Showing papers on "Membrane lipids published in 2008"

Membrane lipids: where they are and how they behave.

Abstract: Throughout the biological world, a 30 A hydrophobic film typically delimits the environments that serve as the margin between life and death for individual cells. Biochemical and biophysical findings have provided a detailed model of the composition and structure of membranes, which includes levels of dynamic organization both across the lipid bilayer (lipid asymmetry) and in the lateral dimension (lipid domains) of membranes. How do cells apply anabolic and catabolic enzymes, translocases and transporters, plus the intrinsic physical phase behaviour of lipids and their interactions with membrane proteins, to create the unique compositions and multiple functionalities of their individual membranes?

Insights into the Mode of Action of Chitosan as an Antibacterial Compound

Abstract: Chitosan is a polysaccharide biopolymer that combines a unique set of versatile physicochemical and biological characteristics which allow for a wide range of applications. Although its antimicrobial activity is well documented, its mode of action has hitherto remained only vaguely defined. In this work we investigated the antimicrobial mode of action of chitosan using a combination of approaches, including in vitro assays, killing kinetics, cellular leakage measurements, membrane potential estimations, and electron microscopy, in addition to transcriptional response analysis. Chitosan, whose antimicrobial activity was influenced by several factors, exhibited a dose-dependent growth-inhibitory effect. A simultaneous permeabilization of the cell membrane to small cellular components, coupled to a significant membrane depolarization, was detected. A concomitant interference with cell wall biosynthesis was not observed. Chitosan treatment of Staphylococcus simulans 22 cells did not give rise to cell wall lysis; the cell membrane also remained intact. Analysis of transcriptional response data revealed that chitosan treatment leads to multiple changes in the expression profiles of Staphylococcus aureus SG511 genes involved in the regulation of stress and autolysis, as well as genes associated with energy metabolism. Finally, a possible mechanism for chitosan's activity is postulated. Although we contend that there might not be a single classical target that would explain chitosan's antimicrobial action, we speculate that binding of chitosan to teichoic acids, coupled with a potential extraction of membrane lipids (predominantly lipoteichoic acid) results in a sequence of events, ultimately leading to bacterial death.

Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress

Abstract: Stress acclimating plants respond to abiotic and biotic stress by remodeling membrane fluidity and by releasing α-linolenic (18:3) from membrane lipids. The modification of membrane fluidity is mediated by changes in unsaturated fatty acid levels, a function provided in part by the regulated activity of fatty acid desaturases. Adjustment of membrane fluidity maintains an environment suitable for the function of critical integral proteins during stress. α-Linolenic acid, released from membrane lipid by regulated lipase activity, is the precursor molecule for phyto-oxylipin biosynthesis. The modulation of chloroplast oleic acid (18:1) levels is central to the normal expression of defense responses to pathogens in Arabidopsis. Oleic (18:1) and linolenic (18:2) acid levels, in part, regulate development, seed colonization, and mycotoxin production by Aspergillus spp.

Functional genomic screen reveals genes involved in lipid-droplet formation and utilization

Abstract: Eukaryotic cells store neutral lipids in cytoplasmic lipid droplets enclosed in a monolayer of phospholipids and associated proteins. These dynamic organelles serve as the principal reservoirs for storing cellular energy and for the building blocks for membrane lipids. Excessive lipid accumulation in cells is a central feature of obesity, diabetes and atherosclerosis, yet remarkably little is known about lipid-droplet cell biology. Here we show, by means of a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells that about 1.5% of all genes function in lipid-droplet formation and regulation. The phenotypes of the gene knockdowns sorted into five distinct phenotypic classes. Genes encoding enzymes of phospholipid biosynthesis proved to be determinants of lipid-droplet size and number, suggesting that the phospholipid composition of the monolayer profoundly affects droplet morphology and lipid utilization. A subset of the Arf1-COPI vesicular transport proteins also regulated droplet morphology and lipid utilization, thereby identifying a previously unrecognized function for this machinery. These phenotypes are conserved in mammalian cells, suggesting that insights from these studies are likely to be central to our understanding of human diseases involving excessive lipid storage.

Membranes: a meeting point for lipids, proteins and therapies

Abstract: Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy.

Protein area occupancy at the center of the red blood cell membrane

Abstract: In the Fluid Mosaic Model for biological membrane structure, proposed by Singer and Nicolson in 1972, the lipid bilayer is represented as a neutral two-dimensional solvent in which the proteins of the membrane are dispersed and distributed randomly. The model portrays the membrane as dominated by a membrane lipid bilayer, directly exposed to the aqueous environment, and only occasionally interrupted by transmembrane proteins. This view is reproduced in virtually every textbook in biochemistry and cell biology, yet some critical features have yet to be closely examined, including the key parameter of the relative occupancy of protein and lipid at the center of a natural membrane. Here we show that the area occupied by protein and lipid at the center of the human red blood cell (RBC) plasma membrane is at least ≈23% protein and less than ≈77% lipid. This measurement is in close agreement with previous estimates for the RBC plasma membrane and the recently published measurements for the synaptic vesicle. Given that transmembrane proteins are surrounded by phospholipids that are perturbed by their presence, the occupancy by protein of more than ≈20% of the RBC plasma membrane and the synaptic vesicle plasma membrane implies that natural membrane bilayers may be more rigid and less fluid than has been thought for the past several decades, and that studies of pure lipid bilayers do not fully reveal the properties of lipids in membranes. Thus, it appears to be the case that membranes may be more mosaic than fluid, with little unperturbed phospholipid bilayer.

Phospholipase C5 (NPC5) is involved in galactolipid accumulation during phosphate limitation in leaves of Arabidopsis

Abstract: The replacement of phospholipids by galacto- and sulfolipids in plant membranes represents an important adaptive process for growth on phosphate-limiting soils. Gene expression and lipid analyses revealed that the MYB transcription factor PHR1 that has been previously shown to regulate phosphate responses is not a major factor controlling membrane lipid changes. Candidate genes for phospholipid degradation were selected based on induction of expression during phosphate deprivation. Lipid measurements in the corresponding Arabidopsis mutants revealed that the non-specific phospholipase C5 (NPC5) is required for normal accumulation of digalactosyldiacylglycerol (DGDG) during phosphate limitation in leaves. The growth and DGDG content of a double mutant npc5 pho1 (between npc5 and the phosphate-deficient pho1 mutant) are reduced compared to parental lines. The amount of DGDG increases from approximately 15 mol% to 22 mol% in npc5, compared to 28 mol% in wild-type, indicating that NPC5 is responsible for approximately 50% of the DGDG synthesized during phosphate limitation in leaves. Expression in Escherichia coli revealed that NPC5 shows phospholipase C activity on phosphatidylcholine and phosphatidylethanolamine. A double mutant of npc5 and pldzeta2 (carrying a mutation in the phospholipase Dzeta2 gene) was generated. Lipid measurements in npc5 pldzeta2 indicated that the contribution of PLDzeta2 to DGDG production in leaves is negligible. In contrast to the chloroplast envelope localization of galactolipid synthesis enzymes, NPC5 localizes to the cytosol, suggesting that, during phosphate limitation, soluble NPC5 associates with membranes where it contributes to the conversion of phospholipids to diacylglycerol, the substrate for galactolipid synthesis.

Amyloid- Peptide (A ) Neurotoxicity Is Modulated by the Rate of Peptide Aggregation: A Dimers and Trimers Correlate with Neurotoxicity

Abstract: Alzheimer9s disease is an age-related neurodegenerative disorder with its toxicity linked to the generation of amyloid-β peptide (Aβ). Within the Aβ sequence, there is a systemic repeat of a GxxxG motif, which theoretical studies have suggested may be involved in both peptide aggregation and membrane perturbation, processes that have been implicated in Aβ toxicity. We synthesized modified Aβ peptides, substituting glycine for leucine residues within the GxxxG repeat motif (GSL peptides). These GSL peptides undergo β-sheet and fibril formation at an increased rate compared with wild-type Aβ. The accelerated rate of amyloid fibril formation resulted in a decrease in the presence of small soluble oligomers such as dimeric and trimeric forms of Aβ in solution, as detected by mass spectrometry. This reduction in the presence of small soluble oligomers resulted in reduced binding to lipid membranes and attenuated toxicity for the GSL peptides. The potential role that dimer and trimer species binding to lipid plays in Aβ toxicity was further highlighted when it was observed that annexin V, a protein that inhibits Aβ toxicity, specifically inhibited Aβ dimers from binding to lipid membranes.

Sterols are mainly in the cytoplasmic leaflet of the plasma membrane and the endocytic recycling compartment in CHO cells.

Abstract: The transbilayer distribution of many lipids in the plasma membrane and in endocytic compartments is asymmetric, and this has important consequences for signaling and membrane physical properties. The transbilayer distribution of cholesterol in these membranes is not properly established. Using the fluorescent sterols, dehydroergosterol and cholestatrienol, and a variety of fluorescence quenchers, we studied the transbilayer distribution of sterols in the plasma membrane (PM) and the endocytic recycling compartment (ERC) of a CHO cell line. A membrane impermeant quencher, 2,4,6-trinitrobenzene sulfonic acid, or lipid-based quenchers that are restricted to the exofacial leaflet of the plasma membrane only reduce the fluorescence intensity of these sterols in the plasma membrane by 15-32%. When the same quenchers have access to both leaflets, they quench 70-80% of the sterol fluorescence. Sterol fluorescence in the ERC is also quenched efficiently in the permeabilized cells. In microinjection experiments, delivery of quenchers into the cytosol efficiently quenched the fluorescent sterols associated with the PM and with the ERC. Quantitative analysis indicates that 60-70% of the PM sterol is in the cytoplasmic leaflet. This means that cholesterol constitutes approximately 40 mol% of cytoplasmic leaflet lipids, which may have important implications for intracellular cholesterol transport and membrane domain formation.

Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K + channels

Abstract: A fundamental question about the gating mechanism of voltage-activated K+ (Kv) channels is how five positively charged voltage-sensing residues1,2 in the fourth transmembrane segment are energetically stabilized, because they operate in a low-dielectric cell membrane. The simplest solution would be to pair them with negative charges3. However, too few negatively charged channel residues are positioned for such a role4,5. Recent studies suggest that some of the channel’s positively charged residues are exposed to cell membrane phospholipids and interact with their head groups5,6,7,8,9. A key question nevertheless remains: is the phospho-head of membrane lipids necessary for the proper function of the voltage sensor itself? Here we show that a given type of Kv channel may interact with several species of phospholipid and that enzymatic removal of their phospho-head creates an insuperable energy barrier for the positively charged voltage sensor to move through the initial gating step(s), thus immobilizing it, and also raises the energy barrier for the downstream step(s).

Asymmetric Tethering of Flat and Curved Lipid Membranes by a Golgin

Abstract: Golgins, long stringlike proteins, tether cisternae and transport vesicles at the Golgi apparatus. We examined the attachment of golgin GMAP-210 to lipid membranes. GMAP-210 connected highly curved liposomes to flatter ones. This asymmetric tethering relied on motifs that sensed membrane curvature both in the N terminus of GMAP-210 and in ArfGAP1, which controlled the interaction of the C terminus of GMAP-210 with the small guanine nucleotide-binding protein Arf1. Because membrane curvature constantly changes during vesicular trafficking, this mode of tethering suggests a way to maintain the Golgi architecture without compromising membrane flow.

Reactive Oxygen Species and Oxidative Stress

Abstract: ion of an allylic hydrogen by an oxygen radical comprises the initiation step of lipid peroxidation (Porter et al., 1995; Wagner et al., 1994), which is included in the summary schematic of lipid peroxidation (Figure 6.5); susceptibility of different PUFAs to lipid peroxidation increases with increasing number of unsaturated carbon double bonds. Initiation results in a lipid radical (R·), which then undergoes rearrangement to a conjugated diene radical; under typical aerobic conditions, this lipid radical will readily react with O2, yielding a lipid peroxyl radical (ROO·). The peroxyl radical can react with another PUFA, thereby abstracting hydrogen, becoming a lipid peroxide (LOOH), and generating another R·. This second R· can also react with O2 to yield ROO·, and this process can be repeated many times, constituting a free-radical chain reaction termed propagation of lipid peroxidation; thus, initiation by one molecule of an oxygen radical can potentially result in the peroxidation of many PUFA molecules. Propagation is an important feature of many free-radical reactions whereby one radical can stimulate a cascade of potentially deleterious reactions in biological systems; this phenomenon is addressed again later. A number of other important reactions are associated with the process of lipid peroxidation; for example, the peroxyl radical can react with other membrane lipids (e.g., cholesterol) or proteins, in addition to PUFA, thus altering these molecules while forming ROOH. Transition metals such as iron and copper, in addition to enhancing production of the powerful initiator ·OH through Fenton chemistry, − = − − = CH CH C C CH H2 H – © 2008 by Taylor & Francis Group, LLC Reactive Oxygen Species and Oxidative Stress 293 can also react directly with ROOH to produce RO· and ROO·, which can initiate new radical chain reactions. In fact, it is thought that transition metals are required for lipid peroxidation to proceed at a significant rate (Sevanian and Ursini, 2000). Lipid peroxidation can be terminated by the reaction of two lipid radicals to produce nonradical products. Additionally, lipid peroxidation can be slowed by the action of α-tocopherol, as described earlier. While stopping a particular radical chain reaction, donation of hydrogen to ROO· by α-tocopherol (yielding the relatively unreactive α-tocopherol radical) results in ROOH, which is still subject to metal-catalyzed radical generation. This is repaired by the action of glutathione peroxidases, discussed earlier, that reduces ROOH to the corresponding alcohol (ROH), effectively preventing further lipid peroxidation. Major consequences of membrane lipid peroxidation include decreased membrane fluidity, increased permeability resulting in inappropriate leakiness to some molecules, and inhibition of membrane-bound enzymes (Richter, 1987).

A defect of sphingolipid metabolism modifies the properties of normal appearing white matter in multiple sclerosis

Abstract: Maintaining the appropriate complement and content of lipids in cellular membranes is critical for normal neural function. Accumulating evidence suggests that even subtle perturbations in the lipid content of neurons and myelin can disrupt their function and may contribute to myelin and axonal degradation. In this study, we determined the composition and quantified the content of lipids and sterols in normal appearing white matter (NAWM) and normal appearing grey matter (NAGM) from control and multiple sclerosis brain tissues by electrospray ionization tandem mass spectrometry. Our results suggest that in active-multiple sclerosis, there is a shift in the lipid composition of NAWM and NAGM to a higher phospholipid and lower sphingolipid content. We found that this disturbance in lipid composition was reduced in NAGM but not in NAWM of inactive-multiple sclerosis. The pattern of disturbance in lipid composition suggests a metabolic defect that causes sphingolipids to be shuttled to phospholipid production. Modelling the biophysical consequence of this change in lipid composition of NAWM indicated an increase in the repulsive force between opposing bilayers that could explain decompaction and disruption of myelin structure.

Membrane Structural Biology: With Biochemical and Biophysical Foundations

Abstract: Dedication Preface 1. Introduction 2. The diversity of membrane lipids 3. Tools for studying membrane components 4. Proteins in or at the bilayer 5. Bundles and barrels 6. Functions and families 7. Protein folding and biogenesis 8. Diffraction and simulation 9. Membrane enzymes 10. Membrane receptors 11. Transporters 12. Channels 13. Electron transport and energy transduction 14. In pursuit of complexity Appendix I. Abbreviations Appendix II. Single-letter codes for amino acids Index.

To flip or not to flip: lipid-protein charge interactions are a determinant of final membrane protein topology.

Abstract: The molecular details of how lipids influence final topological organization of membrane proteins are not well understood. Here, we present evidence that final topology is influenced by lipid–protein interactions most likely outside of the translocon. The N-terminal half of Escherichia coli lactose permease (LacY) is inverted with respect to the C-terminal half and the membrane bilayer when assembled in mutants lacking phosphatidylethanolamine and containing only negatively charged phospholipids. We demonstrate that inversion is dependent on interactions between the net charge of the cytoplasmic surface of the N-terminal bundle and the negative charge density of the membrane bilayer surface. A transmembrane domain, acting as a molecular hinge between the two halves of the protein, must also exit from the membrane for inversion to occur. Phosphatidylethanolamine dampens the translocation potential of negative residues in favor of the cytoplasmic retention potential of positive residues, thus explaining the dominance of positive over negative amino acids as co- or post-translational topological determinants.

Biosynthesis of Membrane Lipids.

Abstract: The pathways in Escherichia coli and (largely by analogy) S. enterica remain the paradigm of bacterial lipid synthetic pathways, although recently considerable diversity among bacteria in the specific areas of lipid synthesis has been demonstrated. The structural biology of the fatty acid synthetic proteins is essentially complete. However, the membrane-bound enzymes of phospholipid synthesis remain recalcitrant to structural analyses. Recent advances in genetic technology have allowed the essentialgenes of lipid synthesis to be tested with rigor, and as expected most genes are essential under standard growth conditions. Conditionally lethal mutants are available in numerous genes, which facilitates physiological analyses. The array of genetic constructs facilitates analysis of the functions of genes from other organisms. Advances in mass spectroscopy have allowed very accurate and detailed analyses of lipid compositions as well as detection of the interactions of lipid biosynthetic proteins with one another and with proteins outside the lipid pathway. The combination of these advances has resulted in use of E. coli and S. enterica for discovery of new antimicrobials targeted to lipid synthesis and in deciphering the molecular actions of known antimicrobials. Finally,roles for bacterial fatty acids other than as membrane lipid structural components have been uncovered. For example, fatty acid synthesis plays major roles in the synthesis of the essential enzyme cofactors, biotin and lipoic acid. Although other roles for bacterial fatty acids, such as synthesis of acyl-homoserine quorum-sensing molecules, are not native to E. coli introduction of the relevant gene(s) synthesis of these foreign molecules readily proceeds and the sophisticated tools available can used to decipher the mechanisms of synthesis of these molecules.

Mutants of Saccharomyces cerevisiae deficient in acyl-CoA synthetases secrete fatty acids due to interrupted fatty acid recycling.

Abstract: In the present study, acyl-CoA synthetase mutants of Saccharomyces cerevisiae were employed to investigate the impact of this activity on certain pools of fatty acids. We identified a genotype responsible for the secretion of free fatty acids into the culture medium. The combined deletion of Faa1p and Faa4p encoding two out of five acyl-CoA synthetases was necessary and sufficient to establish mutant cells that secreted fatty acids in a growth-phase dependent manner. The mutants accomplished fatty acid export during exponential growth-phase followed by fatty acid re-import into the cells during the stationary phase. The data presented suggest that the secretion is driven by an active component. The fatty acid re-import resulted in a severely altered ultrastructure of the mutant cells. Additional strains deficient of any cellular acyl-CoA synthetase activity revealed an almost identical phenotype, thereby proving transfer of fatty acids across the plasma membrane independent of their activation with CoA. Further experiments identified membrane lipids as the origin of the observed free fatty acids. Therefore, we propose the recycling of endogenous fatty acids generated in the course of lipid remodelling as a major task of both acyl-CoA synthetases Faa1p and Faa4p.

Evidence that the Major Membrane Lipids, Except Cholesterol, Are Made in Axons of Cultured Rat Sympathetic Neurons

Abstract: Membrane lipids and proteins required for axonal growth and regeneration are generally believed to be synthesized in the cell bodies of neurons and transported into the axons However, we have demonstrated recently that, in cultured rat sympathetic neurons, axons themselves have the capacity to synthesize phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine In these experiments, we employed a compartment model of neuron culture in which pure axons grow in a fluid environment separate from that containing the cell bodies In the present study, we again used compartmented cultures to confirm and extend the previous results We have shown that three enzymes of phosphatidylcholine biosynthesis via the CDP-choline pathway are present in axons We have also shown that the rate-limiting step in the biosynthesis of phosphatidylcholine by this route in neurons, and locally in axons, is catalyzed by the enzyme CTP:phosphocholine cytidylyltransferase The biosynthesis of other membrane lipids, such as phosphatidylserine, phosphatidylethanolamine derived by decarboxylation of phosphatidylserine, phosphatidylinositol, and fatty acids, also occurs in axons However, the methylation pathway for the conversion of phosphatidylethanolamine into phosphatidylcholine appears to be a quantitatively insignificant route for phosphatidylcholine synthesis in neurons Moreover, our data provided no evidence for the biosynthesis of another important membrane lipid, cholesterol, in axons