Glycerophospholipid polyunsaturation modulates resveratrol action on biomimetic membranes.

The phytoalexin resveratrol has received increasing attention for its potential to prevent oxidative damages in human organism. To shed further light on molecular mechanisms of its interaction with lipid membranes we study resveratrol influence on the organisation and mechanical properties of biomimetic lipid systems composed of synthetic phosphatidylcholines with mixed aliphatic chains and different degree of unsaturation at sn-2 position (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC, and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, PDPC). High-sensitivity isothermal titration calorimetric measurements reveal stronger spontaneous resveratrol association to polyunsaturated phosphatidylcholine bilayers compared to the monounsaturated ones resulting from hydrophobic interactions, conformational changes of the interacting species and desolvation of molecular surfaces. The latter is supported by the results from Laurdan spectroscopy of large unilamellar vesicles providing data on hydration at the glycerol backbones of glycerophospholipides. Higher degree of lipid order is reported for POPC membranes compared to PDPC. While resveratrol mostly enhances the hydration of PDPC membranes, increasing POPC dehydration is reported upon treatment with the polyphenol. Dehydration of the polyunsaturated lipid bilayers is measured only at the highest phytoalexin content studied (resveratrol/lipid 0.5 mol/mol) and is less pronounced than the effect reported for POPC membranes. The polyphenol effect on membrane mechanics is probed by thermal shape fluctuation analysis of quasispherical giant unilamellar vesicles. Markedly different trend of the bending elasticity with increasing resveratrol concentration is reported for the two types of phospholipid bilayers studied. POPC membranes become more rigid in the presence of resveratrol, whereas PDPC-containing bilayers exhibit softening at lower concentrations of the polyphenol followed by a slight growth without bilayer stiffening even at the highest resveratrol content explored. The new data on the structural organization and membrane properties of resveratrol-treated phosphatidylcholine membranes may underpin the development of future liposomal applications of the polyphenol in medicinal chemistry.

Pre-mixing of omega-3 fatty acid-containing liposomes enhances the drug release rate and therapeutic efficacy of anticancer drugs loaded in liposomes.

The therapeutic efficacy of anticancer drugs loaded in liposomes composed of rigid phosphatidylcholine (PC) is hindered by the limited release of these drugs at the tumor site, which in turn hampers delivery of the drug to its intracellular target. In an attempt to improve the therapeutic efficacy of liposomal anticancer drugs, we here explored the use of empty liposomes as "trigger" vehicles to induce drug release from drug-loaded liposomes through liposome-liposome interactions. Empty liposomes containing PC in which omega-3 fatty acids comprised both fatty acid strands (Omega-L) showed a triggering effect on drug release from doxorubicin (DOX)-loaded liposomes (Caelyx). The effectiveness of this triggered-release effect was dependent on the Omega-L composition as well as the mixing ratio of Omega-L to Caelyx. Cryo-TEM and differential calorimetry studies revealed that the Omega-L effect was associated with liposome-liposome interactions that led to loosened membrane packing and increased fluidity of Caelyx. In cultured cells, the intracellular/intranuclear DOX uptake and anticancer efficacy of Caelyx was greatly improved by Omega-L pre-mixing. Intravenous injection of rats with Caelyx, premixed with Omega-L, decreased the area under the plasma concentration-time curve from time zero to time infinity and increased clearance without significantly changing the mean residence time or terminal half-life of DOX compared with Caelyx alone. Ex vivo bioimaging showed that DOX fluorescence in tumors, but not in other organs, was significantly increased by Omega-L premixing. In the mouse xenograft model, premixing of Omega-L with Caelyx suppressed tumor growth 2.5-fold compared with Caelyx. Collectively, the data provide preliminary evidence that the Omega-L-triggered drug release that occurs before and after dosing, particularly at tumor site, improved the therapeutic efficacy of Caelyx. The simple approach described here could enhance the therapeutic value of Caelyx and other anticancer drug-loaded liposomes.

Lipid- and phospho-regulation of CTP:Phosphocholine Cytidylyltransferase α association with nuclear lipid droplets.

Fatty acids stored in triacylglycerol-rich lipid droplets are assembled with a surface monolayer composed primarily of phosphatidylcholine (PC). Fatty acids stimulate PC synthesis by translocating CTP:phosphocholine cytidylyltransferase (CCT) α to the inner nuclear membrane, nuclear lipid droplets (nLD) and lipid associated promyelocytic leukemia (PML) structures (LAPS). Huh7 cells were used to identify how CCTα translocation onto these nuclear structures are regulated by fatty acids and phosphorylation of its serine-rich P-domain. Oleate treatment of Huh7 cells increased nLDs and LAPS that became progressively enriched in CCTα. In cells expressing the phosphatidic acid phosphatase Lipin1α or 1ß, the expanded pool of nLDs and LAPS had a proportional increase in associated CCTα. In contrast, palmitate induced few nLDs and LAPS and inhibited the oleate-dependent translocation of CCTα without affecting total nLDs. Phospho-memetic or phospho-null mutations in the P-domain revealed that a 70% phosphorylation threshold, rather than site-specific phosphorylation, regulated CCTα association with nLDs and LAPS. In vitro candidate kinase and inhibitor studies in Huh7 cells identified cyclin-dependent kinase (CDK) 1 and 2 as putative P-domain kinases. In conclusion, CCTα translocation onto nLDs and LAPS is dependent on available surface area and fatty acid composition, as well as threshold phosphorylation of the P-domain potentially involving CDKs.

Oxidation-resistant nanoliposomes loaded with osteogenic peptides: Characteristics, stability and bioaccessibility.

Phosphatidylcholine (PC) oxidation leads to the fusion of nanoliposomes and leakage of containment compounds during the storage period. This study aims to improve the oxidation resistance by partially substituting PC in the osteogenic peptides (OPs) loaded nanoliposomes with hydrogenated phosphatidylcholine (HPC). The investigation assessed the characteristics, stability, and bioaccessibility of these novel nanoliposomes. By altering the PC/HPC mass ratio from 1:0 to 0:1, an increase in the encapsulation efficiency (EE), loading capacity (LC), polydispersity index (PDI), and bioaccessibility of OPs-loaded nanoliposomes was observed. Additionally, there was a decrease in thiobarbituric acid reactive substances (TBARS), peroxide value (POV), non-volatile aldehyde, and ketone. The stability of salt decreased when using HPC alone (0:1). The performance of OPs-loaded nanoliposomes with a PC/HPC mass ratio of 1:3 was found to be satisfactory in terms of storage and pH stability. Fluorescence spectroscopy, Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared spectroscopy (FTIR) revealed a tighter lipid aggregation, enhanced intermolecular van der Waals forces, and hydrogen bond formation in membranes of nanoliposomes containing HPC. The addition of HPC to the nanoliposomes delayed the release of peptides in simulated without affecting osteogenic activity. These results provide guidance for the development of oxidation-resistant nanoliposomes loaded with OPs products.

Probing the interactions of the HIV-1 matrix protein-derived polybasic region with lipid bilayers: insights from AFM imaging and force spectroscopy.

The human immunodeficiency virus type 1 (HIV-1) matrix protein contains a highly basic region, MA-HBR, crucial for various stages of viral replication. To elucidate the interactions between the polybasic peptide MA-HBR and lipid bilayers, we employed liquid-based atomic force microscopy (AFM) imaging and force spectroscopy on lipid bilayers of differing compositions. In 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, AFM imaging revealed the formation of annulus-shaped protrusions upon exposure to the polybasic peptide, accompanied by distinctive mechanical responses characterized by enhanced bilayer puncture forces. Importantly, our AFM-based force spectroscopy measurements unveiled that MA-HBR induces interleaflet decoupling within the cohesive bilayer organization. This is evidenced by a force discontinuity observed within the bilayer's elastic deformation regime. In POPC/cholesterol bilayers, MA-HBR caused similar yet smaller annular protrusions, demonstrating an intriguing interplay with cholesterol-rich membranes. In contrast, in bilayers containing anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) lipids, MA-HBR induced unique annular protrusions, granular nanoparticles, and nanotubules, showcasing its distinctive effects in anionic lipid-enriched environments. Notably, our force spectroscopy data revealed that anionic POPS lipids weakened interleaflet adhesion within the bilayer, resulting in interleaflet decoupling, which potentially contributes to the specific bilayer perturbations induced by MA-HBR. Collectively, our findings highlight the remarkable variations in how the polybasic peptide, MA-HBR, interacts with lipid bilayers of differing compositions, shedding light on its role in host membrane restructuring during HIV-1 infection.

Thermodynamic study on hydrated bilayers of ether-linked phosphatidylcholines with terminal perfluorobutyl group.

Novel terminally perfluorobutyl group-containing ether-linked phosphatidylcholines with different alkyl chain lengths (di-O-F4-Cn-PCs, n = 14,16 and 18) were developed as possible materials for stable liposomes aiming at applications of structural and functional analyses of membrane proteins. Differential scanning calorimetric investigations of the thermotropic transition of hydrated di-O-F4-Cn-PC bilayers demonstrated that the transition temperature of every di-O-F4-Cn-PC decreases by ~20 °C compared to their corresponding non-fluorinated PCs, di-O-Cn-PCs. With the elongation of the hydrophobic chain, on the other hand, the transition enthalpy (ΔH) and entropy (ΔS) increased in a linear manner. Comparison of ΔH and ΔS values against the net hydrocarbon chain length between di-O-F4-Cn-PCs and di-O-Cn-PCs strongly suggests that in the thermotropic transition of the di-O-F4-Cn-PC membrane, the perfluorobutyl segments undergo very limited structural changes; therefore, the hydrocarbon segments are mainly responsible for the phase transition.

Cryo-TEM Reveals the Influence of Multivalent Charge and PEGylation on Shape Transitions in Fluid Lipid Assemblies: From Vesicles to Discs, Rods, and Spheres.

Lipids, and cationic lipids in particular are of interest as delivery vectors for hydrophobic drugs such as the cancer therapeutic paclitaxel, and the structures of lipid assemblies affect their efficacy. We investigated the effect of incorporating the multivalent cationic lipid MVL5 (+5e) and poly(ethylene glycol)-lipids (PEG-lipids), alone and in combination, on the structure of fluid-phase lipid assemblies of the charge-neutral lipid 1,2-dioleoyl-sn-glycero-phosphocholine (DOPC). This allowed us to elucidate lipid-assembly structure correlations in sonicated formulations with high charge density, which are not accessible with univalent lipids such as the well-studied DOTAP (+1e). Cryogenic transmission electron microscopy (cryo-TEM) allowed us to determine the structure of the lipid assemblies, revealing diverse combinations of vesicles and disc-shaped, worm-like, and spherical micelles. Remarkably, MVL5 forms an essentially pure phase of disc micelles at 50 mol % MVL5. At a higher (75 mol %) content of MVL5, short- and intermediate-length worm-like micellar rods were observed, and in ternary mixtures with PEG-lipid, longer and highly flexible worm-like micelles formed. Independent of their length, the worm-like micelles coexisted with spherical micelles. In stark contrast, DOTAP forms mixtures of vesicles, disc micelles, and spherical micelles at all studied compositions, even when combined with PEG-lipids. The observed similarities and differences in the effects of charge (multivalent versus univalent) and high curvature (multivalent charge versus PEG-lipid) on the assembly structure provide insights into parameters that control the size of fluid lipid nanodiscs, relevant for future applications.

Relative Quantification of Lipid Isomers in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions and Infrared Multiphoton Dissociation.

Accurate structural identification of lipids in imaging mass spectrometry is critical to properly contextualizing spatial distributions with tissue biochemistry. Gas-phase charge inversion ion/ion reactions alter the ion type prior to dissociation to allow for more structurally informative fragmentation and improve lipid identification at the isomeric level. In this work, infrared multiphoton dissociation (IRMPD) was interfaced with a commercial hybrid Qh-FT-ICR mass spectrometer to enable the rapid fragmentation of gas-phase charge inversion ion/ion reaction products at every pixel in imaging mass spectrometry experiments. An ion/ion reaction between phosphatidylcholine (PC) monocations generated from rat brain tissue via matrix-assisted laser desorption/ionization (MALDI) and 1,4-phenylenediproprionic acid reagent dianions generated via electrospray ionization (ESI) followed by IRMPD of the resulting product ion complex produces selective fatty acyl chain cleavages indicative of fatty acyl carbon compositions in the lipid. Ion/ion reaction images using this workflow allow for mapping of the relative spatial distribution of multiple PC isomers under a single sum composition lipid identification. Lipid isomers display significantly different relative spatial distributions within rat brain tissue, highlighting the importance of resolving isomers in imaging mass spectrometry experiments.

Investigating the Effects of the POPC-POPG Lipid Bilayer Composition on PAP248-286 Binding Using CG Molecular Dynamics Simulations.

PAP248-286 is a fusogenic peptide derived from prostatic acid phosphatase, commonly found in human semen, and is known to mediate HIV fusion with cell membranes. In this study, we performed 120 independent coarse-grained molecular dynamics simulations to investigate the spontaneous binding of PAP248-286 monomers, considering both charged and neutral histidine (His) residues, to membrane bilayers composed of different lipid compositions: 100% POPC, 70% POPC-30% POPG, and 50% POPC-50% POPG. Our simulations revealed that PAP248-286 displayed spontaneous binding to the membrane, with increased binding observed in the presence of anionic lipid POPG. Specifically, in systems containing 30% and 50% POPG lipids, monomer residues, particularly in the systems containing charged histidine (His) residues, exhibited prolonged binding with the membrane. Furthermore, our simulations indicated that PAP248-286 adopted a parallel orientation with the membrane, exposing its positively charged residues to the lipid bilayer. Interestingly, systems containing charged His residues showed a higher lipid occupancy around the peptide. These findings are consistent with previous experimental data, suggesting that PAP248-286 binding is enhanced in membranes with charged His residues, resembling the conditions found in the acidic vaginal pH environment. The results of our study provide further insights into the molecular mechanisms underlying the membrane binding of PAP248-286, contributing to our understanding of its potential role in HIV fusion and infection.

Understanding the Mechanical Properties of Ultradeformable Liposomes Using Molecular Dynamics Simulations.

Improving drug delivery efficiency to solid tumor sites is a central challenge in anticancer therapeutic research. Our previous experimental study (Guo et al., Nat. Commun. 2018, 9, 130) showed that soft, elastic liposomes had increased uptake and accumulation in cancer cells and tumors in vitro and in vivo respectively, relative to rigid particles. As a first step toward understanding how liposomes' molecular structure and composition modulates their elasticity, we performed all-atom and coarse-grained classical molecular dynamics (MD) simulations of lipid bilayers formed by mixing a long-tailed unsaturated phospholipid with a short-tailed saturated lipid with the same headgroup. The former types of phospholipids considered were 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (termed here DPMPC). The shorter saturated lipids examined were 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Several lipid concentrations and surface tensions were considered. Our results show that DOPC or DPMPC systems having 25-35 mol % of the shortest lipids DHPC or DDPC are the least rigid, having area compressibility moduli KA that are ∼10% smaller than the values observed in pure DOPC or DPMPC bilayers. These results agree with experimental measurements of the stretching modulus and lysis tension in liposomes with the same compositions. These mixed systems also have lower areas per lipid and form more uneven x-y interfaces with water, the tails of both primary and secondary lipids are more disordered, and the terminal methyl groups in the tails of the long lipid DOPC or DPMPC wriggle more in the vertical direction, compared to pure DOPC or DPMPC bilayers or their mixtures with the longer saturated lipid DLPC or DMPC. These observations confirm our hypothesis that adding increasing concentrations of the short unsaturated lipid DHPC or DDPC to DOPC or DPMPC bilayers alters lipid packing and thus makes the resulting liposomes more elastic and less rigid. No formation of lipid nanodomains was noted in our simulations, and no clear trends were observed in the lateral diffusivities of the lipids as the concentration, type of secondary lipid, and surface tension were varied.

Visualization of Molecular Permeation into a Multi-compartment Phospholipid Vesicle.

Passive permeation of small molecules into vesicles with multiple compartments is a critical event in many chemical and biological processes. We consider the translocation of the peptide NAF-144-67 labeled with a fluorescent fluorescein dye across membranes of rhodamine-labeled 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) into liposomes with internal vesicles. Time-resolved microscopy revealed a sequential absorbance of the peptide in both the outer and inner micrometer vesicles that developed over a time period of minutes to hours, illustrating the spatial and temporal progress of the permeation. There is minimal perturbation of the membrane structure and no evidence for pore formation. On the basis of molecular dynamics simulations of NAF-144-67, we extended a local defect model to migration processes that include multiple compartments. The model captures the long residence time of the peptide within the membrane and the rate of permeation through the liposome and its internal compartments. Imaging experiments confirm the semi-quantitative description of the permeation of the model by activated diffusion and open the way for studies of more complex systems.

Separation of Isobaric Lipids in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions.

The diverse array of chemical compounds present in tissue samples results in many isobaric (i.e., same nominal mass) compounds in imaging mass spectrometry experiments. Adequate separation and differentiation of these compounds is necessary to ensure accurate analyte identification and avoid composite images comprising multiple compounds. High-resolution accurate mass (HRAM) measurements are able to resolve these compounds in some instances, but HRAM measurements are not always feasible depending on the instrument platform and the desired experimental time scale. Alternatively, tandem mass spectrometry (MS/MS) can be used to perform gas-phase transformations that improve molecular specificity. While conventional MS/MS methods employ collision induced dissociation (CID) to fragment compounds of interest and then analyze the product masses, gas-phase ion/ion reactions can be used to instead selectively react with desired classes of analytes. Herein, we have used gas-phase charge inversion ion/ion reactions to selectively resolve phosphatidylcholines (PCs) in isobaric lipid mixtures. A 1,4-phenylenedipropionic acid (PDPA) reagent dianion readily reacts with [M + H]+, [M + Na]+, and [M + K]+ ion types to produce demethylated product anions for each PC, [PC - CH3]-. These product anions are no longer isobaric and now differ in mass by 22 Da (protonated versus sodiated) and 16 Da (sodiated versus potassiated), respectively. This reaction has been used to differentiate isobaric lipids in the imaging mass spectrometry analysis of rat brain tissue.

Impact of transmembrane peptides on individual lipid motions and collective dynamics of lipid bilayers.

The fluid nature of lipid bilayers is indispensable for the dynamic regulation of protein function and membrane morphology in biological membranes. Membrane-spanning domains of proteins interact with surrounding lipids and alter the physical properties of lipid bilayers. However, there is no comprehensive view of the effects of transmembrane proteins on the membrane's physical properties. Here, we investigated the effects of transmembrane peptides with different flip-flop-promoting abilities on the dynamics of a lipid bilayer employing complemental fluorescence and neutron scattering techniques. The quasi-elastic neutron scattering and fluorescence experiments revealed that lateral diffusion of the lipid molecules and the acyl chain motions were inhibited by the inclusion of transmembrane peptides. The neutron spin-echo spectroscopy measurements indicated that the lipid bilayer became more rigid but more compressible and the membrane viscosity increased when the transmembrane peptides were incorporated into the membrane. These results suggest that the inclusion of rigid transmembrane structures hinders individual and collective lipid motions by slowing down lipid diffusion and increasing interleaflet coupling. The present study provides a clue for understanding how the local interactions between lipids and proteins change the collective dynamics of the lipid bilayers, and therefore, the function of biological membranes.

Transesterification of phosphatidylcholine with DHA-rich algal oil using immobilized Candida antarctica lipase B to produce DHA-phosphatidylcholine.

Docosahexaenoic acid (DHA) enriched with phospholipids (PLs) (DHA-PLs) is a type of structured PL with good physicochemical and nutritional properties. Compared to PLs and DHA, DHA-PLs has higher bioavailability and structural stability and many nutritional benefits. To improve the enzymatic synthesis of DHA-PLs, this study investigated the preparation of phosphatidylcholine (PC) enriched with DHA (DHA-PC) via enzymatic transesterification of algal oil, which is rich in DHA-triglycerides, using immobilized Candida antarctica lipase B (CALB). The optimized reaction system incorporated 31.2% DHA into the acyl chain of PC and converted 43.6% PC to DHA-PC within 72 h at 50 °C, 1:8 PC: algal oil mass ratio, 25% enzyme load (based on total substrate mass), and 0.02 g/mL molecular sieve concentration. Consequently, the side reactions of PC hydrolysis were effectively suppressed and products with high PC content (74.8%) were produced. Molecular structure analysis showed that exogenous DHA was specifically incorporated into the sn-1 site of the PC by immobilized CALB. Furthermore, the evaluation of reusability with eight cycles showed that the immobilized CALB had good operational stability in the present reaction system. Collectively, this study demonstrated the applicability of immobilized CALB as a biocatalyst for synthesizing DHA-PC and provided an improved enzyme-catalyzed method for future DHA-PL synthesis.

Phospholipid lateral diffusion in the presence of cationic peptides as measured via 31P CODEX NMR.

The effects of two cationic peptides on phospholipid lateral diffusion in binary mixtures of POPC with various anionic phospholipids were measured via 31P CODEX NMR. Large unilamellar vesicles composed of POPC/POPG (70/30 mol/mol), or POPC/DOPS (70/30 mol/mol), or POPC/TOCL (85/15 mol/mol), or POPC/DOPA (50/50 mol/mol) were exposed to either polylysine (pLYS, N = 134 monomers) or KL-14 (KKLL KKAKK LLKKL), a model amphipathic helical peptide, in an amount corresponding to 80% neutralization of the anionic phospholipid charge by the cationic lysine residues. In the absence of added peptide, phospholipid lateral diffusion coefficients (all measured at 10 °C) increased with increasing reduced temperature (T-Tm). The POPC/DOPA mixture was an exception to this generalization, in that lateral diffusion for both components was far slower than any other mixture investigated, an effect attributed to intermolecular hydrogen bonding. The addition of pLYS or KL-14 decreased lateral diffusion in the POPC/DOPS LUV, but had minimal effects in the POPC/POPG LUV, indicating that ease of access of the cationic peptide residues to the anionic phospholipid groups was important. Both cationic peptides produced the opposite effect in the POPC/DOPA case, in that lateral diffusion increased significantly in their presence, with KL-14 being most effective. This latter observation was interpreted in terms of the electrostatic / H-bond model proposed by Kooijman et al. [Journal of Biological Chemistry, 282:11356-11,364, 2007] to describe the mechanism of interaction between the phosphomonoester head group of PA and the tertiary amine of lysine.

Lipid-Specific Direct Translocation of the Cell-Penetrating Peptide NAF-144-67 across Bilayer Membranes.

The cell-penetrating peptide NAF-1 has recently emerged as a promising candidate for selective penetration and destruction of cancer cells. It displays numerous membrane-selective behaviors including cell-specific uptake and organelle-specific degradation. In this work, we explore membrane penetration and translocation of NAF-1 in model lipid bilayer vesicles as a function of lipid identity in zwitterionic phosphatidylcholine lipids mixed with anionic phosphatidylserine, phosphatidylglycerol, and phosphatidic acid lipids. By monitoring the digestion of NAF-1 using the protease trypsin located inside but not outside the vesicles, we determined that the translocation of NAF-1 was significantly enhanced by the presence of phosphatidic acid in the membrane compared to the other three anionic or zwitterionic lipids. These findings were correlated to fluorescence measurements of dansyl-labeled NAF-1, which revealed whether noncovalent interactions between NAF-1 and the bilayer were most stable either at the membrane/solution interface or within the membrane interior. Phosphatidic acid promoted interactions with fatty acid tails, while phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol stabilized interactions with polar lipid headgroups. Interfacial vibrational sum frequency spectroscopy experiments revealed that the phosphate moiety on phosphatidic acid headgroups was better hydrated than on the other three lipids, which helped to shuttle NAF-1 into the hydrophobic region. Our findings demonstrate that permeation does not depend on the net charge on phospholipid lipid headgroups in these model vesicles and suggest a model wherein NAF-1 crosses membranes selectively due to lipid-specific interactions at bilayer surfaces.

Structural Characterization and Quantitation of Ether-Linked Glycerophospholipids in Peroxisome Biogenesis Disorder Tissue by Ultraviolet Photodissociation Mass Spectrometry.

The biological impact of ether glycerophospholipids (GP) in peroxisomal disorders and other diseases makes them significant targets as biomarkers for diagnostic assays or deciphering pathology of the disorders. Ether lipids include both plasmanyl and plasmenyl lipids, which each contain an ether or a vinyl ether bond at the sn-1 linkage position, respectively. This linkage, in contrast to traditional diacyl GPs, precludes their detailed characterization by mass spectrometry via traditional collisional-based MS/MS techniques. Additionally, the isomeric nature of plasmanyl and plasmenyl pairs of ether lipids introduces a further level of complexity that impedes analysis of these species. Here, we utilize 213 nm ultraviolet photodissociation mass spectrometry (UVPD-MS) for detailed characterization of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) plasmenyl and plasmanyl lipids in mouse brain tissue. 213 nm UVPD-MS enables the successful differentiation of these four ether lipid subtypes for the first time. We couple this UVPD-MS methodology to reversed-phase liquid chromatography (RPLC) for characterization and relative quantitation of ether lipids from normal and diseased (Pex7 deficiency modeling the peroxisome biogenesis disorder, RCDP) mouse brain tissue, highlighting the ability to pinpoint specific structural features of ether lipids that are important for monitoring aberrant lipid metabolism in peroxisomal disorders.

Lipid Composition Is Critical for Accurate Membrane Permeability Prediction of Cyclic Peptides by Molecular Dynamics Simulations.

Cyclic peptides have attracted attention as a promising pharmaceutical modality due to their potential to selectively inhibit previously undruggable targets, such as intracellular protein-protein interactions. Poor membrane permeability is the biggest bottleneck hindering successful drug discovery based on cyclic peptides. Therefore, the development of computational methods that can predict membrane permeability and support elucidation of the membrane permeation mechanism of drug candidate peptides is much sought after. In this study, we developed a protocol to simulate the behavior in membrane permeation steps and estimate the membrane permeability of large cyclic peptides with more than or equal to 10 residues. This protocol requires the use of a more realistic membrane model than a single-lipid phospholipid bilayer. To select a membrane model, we first analyzed the effect of cholesterol concentration in the model membrane on the potential of mean force and hydrogen bonding networks along the direction perpendicular to the membrane surface as predicted by molecular dynamics simulations using cyclosporine A. These results suggest that a membrane model with 40 or 50 mol % cholesterol was suitable for predicting the permeation process. Subsequently, two types of membrane models containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 40 and 50 mol % cholesterol were used. To validate the efficiency of our protocol, the membrane permeability of 18 ten-residue peptides was predicted. Correlation coefficients of R > 0.8 between the experimental and calculated permeability values were obtained with both model membranes. The results of this study demonstrate that the lipid membrane is not just a medium but also among the main factors determining the membrane permeability of molecules. The computational protocol proposed in this study and the findings obtained on the effect of membrane model composition will contribute to building a schematic view of the membrane permeation process. Furthermore, the results of this study will eventually aid the elucidation of design rules for peptide drugs with high membrane permeability.

Effects of cholesterol on lipid vesicle fusion mediated by infectious salmon anaemia virus fusion peptides.

Studying the variables that affect the membrane fusion mechanism of enveloped viruses is important for developing new strategies to combat viral infections. We analysed the effects of lipid vesicle cholesterol content on membrane fusion that is facilitated by infectious salmon anaemia virus (ISAV) fusion peptides. Lipid mixing assays were performed to study membrane fusion in large unilamellar vesicles (LUV) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoylphosphatidylcholine (DPPC) and cholesterol. Lipid mixing (%) increased more over time when 0.2 µm LUV contained no cholesterol or when the LUV membranes contained 15 mol% cholesterol. The secondary structure of the ISAV fusion peptides consistently remained a ß-sheet both in water and in the presence of vesicles. Additionally, the dissociation constant (Kd) between the peptides and the lipid vesicles was obtained with different cholesterol contents. In the tests performed with lipid vesicles (0.2 µm or 0.4 µm LUV), cholesterol was found to influence membrane fusion that was facilitated by ISAV fusion peptides; however, the peptides studied did not require cholesterol in their membranes to facilitate membrane fusion in the smallest lipid vesicles (0.2 µm LUV).

Domain Size Regulation in Phospholipid Model Membranes Using Oil Molecules and Hybrid Lipids.

The formation of domains in multicomponent lipid mixtures has been suggested to play a role in moderating signal transduction in cells. Understanding how domain size may be regulated by both hybrid lipid molecules and impurities is important for understanding real biological processes; at the same time, developing model systems where domain size can be regulated is crucial to enable systematic studies of domain formation kinetics and thermodynamics. Here, we perform a model study of the effects of oil molecules, which swell the bilayer, and line-active hybrid phospholipids using a thermally induced liquid-solid phase separation in planar, free-standing lipid bilayers consisting of DOPC and DPPC (1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, respectively). The experiments show that the kinetics of domain growth are significantly affected by the type and molecular structure of the oil (squalene, hexadecane, or decane), with the main contributing factors being the degree of swelling of the bilayer and the changes in line tension induced by the different oils, with smaller domains resulting from systems with smaller values of the line tension. POPC (1-palmitoyl-sn-2-oleoyl-glycero-3-phosphocholine), on the other hand, acts as a line-active hybrid lipid, reducing the domain size when added in small amounts and slowing down domain coarsening. Finally, we show that despite the regulation of domain size by both methods, the phase transition temperature is influenced by the presence of oil molecules but not significantly by the presence of hybrid lipids. Overall, our results show how to regulate domain size in binary membrane model systems, over a wide range of length scales, by incorporating oil molecules and hybrid lipids.