, did not mitigate the reduction of cholesterol esterification induced by antipsychotics.

Conclusion

Curcumin stimulates exosome release to remove cholesterol (and presumably other lipids) accumulated within the endolysosomal compartment, thereby normalizing intracellular lipid homeostasis. This action may help minimize the adverse metabolic effects of antipsychotic treatment, which should now be evaluated in clinical trials.     

Surface Electrostatics of Lipid Bilayers by EPR of a pH-Sensitive Spin-Labeled Lipid

Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids’ polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes.     

Penetration of Cell Membranes and Synthetic Lipid Bilayers by Nanoprobes

Nanoscale devices have been proposed as tools for measuring and controlling intracellular activity by providing electrical and/or chemical access to the cytosol. Unfortunately, nanostructures with diameters of 50–500 nm do not readily penetrate the cell membrane, and rationally optimizing nanoprobes for cell penetration requires real-time characterization methods that are capable of following the process of membrane penetration with nanometer resolution. Although extensive work has examined the rupture of supported synthetic lipid bilayers, little is known about the applicability of these model systems to living cell membranes with complex lipid compositions, cytoskeletal attachment, and membrane proteins. Here, we describe atomic force microscopy (AFM) membrane penetration experiments in two parallel systems: live HEK293 cells and stacks of synthetic lipid bilayers. By using the same probes in both systems, we were able to clearly identify membrane penetration in synthetic bilayers and compare these events with putative membrane penetration events in cells. We examined membrane penetration forces for three tip geometries and 18 chemical modifications of the probe surface, and in all cases the median forces required to penetrate cellular and synthetic lipid bilayers with nanoprobes were greater than 1 nN. The penetration force was sensitive to the probe's sharpness, but not its surface chemistry, and the force did not depend on cell surface or cytoskeletal properties, with cells and lipid stacks yielding similar forces. This systematic assessment of penetration under various mechanical and chemical conditions provides insights into nanoprobe-cell interactions and informs the design of future intracellular nanoprobes.     

Penetration of Cell Membranes and Synthetic Lipid Bilayers by Nanoprobes

Nanoscale devices have been proposed as tools for measuring and controlling intracellular activity by providing electrical and/or chemical access to the cytosol. Unfortunately, nanostructures with diameters of 50–500 nm do not readily penetrate the cell membrane, and rationally optimizing nanoprobes for cell penetration requires real-time characterization methods that are capable of following the process of membrane penetration with nanometer resolution. Although extensive work has examined the rupture of supported synthetic lipid bilayers, little is known about the applicability of these model systems to living cell membranes with complex lipid compositions, cytoskeletal attachment, and membrane proteins. Here, we describe atomic force microscopy (AFM) membrane penetration experiments in two parallel systems: live HEK293 cells and stacks of synthetic lipid bilayers. By using the same probes in both systems, we were able to clearly identify membrane penetration in synthetic bilayers and compare these events with putative membrane penetration events in cells. We examined membrane penetration forces for three tip geometries and 18 chemical modifications of the probe surface, and in all cases the median forces required to penetrate cellular and synthetic lipid bilayers with nanoprobes were greater than 1 nN. The penetration force was sensitive to the probe''s sharpness, but not its surface chemistry, and the force did not depend on cell surface or cytoskeletal properties, with cells and lipid stacks yielding similar forces. This systematic assessment of penetration under various mechanical and chemical conditions provides insights into nanoprobe-cell interactions and informs the design of future intracellular nanoprobes.     

Peptide Nanopores and Lipid Bilayers: Interactions by Coarse-Grained Molecular-Dynamics Simulations

A set of 49 protein nanopore-lipid bilayer systems was explored by means of coarse-grained molecular-dynamics simulations to study the interactions between nanopores and the lipid bilayers in which they are embedded. The seven nanopore species investigated represent the two main structural classes of membrane proteins (α-helical and β-barrel), and the seven different bilayer systems range in thickness from ∼28 to ∼43 Å. The study focuses on the local effects of hydrophobic mismatch between the nanopore and the lipid bilayer. The effects of nanopore insertion on lipid bilayer thickness, the dependence between hydrophobic thickness and the observed nanopore tilt angle, and the local distribution of lipid types around a nanopore in mixed-lipid bilayers are all analyzed. Different behavior for nanopores of similar hydrophobic length but different geometry is observed. The local lipid bilayer perturbation caused by the inserted nanopores suggests possible mechanisms for both lipid bilayer-induced protein sorting and protein-induced lipid sorting. A correlation between smaller lipid bilayer thickness (larger hydrophobic mismatch) and larger nanopore tilt angle is observed and, in the case of larger hydrophobic mismatches, the simulated tilt angle distribution seems to broaden. Furthermore, both nanopore size and key residue types (e.g., tryptophan) seem to influence the level of protein tilt, emphasizing the reciprocal nature of nanopore-lipid bilayer interactions.     

Membrane Permeabilization Induced by Sphingosine: Effect of Negatively Charged Lipids

Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1, 3-diol] is the most common sphingoid long chain base in sphingolipids. It is the precursor of important cell signaling molecules, such as ceramides. In the last decade it has been shown to act itself as a potent metabolic signaling molecule, by activating a number of protein kinases. Moreover, sphingosine has been found to permeabilize phospholipid bilayers, giving rise to vesicle leakage. The present contribution intends to analyze the mechanism by which this bioactive lipid induces vesicle contents release, and the effect of negatively charged bilayers in the release process. Fluorescence lifetime measurements and confocal fluorescence microscopy have been applied to observe the mechanism of sphingosine efflux from large and giant unilamellar vesicles; a graded-release efflux has been detected. Additionally, stopped-flow measurements have shown that the rate of vesicle permeabilization increases with sphingosine concentration. Because at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g., bilayers containing phosphatidic acid together with sphingomyelins, phosphatidylethanolamine, and cholesterol) gives rise to a release of vesicular contents, faster than with electrically neutral bilayers. Furthermore, phosphorous 31-NMR and x-ray data show the capacity of sphingosine to facilitate the formation of nonbilayer (cubic phase) intermediates in negatively charged membranes. The data might explain the pathogenesis of Niemann-Pick type C1 disease.     

Lipid Clustering Correlates with Membrane Curvature as Revealed by Molecular Simulations of Complex Lipid Bilayers

Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP₂), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP₂, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.     

Membrane Permeabilization Induced by Sphingosine: Effect of Negatively Charged Lipids

Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1, 3-diol] is the most common sphingoid long chain base in sphingolipids. It is the precursor of important cell signaling molecules, such as ceramides. In the last decade it has been shown to act itself as a potent metabolic signaling molecule, by activating a number of protein kinases. Moreover, sphingosine has been found to permeabilize phospholipid bilayers, giving rise to vesicle leakage. The present contribution intends to analyze the mechanism by which this bioactive lipid induces vesicle contents release, and the effect of negatively charged bilayers in the release process. Fluorescence lifetime measurements and confocal fluorescence microscopy have been applied to observe the mechanism of sphingosine efflux from large and giant unilamellar vesicles; a graded-release efflux has been detected. Additionally, stopped-flow measurements have shown that the rate of vesicle permeabilization increases with sphingosine concentration. Because at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g., bilayers containing phosphatidic acid together with sphingomyelins, phosphatidylethanolamine, and cholesterol) gives rise to a release of vesicular contents, faster than with electrically neutral bilayers. Furthermore, phosphorous 31-NMR and x-ray data show the capacity of sphingosine to facilitate the formation of nonbilayer (cubic phase) intermediates in negatively charged membranes. The data might explain the pathogenesis of Niemann-Pick type C1 disease.     

The Mechanism of Detergent Solubilization of Lipid Bilayers

Multiple data are available on the self-assembly of mixtures of bilayer-forming amphiphiles, particularly phospholipids and micelle-forming amphiphiles, commonly denoted detergents. The structure of such mixed assemblies has been thoroughly investigated, described in phase diagrams, and theoretically rationalized in terms of the balance between the large spontaneous curvature of the curvophilic detergent and the curvophobic phospholipids. In this critical review, we discuss the mechanism of this process and try to explain the actual mechanism involved in solubilization. Interestingly, membrane solubilization by some detergents is relatively slow and the common attribute of these detergents is that their trans-bilayer movement, commonly denoted flip-flop, is very slow. Only detergents that can flip into the inner monolayer cause relatively rapid solubilization of detergent-saturated bilayers. This occurs via the following sequence of events: 1), relatively rapid penetration of detergent monomers into the outer monolayer; 2), trans-membrane equilibration of detergent monomers between the two monolayers; 3), saturation of the bilayer by detergents and consequent permeabilization of the membrane; and 4), transition of the whole bilayer to thread-like mixed micelles. When the detergent cannot flip to the inner monolayer, the outer monolayer becomes unstable due to mass imbalance between the monolayers and inclusion of the curvophilic detergent molecules in a flat surface. Consequently, the outer monolayer forms mixed micellar structures within the outer monolayer. Shedding of these micelles into the aqueous solution results in partial solubilization. The consequent leakage of detergent into the liposome results in trans-membrane equilibration of detergent and subsequent micellization through the rapid bilayer-saturation mechanism.     

Interaction of Tea Catechin (—)-Epigallocatechin Gallate with Lipid Bilayers

A major component of green tea extracts, catechin (—)-Epigallocatechin gallate (EGCg), has been reported to be biologically active and interacting with membranes. A recent study reported drastic effects of EGCg on giant unilamellar vesicles (GUVs). In particular, EGCg above 30 μM caused GUVs to burst. Here we investigated the effect of EGCg on single GUVs at lower concentrations, believing that its molecular mechanism would be more clearly revealed. We used the micropipette aspiration method, by which the changes of surface area and volume of a GUV could be measured as a result of interaction with EGCg. We also used x-ray diffraction to measure the membrane thinning effect by EGCg. To understand the property of EGCg, we compared its effect with other membrane-active molecules, including pore-forming peptide magainin, the turmeric (curry) extract curcumin, and detergent Triton X100. We found the effect of EGCg somewhat unique. Although EGCg readily binds to lipid bilayers, its membrane area expansion effect is one order of magnitude smaller than curcumin. EGCg also solubilizes lipid molecules from lipid bilayers without forming pores, but its effect is different from that of Triton X100.