Lipid Exchange Research Articles

Endoplasmic reticulum-plasma membrane contact sites integrate sterol and phospholipid regulation.

Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

An EPR spin probe study of the interactions between PC liposomes and stratum corneum membranes

The electron paramagnetic resonance (EPR) spin labeling methodology was used to analyze the interactions of phosphatidylcholine (PC) liposomal formulations that are commonly used as transepidermal drug delivery systems with stratum corneum (SC) membranes. The lipid dynamics of five liposome formulations were evaluated to study the influences of sorbitan monooleate (Span80), cholesterol, and cholesterol with the charged lipids 2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG) on the molecular dynamics of PC vesicles. The EPR spectra of 5-doxyl-stearic acid (5-DSA) showed that the addition of Span80 to the liposomes increased the lipid fluidity, whereas cholesterol had the opposite effect, and the combination of charged lipids and cholesterol did not additionally influence the lipid bilayer dynamics. Fatty acid spin-labeled SC membranes were treated with the liposome formulations, leading to migration of the spin label to the molecular environment of the formulation and the presence of two spectral components representing distinct mobility states. In terms of molecular dynamics, these environments correspond to the lipid domains of the untreated SC and the liposome, indicating a poor interaction between the liposome and SC membranes. However, the contact was sufficient to allow a pronounced exchange of the spin-labeled fatty acid. Our data suggest that flexible liposomes may access the inner intercellular membranes of the SC and facilitate mutual lipid exchange without losing their relative liposomal integrity.

Calcium and lipid exchange machinery in Sertoli cells

Sperm production (spermatogenesis) takes place in the seminiferous tubules of the testis. Specialized junction complexes termed ‘ectoplasmic specializations' (ESs) in the seminiferous epithelium have several key functions. ESs between Sertoli cells disassemble during the movement of early spermatogenic cells towards the apex of the epithelium and those ESs between a Sertoli cell and late stage spermatids disassemble during sperm release. There is evidence that structures called ‘Tubulobulbar Complexes' (TBCs) are endocytic structures responsible for the internalization and eventual degradation of junctions associated with ESs, allowing for early spermatocyte translocation from the basal epithelium and release of late spermatids from the apical epithelium. One key feature of both ESs and TBCs is their extensive association with peripheral ER. ESs are tripartite in structure consisting of Sertoli cell plasma membrane (PM), a layer of actin bundles, and an overlying leaflet of ER. TBCs are double membrane invaginations that appear as ESs are beginning to break down. They are long, endocytic in‐growths, with a swelled bulb region that contains an extensive ER‐PM contact site. TBC bulbs eventually ‘bud’, fuse, and become endosomes. Little is understood about the function of the ER at both ESs and TBCs, though it is well established that ER contact sites in other cells function in both calcium signalling and lipid exchange. If ER in ESs and ER‐TBC contacts function in calcium and lipid exchange, then known calcium signalling machinery and lipid exchange proteins should be localized to these structures. Using confocal imaging, we localized the CACNA1H voltage gated calcium channel to the PM of apical ESs, and the VAPA integral ER protein, which is associated with lipid synthesis and exchange machinery, to the apical ER as well as the ER in basal regions known to contain ESs. This data adds further weight to the hypothesis that calcium exchange is integral to regulation of ES and TBC structure, as well as begins our exploration into the possible role of lipid exchange at these sites.Support or Funding InformationThis work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada (155397‐2013 to A. Wayne Vogl)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reproducibility of Phosphoinositide Lipid Extraction & Presentation in Bioanalytical Assays

Phosphatidylinositol 4‐phosphate (PI4P) produced by Phosphatidylinositol 4‐Phosphate kinases is an abundant Golgi phosphoinositide functioning in lipid exchange and vesicle transport within mammalian cells. Researchers wanting to quantify PI4P use the Bligh and Dyer method for extracting and separating phospholipids in cells and biological tissues. This common lipid extraction procedure is known to have inherent variation. Using a human promyelocytic leukemia cell line and an ELISA that is sensitive and specific for PI4P, we examine sources of variation in bioassays including sample (HL‐60 cells), the extraction procedure, and lipid detection. We determined a PI4P Mass ELISA has low coefficient of variation (<10%) if the extracted sample volume is held constant and samples are prepped and lipids presented with standardized procedures. Thus, the organic solvent extraction process is the major source of variation in typical Bligh‐Dyer preparations.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulatory network analysis of endoplasmic reticulum–mitochondrial contacts

The contact of subcellular organelles serves as molecular signaling hubs to maintain cellular homeostasis. Endoplasmic Reticulum‐Mitochondria Contacts (ERMC) are one of these signaling hubs. They regulate diverse cellular activities such as Ca2+ or lipid exchange, and mitochondria dynamics including metabolism, apoptosis and autophagy. The significance of ERMCs has recently been highlighted its dysregulation is linked to metabolic disease and neurodegenerative diseases. We previously identified multiple physical components of ERMCs using engineered ascorbate peroxidase (APEX) to map the ERMCs proteome. The identified 405 proteins with a 2.0‐fold cut‐off ratio (log base 2) in culture (SILAC)‐LC/MS‐MS quantification are classified by gene set enrichment analysis (GSEA). The analysis shows protein biosynthesis‐related proteins are enriched along with several Rho/kinase proteins. To investigate ERMC regulatory network, we screened 1126 compounds and ORFs identified in the proteomic analysis with a Split‐Rluc8 complementation assay; a biosensor that measures ERMCs. We found several modulators of adrenergic signaling associated with increased ERMC. These results may be useful to elucidate the regulation of ERMCs, understand ERMC physiology and unravel the disease mechanism.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential Roles of CIDE Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes

Liver plays a major regulatory role in whole-body lipid metabolism. Disruption of the hepatic lipid metabolism could lead to the initiation and progression of several metabolic disorders. The accumulation of fat in the form of lipid droplets (LDs) is an early pathophysiological feature of altered liver metabolism that is linked to insulin resistance and the potential progression of severe liver diseases, such as liver steatosis, liver cirrhosis and hepatocellular carcinoma. CIDE proteins including Cidea, Cideb and Cidec (also called Fsp27) play important roles in lipid metabolism. Cidea and Cidec are predominantly expressed in adipocytes, while Cideb is specifically expressed in the liver of wild-type mice. Cidea and Cidec are upregulated in the steatotic liver. Overexpression of Cidea/Cidec in mouse liver resulted in increased hepatic lipid accumulation and the formation of large LDs. Cidea expression in hepatocytes was specifically induced by saturated fatty acids, and such induction was reduced when SREBP1c was knocked down. Next, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Fasting or High-fat-diet dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, while Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions. Support or Funding Information This work was supported by grants from the National Basic Research Program, the National Natural Science Foundation of China and the China Postdoctoral Science Foundation. Proposed model of CIDEs mediated lipid droplets fusion in hepatocytes. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Oxysterol-Binding Protein Cycle: Burning Off PI(4)P to Transport Cholesterol.

To maintain an asymmetric distribution of ions across membranes, protein pumps displace ions against their concentration gradient by using chemical energy. Here, we describe a functionally analogous but topologically opposite process that applies to the lipid transfer protein (LTP) oxysterol-binding protein (OSBP). This multidomain protein exchanges cholesterol for the phosphoinositide phosphatidylinositol 4-phosphate [PI(4)P] between two apposed membranes. Because of the subsequent hydrolysis of PI(4)P, this counterexchange is irreversible and contributes to the establishment of a cholesterol gradient along organelles of the secretory pathway. The facts that some natural anti-cancer molecules block OSBP and that many viruses hijack the OSBP cycle for the formation of intracellular replication organelles highlight the importance and potency of OSBP-mediated lipid exchange. The architecture of some LTPs is similar to that of OSBP, suggesting that the principles of the OSBP cycle-burning PI(4)P for the vectorial transfer of another lipid-might be general.

Lipid Monolayer Formation and Lipid Exchange Monitored by a Graphene Field-Effect Transistor.

Anionic and cationic lipids are key molecules involved in many cellular processes; their distribution in biomembranes is highly asymmetric, and their concentration is well-controlled. Graphene solution-gated field-effect transistors (SGFETs) exhibit high sensitivity toward the presence of surface charges. Here, we establish conditions that allow the observation of the formation of charged lipid layers on solution-gated field-effect transistors in real time. We quantify the electrostatic screening of electrolyte ions and derive a model that explains the influence of charged lipids on the ion sensitivity of graphene SGFETs. The electrostatic model is validated using structural information from X-ray reflectometry measurements, which show that the lipid monolayer forms on graphene. We demonstrate that SGFETs can be used to detect cationic lipids by self-exchange of lipids. Furthermore, SGFETs allow measuring the kinetics of layer formation induced by vesicle fusion or spreading from a reservoir. Because of the high transconductance and low noise of the electrical readout, we can observe characteristic conductance spikes that we attribute to bouncing-off events of lipid aggregates from the SGFET surface, suggesting a great potential of graphene SGFETs to measure the on-off kinetics of small aggregates interacting with supported layers.

Role of Coulombic Repulsion in Collisional Lipid Transfer Among SMA(2:1)-Bounded Nanodiscs.

Styrene/maleic acid (SMA) and related copolymers are attracting great interest because they solubilise membrane proteins and lipids to form polymer-encapsulated nanodiscs. These nanodiscs retain a lipid-bilayer core surrounded by a polymer rim and can harbour a membrane protein or a membrane-protein complex. SMA exists in different styrene/maleic acid molar ratios, which results in differences in hydrophobicity and solubilisation properties. We have recently demonstrated fast collisional lipid transfer among nanodiscs encapsulated by the relatively hydrophobic copolymer SMA(3:1). Here, we used time-resolved Förster resonance energy transfer to quantify the lipid-transfer kinetics among nanodiscs bounded by SMA(2:1), a less hydrophobic copolymer that is superior in terms of lipid and membrane-protein solubilisation. Moreover, we assessed the effects of ionic strength and, thereby, the role of Coulombic repulsion in the exchange of lipid molecules among these polyanionic nanodiscs. Collisional lipid transfer was slower among SMA(2:1) nanodiscs (kcol = 5.9M-1s-1) than among SMA(3:1) nanodiscs (kcol = 222M-1 s-1) but still two to three orders of magnitude faster than diffusional lipid exchange among protein-encapsulated nanodiscs or vesicles. Increasing ionic strength accelerated lipid transfer in a manner predicted by the Davies equation, an empirical extension of the Debye-Hückel limiting law, or an extended equation taking into account the finite size of the nanodiscs. Using the latter approach, quantitative agreement between experiment and theory was achieved for an effective nanodisc charge number of z ≈ -33, which is an order of magnitude less than their nominal overall charge.

The Phase Behavior and Organization of Sphingomyelin/Cholesterol Membranes: A Deuterium NMR Study

We have studied the dependence of the phase and domain characteristics of sphingomyelin (SM)/cholesterol model membranes on sterol content and temperature using deuterium nuclear magnetic resonance. NMR spectra of N-palmitoyl(D31)-D-erythro-sphingosylphosphorylcholine (PSM-d31) were taken for temperatures from 25 to 70°C and cholesterol concentrations of 0–40%. Analogous experiments were performed using 1-palmitoyl,2-palmitoyl(D31)-sn-glycero-3-phosphocholine (DPPC-d31)/cholesterol membranes to carefully compare the data obtained using palmitoyl chains that have similar “kinked” conformations. The constructed phase diagrams exhibit both solid-ordered (so) + liquid-ordered (lo) and liquid-disordered (ld) + lo phase-coexistence regions with a clear three-phase line. Macroscopic (micron-sized) coexistence of ld and lo phases was not observed; instead, line-broadening in the ld+lo region was characterized by intermediate exchange of lipids between the two types of domains. The length scales associated with the domains were estimated to be 75–150 nm for PSM-d31/cholesterol and DPPC-d31/cholesterol model membranes.

The impact of lipid metabolism on the rate of aging of patients with arterial hypertension.

Now atherosclerosis is even more often positioned as the natural hereditary determined process determining the rate of aging of an organism and life expectancy. Research objective: to study correlation interrelations between indicators of a lipidic profile and rate of aging of men with an arterial hypertension. We have conducted a one-stage research of 123 male patients (middle age 52,4±0,60 years). The main group (I) was made by patients with the arterial hypertension (AH) of the II stage of high risk. The control (II) group was made by 60 patients without kliniko-tool symptoms of cardiovascular pathology. The research of a lipid profile was conducted on the biochemical autoanalyzer «Humastar 600» (Human GMBH, Germany) with studying of the general cholesterol (OHS), lipoproteid of the low density (LDL), lipoproteid of the high density (LPVP), triglycerides (TG), calculation of the aterogennost coefficient (AC) was also carried out. Rate of aging decided on the basis of integrated assessment of the biological age (BA) on use of a technique of the Kiev Scientific Research Institute Gerontology. Patients with AG have a direct correlation dependence of moderate force between biological age and lipid exchange traces with OHS indicators (r=0,364, p=0,003), LDL (r=0,359, p=0,004), TG (r=0,324, p=0,010) and KA (r=0,488, p<0,001). The return correlation communication is noted with LPVP (-0,446, р<0,001). Patients have groups of control the direct correlation dependence of moderate force of BV and lipidic range is noted also for atherogenous fractions of lipoproteid and KA, feedback - for LPVP (-0,623, р<0,001). On the basis of the carried-out mathematical modeling formulas for determination of biological age of the considered groups of patients taking into account indicators of a lipidic profile have been made. The correlation analysis which is carried out by us reflects a dislipidemiya contribution to development of the accelerated aging of an organism, and the executed mathematical modeling allows to carry out diagnostics of the accelerated aging of patients by means of indicators of lipid exchange.

Cyclodextrin-Mediated Lipid Exchange Monitored with FRET

Cyclodextrin-Mediated Lipid Exchange Monitored with FRET

Correlated Cryo-Fluorescence and Cryo-Electron Microscopy can Identify Sites of Membrane Fusion

Advances in cryo-electron microscopy have recently been coupled with correlated light and electron microscopy (CLEM), where fluorescence is used to locate regions of interest and characterize their molecular composition prior to data collection. While the goal of cryo-CLEM is typically the localization of a cell, organelle or protein complex of interest within a vitrified sample, recent advances of cryo-fluorescence microscopy into single-molecule regimes [Wolff et al., Biol. Cell. 2016] and the thin depth of samples suggest that fluorescence could also be used to probe function or dynamics similar to TIRF-based single-particle assays. In this way, cryo-CLEM has the ability to assist not only in efficient data collection, but also a description of functional state that might not be apparent by visualization alone. We have established that resonant energy transfer is possible at cryo temperatures, highlighting the potential of cryo-CLEM for applications requiring fluorophore autoquenching or FRET. Using autoquenching, we have adapted an influenza fusion assay for cryo-CLEM. During fusion between an influenza virus-like particle and a unilamellar vesicle, the two lipid bilayers enter into apposition before beginning lipid exchange, progressing to hemifusion and finally opening a fusion pore. By incorporating cryo-CLEM, fusion events are localized and identified based on the fluorescent signatures for these three stages, before being targeted for cryo-electron tomography. The fluorescence can then place fusion events from the cryo-CLEM experiment within either a bulk kinetic fusion assay time course completed with the same sample batch, or into published single-molecule time courses.

Investigating the Transbilayer Distribution of Cholesterol in Asymmetric Unilamellar Vesicles using Small-Angle Scattering

Cholesterol is the most abundant component of eukaryotic plasma membranes (PMs), accounting for 30-50% of the total lipid. In addition to modulating the structural and mechanical properties of lipid bilayers, cholesterol promotes the formation of ordered+disordered fluid phase separation in model membranes, and may similarly contribute to microdomain or “raft” formation in the PM. A major unresolved question is how PM properties and functions are influenced by the asymmetric transbilayer distribution of lipids, including cholesterol. While the distribution of most phospholipids and sphingolipids is known, determining the average transbilayer distribution of cholesterol has been challenging due to its rapid translocation between the PM leaflets. Previous attempts to probe the cholesterol distribution in cell membranes using fluorescence-based methods have yielded conflicting results. Moreover, studies using model vesicles have been limited due to the difficulty of preparing asymmetric liposomes. Making use of recent advancements enabling the production of asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange, we prepared cholesterol-containing aLUVs in which one leaflet was enriched in egg sphingomyelin (eSM), and the other in phosphatidylethanolamine (POPE). We then used small-angle neutron and x-ray scattering combined with isotopic labeling strategies to probe the average cholesterol location within these bilayers. An analysis of scattering data, together with complementary Molecular Dynamics simulations, provides insight into the transbilayer distribution of cholesterol in asymmetric membranes.

Cytochrome P450 Prefers to be in Liquid-Ordered Domains in the Endoplasmic Reticulum

High-resolution mapping of protein/lipid interactions is essential to fully understand the biological function of a membrane protein, but poses tremendous challenges to most biophysical and biochemical approaches. These challenges are more severe for membrane proteins like cytochrome P450 that contain a very large soluble domain and exhibit a completely different time scale of dynamics as compared to its transmembrane domain [Durr et al., BBA Biomemb, 1768(12), 2007; Yamamoto et al., Sci Rep, 3, 2013; Yamamoto et al., Sci Rep, 7, 2017]. In this study, we use peptide-based lipid nanodiscs to “trap” the microsomal cytochrome P450 2B4 alone or also in complex with its redox partners [Zhang et al., Angew Chem, 55(14), 2016]. We effectively utilized the unique nature of peptide-based nanodiscs, that allows for the lipid exchange among nanodiscs, in order to characterize the immediate membrane environment preferred/stabilized by cytochrome P450. We report the first evidence that CYP2B4 is able to induce the formation of “raft”-like domains in a biomimetic of the endoplasmic reticulum (ER). NMR experiments were used to identify and quantitatively determine the lipids present in the nanodiscs. In addition, biophysical experiments were supported by molecular dynamics simulations to identify a sphingomyelin binding motif in P450, which is largely conserved among microsomal P450s. The protein-induced raft formation increased the thermal stability of P450 and also dramatically altered the ligand binding kinetics. These results unveil lipid/protein dynamics which can possibly contribute to the delicate mechanism of redox catalysis in lipid membrane systems [Barnaba et al., F1000Research, 6, 2017].

Effect of bilayer charge on lipoprotein lipid exchange.

Lipoproteins play a key role in the onset and development of atherosclerosis, the formation of lipid plaques at blood vessel walls. The plaque formation, as well as subsequent calcification, involves not only endothelial cells but also connective tissue, and is closely related to a wide range of cardiovascular syndromes, that together constitute the number one cause of death in the Western World. High (HDL) and low (LDL) density lipoproteins are of particular interest in relation to atherosclerosis, due to their protective and harmful effects, respectively. In an effort to elucidate the molecular mechanisms underlying this, and to identify factors determining lipid deposition and exchange at lipid membranes, we here employ neutron reflection (NR) and quartz crystal microbalance with dissipation (QCM-D) to study the effect of membrane charge on lipoprotein deposition and lipid exchange. Dimyristoylphosphatidylcholine (DMPC) bilayers containing varying amounts of negatively charged dimyristoylphosphatidylserine (DMPS) were used to vary membrane charge. It was found that the amount of hydrogenous material deposited from either HDL or LDL to the bilayer depends only weakly on membrane charge density. In contrast, increasing membrane charge resulted in an increase in the amount of lipids removed from the supported lipid bilayer, an effect particularly pronounced for LDL. The latter effects are in line with previously reported observations on atherosclerotic plaque prone regions of long-term hyperlipidaemia and type 2 diabetic patients, and may also provide some molecular clues into the relation between oxidative stress and atherosclerosis.

СОСТОЯНИЕ ПРОЦЕССОВ ЛИПОПЕРОКСИДАЦИИ У МАЛЬЧИКОВ-ПОДРОСТКОВ С ОЖИРЕНИЕМ

The problem of obesity is extremely relevant for modern medical science and healthcare, and child and adolescent&#x0D; obesity deserves special attention. It seems to be interesting to trace the interrelation of pathological processes in&#x0D; obesity in the form of metabolic disorders at the level of the organism and at the cell level - disorders in the system&#x0D; of lipid peroxidation. Studies in adolescent boys are relevant, as there has been a general increase in the incidence in&#x0D; this age category. The purpose of the research was to study the state of the system “lipid peroxidation – antioxidant&#x0D; protection” in adolescent boys with exogenous-constitutional obesity. 19 adolescent boys with obesity were examined&#x0D; in comparison with the control group. Spectrophotometric and fluorometric methods of examination were used. An&#x0D; increase in the level of secondary lipid peroxidation products – ketodienes and conjugated trienes (1.65 times) – was&#x0D; revealed in adolescent boys with obesity. In the antioxidant defense system, differences in this group included reduced&#x0D; values of α-tocopherol (1.42 times), retinol (1.51 times) and superoxide dismutase activity (1.19 times), in the absence&#x0D; of statistically significant changes in the total antioxidant activity (AOA) in blood and components of glutathione status.&#x0D; The revealed disorders allow to recommend the complex therapy including prescription of antioxidants and medicines&#x0D; directed on normalization of lipid exchange at the given category of patients.

Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites

Membrane contact sites (MCSs) in eukaryotic cells are hotspots for lipid exchange, which is essential for many biological functions, including regulation of membrane properties and protein trafficking. Lipid transfer proteins anchored at membrane contact sites (LAMs) contain sterol-specific lipid transfer domains [StARkin domain (SD)] and multiple targeting modules to specific membrane organelles. Elucidating the structural mechanisms of targeting and ligand recognition by LAMs is important for understanding the interorganelle communication and exchange at MCSs. Here, we determined the crystal structures of the yeast Lam6 pleckstrin homology (PH)-like domain and the SDs of Lam2 and Lam4 in the apo form and in complex with ergosterol. The Lam6 PH-like domain displays a unique PH domain fold with a conserved N-terminal α-helix. The Lam6 PH-like domain lacks the basic surface for phosphoinositide binding, but contains hydrophobic patches on its surface, which are critical for targeting to endoplasmic reticulum (ER)-mitochondrial contacts. Structures of the LAM SDs display a helix-grip fold with a hydrophobic cavity and a flexible Ω1-loop as a lid. Ergosterol is bound to the pocket in a head-down orientation, with its hydrophobic acyl group located in the tunnel entrance. The Ω1-loop in an open conformation is essential for ergosterol binding by direct hydrophobic interaction. Structural comparison suggested that the sterol binding mode of the Lam2 SD2 is likely conserved among the sterol transfer proteins of the StARkin superfamily. Structural models of full-length Lam2 correlated with the sterol transport function at the membrane contact sites.

ПРОЯВЛЕНИЕ ПОЛИМОРФИЗМА ГЕНОВ ЛИПИДНОГО ОБМЕНА ДЛЯ ВЫЯВЛЕНИЯ ИХ РОЛИ В РАЗВИТИИ СОСУДИСТЫХ ЗАБОЛЕВАНИЙ У ДЕТЕЙ, РОЖДЁННЫХ ОТ МАТЕРЕЙ С МЕТАБОЛИЧЕСКИМ СИНДРОМОМ

Objective: To study polymorphism of lipid metabolism genes as a predictor of the development of vascular disorders in newborns from the mothers with metabolic syndrome. Methods: 48 people were surveyed: 24 mothers and their newborn's children (24 children), of whom 18 (9 mothers and 9 children) went to the control group. A biochemical blood test with the study of carbohydrate, lipid and protein metabolism on the automatic biochemical analyzer Olympus AU 680 was conducted in all the blood serum. The material for genetic research was venous blood. Genetic polymorphisms determined using the reagent kit “Lipid exchange. Additional profile” with the definition of polymorphisms АВСА1, АРОС3, LPL, PON1 by pyrosequencing method. Results: According to the research in the blood serum of children in the violations of protein and carbohydrate metabolism is not revealed. The increase in bilirubin is explained by the physiological jaundice of the newborns, which show extracting of children from the maternity hospital. Women in childbirth and their newborn children, despite the permissible values of the studied indices of lipid metabolism, a high level of atherogenicity coefficient was observed. In the conduct of molecular genetic research “Lipid exchange. Additional profile” by the method of pyrosequencing women in childbirth and their newborn children in groups 2 and 3 it turned out that the examined children of the protective allele in polymorphism rs2230806 (variant GG) was not identified, that is associated with the risk of cardiovascular diseases, while mothers were present. Women in childbirth and their newborn babies were indentified allele risk in polymorphism rs5128, a homozygote that is associated with the development of the metabolic syndrome. A protective allele in polymorphism rs328 was not found, which indicates the possible development of coronary heart disease, ischemic and athero-thrombotic stroke. In children, a risk allele in polymorphism rs662 was identified in the heterozygous state – a marker of increased risk of cardiovascular and atherosclerotic diseases, which was absent in mothers. Conclusion: In the case of newborn children, the alleles of risk or the absence of protective alleles of the above-mentioned polymorphisms, should be classified as a risk group if possible development of cardiovascular pathology. Keywords: Metabolic syndrome, genetic polymorphisms, newborn children, cardiovascular pathology.

Perspectives of Lipid-Based Drug Carrier Systems for Transdermal Delivery.

Transdermal delivery serves as non-invasive and effortless terminable means for systemic as well as topical drug delivery and finds itself as an option to conventional delivery route. Significant impervious nature of skin is the greatest hurdle for successful delivery of drug molecules to the deeper layers of skin for systemic absorption. Many approaches have been carried out for delivery of a medicament across skin barrier to enhance the efficacy. Among them lipid-based colloidal carriers have gained a unique position for transdermal delivery of drugs and bioactives owing to the presence of epidermal lipids as the chief component within the penetration barrier in high amount. Skin-carrier interaction involves attachment of these carriers to skin with a view to permit exchange of lipid between the outermost layers of the stratum corneum. Based on extensive literature search, although numerous reviews are available on lipid-based systems, but none of them relates exclusively to their transdermal uptake and toxicity. This review specifically focuses on the hurdles of transdermal drug delivery, role of lipid vehicular systems in transdermal drug delivery, uptake pathways, sequential uptake mechanism and cytotoxicity issues of lipid-based carriers which although considered safe, are not completely free from toxicity.