Lipid complexes with cationic peptides and OAKs; their role in antimicrobial action and in the delivery of antimicrobial agents

Antimicrobial agents are toxic to bacteria by a variety of mechanisms. One mechanism that is very dependent on the lipid composition of the bacterial membrane is the clustering of anionic lipid by cationic antimicrobial agents. Certain species of oligo-acyl-lysine (OAK) antimicrobial agents are particularly effective in clustering anionic lipids in mixtures mimicking the composition of bacterial membranes. The clustering of anionic lipids by certain cationic antimicrobial agents contributes to the anti-bacterial action of these agents. Bacterial membrane lipids are a determining factor, resulting in some species of bacteria being more susceptible than others. In addition, lipids can be used to increase the effectiveness of antimicrobial agents when administered in vivo. Therefore, we review some of the structures in which lipid mixtures can assemble, to more effectively be utilized as antimicrobial delivery systems. We describe in more detail the complexes formed between mixtures of lipids mimicking bacterial membranes and an OAK and their usefulness in synergizing with antibiotics to overcome bacterial multidrug resistance.     

A dynamic view of peptides and proteins in membranes

Biological membranes are highly dynamic supramolecular arrangements of lipids and proteins, which fulfill key cellular functions. Relatively few high-resolution membrane protein structures are known to date, although during recent years the structural databases have expanded at an accelerated pace. In some instances the structures of reaction intermediates provide a stroboscopic view on the conformational changes involved in protein function. Other biophysical approaches add dynamic aspects and allow one to investigate the interactions with the lipid bilayers. Membrane-active peptides fulfill many important functions in nature as they act as antimicrobials, channels, transporters or hormones, and their studies have much increased our understanding of polypeptide-membrane interactions. Interestingly several proteins have been identified that interact with the membrane as loose arrays of domains. Such conformations easily escape classical high-resolution structural analysis and the lessons learned from peptides may therefore be instructive for our understanding of the functioning of such membrane proteins. Received 11 March 2008; received after revision 2 May 2008; accepted 5 May 2008     

Lipid transport pathways in mammalian cells

Summary A major deficit in our understanding of membrane biogenesis in eukaryotes is the definition of mechanisms by which the lipid constituents of cell membranes are transported from their sites of intracellular synthesis to the multiplicity of membranes that constitute a typical cell. A variety of approaches have been used to examine the transport of lipids to different organelles. In many cases the development of new methods has been necessary to study the problem. These methods include cytological examination of cells labeled with fluorescent lipid analogs, improved methods of subcellular fractionation, in situ enzymology that demonstrates lipid translocation by changes in lipid structure, and cell-free reconstitution with isolated organelles. Several general patterns of lipid transport have emerged but there does not appear to be a unifying mechanism by which lipids move among different organelles. Significant evidence now exists for vesicular and metabolic energy-dependent mechanisms as well as mechanisms that are clearly independent of cellular ATP content.     

Antigen presentation by CD1 molecules and the generation of lipid-specific T cell immunity

It is now well demonstrated that the repertoire of T cells includes not only cells that recognize specific MHC-presented peptide antigens, but also cells that recognize specific self and foreign lipid antigens. This T cell recognition of lipid antigens is mediated by a family of conserved MHC class I-like cell surface glycoproteins known as CD1 molecules. These are specialized antigen-presenting molecules that directly bind a wide variety of lipids and present them for T cell recognition at the surface of antigen-presenting cells. Distinct populations of T cells exist that recognize CD1-presented lipids of microbial, environmental or self origin, and these T cells participate in immune responses associated with infectious, neoplastic, autoimmune and allergic diseases. Here we review the current knowledge of the biology of the CD1 system, including the structure, biosynthesis and trafficking of CD1 molecules, the structures of defined lipid antigens and the types of functional responses mediated by T cells specific for CD1-presented lipids.     

Recombination of integral and peripheral protein fractions from human red cell membrane with homologous lipids

Summary Integral and peripheral protein fractions from human red cell membranes were recombined with a total red cell lipid extract and with homologous lipids in varying mixtures, by dialysis from 2-chlorethanol solutions. The 2 protein fractions were compared for lipid binding capacity and for selectivity towards individual lipids.     

Currents through ionic channels in multicellular cardiac tissue and single heart cells

Ionic channels are elementary excitable elements in the cell membranes of heart and other tissues. They produce and transduce electrical signals. After decades of trouble with quantitative interpretation of voltage-clamp data from multicellular heart tissue, due to its morphological complexness and methodological limitations, cardiac electrophysiologists have developed new techniques for better control of membrane potential and of the ionic and metabolic environment on both sides of the plasma membrane, by the use of single heart cells. Direct recordings of the behavior of single ionic channels have become possible by using the patch-clamp technique, which was developed simultaneously. Biochemists have made excellent progress in purifying and characterizing ionic channel proteins, and there has been initial success in reconstituting some partially purified channels into lipid bilayers, where their function can be studied.     

Sticky-finger interaction sites on cytosolic lipid-binding proteins?

The cytosolic lipid-binding proteins (cLBPs) comprise a large family of small (14-15 kDa) intracellular proteins involved in the transport of small lipids, including fatty acids and retinoids within cells. Their presumed function is to solubilise, protect from chemical damage and deliver to the correct destination lipids for purposes ranging from energy metabolism (e.g. fatty acids) to signalling, gene activation and cellular differentiation (e.g. retinoids and eicosanoids). It is therefore probable that cLBPs interact directly with cellular components (membranes and/or proteins) to collect and deposit their ligands, and some external features of the different cLBPs may be involved in such interactions and determine which cellular component (integral membrane or cytosolic proteins, or membranes of different lipid compositions or domain structures) with which a given cLBP will interact. Here we have focussed on a previously unrecognised feature of cLBPs which descriminates between those for which there is empiral evidence for direct interaction with membranes, and those which do not. This is a group of bulky hydrophobic amino acid side chains (e.g. tryptophans, phenylalanines, leucines) which project directly into solvent adjacent to the portal of entry and exit of the lipid ligands. Such side chains are usually found internal to proteins, but are common at sites of protein:protein or protein:membrane interactions. These 'sticky fingers' could therefore be critical to the nature and specificity of the interactions cLBPs undergo in the web of cross-traffic in lipid movements within cells.     

Membrane fusion: the process and its energy suppliers

Membrane fusion constitutes a pivotal process in eukaryotic cell physiology. Both specialized proteins and membrane lipids play key roles in fusion. Here, our current understanding of the mechanism of membrane fusion is reviewed. The focus is on the relatively simple and well-understood proteinaceous fusion machinery of enveloped viruses and the physical properties of lipids that appear to be of great relevance for fusion progression. Recent observations suggest that viral fusion proteins use packed conformational energy and bilayer-destabilizing domains to (i) bring participating membranes into intimate contact, (ii) merge proximal lipid monolayers through highly curved stalk/hemifusion intermediates, and (iii) generate a lipid-containing fusion pore, thereby terminating the fusion process. Received 4 January 2002; received after revision 3 April 2002; accepted 5 April 2002     

Functional significance of the lipid-protein interface in photosynthetic membranes

The functional significance of the lipid-protein interface in photosynthetic membranes, mainly in thylakoids, is reviewed with emphasis on membrane structure and dynamics. The lipid-protein interface is identified primarily by the restricted molecular dynamics of its lipids as compared with the dynamics in the bulk lipid phase of the membrane. In a broad sense, lipid-protein interfaces comprise solvation shell lipids that are weakly associated with the hydrophobic surface of transmembrane proteins but also include lipids that are strongly and specifically bound to membrane proteins or protein assemblies. The relation between protein-associated lipids and the overall fluidity of the thylakoid membrane is discussed. Spin label electron paramagnetic resonance spectroscopy has been identified as the technique of choice to characterize the protein solvation shell in its highly dynamic nature; biochemical and direct structural methods have revealed an increasing number of protein-bound lipids. The structural and functional roles of these protein-bound lipids are mustered, but in most cases they remain to be determined. As suggested by recent data, the interaction of the non-bilayer-forming lipid, monogalactosyldyacilglycerol (MGDG), with the main light-harvesting chlorophyll a/b-binding protein complexes of photosystem-II (LHCII), the most abundant lipid and membrane protein components on earth, play multiple structural and functional roles in developing and mature thylakoid membranes. A brief outlook to future directions concludes this review.     

DiC14-amidine confers new anti-inflammatory properties to phospholipids

The inflammatory effect of unmethylated CpG DNA sequences represents a major obstacle to the use of cationic lipids for in vivo gene therapy. Although the mechanism of CpG-induced inflammatory response is rather well understood nowadays, few solutions have been designed to circumvent this effect in gene therapy experiments. Our previous work has shown that a refractory state towards inflammation can be elicited by preinjecting cationic liposomes. Here, we present evidence that diC14-amidine liposomes confer new anti-inflammatory properties to phospholipids from low-density lipoprotein (LDL) and even to synthetic phospholipids for which such an observation has not been reported so far. Whereas oxidation of LDL lipids was a prerequisite for any anti-inflammatory activity, lipid oxidation is no longer required in our experiments, suggesting that cationic lipids transport phospholipids through a different route and affect different pathways.This opens up new possibilities for manipulating inflammatory responses in gene therapy protocols but also in a general manner in immunological experiments. Received 12 November 2007; received after revision 4 December 2007; accepted 4 December 2007     

The Sushi peptides: structural characterization and mode of action against Gram-negative bacteria

The compositional difference in microbial and human cell membranes allows antimicrobial peptides to preferentially bind microbes. Peptides which specifically target lipopolysaccharide (LPS) and palmitoyl-oleoyl-phosphatidylglycerol (POPG) are efficient antibiotics. From the core LPS-binding region of Factor C, two 34-mer Sushi peptides, S1 and S3, were derived. S1 functions as a monomer, while S3 is active as a dimer. Both S1 and S3 display detergent-like properties in disrupting LPS aggregates, with specificity for POPG resulting from electrostatic and hydrophobic forces between the peptides and the bacterial lipids. During interaction with POPG, the S1 transitioned from a random coil to an α-helix, while S3 resumed a mixture of α-helix and β-sheet structures. The unsaturated nature of POPG confers fluidity and enhances insertion of the peptides into the lipid bilayer, causing maximal disruption of the bacterial membrane. These parameters should be considered in designing and developing new generations of peptide antibiotics with LPS-neutralizing capability. Received 2 October 2007; received after revision 2 November 2007; accepted 4 December 2007 J. L. Ding, B. Ho: Co-senior authors.     

Protein folding in membranes

Separation of cells and organelles by bilayer membranes is a fundamental principle of life. Cellular membranes contain a baffling variety of proteins, which fulfil vital functions as receptors and signal transducers, channels and transporters, motors and anchors. The vast majority of membrane-bound proteins contain bundles of α-helical transmembrane domains. Understanding how these proteins adopt their native, biologically active structures in the complex milieu of a membrane is therefore a major challenge in today’s life sciences. Here, we review recent progress in the folding, unfolding and refolding of α-helical membrane proteins and compare the molecular interactions that stabilise proteins in lipid bilayers. We also provide a critical discussion of a detergent denaturation assay that is increasingly used to determine membrane-protein stability but is not devoid of conceptual difficulties.     

Membrane protein folding on the example of outer membrane protein A of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The biophysical principles and mechanisms by which membrane proteins insert and fold into a biomembrane have mostly been studied with bacteriorhodopsin and outer membrane protein A (OmpA). This review describes the assembly process of the monomeric outer membrane proteins of Gram-negative bacteria, for which OmpA has served as an example. OmpA is a two-domain outer membrane protein composed of a 171-residue eight-stranded -barrel transmembrane domain and a 154-residue periplasmic domain. OmpA is translocated in an unstructured form across the cytoplasmic membrane into the periplasm. In the periplasm, unfolded OmpA is kept in solution in complex with the molecular chaperone Skp. After binding of periplasmic lipopolysaccharide, OmpA insertion and folding occur spontaneously upon interaction of the complex with the phospholipid bilayer. Insertion and folding of the -barrel transmembrane domain into the lipid bilayer are highly synchronized, i.e. the formation of large amounts of -sheet secondary structure and -barrel tertiary structure take place in parallel with the same rate constants, while OmpA inserts into the hydrophobic core of the membrane. In vitro, OmpA can successfully fold into a range of model membranes of very different phospholipid compositions, i.e. into bilayers of lipids of different headgroup structures and hydrophobic chain lengths. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers. Their formation was monitored by time-resolved distance determinations by fluorescence quenching, and they were structurally distinguished by the relative positions of the five tryptophan residues of OmpA in projection to the membrane normal. Recent studies indicate a chaperone-assisted, highly synchronized mechanism of secondary and tertiary structure formation upon membrane insertion of -barrel membrane proteins such as OmpA that involves at least three structurally distinct folding intermediates.     

Membrane fusion

Summary The factors involved in the regulation of biological membrane fusion and models proposed for the molecular mechanism of biomembrane fusion are reviewed. The results obtained in model systems are critically discussed in the light of the known properties of biomembranes and characteristics of biomembrane fusion. Biological membrane fusion is a local-point event; extremely fast, non-leaky, and under strict control. Fusion follows on a local and most probably protein-modulated destabilization, and a transition of the interacting membranes from a bilayer to a non-bilayer lipid structure. The potential role of type II non-bilayer preferring lipids and of proteins in the local destabilization of the membranes is evaluated. Proteins are not only responsible for the mutual recognition of the fusion partners, but are most likely also to be involved in the initiation of biomembrane fusion, by locally producing or activating fusogens, or by acting as fusogens.     

Membrane fusion

The factors involved in the regulation of biological membrane fusion and models proposed for the molecular mechanism of biomembrane fusion are reviewed. The results obtained in model systems are critically discussed in the light of the known properties of biomembranes and characteristics of biomembrane fusion. Biological membrane fusion is a local-point event; extremely fast, non-leaky, and under strict control. Fusion follows on a local and most probably protein-modulated destabilization, and a transition of the interacting membranes from a bilayer to a non-bilayer lipid structure. The potential role of type II non-bilayer preferring lipids and of proteins in the local destabilization of the membranes is evaluated. Proteins are not only responsible for the mutual recognition of the fusion partners, but are most likely also to be involved in the initiation of biomembrane fusion, by locally producing or activating fusogens, or by acting as fusogens.     

Membrane-shed vesicles from the parasite <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>: characterization and their association with cell interaction

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor–ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions.     

Properties and modes of action of specific and non-specific phospholipid transfer proteins

We have described the mode of action of the phosphatidylcholine transfer protein (PC-TP), the phosphatidylinositol transfer protein (PI-TP) and the non-specific lipid transfer protein (nsL-TP) isolated from bovine and rat tissues. PC-TP and PI-TP specifically bind one phospholipid molecule to be carried between membranes. PC-TP, and most likely PI-TP as well, have independent binding sites for the sn-1- and sn-2-fatty acyl chains. These sites have different properties, which may explain the ability of PC-TP and PI-TP to discriminate between positional phospholipid isomers. nsL-TP, which is identical to sterol carrier protein 2, transfers all common phospholipids, cholesterol and oxysterol derivatives between membranes. This protein is very efficient in mediating a net mass transfer of lipids to lipid-deficient membranes. Models for its mode of action, which is clearly different from that of PC-TP and PI-TP, are presented.     

Transmembrane movements of lipids

Summary Membranes allow the rapid passage of uncharged lipids. Phospholipids on the other hand diffuse very slowly from one monolayer to another with a half-time of several hours. This slow spontaneous movement in a pure lipid bilayer can be selectively modulated in biological membranes by intrinsic proteins. In microsomes, and probably in bacterial membranes, non-specific phospholipid flippases allow the rapid redistribution of newly synthesized phospholipids. In eukaryotic plasma membranes, aminophospholipid translocase selectively pumps phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the outer to the inner leaflet and establishes a permanent lipid asymmetry. The discovery of an aminophospholipid translocase in chromaffin granules proves that eukaryotic organelles may also contain lipid translocators.     

The assembly of lipids into lipoproteins during secretion

The process of assembly and secretion of lipoproteins is discussed with particular reference to the role of lipids. The majority of circulating lipoproteins is produced by the liver (80%) with the remainder being supplied by the intestine. The liver secretes both very low density lipoproteins and high density lipoproteins, but the assembly and secretion of these two types of particles may follow different routes. The major lipid components of lipoproteins are triacylglycerols, cholesterol, cholesterol esters and phospholipids. The biosynthesis of these lipids occurs on membranes of the endoplasmic reticulum, with many of the enzymes also being present in the Golgi; the roles of these two subcellular organelles in the assembly of lipoproteins are discussed. There appears to be a compartmentalization of lipids in cells, such that defined pools, often those newly-synthesized, are preferred, or even required, for lipoprotein assembly. The process of hepatic very low density lipoprotein secretion appears to be regulated by the supply of lipids. Indeed, the synthesis of new lipid may be a major driving force in lipoprotein assembly and secretion.     

Lipid transfer in plants

Summary Plant cells contain cytosolic proteins, called lipid transfer proteins (LTP), which are able to facilitate in vitro intermembrane transfer of phospholipids. Proteins of this kind from three plants, purified to homogeneity, have several properties in common: molecular mass around 9 kDa, high isoelectric point, lack of specificity for phospholipids, and binding ability for fatty acids. The comparison of their amino acid sequences revealed striking homologies and conserved domains which are probably involved in their function as LTPs. These proteins could play a major role in membrane biogenesis by conveying phospholipids from their site of biosynthesis to membranes unable to form these lipids. Immunochemical methods were used to establish an in vivo correlation between membrane biogenesis and the level of LTP or the amount of LTP synthesized in vitro from mRNAs. The recent isolation of a full-length cDNA allows novel approaches to studying the participation of LTPs in the biogenesis of plant cell membranes.