Structural basis of lipid head group entry to the Kennedy pathway by FLVCR1.

Phosphatidylcholine and phosphatidylethanolamine, the two most abundant phospholipids in mammalian cells, are synthesized de novo by the Kennedy pathway from choline and ethanolamine, respectively1-6. Despite the essential roles of these lipids, the mechanisms that enable the cellular uptake of choline and ethanolamine remain unknown. Here we show that the protein encoded by FLVCR1, whose mutation leads to the neurodegenerative syndrome posterior column ataxia and retinitis pigmentosa7-9, transports extracellular choline and ethanolamine into cells for phosphorylation by downstream kinases to initiate the Kennedy pathway. Structures of FLVCR1 in the presence of choline and ethanolamine reveal that both metabolites bind to a common binding site comprising aromatic and polar residues. Despite binding to a common site, FLVCR1 interacts in different ways with the larger quaternary amine of choline in and with the primary amine of ethanolamine. Structure-guided mutagenesis identified residues that are crucial for the transport of ethanolamine, but dispensable for choline transport, enabling functional separation of the entry points into the two branches of the Kennedy pathway. Altogether, these studies reveal how FLVCR1 is a high-affinity metabolite transporter that serves as the common origin for phospholipid biosynthesis by two branches of the Kennedy pathway.

Variable Non-Gaussian Transport of Nanoplastic on Supported Lipid Bilayers in Saline Conditions.

Nanoplastic-lipid interaction is vital to understanding the nanoscale mechanism of plastic adsorption and aggregation on a lipid membrane surface. However, a single-particle mechanistic picture of the nanoplastic transport process on a lipid surface remains unclear. Here, we report a salt-dependent non-Gaussian transport mechanism of polystyrene particles on a supported 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) lipid bilayer surface. Particle stickiness on the POPC surface increases with salt concentration, where the particles stay longer at the surface and diffuse to shorter distances. Additionally, a non-Gaussian diffusion state dominates the transport process at high salt concentrations. Our current study provides insight into the transport mechanism of polystyrene (PS) particles on supported lipid membranes, which is essential to understanding fundamental questions regarding the adsorption mechanisms of nanoplastics on lipid surfaces.

Relative biomembrane fusogenicities of the tumor-selective liposomes of RGDK- and CGKRK-lipopeptides.

Cancer is the second leading cause of death globally after heart diseases. Currently used highly cytotoxic anti-cancer drugs not only kill cancer cells but also often kill non-cancerous healthy body cells, causing adverse side effects. Efforts are now being directed towards developing tumor-selective chemotherapy. Tumor/tumor endothelial cell selective peptide ligands are being covalently grafted onto the exo-surfaces of drug carriers such as liposomes, polymers, etc. A number of prior studies used conjugation of tumor/tumor endothelial cell-selective RGDK- or CGKRK-peptide ligands on the outer surfaces of liposomes, metal-based nanoparticles, single walled carbon nanotubes (SWNTs), etc. However, studies aimed at examining the relative cell membrane fusogenicities and the relative degrees of cellular uptake for the RGDK- and CGKRK-ligand-grafted nanometric drug carriers have not yet been undertaken. Herein, using the widely used liposomes of DOPC, DOPE, DOPS and cholesterol (45 : 25 : 20 : 15, w/w ratio) as the model biomembranes and the fluorescence resonance energy transfer (FRET) assay for measuring membrane fusogenicities, we show that the liposomes of the RGDK-lipopeptide are more biomembrane fusogenic than the liposomes of the CGKRK-lipopeptide. Notably, such FRET assay-derived relative biomembrane fusogenicities of the liposomes of RGDK- and CGKRK-lipopeptides were found to be consistent with their relative degrees of cellular uptake in cultured cancer cells. The present findings open the door for undertaking in-depth in vivo studies aimed at evaluating the relative therapeutic potential of different nanocarriers of drugs/genes/siRNA having tumor-targeting RGDK- and CGKRK-peptides on their exo-surfaces.

Spatial Arrangement of the Drug Ibuprofen in a Model Membrane in the Presence of Lipid Rafts.

Many pharmaceutical drugs are known to interact with lipid membranes through nonspecific molecular interactions, which affect their therapeutic effect. Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID) and one of the most commonly prescribed. In the presence of cholesterol, lipid bilayers can separate into nanoscale liquid-disordered and liquid-ordered structures, the latter known as lipid rafts. Here, we study spin-labeled ibuprofen (ibuprofen-SL) in the model membrane consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol in the molar ratio of (0.5-0.5xchol)/(0.5-0.5xchol)/xchol. Electron paramagnetic resonance (EPR) spectroscopy is employed, along with its pulsed version of double electron-electron resonance (DEER, also known as PELDOR). The data obtained indicate lateral lipid-mediated clustering of ibuprofen-SL molecules with a local surface density noticeably larger than that expected for random lateral distribution. In the absence of cholesterol, the data can be interpreted as indicating alternating clustering in two opposing leaflets of the bilayer. In the presence of cholesterol, for xchol ≥ 20 mol %, the results show that ibuprofen-SL molecules have a quasi-regular lateral distribution, with a "superlattice" parameter of ∼3.0 nm. This regularity can be explained by the entrapment of ibuprofen-SL molecules by lipid rafts known to exist in this system with the additional assumption that lipid rafts have a nanoscale substructure.

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.

Pulsed Direct Current Arc-Induced Nanoelectrospray Ionization Mass Spectrometry.

This study explores the innovative field of pulsed direct current arc-induced nanoelectrospray ionization mass spectrometry (DCAI-nano-ESI-MS), which utilizes a low-temperature direct current (DC) arc to induce ESI during MS analyses. By employing a 15 kV output voltage, the DCAI-nano-ESI source effectively identifies various biological molecules, including angiotensin II, bradykinin, cytochrome C, and soybean lecithin, showcasing impressive analyte signals and facilitating multicharge MS in positive- and negative-ion modes. Notably, results show that the oxidation of fatty acids using a DC arc produces [M + O - H]- ions, which aid in identifying the location of C═C bonds in unsaturated fatty acids and distinguishing between isomers based on diagnostic ions observed during collision-induced dissociation tandem MS. This study presents an approach for identifying the sn-1 and sn-2 positions in phosphatidylcholine using phosphatidylcholine and nitrate adduct ions, accurately determining phosphatidylcholine molecular configurations via the Paternò-Büchi reaction. With all the advantages above, DCAI-nano-ESI holds significant promise for future analytical and bioanalytical applications.

Cholesterol Changes Interfacial Water Alignment in Model Cell Membranes.

The nanoscopic layer of water that directly hydrates biological membranes plays a critical role in maintaining the cell structure, regulating biochemical processes, and managing intermolecular interactions at the membrane interface. Therefore, comprehending the membrane structure, including its hydration, is essential for understanding the chemistry of life. While cholesterol is a fundamental lipid molecule in mammalian cells, influencing both the structure and dynamics of cell membranes, its impact on the structure of interfacial water has remained unknown. We used surface-specific vibrational sum-frequency generation spectroscopy to study the effect of cholesterol on the structure and hydration of monolayers of the lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and egg sphingomyelin (SM). We found that for the unsaturated lipid DOPC, cholesterol intercalates in the membrane without significantly changing the orientation of the lipid tails and the orientation of the water molecules hydrating the headgroups of DOPC. In contrast, for the saturated lipids DPPC and SM, the addition of cholesterol leads to clearly enhanced packing and ordering of the hydrophobic tails. It is also observed that the orientation of the water hydrating the lipid headgroups is enhanced upon the addition of cholesterol. These results are important because the orientation of interfacial water molecules influences the cell membranes' dipole potential and the strength and specificity of interactions between cell membranes and peripheral proteins and other biomolecules. The lipid nature-dependent role of cholesterol in altering the arrangement of interfacial water molecules offers a fresh perspective on domain-selective cellular processes, such as protein binding.

How Does the Antibiotic Amphotericin B Enter Membranes and What Does It Do There?

Amphotericin B is a popular antifungal antibiotic, but the exact way it works is still a matter of debate. Here, we used monolayers composed of phosphatidylcholine with ergosterol as a model of fungal lipid membranes to study drug incorporation from the aqueous phase and analyze the molecular reorganization of membranes underlying the biological activity of the antibiotic. The results show that the internalization of antibiotic molecules into membranes occurs only in the presence of ergosterol in the lipid phase. Comparison of images of solid-supported monolayers obtained by atomic force microscopy and lifetime imaging fluorescence microscopy shows the formation of intramembrane clusters of various sizes in the lipid phase, consisting mainly of antibiotic dimers and relatively large membrane pores (∼15 nm in diameter). The results reveal multiple modes of action of amphotericin B, acting simultaneously, each of which adversely affects the structural properties of the lipid membranes and their physiological functionality.

Effects of the Molecular Structure of Malodor Substances and Their Masking on 1,2-Dioleoyl-sn-glycero-3-phosphocholine Molecular Layers.

Certain odors have been shown not only to cause health problems and stress but also to affect skin barrier function. Therefore, it is important to understand olfactory masking to develop effective fragrances to mask malodors. However, olfaction and olfactory masking mechanisms are not yet fully understood. To understand the mechanism of the masking effect that has been studied, the responses of several target substance (TS) molecules-1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) mixed molecular layers to odorant (OD) molecules were examined as a simple experimental model of epithelial cellular membranes injured by TS molecules. Here, we examined trans-2-nonenal, 1-nonanal, trans-2-decenal, and 1-decanal as TS molecules to clarify the effects of double bonds and hydrocarbon chain lengths on the phospholipid molecular layer. In addition, benzaldehyde and cyclohexanecarboxaldehyde were utilized as OD molecules to clarify the masking effect of the aromatic ring. Surface pressure (Π)-area (A) isotherms were measured to clarify the adsorption or desorption of TS and OD molecules on the DOPC molecular layer. In addition, Fourier transform infrared spectroscopy was performed to clarify the interactions among DOPC, TS, and OD molecules. We found that TS molecules with and without double bonds had different effects on the DOPC molecular layer and that molecules with shorter chain lengths had greater effects on the DOPC molecular layer. Furthermore, OD molecules with aromatic rings counteracted the effects of the TS molecules. On the basis of this research, not only a detailed mechanism by which odor molecules affect lipid membranes without mediating olfactory receptors is elucidated but also more effective OD molecules with masking effects are proposed.

Hydrogenated Soy Phosphatidylcholine Liposomes Stimulate Differential Expression of Chemokines And Cytokines by Rat Alveolar Macrophages In Vitro.

Liposomes are being developed as inhalable drug delivery systems, but concerns remain about their impact on the lungs. To better understand the impact of liposomes and their physicochemical properties on alveolar macrophages, the cytokine and chemokine expression profile of rat alveolar Nr8383 macrophages exposed to 0.1 and 1 mg/ml hydrogenated soy phosphatidylcholine (HSPC) liposomes was examined. Expression patterns varied considerably between liposomes in a concentration-dependent manner, with both anti- and pro-inflammatory chemokines/cytokines produced. Uncharged liposomes induce the greatest production of cytokines and chemokines, followed by PEGylated liposomes. The most significant increase in cytokine/chemokine expression was seen for IL-2 (up to 24-fold), IL-4 (up to 5-fold), IL-18 and VEGF (up to 10-fold), while liposome exposure significantly reduced MIP1 expression (5-fold). In summary, we demonstrate that liposome surface properties promote variable patterns of cytokine and chemokine secretion by alveolar macrophages. This suggests that the type of liposome employed may influence the type of immune response generated in the lung and by extension, dictate how inhaled liposomal nanomedicines affect the lungs response to inhaled toxicants and local infections.

Exploring the Physical Properties of Lipid Membranes with Polyhydroxy Oxanorbornane Head Group Using NBD-Conjugated and DPH Fluorescent Probes.

The present study focuses on exploring the physical properties of lipid membranes based on the polyhydroxy oxanorbornane (PH-ONB) headgroup, designed as synthetic analogues of naturally occurring archaeal lipid membranes. Specifically, we study two variants of PH-ONB headgroup-based lipids differing in the number of hydroxy groups present in the headgroup, with one having two hydroxy groups (ONB-2OH) and the other having three (ONB-3OH). These lipids form stable bilayer membranes. The study begins with a comprehensive analysis of the fluorescence characteristics of nitrobenzoxadiazole (NBD)-tagged ONB-based lipids in different solvent environments and within a model lipid membrane 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Subsequently, the physical properties of the ONB-based membranes were examined by using an NBD-tagged ONB-based probe and a commonly used extrinsic 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescent probe. The steady-state and time-resolved fluorescence properties of the NBD-tagged ONB-based probe and DPH were used to compare the physical properties of the ONB-based membranes, including polarity, fluidity, phase transition, order, hydration, location, heterogeneity, and rotational diffusion. The solid gel to liquid crystalline phase transition temperatures of ONB-2OH and ONB-3OH lipid membranes are found to be (68 ± 1) °C and (74 ± 1) °C, respectively. The variation in organization (size), fluidity, and phase transition temperature of ONB-based lipid membranes is explained by the extent of hydrogen bonding interactions between lipid head groups. ONB-based membranes exhibit characteristics similar to those of phospholipid membranes and possess a notably high phase transition temperature. These properties make them a promising and cost-effective synthetic alternative to archaeal lipid membranes with a wide range of potential applications.

Comparison of structural effects of cholesterol, lanosterol, and oxysterol on phospholipid (POPC) bilayers.

Membrane sterols contribute to the function of biomembranes by regulating the physical properties of the lipid bilayers. Cholesterol, a typical mammalian sterol, is biosynthesized by oxidation of lanosterol. From a molecular evolutionary perspective, lanosterol is considered the ancestral molecule of cholesterol. Here, we studied whether cholesterol is superior to lanosterol in regulating the physical properties of the lipid bilayer in terms of the structural effect on model biomembranes composed of a phospholipid. For comparison, oxysterol, which is formed by oxidation of cholesterol, was also studied. The phospholipid used was 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which is abundantly found in mammalian biomembranes, and 7ß-hydroxycholesterol, which is highly cytotoxic, was used as the oxysterol. The apparent molecular volume was calculated from the mass density determined by the flotation method using H2O and D2O, and the bilayer thickness was determined by reconstructing the electron density distribution from X-ray diffraction data of the POPC/sterol mixtures at a sterol concentration of 30 mol%. The apparent occupied area at the bilayer surface was calculated from the above two structural data. The cholesterol system had the thickest bilayer thickness and the smallest occupied area of the three sterols studied here. This indicates that the POPC/cholesterol bilayer has a better barrier property than the other two systems. Compared to cholesterol, the effects of lanosterol and 7ß-hydroxycholesterol on lipid bilayer properties can be interpreted as suboptimal for the function of mammalian biomembranes.

Insight into the Molecular Mechanism of Surface Interactions of Phosphatidylcholines─Langmuir Monolayer Study Complemented with Molecular Dynamics Simulations.

Mutual interactions between components of biological membranes are pivotal for maintaining their proper biophysical properties, such as stability, fluidity, or permeability. The main building blocks of biomembranes are lipids, among which the most important are phospholipids (mainly phosphatidylcholines (PCs)) and sterols (mainly cholesterol). Although there is a plethora of reports on interactions between PCs, as well as between PCs and cholesterol, their molecular mechanism has not yet been fully explained. Therefore, to resolve this issue, we carried out systematic investigations based on the classical Langmuir monolayer technique complemented with molecular dynamics simulations. The studies involved systems containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) analogues possessing in the structure one or two polar functional groups similar to those of DPPC. The interactions and rheological properties of binary mixtures of DPPC analogues with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol were compared with reference systems (DPPC/POPC and DPPC/cholesterol). This pointed to the importance of the ternary amine group in PC/cholesterol interactions, while in PC mixtures, the phosphate group played a key role. In both cases, the esterified glycerol group had an effect on the magnitude of interactions. The obtained results are crucial for establishing structure-property relationships as well as for designing substitutes for natural lipids.

Exploring the synergistic effects of amoxicillin and methylene blue on unsaturated lipid structures: A study of Langmuir monolayers and giant unilamellar vesicles.

The potentially toxic effects of emerging pollutant mixtures often deviate from the individual compound effects, presenting additive, synergistic, or agonistic interactions. This study delves into the complex world of emerging pollutants' mixtures, with a particular focus on their potential impact on unsaturated lipid DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine) structured as both monolayers and bilayers, which are valuable tools for mimicking cell membranes. Specifically, we examine the effects of two common types of pollutants: antibiotics (amoxicillin) and dyes (methylene blue). Utilizing Langmuir monolayers, our research reveals a synergistic effect within the pollutant mixture, as evidenced by pressure-area isotherms and polarization-modulated infrared reflection absorption spectroscopy. We identify the specific chemical interactions contributing to this synergistic effect. Furthermore, through contrast phase microscopy experiments on giant unilamellar vesicles (bilayer system), we find that the individual pollutants and the mixture exhibit similar molecular effects on the bilayer, revealing that the molecular size is a key factor in the bilayer-mixture of pollutant interaction. This highlights the importance of considering molecular size in the interactions with bilayer systems. In summary, our research dissects the critical factors of chemical interactions and molecular size concerning the effects of pollutants on DOPC, serving as simplified models of cell membranes. This study underscores the significance of comprehending the molecular effects of emerging pollutants on human health and the development of models for exploring their intricate interactions with cell membranes.

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.

Bottom-up approach to explore alpha-amylase assisted membrane remodelling.

Soluble alpha-amylases play an important role in the catabolism of polysaccharides. In this work, we show that the malt α -amylase can interact with the lipid membrane and further alter its mechanical properties. Vesicle fluctuation spectroscopy is used for quantitative measurement of the membrane bending rigidity of phosphatidylcholines lipid vesicles from the shape fluctuation based on the whole contour of Giant Unilamellar Vesicles (GUVs). The bending rigidity of the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid vesicles in water increases significantly with the presence of 0.14 micromolar alpha-amylase (AA) in the exterior solution. It appears that the enzyme present in the external solution interacts with the outer layer of the bilayer membrane, leading to an asymmetry of the solution on either side of the bilayer membrane and altering its elasticity. At AA concentration of 1.5 micromolars and above, changes in the morphology of the GUV membrane are observed. The interaction between AA in the external solution and the external leaflet causes the bilayer membrane to curve spontaneously, leading to the formation of outbuds, giving a positive spontaneous curvature of C0 ≤ 0.05 µm-1 at ≈ 1 mg / ml of the AA concentration. We validate and characterize its concentration-dependent role in stabilizing the membrane curvature. Our findings indicate that the involvement of the enzyme, depending on the concentration, can have a considerable effect on the mechanical characteristics of the membrane.

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.

Antivesiculation and Complete Unbinding of Tail-Tethered Lipids.

We report the effect of tail-tethering on vesiculation and complete unbinding of bilayered membranes. Amphiphilic molecules of a bolalipid, resembling the tail-tethered molecular structure of archaeal lipids, with two identical zwitterionic phosphatidylcholine headgroups self-assemble into a large flat lamellar membrane, in contrast to the multilamellar vesicles (MLVs) observed in its counterpart, monopolar nontethered zwitterionic lipids. The antivesiculation is confirmed by small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cyro-TEM). With the net charge of zero and higher bending rigidity of the membrane (confirmed by neutron spin echo (NSE) spectroscopy), the current membrane theory would predict that membranes should stack with each other (aka "bind") due to dominant van der Waals attraction, while the outcome of the nonstacking ("unbinding") membrane suggests that the theory needs to include entropic contribution for the nonvesicular structures. This report pioneers an understanding of how the tail-tethering of amphiphiles affects the structure, enabling better control over the final nanoscale morphology.