Dispatched uses Na+ flux to power release of lipid-modified Hedgehog.

The Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters1,2 and to H+-driven transporters of the RND family3,4, enables tissue-patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly -localized sites of embryonic expression5-10. Here we determine a cryo-electron microscopy structure of the mouse protein Dispatched homologue 1 (DISP1), revealing three Na+ ions coordinated within a channel that traverses its transmembrane domain. We find that the rate of Hedgehog export is dependent on the Na+ gradient across the plasma membrane. The transmembrane channel and Na+ binding are disrupted in DISP1-NNN, a variant with asparagine substitutions for three intramembrane aspartate residues that each coordinate and neutralize the charge of one of the three Na+ ions. DISP1-NNN and variants that disrupt single Na+ sites retain binding to, but are impaired in export of the lipid-modified Hedgehog protein to the SCUBE2 acceptor. Interaction of the amino-terminal signalling domain of the Sonic hedgehog protein (ShhN) with DISP1 occurs via an extensive buried surface area and contacts with an extended furin-cleaved DISP1 arm. Variability analysis reveals that ShhN binding is restricted to one extreme of a continuous series of DISP1 conformations. The bound and unbound DISP1 conformations display distinct Na+-site occupancies, which suggests a mechanism by which transmembrane Na+ flux may power extraction of the lipid-linked Hedgehog signal from the membrane. Na+-coordinating residues in DISP1 are conserved in PTCH1 and other metazoan RND family members, suggesting that Na+ flux powers their conformationally driven activities.

First Isolation and Structure Elucidation of GDNT-ß-Glu - Tetraether Lipid Fragment from Archaeal Sulfolobus Strains.

Due to their special chemical structure, tetraether lipids (TEL) represent essential elements of archaeal membranes, providing these organisms with extraordinary properties. Here we describe the characterization of a newly isolated structural element of the main lipids. The TEL fragment GDNT-ß-Glu was isolated from Sulfolobus metallicus and characterized in terms of its chemical structure by NMR- and MS-investigations. The obtained data are dissimilar to analogically derived established structures - in essence, the binding relationships in the polar head group are re-determined and verified. With this work, we provide an important contribution to the structure elucidation of intact TEL also contained in other Sulfolobus strains such as Solfulobus acidocaldarius and Sulfolobus solfataricus.

Non-detergent Isolation of Membrane Structures from Beet Plasmalemma and Tonoplast Having Lipid Composition Characteristic of Rafts.

Vacuolar and plasma membranes were isolated by a detergent-free method from beet roots (Beta vulgaris L.), and were fractionated in a sucrose density gradient of 15-60% by high-speed centrifugation at 200,000×g during 18 h. The membrane material distributed over the sucrose density gradient was analyzed for the presence of lipids characteristic of raft structures in different zones of the gradient. The quantitative and qualitative content of lipids and sterols, and the composition of fatty acids were analyzed. Some membrane structures differing in their biochemical characteristics were revealed to be located in different zones of the sucrose gradient. The results of the analysis allowed us to identify three zones in the sucrose gradient after the vacuolar membrane fractionation and two zones in the plasma membrane where membrane structures, which may be defined as rafts for their lipid composition, were presented.

An Ionic Liquid Extraction That Preserves the Molecular Structure of Cutin Shown by Nuclear Magnetic Resonance.

The biopolyester cutin is ubiquitous in land plants, building the polymeric matrix of the plant's outermost defensive barrier, the cuticle. Cutin influences many biological processes in planta; however, due to its complexity and highly branched nature, the native structure remains partially unresolved. Our aim was to define an original workflow for the purification and systematic characterization of the molecular structure of cutin. To purify cutin we tested the ionic liquids cholinium hexanoate and 1-butyl-3-methyl-imidazolium acetate. The ensuing polymeric materials are highly esterified, amorphous, and have a typical monomeric composition as demonstrated by solid-state NMR, complemented by spectroscopic, thermal, and x-ray scattering analyses. We performed a systematic study by solution-state NMR of cryogenically milled cutins extracted from tomatoes (Solanum lycopersicum 'Micro-Tom'; the wild type and the GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE [GPAT6] and CUTIN SYNTHASE [CUS1] mutants). We resolved their molecular structures, relative distribution of ester aliphatics, free acid end-groups and free hydroxyl groups, differentiating between those derived from primary and secondary esters. Our data demonstrate the existence of free hydroxyl groups in cutin and provide insight into how the mutations affect the esterification arrangement of cutin. The usage of ionic liquids for studying plant polyesters has advantages over conventional approaches, since simple modifications can be applied to recover a biopolymer carrying distinct types/degrees of modifications (e.g. preservation of esters or cuticular polysaccharides), which in combination with the solution NMR methodologies developed here, constitutes essential tools to fingerprint the multifunctionality and the structure of cutin in planta.

Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells.

This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4-6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30-32 C atoms in their acyl chains but were relatively poor in those containing 34-40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes.

Styrene maleic-acid lipid particles (SMALPs) into detergent or amphipols: An exchange protocol for membrane protein characterisation.

Membrane proteins are traditionally extracted and purified in detergent for biochemical and structural characterisation. This process is often costly and laborious, and the stripping away of potentially stabilising lipids from the membrane protein of interest can have detrimental effects on protein integrity. Recently, styrene-maleic acid (SMA) co-polymers have offered a solution to this problem by extracting membrane proteins directly from their native membrane, while retaining their naturally associated lipids in the form of stable SMA lipid particles (SMALPs). However, the inherent nature and heterogeneity of the polymer renders their use challenging for some downstream applications - particularly mass spectrometry (MS). While advances in cryo-electron microscopy (cryo-EM) have enhanced our understanding of membrane protein:lipid interactions in both SMALPs and detergent, the resolution obtained with this technique is often insufficient to accurately identify closely associated lipids within the transmembrane annulus. Native-MS has the power to fill this knowledge gap, but the SMA polymer itself remains largely incompatible with this technique. To increase sample homogeneity and allow characterisation of membrane protein:lipid complexes by native-MS, we have developed a novel SMA-exchange method; whereby the membrane protein of interest is first solubilised and purified in SMA, then transferred into amphipols or detergents. This allows the membrane protein and endogenously associated lipids extracted by SMA co-polymer to be identified and examined by MS, thereby complementing results obtained by cryo-EM and creating a better understanding of how the lipid bilayer directly affects membrane protein structure and function.

Nontuberculous Mycobacteria Show Differential Infectivity and Use Phospholipids to Antagonize LL-37.

Comparisons of infectivity among the clinically important nontuberculous mycobacteria (NTM) species have not been explored in great depth. Rapid-growing mycobacteria, including Mycobacterium abscessus and M. porcinum, can cause indolent but progressive lung disease. Slow-growing members of the M. avium complex are the most common group of NTM to cause lung disease, and molecular approaches can now distinguish between several distinct species of M. avium complex including M. intracellulare, M. avium, M. marseillense, and M. chimaera. Differential infectivity among these NTM species may, in part, account for differences in clinical outcomes and response to treatment; thus, knowing the relative infectivity of particular isolates could increase prognostication accuracy and enhance personalized treatment. Using human macrophages, we investigated the infectivity and virulence of nine NTM species, as well as multiple isolates of the same species. We also assessed their capacity to evade killing by the antibacterial peptide cathelicidin (LL-37). We discovered that the ability of different NTM species to infect macrophages varied among the species and among isolates of the same species. Our biochemical assays implicate modified phospholipids, which may include a phosphatidylinositol or cardiolipin backbone, as candidate antagonists of LL-37 antibacterial activity. The high variation in infectivity and virulence of NTM strains suggests that more detailed microbiological and biochemical characterizations are necessary to increase our knowledge of NTM pathogenesis.

Quantitative molecular tissue atlas of Bis(monoacylglycero)phosphate and phosphatidylglycerol membrane lipids in rodent organs generated by methylation assisted high resolution mass spectrometry.

Bis(monoacylglycero)phosphate (BMP) and phosphatidylglycerol (PG) are structural isomeric phospholipids with very different properties and biological functions. Due to their isomeric nature, it has thus far been challenging to simultaneously quantify BMP and PG lipids in tissue samples by mass spectrometry. Therefore, we have developed a sensitive LC-MS/MS based approach with prior methylation derivatization that is able to handle large batches of samples. Using this high throughput platform, a simulated MS/MS database was established for confident lipid assignment. In this work, we have simultaneously identified and quantified BMP and PG lipid molecules in different body tissues of rats and mice. We report for the first time a quantitative molecular atlas of BMP and PG lipids for 14 different tissues and organs in Wistar rats, NMRI and CD1 mice. Organ- and species-specificity was analyzed and compared for both lipid molecule classes. A total of 34 BMP and 10 PG molecules were quantified, with PG concentrations being generally much higher across tissues than BMP, but BMP lipids showing a much higher molecular diversity between animal organs. The large diversity of the BMP lipids with regard to their abundance and molecular composition suggests distinct biological function(s) of the individual BMP molecules in different tissues and organs of body. Particularly high tissue levels of BMP were seen in spleen, lung, liver, kidney and small intestines, i.e. tissues that are known for their high abundance and/or activity level of lysosomes late and endosomes. Elevated BMP levels in brain tissue of APP/PSEN transgenic compared to age matched wild-type mice were also observed using this platform. This analytical methodology presented a high throughput LC-based approach incorporating simulated MS/MS database to identify and quantify BMP lipids as well as PG molecules.

Direct Depolymerization Coupled to Liquid Extraction Surface Analysis-High-Resolution Mass Spectrometry for the Characterization of the Surface of Plant Tissues.

The cuticle, the outermost layer covering the epidermis of most aerial organs of land plants, can have a heterogeneous composition even on the surface of the same organ. The main cuticle component is the polymer cutin which, depending on its chemical composition and structure, can have different biophysical properties. In this study, we introduce a new on-surface depolymerization method coupled to liquid extraction surface analysis (LESA) high-resolution mass spectrometry (HRMS) for a fast and spatially resolved chemical characterization of the cuticle of plant tissues. The method is composed of an on-surface saponification, followed by extraction with LESA using a chloroform-acetonitrile-water (49:49:2) mixture and direct HRMS detection. The method is also compared with LESA-HRMS without prior depolymerization for the analysis of the surface of the petals of Hibiscus richardsonii flowers, which have a ridged cuticle in the proximal region and a smooth cuticle in the distal region. We found that on-surface saponification is effective enough to depolymerize the cutin into its monomeric constituents thus allowing detection of compounds that were not otherwise accessible without a depolymerization step. The effect of the depolymerization procedure was more pronounced for the ridged/proximal cuticle, which is thicker and richer in epicuticular waxes compared with the cuticle in the smooth/distal region of the petal.

Protein and lipid fingerprinting of native-like membrane complexes by combining TLC and protein electrophoresis.

TLC has traditionally been used to analyze lipids isolated from membrane complexes. Here, we describe a method based on the combination of TLC and SDS-PAGE to qualitatively analyze the protein/lipid profile of membrane complexes such as those of lung surfactant. For this purpose, native lung surfactant was applied onto a silica TLC plate in the form of an aqueous suspension, preserving not only hydrophilic proteins associated with lipids but also native protein-lipid interactions. Using native membrane complexes in TLC allows the differential migration of lipids and their separation from the protein components. As a result, (partly) delipidated protein-enriched bands can be visualized and analyzed by SDS-PAGE to identify proteins originally associated with lipids. Interestingly, the hydrophobic surfactant protein C, which interacts tightly with lipids in native membrane complexes, migrates through the TLC plate, configuring specific bands that differ from those corresponding to lipids or proteins. This method therefore allows the detection and analysis of strong native-like protein-lipid interactions.

Fatty Acid Composition and Thermotropic Behavior of Glycolipids and Other Membrane Lipids of Ulva lactuca (Chlorophyta) Inhabiting Different Climatic Zones.

Increasing global temperatures are expected to increase the risk of extinction of various species due to acceleration in the pace of shifting climate zones. Nevertheless, there is no information on the physicochemical properties of membrane lipids that enable the adaptation of the algae to different climatic zones. The present work aimed to compare fatty acid composition and thermal transitions of membrane lipids from green macroalgae Ulva lactuca harvested in the Sea of Japan and the Adriatic Sea in summer. U. lactuca inhabiting the Adriatic Sea had bleached parts of thalli which were completely devoid of chloroplast glycolipids. The adaptation to a warmer climatic zone was also accompanied by a significant decrease in the ratio between unsaturated and saturated fatty acids (UFA/SFA) of membrane lipids, especially in bleached thalli. Hence, bleaching of algae is probably associated with the significant decrease of the UFA/SFA ratio in glycolipids. The decreasing ratio of n-3/n-6 polyunsaturated fatty acids (PUFAs) was observed in extra-plastidial lipids and only in the major glycolipid, non-lamellar monogalactosyldiacylglycerol. The opposite thermotropic behavior of non-lamellar and lamellar glycolipids can contribute to maintenance of the highly dynamic structure of thylakoid membranes of algae in response to the increasing temperatures of climatic zones.

Molecular structural diversity of mitochondrial cardiolipins.

Current strategies used to quantitatively describe the biological diversity of lipids by mass spectrometry are often limited in assessing the exact structural variability of individual molecular species in detail. A major challenge is represented by the extensive isobaric overlap present among lipids, hampering their accurate identification. This is especially true for cardiolipins, a mitochondria-specific class of phospholipids, which are functionally involved in many cellular functions, including energy metabolism, cristae structure, and apoptosis. Substituted with four fatty acyl side chains, cardiolipins offer a particularly high potential to achieve complex mixtures of molecular species. Here, we demonstrate how systematically generated high-performance liquid chromatography-mass spectral data can be utilized in a mathematical structural modeling approach, to comprehensively analyze and characterize the molecular diversity of mitochondrial cardiolipin compositions in cell culture and disease models, cardiolipin modulation experiments, and a broad variety of frequently studied model organisms.

Temperature-Dependent Lipid Extraction from Membranes by Boron Nitride Nanosheets.

Two-dimensional (2D) materials can mechanically insert into cell membranes and extract lipids out, thus leading to the destruction of cell integrity. On the one hand, the cytotoxicity of 2D materials can be harnessed in surface engineering to resist biofouling, and on the other hand, it causes great concern with in vivo biomedical applications ranging from drug delivery to nanoimaging. To understand the nature of this cytotoxic behavior and find strategies to control it, we performed molecular dynamics (MD) simulations on the lipid extraction of hexagonal boron nitride (BN) nanosheets from lipid membranes. Interestingly, we observed that the lipid extraction behavior suddenly disappears as temperature decreases. Structural analyses revealed that this temperature dependence is related to the lipid membrane phase transition, which was confirmed by an additional membrane model with phase state regulated by cholesterol. The potential of mean force calculation was adopted to clarify the thermodynamic origin of these results, which also indicates directions to adjust the lipid extraction behavior of nanomaterials. Overall, this work suggests that the cytotoxic mechanical interactions between 2D materials and cell membranes can be controlled by temperature and other factors which can induce phase transitions of lipid membranes and that the thermodynamic threshold of the lipid extraction varies for surfaces with different curvature. This work clarifies the thermodynamics in the lipid extraction phenomenon of 2D materials and indicates possible strategies to adjust the antibacterial performance or cytotoxicity of 2D materials.

Inorganic mercury and cadmium induce rigidity in eukaryotic lipid extracts while mercury also ruptures red blood cells.

Hg and Cd are non-essential toxic heavy metals that bioaccumulate in the tissues of living systems but less is known about their interactions with Eukaryotic lipid bilayers. Microscopy experiments showed that Hg and Cd changed the cell morphology of rabbit erythrocytes while Hg also induced cell rupture. As membranes are one of the first available targets, our study aimed to better understand metal-lipid interactions that could lead to toxic effects. Fluorescence spectroscopy (Laurdan Generalized Polarization) and dynamic light scattering were used to analyze metal-induced changes in membrane fluidity and the size of liposomes composed of Brain (Porcine), Liver (Bovine), Heart (Bovine) and Yeast (S. cerevisiae) lipid extracts. Under physiological chloride and pH levels, Hg irreversibly cleaves plasmalogens resulting in an increase in membrane rigidity. These lipids are enriched in Brain, Heart and Erythrocyte membranes and are important in signalling and the protection against oxidative stress. Interestingly, Hg had a heavily reduced effect on the plasmalogen-free Yeast extract membrane. In contrast, Cd induced rigidity by targeting negatively charged phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol and cardiolipin in these extracts. Metal-induced liposome aggregation depended on the proportion of negatively charged lipids/plasmalogen and even the order of metal addition. Our results show that data from model systems correlate with trends observed in complex biological extracts and red blood cells and serve as a predictive tool for analyzing metal-lipid interactions. The determination of the specific lipid targets for Hg and Cd provides new insights how these metals exert toxic effects on cell membranes.

Analysis of Lipids and Lipid Rafts in Borrelia.

Lipid rafts are membrane microdomains that are involved in cellular processes such as protein trafficking and signaling processes, and which play a fundamental role in membrane fluidity and budding. The lipid composition of the membrane and the biochemical characteristics of the lipids found within rafts define the ability of cells to form microdomains and compartmentalize the membrane. In this chapter, we describe the biophysical, biochemical, and molecular approaches used to define and characterize lipid rafts in the Lyme disease agent, Borrelia burgdorferi.

ER Membrane Lipid Composition and Metabolism: Lipidomic Analysis.

Plant ER membranes are the major site of biosynthesis of several lipid families (phospholipids, sphingolipids, neutral lipids such as sterols and triacylglycerols). The structural diversity of lipids presents considerable challenges to comprehensive lipid analysis. This chapter will briefly review the various biosynthetic pathways and will detail several aspects of the lipid analysis: lipid extraction, handling, separation, detection, identification, and data presentation. The different tools/approaches used for lipid analysis will also be discussed in relation to the studies to be carried out on lipid metabolism and function.

Lipophilic fractions from the marine sponge Halichondria sitiens decrease secretion of pro-inflammatory cytokines by dendritic cells and decrease their ability to induce a Th1 type response by allogeneic CD4+ T cells.

CONTEXT: Halichondria (Halichondriidae) marine sponges contain components possessing various biological activities, but immunomodulation is not among the ones reported. OBJECTIVE: This study evaluated the immunomodulatory effects of fractions/compounds from Halichondria sitiens Schmidt. MATERIALS AND METHODS: Crude dichloromethane/methanol extracts of H. sitiens were subjected to various chromatographic techniques to obtain fractions/compounds with immunomodulatory activity, using bioassay-guided isolation. The effects of the fractions/compounds were determined by measuring secretion of cytokines and expression of surface molecules by dendritic cells (DCs) and their ability to stimulate and modify cytokine secretion by allogeneic CD4+ T cells. The bioactive fractions were chemically analyzed to identify the immunomodulatory constituents by 1D, 2D NMR, and HRMS data. RESULTS: Several lipophilic fractions from H. sitiens at 10 µg/mL decreased secretion of the pro-inflammatory cytokines IL-12p40 and IL-6 by the DCs, with maximum inhibition being 64% and 25%, respectively. In addition, fractions B3b3F and B3b3J decreased the ability of DCs to induce T cell secretion of IFN-γ. Fraction B3b3 induced morphological changes in DCs, characterized by extreme elongation of dendrites and cell clustering. Chemical screening revealed the presence of glycerides and some minor unknown constituents in the biologically active fractions. One new glyceride, 2,3-dihydroxypropyl 2-methylhexadecanoate (1), was isolated from one fraction and two known compounds, 3-[(1-methoxyhexadecyl)oxy]propane-1,2-diol (2) and monoheptadecanoin (3), were identified in another, but none of them had immunomodulatory activity. DISCUSSION AND CONCLUSIONS: These results demonstrate that several lipophilic fractions from H. sitiens have anti-inflammatory effects on DCs and decrease their ability to induce a Th1 type immune response.

Comparative studies of cutins from lime (Citrus aurantifolia) and grapefruit (Citrus paradisi) after TFA hydrolysis.

Grapefruit and lime cutins were analyzed and compared in order to obtain information about their cutin architecture. This was performed using a sequential hydrolysis, first with trifluoroacetic acid to remove most of the polysaccharides present in the cutins, followed by an alkaline hydrolysis in order to obtain the main aliphatic compounds. Analysis by CPMAS 13C NMR and ATR FT-IR of the cutins after 2.0 M TFA revealed that grapefruit cutin has independent aliphatic and polysaccharide domains while in the lime cutin these components could be homogeneously distributed. These observations were in agreement with an AFM analysis of the cutins obtained in the hydrolysis reactions. The main aliphatic compounds were detected and characterized as 16-hydroxy-10-oxo-hexadecanoic acid and 10,16-dihydroxyhexadecanoic acid. These were present in grapefruit cutin at 35.80% and 21.86% and in lime cutin at 20.44% and 40.36% respectively.

Identification of the ESKAPE pathogens by mass spectrometric analysis of microbial membrane glycolipids.

Rapid diagnostics that enable identification of infectious agents improve patient outcomes, antimicrobial stewardship, and length of hospital stay. Current methods for pathogen detection in the clinical laboratory include biological culture, nucleic acid amplification, ribosomal protein characterization, and genome sequencing. Pathogen identification from single colonies by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of high abundance proteins is gaining popularity in clinical laboratories. Here, we present a novel and complementary approach that utilizes essential microbial glycolipids as chemical fingerprints for identification of individual bacterial species. Gram-positive and negative bacterial glycolipids were extracted using a single optimized protocol. Extracts of the clinically significant ESKAPE pathogens: E nterococcus faecium, S taphylococcus aureus, K lebsiella pneumoniae, A cinetobacter baumannii, P seudomonas aeruginosa, and E nterobacter spp. were analyzed by MALDI-TOF-MS in negative ion mode to obtain glycolipid mass spectra. A library of glycolipid mass spectra from 50 microbial entries was developed that allowed bacterial speciation of the ESKAPE pathogens, as well as identification of pathogens directly from blood bottles without culture on solid medium and determination of antimicrobial peptide resistance. These results demonstrate that bacterial glycolipid mass spectra represent chemical barcodes that identify pathogens, potentially providing a useful alternative to existing diagnostics.

Isolation of Neuronal Synaptic Membranes by Sucrose Gradient Centrifugation.

Sucrose gradient centrifugation is a very useful technique for isolating specific membrane types based on their size and density. This is especially useful for detecting fatty acids and lipid molecules that are targeted to specialized membranes. Without fractionation, these types of molecules could be below the levels of detection after being diluted out by the more abundant lipid molecules with a more ubiquitous distribution throughout the various cell membranes. Isolation of specific membrane types where these lipids are concentrated allows for their detection and analysis. We describe herein our synaptic membrane isolation protocol that produces excellent yield and clear resolution of five major membrane fractions from a starting neural tissue homogenate: P1 (Nuclear), P2 (Cytoskeletal), P3 (Neurosynaptosomal), PSD (Post-synaptic Densities), and SV (Synaptic Vesicle).