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A vital function of the cell membrane in all living organism is to maintain the membrane permeability barrier and fluidity. The composition of the phospholipid bilayer is distinct in archaea when compared to bacteria and eukarya. In archaea, isoprenoid hydrocarbon side chains are linked via an ether bond to the sn-glycerol-1-phosphate backbone. In bacteria and eukarya on the other hand, fatty acid side chains are linked via an ester bond to the sn-glycerol-3-phosphate backbone. The polar head groups are globally shared in the three domains of life. The unique membrane lipids of archaea have been implicated not only in the survival and adaptation of the organisms to extreme environments but also to form the basis of the membrane composition of the last universal common ancestor (LUCA). In nature, a diverse range of archaeal lipids is found, the most common are the diether (or archaeol) and the tetraether (or caldarchaeol) lipids that form a monolayer. Variations in chain length, cyclization and other modifications lead to diversification of these lipids. The biosynthesis of these lipids is not yet well understood however progress in the last decade has led to a comprehensive understanding of the biosynthesis of archaeol. This review describes the current knowledge of the biosynthetic pathway of archaeal ether lipids; insights on the stability and robustness of archaeal lipid membranes; and evolutionary aspects of the lipid divide and the LUCA. It examines recent advances made in the field of pathway reconstruction in bacteria.
The longevity-homeoviscous adaptation (LHA) theory of ageing states that lipid composition of cell membranes is linked to metabolic rate and lifespan, which has been widely shown in mammals and birds but not sufficiently in fish. In this study, two species of the genus Amphiprion (Amphiprion percula and Amphiprion clarkii, with estimated maximum lifespan potentials [MLSP] of 30 and 9-16 years, respectively) and the damselfish Chromis viridis (estimated MLSP of 1-2 years) were chosen to test the LHA theory of ageing in a potential model of exceptional longevity. Brain, livers and samples of skeletal muscle were collected for lipid analyses and integral part in the computation of membrane peroxidation indexes (PIn) from phospholipid (PL) fractions and PL fatty acid composition. When only the two Amphiprion species were compared, results pointed to the existence of a negative correlation between membrane PIn value and maximum lifespan, well in line with the predictions from the LHA theory of ageing. Nevertheless, contradictory data were obtained when the two Amphiprion species were compared to the shorter-lived C. viridis. These results along with those obtained in previous studies on fish denote that the magnitude (and sometimes the direction) of the differences observed in membrane lipid composition and peroxidation index with MLSP cannot explain alone the diversity in longevity found among fishes.
Like several other large, multimeric bacterial outer membrane proteins (OMPs), the assembly of the Klebsiella oxytoca OMP PulD does not rely on the universally conserved β-barrel assembly machinery (BAM) that catalyses outer membrane insertion. The only other factor known to interact with PulD prior to or during outer membrane targeting and assembly is the cognate chaperone PulS. Here, in vitro translation-transcription coupled PulD folding demonstrated that PulS does not act during the membrane insertion of PulD, and engineered in vivo site-specific cross-linking between PulD and PulS showed that PulS binding does not prevent membrane insertion. In vitro folding kinetics revealed that PulD is atypical compared to BAM-dependent OMPs by inserting more rapidly into membranes containing E. coli phospholipids than into membranes containing lecithin. PulD folding was fast in diC14:0-phosphatidylethanolamine liposomes but not diC14:0-phosphatidylglycerol liposomes, and in diC18:1-phosphatidylcholine liposomes but not in diC14:1-phosphatidylcholine liposomes. These results suggest that PulD efficiently exploits the membrane composition to complete final steps in insertion and explain how PulD can assemble independently of any protein-assembly machinery. Lipid-assisted assembly in this manner might apply to other large OMPs whose assembly is BAM-independent.
We recently introduced a MαCD-based method to efficiently replace virtually the entire population of plasma membrane outer leaflet phospholipids and sphingolipids of cultured mammalian cells with exogenous lipids (Li et al, (2016) Proc. Natl. Acad. Sci USA 113:14025-14030). Here, we show if the lipid-to- MαCD ratio is too high or low, cells can round up and develop membrane leakiness. We found that this cell damage can be reversed/prevented if cells are allowed to recover from the exchange step by incubation in complete growth medium. After exchange and transfer to complete growth medium cell growth was similar to that of untreated cells. In some cases, cell damage was also prevented by carrying out exchange at close to room temperature (rather than at 37°C). Exchange with lipids that do (sphingomyelin) or do not (unsaturated phosphatidylcholine) support a high level of membrane order in lipid vesicles had the analogous effect on plasma membrane order, confirming exogenous lipid localization in the plasma membrane. Importantly, changes in lipid composition and plasma membrane properties after exchange and recovery persisted for several hours. Thus, it should be possible to use lipid exchange to investigate the effect of plasma membrane lipid composition upon several aspects of membrane structure and function.
β Barrel outer membrane proteins (OMPs) cluster into supramolecular assemblies that give function to the outer membrane (OM) of Gram-negative bacteria. How such assemblies form is unknown. Here, through photoactivatable cross-linking into the Escherichia coli OM, coupled with simulations, and biochemical and biophysical analysis, we uncover the basis for OMP clustering in vivo. OMPs are typically surrounded by an annular shell of asymmetric lipids that mediate higher-order complexes with neighboring OMPs. OMP assemblies center on the abundant porins OmpF and OmpC, against which low-abundance monomeric β barrels, such as TonB-dependent transporters, are packed. Our study reveals OMP-lipid-OMP complexes to be the basic unit of supramolecular OMP assembly that, by extending across the entire cell surface, couples the requisite multifunctionality of the OM to its stability and impermeability.
Photoactivation of caged biomolecules has become a powerful approach to study cellular signalling events. Here we report a method for anchoring and uncaging biomolecules exclusively at the outer leaflet of the plasma membrane by employing a photocleavable, sulfonated coumarin derivative. The novel caging group allows quantifying the reaction progress and efficiency of uncaging reactions in a live-cell microscopy setup, thereby greatly improving the control of uncaging experiments. We synthesized arachidonic acid derivatives bearing the new negatively charged or a neutral, membrane-permeant coumarin caging group to locally induce signalling either at the plasma membrane or on internal membranes in β-cells and brain slices derived from C57B1/6 mice. Uncaging at the plasma membrane triggers a strong enhancement of calcium oscillations in β-cells and a pronounced potentiation of synaptic transmission while uncaging inside cells blocks calcium oscillations in β-cells and causes a more transient effect on neuronal transmission, respectively. The precise subcellular site of arachidonic acid release is therefore crucial for signalling outcome in two independent systems.
Rapid conduction of nerve impulses requires coating of axons by myelin sheaths, which are multilamellar, lipid-rich membranes produced by oligodendrocytes in the central nervous system. To act as an insulator, myelin has to form a stable and firm membrane structure. In this study, we have analyzed the biophysical properties of myelin membranes prepared from wild-type mice and from mouse mutants that are unable to form stable myelin. Using C-Laurdan and fluorescence correlation spectroscopy, we find that lipids are tightly organized and highly ordered in myelin isolated from wild-type mice, but not from shiverer and ceramide synthase 2 null mice. Furthermore, only myelin lipids from wild-type mice laterally segregate into physically distinct lipid phases in giant unilamellar vesicles in a process that requires very long chain glycosphingolipids. Taken together, our findings suggest that oligodendrocytes exploit the potential of lipids to self-segregate to generate a highly ordered membrane for electrical insulation of axons.
An enigmatic step in de novo formation of the autophagosome membrane compartment is the expansion of the precursor membrane phagophore, which requires the acquisition of lipids to serve as building blocks. Autophagy-related 2 (ATG2), the rod-shaped protein that tethers phosphatidylinositol 3-phosphate (PI3P)-enriched phagophores to the endoplasmic reticulum (ER), is suggested to be essential for phagophore expansion, but the underlying mechanism remains unclear. Here, we demonstrate that human ATG2A is a lipid transfer protein. ATG2A can extract lipids from membrane vesicles and unload them to other vesicles. Lipid transfer by ATG2A is more efficient between tethered vesicles than between untethered vesicles. The PI3P effectors WIPI4 and WIPI1 associate ATG2A stably to PI3P-containing vesicles, thereby facilitating ATG2A-mediated tethering and lipid transfer between PI3P-containing vesicles and PI3P-free vesicles. Based on these results, we propose that ATG2-mediated transfer of lipids from the ER to the phagophore enables phagophore expansion.
Lipid peroxidation in rat liver microsomes, induced by nitrogen dioxide (NO2), a free radical toxin, was examined in the absence and in the presence of varying concentrations of beta-carotene. The extent of peroxidation was assayed by determining the malonaldehyde formed as thiobarbituric acid reactive substances (TBARS). When the concentration of beta-carotene was 13.8 and 43.1 nmol/mg protein, no protection was seen, rather an increase of 10% and 30%, respectively, in TBARS was observed as compared with the normal microsomes containing no beta-carotene. However, at beta-carotene concentrations of 66.5 and 89.4 nmol/mg protein, only a marginal increase of 9% and 4% in TBARS, respectively, was observed. The amount of beta-carotene consumed during peroxidation, determined by following the absorbance at 450 nm, was found to increase linearly with increased exposure to NO2. The direct reaction of NO2 with beta-carotene was studied in an inert organic solvent, acetonitrile, by following the absorption spectrum of beta-carotene in the wavelength region 220-600 nm. The rate of loss of beta-carotene was found to be much faster than that in microsomes. The results suggest that in in vitro systems, the reaction of secondary lipid-derived radicals with beta-carotene and their relative competition for NO2 plays an important role in the actual function of beta-carotene as a prooxidant or an antioxidant. Another lipid soluble antioxidant, alpha-tocopherol (vitamin-E), showed significant protection against NO2-induced lipid peroxidation at a concentration of 45 nmol/mg protein under these conditions.
Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1, 3-diol] is the most common sphingoid long chain base in sphingolipids. It is the precursor of important cell signaling molecules, such as ceramides. In the last decade it has been shown to act itself as a potent metabolic signaling molecule, by activating a number of protein kinases. Moreover, sphingosine has been found to permeabilize phospholipid bilayers, giving rise to vesicle leakage. The present contribution intends to analyze the mechanism by which this bioactive lipid induces vesicle contents release, and the effect of negatively charged bilayers in the release process. Fluorescence lifetime measurements and confocal fluorescence microscopy have been applied to observe the mechanism of sphingosine efflux from large and giant unilamellar vesicles; a graded-release efflux has been detected. Additionally, stopped-flow measurements have shown that the rate of vesicle permeabilization increases with sphingosine concentration. Because at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g., bilayers containing phosphatidic acid together with sphingomyelins, phosphatidylethanolamine, and cholesterol) gives rise to a release of vesicular contents, faster than with electrically neutral bilayers. Furthermore, phosphorous 31-NMR and x-ray data show the capacity of sphingosine to facilitate the formation of nonbilayer (cubic phase) intermediates in negatively charged membranes. The data might explain the pathogenesis of Niemann-Pick type C1 disease.
Scaffolding proteins play important roles in supporting the plasma membrane (sarcolemma) of muscle cells. Among them, dystrophin strengthens the sarcolemma through protein-lipid interactions, and its absence due to gene mutations leads to the severe Duchenne muscular dystrophy. Most of the dystrophin protein consists of a central domain made of 24 spectrin-like coiled-coil repeats (R). Using small angle neutron scattering (SANS) and the contrast variation technique, we specifically probed the structure of the three first consecutive repeats 1-3 (R1-3), a part of dystrophin known to physiologically interact with membrane lipids. R1-3 free in solution was compared to its structure adopted in the presence of phospholipid-based bicelles. SANS data for the protein/lipid complexes were obtained with contrast-matched bicelles under various phospholipid compositions to probe the role of electrostatic interactions. When bound to anionic bicelles, large modifications of the protein three-dimensional structure were detected, as revealed by a significant increase of the protein gyration radius from 42 ± 1 to 60 ± 4 Å. R1-3/anionic bicelle complexes were further analyzed by coarse-grained molecular dynamics simulations. From these studies, we report an all-atom model of R1-3 that highlights the opening of the R1 coiled-coil repeat when bound to the membrane lipids. This model is totally in agreement with SANS and click chemistry/mass spectrometry data. We conclude that the sarcolemma membrane anchoring that occurs during the contraction/elongation process of muscles could be ensured by this coiled-coil opening. Therefore, understanding these structural changes may help in the design of rationalized shortened dystrophins for gene therapy. Finally, our strategy opens up new possibilities for structure determination of peripheral and integral membrane proteins not compatible with different high-resolution structural methods.
Lipid based nanoparticles (LNPs) are clinically successful vectors for hepatic delivery of nucleic acids. These systems are being developed for non-hepatic delivery of mRNA for the treatment of diseases like cystic fibrosis or retinal degeneration as well as infectious diseases. Localized delivery to the lungs requires aerosolization. We hypothesized that structural lipids within LNPs would provide features of integrity which can be tuned for attributes required for efficient hepatic and non-hepatic gene delivery. Herein, we explored whether naturally occurring lipids that originate from the cell membrane of plants and microorganisms enhance mRNA-based gene transfection in vitro and in vivo and whether they assist in maintaining mRNA activity after nebulization.
The evolutionary survival of Mycobacterium tuberculosis, the cause of human tuberculosis, depends on its ability to invade the host, replicate, and transmit infection. At its initial peripheral infection site in the distal lung airways, M. tuberculosis infects macrophages, which transport it to deeper tissues. How mycobacteria survive in these broadly microbicidal cells is an important question. Here we show in mice and zebrafish that M. tuberculosis, and its close pathogenic relative Mycobacterium marinum, preferentially recruit and infect permissive macrophages while evading microbicidal ones. This immune evasion is accomplished by using cell-surface-associated phthiocerol dimycoceroserate (PDIM) lipids to mask underlying pathogen-associated molecular patterns (PAMPs). In the absence of PDIM, these PAMPs signal a Toll-like receptor (TLR)-dependent recruitment of macrophages that produce microbicidal reactive nitrogen species. Concordantly, the related phenolic glycolipids (PGLs) promote the recruitment of permissive macrophages through a host chemokine receptor 2 (CCR2)-mediated pathway. Thus, we have identified coordinated roles for PDIM, known to be essential for mycobacterial virulence, and PGL, which (along with CCR2) is known to be associated with human tuberculosis. Our findings also suggest an explanation for the longstanding observation that M. tuberculosis initiates infection in the relatively sterile environment of the lower respiratory tract, rather than in the upper respiratory tract, where resident microflora and inhaled environmental microbes may continually recruit microbicidal macrophages through TLR-dependent signalling.
The Hedgehog (HH) pathway is critical for development and adult tissue homeostasis. Aberrant HH signaling can lead to congenital malformations and diseases including cancer. Although cholesterol and several oxysterol lipids have been shown to play crucial roles in HH activation, the molecular mechanisms governing their regulation remain unresolved. Here, we identify Canopy4 (CNPY4), a Saposin-like protein, as a regulator of the HH pathway that modulates levels of membrane sterol lipids. Cnpy4-/- embryos exhibit multiple defects consistent with HH signaling perturbations, most notably changes in digit number. Knockdown of Cnpy4 hyperactivates the HH pathway in vitro and elevates membrane levels of accessible sterol lipids, such as cholesterol, an endogenous ligand involved in HH activation. Our data demonstrate that CNPY4 is a negative regulator that fine-tunes HH signal transduction, revealing a previously undescribed facet of HH pathway regulation that operates through control of membrane composition.
The effect of lipid composition on models of the inner leaflet of mammalian cell membranes has been investigated. Grazing incidence X-ray diffraction and X-ray and neutron reflectivity have been used to characterize lipid packing and solvation, while electrochemical and infrared spectroscopic methods have been employed to probe phase behavior in an applied electric field. Introducing a small quantity of the anionic lipid dimyristoylphosphatidylserine (DMPS) into bilayers of zwitterionic dimyristoylphosphatidylethanolamine (DMPE) results in a significant change in the bilayer response to an applied field: the tilt of the hydrocarbon chains increases before returning to the original tilt angle on detachment of the bilayer. Equimolar mixtures, with slightly closer chain packing, exhibit a similar but weaker response. The latter also tend to incorporate more solvent during this electrochemical phase transition, at levels similar to those of pure DMPS. Reflectivity measurements reveal greater solvation of lipid layers for DMPS > 30 mol %, matching the greater propensity for DMPS-rich bilayers to incorporate water. Taken together, the data indicate that the range of 10-35 mol % DMPS provides optimum bilayer properties (in flexibility and function as a barrier), which may explain why the DMPS content of cell membranes tends to be found within this range.
Brain membrane lipid in rats were analyzed after being fed either 30 or 100 ppm fluoride for 3, 5, and 7 months. The protein content of brain with fluorosis decreased, whereas the DNA content remained stable during the entire period of investigation. After 7 months of fluoride treatment, the total brain phospholipid content decreased by 10% and 20% in the 30 and 100 ppm fluoride groups, respectively. The main species of phospholipid influenced by fluorosis were phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine. The fatty acid and aldehyde compositions of individual phospholipid classes were unchanged. No modifications could be detected in the amounts of cholesterol and dolichol. After 3 months of fluoride treatment, ubiquinone contents in brain were lower; however, at 7 months they were obviously increased in both groups of fluoride treatment. The results demonstrate that the contents of phospholipid and ubiquinone are modified in brains affected by chronic fluorosis and these changes of membrane lipids could be involved in the pathogenesis of this disease.
Serotonin (5-hydroxytryptamine, 5-HT) is a well-known neurotransmitter that is involved in a growing number of functions in peripheral tissues. Recent studies have shown nonpharmacological functions of 5-HT linked to its chemical properties. Indeed, it was reported that 5-HT may, on the one hand, bind lipid membranes and, on the other hand, protect red blood cells through a mechanism independent of its specific receptors. To better understand these underevaluated properties of 5-HT, we combined biochemical, biophysical, and molecular dynamics simulations approaches to characterize, at the molecular level, the antioxidant capacity of 5-HT and its interaction with lipid membranes. To do so, 5-HT was added to red blood cells and lipid membranes bearing different degrees of unsaturation. Our results demonstrate that 5-HT acts as a potent antioxidant and binds with a superior affinity to lipids with unsaturation on both alkyl chains. We show that 5-HT locates at the hydrophobic-hydrophilic interface, below the glycerol group. This interfacial location is stabilized by hydrogen bonds between the 5-HT hydroxyl group and lipid headgroups and allows 5-HT to intercept reactive oxygen species, preventing membrane oxidation. Experimental and molecular dynamics simulations using membrane enriched with oxidized lipids converge to further reveal that 5-HT contributes to the termination of lipid peroxidation by direct interaction with active groups of these lipids and could also contribute to limit the production of new radicals. Taken together, our results identify 5-HT as a potent inhibitor of lipid peroxidation and offer a different perspective on the role of this pleiotropic molecule.
Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.
Co-existing disordered and ordered (raft) membrane domains exist in Borrelia burgdorferi, the causative agent of Lyme disease. However, although B. burgdorferi contains cholesterol lipids, it lacks sphingolipids-a crucial component of rafts in eukaryotes. To define the principles of ordered lipid domain formation in Borrelia, the domain forming properties of vesicles composed of its three major lipids, acylated cholesteryl galactoside (ACGal), monogalactosyl diacyglycerol (MGalD), and phosphatidylcholine (PC) and/or their mixtures were studied. Anisotropy and fluorescence resonance energy transfer measurements were used to assay membrane order and ordered-domain formation. ACGal had the highest potential to form ordered domains. Interestingly, mixtures of ACGal with B. burgdorferi PC formed ordered domains more readily than mixtures of ACGal with MGalD. This appears to reflect the relatively high level of saturation observed for B. burgdorferi PC, as vesicles containing ACGal and PC, but in which the unsaturated lipid dioleoyl PC was substituted for Borrelia PC, failed to form ordered domains. In addition, the properties of ACGal were compared to those of cholesterol. Depending on what other lipids were present, ordered-domain formation in the presence of ACGal was greater than or equal to that in the presence of cholesterol. Giant unilamellar vesicles formed from ACGal-containing mixtures showed rounded domain shapes similar to those in analogous vesicles containing cholesterol, indicative of liquid-ordered state rather than solid-like gel-state domain formation. Over all, principles of ordered-domain formation in B. burgdorferi appear to be very similar to those in eukaryotes, with saturated PC taking the place of sphingolipids, but with ACGal being the main lipid component inducing ordered-domain formation.
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