Fatty acid and peptide profiles in plasma membrane and membrane rafts of PUFA supplemented RAW264.7 macrophages

The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.     

Diet-induced docosahexaenoic acid non-raft domains and lymphocyte function

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that generally suppresses the function of T lymphocytes and antigen presenting cells (APCs). An emerging mechanism by which DHA modifies lymphocyte function is through changes in the organization of sphingolipid/cholesterol lipid raft membrane domains. Two contradictory models have been proposed to explain how DHA exerts its effects through changes in raft organization. The biophysical model, developed in model membranes, shows that DHA-containing phospholipids form unique non-raft membrane domains, that are organizationally distinct from lipid rafts, which serve to alter the conformation and/or lateral organization of lymphocyte proteins. In contrast, the cellular model on DHA and rafts shows that DHA suppresses lymphocyte function, in part, by directly incorporating into lipid rafts and altering protein activity. To reconcile opposing biophysical and cellular viewpoints, a major revision to existing models is presented herein. Based largely on quantitative microscopy data, it is proposed that DHA, consumed through the diet, modifies lymphocyte function, in part, through the formation of nanometer scale DHA-rich domains. These nano-scale domains disrupt the optimal raft-dependent clustering of proteins necessary for initial signaling. The data covered in this review highlights the importance of understanding how dietary n-3 PUFAs modify lymphocyte membranes, which is essential toward developing these fatty acids as therapeutic agents for treating inflammatory diseases.     

Dietary docosahexaenoic acid suppresses T cell protein kinase C theta lipid raft recruitment and IL-2 production

To date, the proximal molecular targets through which dietary n-3 polyunsaturated fatty acids (PUFA) suppress the inflammatory process have not been elucidated. Because cholesterol and sphingolipid-enriched rafts have been proposed as platforms for compartmentalizing dynamically regulated signaling assemblies at the plasma membrane, we determined the in vivo effects of fish oil and highly purified docosahexaenoic acid (DHA; 22:6n-3) on T cell microdomain lipid composition and the membrane subdomain distribution of signal-transducing molecules (protein kinase C (PKC)theta;, linker for activation of T cells, and Fas/CD95), before and after stimulation. Mice were fed diets containing 5 g/100 g corn oil (control), 4 g/100 g fish oil (contains a mixture of n-3 PUFA) plus 1 g/100 g corn oil, or 4 g/100 g corn oil plus 1 g/100 g DHA ethyl ester for 14 days. Dietary n-3 PUFA were incorporated into splenic T cell lipid raft and soluble membrane phospholipids, resulting in a 30% reduction in raft sphingomyelin content. In addition, polyclonal activation-induced colocalization of PKCtheta; with lipid rafts was reduced by n-3 PUFA feeding. With respect to PKCtheta; effector pathway signaling, both AP-1 and NF-kappaB activation, IL-2 secretion, and lymphoproliferation were inhibited by fish oil feeding. Similar results were obtained when purified DHA was fed. These data demonstrate for the first time that dietary DHA alters T cell membrane microdomain composition and suppresses the PKCtheta; signaling axis.     

Physical and chemical modulation of lipid rafts by a dietary n-3 polyunsaturated fatty acid increases ethanol-induced oxidative stress

Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported to modulate lipid raft-dependent signaling, but not yet lipid raft-dependent oxidative stress. Previously, we have shown that ethanol-induced membrane remodeling, i.e., an increase in membrane fluidity and alterations in physical and biochemical properties of lipid rafts, participated in the development of oxidative stress. Thus, we decided to study n-3 PUFA effects in this context, by pretreating hepatocytes with eicosapentaenoic acid (EPA), a long-chain n-3 PUFA, before addition of ethanol. EPA was found to increase ethanol-induced oxidative stress through membrane remodeling. Addition of EPA resulted in a marked increase in lipid raft aggregation compared to ethanol alone. In addition, membrane fluidity of lipid rafts was markedly enhanced. Interestingly, EPA was found to preferentially incorporate into nonraft membrane regions, leading to raft cholesterol increase. Lipid raft aggregation by EPA enhanced phospholipase Cγ translocation into these microdomains. Finally, phospholipase Cγ was shown to participate in the potentiation of oxidative stress by promoting lysosome accumulation, a major source of low-molecular-weight iron. To conclude, the ability of EPA to modify lipid raft physical and chemical properties plays a key role in the enhancement, by this dietary n-3 PUFA, of ethanol-induced oxidative stress.     

Biophysical and biochemical mechanisms by which dietary N-3 polyunsaturated fatty acids from fish oil disrupt membrane lipid rafts

N-3 polyunsaturated fatty acids (PUFAs) from fish oil exert their functional effects by targeting multiple mechanisms. One mechanism to emerge in the past decade is the ability of n-3 PUFA acyl chains to perturb the molecular organization of plasma membrane sphingolipid/cholesterol-enriched lipid raft domains. These domains are nanometer-scale assemblies that coalesce to compartmentalize select proteins for optimal function. Here we review recent evidence on how n-3 PUFAs modify lipid rafts from biophysical and biochemical experiments from several different model systems. A central theme emerges from these studies. N-3 PUFA acyl chains display tremendous conformational flexibility and a low affinity for cholesterol and saturated acyl chains. This unique flexibility of n-3 PUFA acyl chains impacts the organization of inner and outer leaflet lipid rafts by disrupting acyl chain packing and molecular order within rafts. Ultimately, the disruption in raft organization has consequences for protein clustering and thereby signaling. Overall, elucidating the complex mechanisms by which n-3 PUFA acyl chains reorganize membrane architecture will enhance the translation of these fatty acids into the clinic for treating several diseases.     

Effect of a 2-hydroxylated fatty acid on Cholesterol-rich membrane domains

Abstract

2-Hydroxyoleic acid (2OHOA) is a synthetic fatty acid with antihypertensive properties that is able to alter structural membranes properties. The main purpose of this study was to analyze the effect of 2OHOA on the membrane architecture in cholesterol (Cho)-rich domains. For this purpose, model membranes mimicking the composition of lipid rafts and PC- or PE-Cho-rich domains were examined in the absence and presence of 2OHOA by synchrotron X-ray diffraction, atomic force microscopy (AFM) and microcalorimetry (DSC) techniques. Our results demonstrate that 2OHOA phase separates from lipid raft domains and affects the lateral organization of lipids in the membrane. In model raft membranes, 2OHOA interacted with the sphingomyelin (SM) gel phase increasing the thickness of the water layer, which should lead to increased bilayer fluidity. The hydrogen binding competition between 2OHOA and Cho could favour the enrichment of 2OHOA in SM domains separated from the SM-Cho domains, resulting in an enhanced phase separation into SM-2OHOA-rich liquid-disordered (non-raft) and SM-Cho-rich liquid-ordered (raft) domains. The segregation into 2OHOA-rich/Cho-poor and 2OHOA-poor/Cho-rich domains was also observed in PC bilayers.     

Molecular and biophysical features of hippocampal “lipid rafts aging” are modified by dietary n-3 long-chain polyunsaturated fatty acids

“Lipid raft aging” in nerve cells represents an early event in the development of aging-related neurodegenerative diseases, such as Alzheimer's disease. Lipid rafts are key elements in synaptic plasticity, and their modification with aging alters interactions and distribution of signaling molecules, such as glutamate receptors and ion channels involved in memory formation, eventually leading to cognitive decline. In the present study, we have analyzed, in vivo, the effects of dietary supplementation of n-3 LCPUFA on the lipid structure, membrane microviscosity, domain organization, and partitioning of ionotropic and metabotropic glutamate receptors in hippocampal lipid raffs in female mice. The results revealed several lipid signatures of “lipid rafts aging” in old mice fed control diets, consisting in depletion of n-3 LCPUFA, membrane unsaturation, along with increased levels of saturates, plasmalogens, and sterol esters, as well as altered lipid relevant indexes. These changes were paralleled by increased microviscosity and changes in the raft/non-raft (R/NR) distribution of AMPA-R and mGluR5. Administration of the n-3 LCPUFA diet caused the partial reversion of fatty acid alterations found in aged mice and returned membrane microviscosity to values found in young animals. Paralleling these findings, lipid rafts accumulated mGluR5, NMDA-R, and ASIC2, and increased their R/NR proportions, which collectively indicate changes in synaptic plasticity. Unexpectedly, this diet also modified the lipidome and dimension of lipid rafts, as well as the domain redistribution of glutamate receptors and acid-sensing ion channels involved in hippocampal synaptic plasticity, likely modulating functionality of lipid rafts in memory formation and reluctance to age-associated cognitive decline.     

The use of styrene-maleic acid copolymer (SMA) for studies on T cell membrane rafts

An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250?nm) than those containing non-raft proteins (<20?nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid L_o phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different – the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.     

Chemical–Physical Changes in Cell Membrane Microdomains of Breast Cancer Cells After Omega-3 PUFA Incorporation

Epidemiologic and experimental studies suggest that dietary fatty acids influence the development and progression of breast cancer. However, no clear data are present in literature that could demonstrate how n?-?3 PUFA can interfere with breast cancer growth. It is suggested that these fatty acids might change the structure of cell membrane, especially of lipid rafts. During this study we treated MCF-7 and MDA-MB-231 cells with AA, EPA, and DHA to assess if they are incorporated in lipid raft phospholipids and are able to change chemical and physical properties of these structures. Our data demonstrate that PUFA and their metabolites are inserted with different yield in cell membrane microdomains and are able to alter fatty acid composition without decreasing the total percentage of saturated fatty acids that characterize these structures. In particular in MDA-MB-231 cells, that displays the highest content of Chol and saturated fatty acids, we observed the lowest incorporation of DHA, probably for sterical reasons; nevertheless DHA was able to decrease Chol and SM content. Moreover, PUFA are incorporated in breast cancer lipid rafts with different specificity for the phospholipid moiety, in particular PUFA are incorporated in PI, PS, and PC phospholipids that may be relevant to the formation of PUFA metabolites (prostaglandins, prostacyclins, leukotrienes, resolvines, and protectines) of phospholipids deriving second messengers and signal transduction activation. The bio-physical changes after n?-?3 PUFA incubation have also been highlighted by atomic force microscopy. In particular, for both cell lines the DHA treatment produced a decrease of the lipid rafts in the order of about 20-30?%. It is worth noticing that after DHA incorporation lipid rafts exhibit two different height ranges. In fact, some lipid rafts have a higher height of 6-6.5?nm. In conclusion n?-?3 PUFA are able to modify lipid raft biochemical and biophysical features leading to decrease of breast cancer cell proliferation probably through different mechanisms related to acyl chain length and unsaturation. While EPA may contribute to cell apoptosis mainly through decrease of AA concentration in lipid raft phospholipids, DHA may change the biophysical properties of lipid rafts decreasing the content of cholesterol and probably the distribution of key proteins.     

Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition

Polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5 (n-3)) inhibit T lymphocyte activation probably by displacing acylated signaling proteins from membrane lipid rafts. Under physiological conditions, saturated fatty acyl residues of such proteins partition into the cytoplasmic membrane lipid leaflet with high affinity for rafts that are enriched in saturated fatty acyl-containing lipids. However, the biochemical alteration causing displacement of acylated proteins from rafts in PUFA-treated T cells is still under debate but could principally be attributed to altered protein acylation or changes in raft lipid composition. We show that treatment of Jurkat T cells with polyunsaturated eicosapentaenoic acid (20:5 (n-3)) results in marked enrichment of PUFAs (20:5; 22:5) in lipids from isolated rafts. Moreover, PUFAs were significantly incorporated into phosphatidylethanolamine that predominantly resides in the cytoplasmic membrane lipid leaflet. Notably, palmitate-labeled Src family kinase Lck and the linker for activation of T cells (LAT) were both displaced from lipid rafts indicating that acylation by PUFAs is not required for protein displacement from rafts in PUFA-treated T cells. In conclusion, these data provide strong evidence that displacement of acylated proteins from rafts in PUFA-treated T cells is predominantly due to altered raft lipid composition.     

Docosahexaenoic and Eicosapentaenoic Acids Segregate Differently between Raft and Nonraft Domains

Omega-3 polyunsaturated fatty acids (n-3 PUFA), enriched in fish oils, are increasingly recognized to have potential benefits for treating many human afflictions. Despite the importance of PUFA, their molecular mechanism of action remains unclear. One emerging hypothesis is that phospholipids containing n-3 PUFA acyl chains modify the structure and composition of membrane rafts, thus affecting cell signaling. In this study the two major n-3 PUFA found in fish oils, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, are compared. Using solid-state (2)H NMR spectroscopy we explored the molecular organization of 1-[(2)H(31)]palmitoyl-2-eicosapentaenoylphosphatidylcholine (PEPC-d(31)) and 1-[(2)H(31)]palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC-d(31)) in mixtures with sphingomyelin (SM) and cholesterol (chol). Our results indicate that whereas both PEPC-d(31) and PDPC-d(31) can accumulate into SM-rich/chol-rich raftlike domains, the tendency for DHA to incorporate into rafts is more than twice as great as for EPA. We propose that DHA may be the more bioactive component of fish oil that serves to disrupt lipid raft domain organization. This mechanism represents an evolution in the view of how PUFA remodel membrane architecture.     

Docosahexaenoic acid alters the size and distribution of cell surface microdomains

We recently generated nutritional data suggesting that chemoprotective dietary n-3 polyunsaturated fatty acids (n-3 PUFA) are capable of displacing acylated proteins from lipid raft microdomains in vivo [D.W. Ma, J. Seo, L.A. Davidson, E.S. Callaway, Y.Y. Fan, J.R. Lupton, R.S. Chapkin, n-3 PUFA alter caveolae lipid composition and resident protein localization in mouse colon, FASEB J. 18 (2004) 1040-1042; Y.Y. Fan, L.H. Ly, R. Barhoumi, D.N. McMurray, R.S. Chapkin, Dietary docosahexaenoic acid suppresses T cell protein kinase Ctheta lipid raft recruitment and IL-2 recruitment, J. Immunol. 173 (2004) 6151-6160]. A primary source of very long chain n-3 PUFA in the diet is derived from fish enriched with docosahexaenoic acid (DHA, 22:6n-3). In this study, we sought to determine the effect of DHA on cell surface microdomain organization in situ. Using immuno-gold electron microscopy of plasma membrane sheets coupled with spatial point analysis of validated microdomain markers, morphologically featureless microdomains were visualized in HeLa cells at high resolution. Clustering of probes within cholesterol-dependent (GFP-tH) versus cholesterol-independent (GFP-tK) nanoclusters was differentially sensitive to n-3 PUFA treatment of cells. Univariate K-function analysis of GFP-tH (5 nm gold) revealed a significant increase in clustering (p<0.05) by pre-treatment with DHA and linoleic acid (LA, 18:2(Delta9,12)) compared to control fatty acids; whereas LA significantly (p<0.05) reduced GFP-tK clustering. These novel data suggest that the plasma membrane organization of inner leaflets is fundamentally altered by PUFA-enrichment. We speculate that our findings may help define a new paradigm to better understand the complexity of n-3 PUFA modulation of signaling networks.     

n-3 polyunsaturated fatty acids suppress the localization and activation of signaling proteins at the immunological synapse in murine CD4+ T cells by affecting lipid raft formation

The molecular properties of immunosuppressive n-3 polyunsaturated fatty acids (PUFA) have not been fully elucidated. Using CD4(+) T cells from wild-type control and fat-1 transgenic mice (enriched in n-3 PUFA), we show that membrane raft accumulation assessed by Laurdan (6-dodecanoyl-2-dimethyl aminonaphthalene) labeling was enhanced in fat-1 cells following immunological synapse (IS) formation by CD3-specific Ab expressing hybridoma cells. However, the localization of protein kinase Ctheta, phospholipase Cgamma-1, and F-actin into the IS was suppressed. In addition, both the phosphorylation status of phospholipase Cgamma-1 at the IS and cell proliferation as assessed by CFSE labeling and [(3)H]thymidine incorporation were suppressed in fat-1 cells. These data imply that lipid rafts may be targets for the development of dietary agents for the treatment of autoimmune and chronic inflammatory diseases.     

Myogenic differentiation and lipid-raft composition of L6 skeletal muscle cells are modulated by PUFAs

Lipid composition and fatty acid analysis of the major classes of membrane phospholipids were determined during myogenic differentiation of L6 skeletal muscle cells. The cholesterol to glycerophospholipids ratio decreased during differentiation, both in total (TM) and detergent-resistant membranes (DRM). Analyses of the membrane lipids showed that differentiation had a major impact on the molecular composition of glycerophospholipids. A significant decrease in the concentration of saturated fatty acids was detected in glycerophospholipid classes, and to a lesser extent in sphingolipids, while the concentration of 16:1n-7, 18:1n-7 and 18:1n-9 increased. At the same time, the concentration of long polyunsaturated fatty acid chains decreased in TM and DRM glycerophospholipids, resulting in a lower saturated to unsaturated fatty acid ratio in myotubes as compared to myoblasts. Interestingly, the observed n-3/n-6 ratio was lower in differentiated cell membranes. PUFA supplementation of L6 cells led to an increase in myogenic differentiation correlated to an incorporation of added PUFAs in TM and DRM glycerophospholipids. As expected after n-3 PUFA supplementation, the n-3/n-6 ratio was clearly increased in TM and, surprisingly, this was also the case in isolated DRM. n-3 and n-6 PUFAs significantly and time-dependently increased the phosphorylation of kinase p70S6K1 during myogenic differentiation, revealing the activation of the upstream kinase mTORC1, a major regulator of cell cycle and protein translation. In contrast, PUFAs did not affect the phosphorylation of the kinase Akt, another pivotal regulator of cell metabolism. These results suggest that PUFA supplementation modified the membrane lipid composition and affected the differentiation of L6 cells.     

n-3 PUFA and membrane microdomains: a new frontier in bioactive lipid research

In recent years, our understanding of the plasma membrane has changed considerably as our knowledge of lipid microdomains has expanded. Lipid microdomains include structures known as lipid rafts and caveolae, which are readily identified by their unique lipid constituents. Cholesterol, sphingolipids and phospholipids with saturated fatty acyl chain moieties are highly enriched in these lipid microdomains. Lipid rafts and caveolae have been shown to play an important role in the compartmentalization, modulation and integration of cell signaling. Therefore, these microdomains may have an influential role in human disease. Dietary n-3 polyunsaturated fatty acids (PUFA) ameliorate a number of human diseases including coronary heart disease, autoimmune and inflammatory disorders, diabetes, obesity and cancer, which has been generally linked to its membrane remodeling properties. Recent in vitro evidence suggests that perturbations in membrane composition alter the function of resident proteins and, consequently, cellular responses. This review examines the role of n-3 PUFA in modulating the lipid composition and functionality of lipid microdomains and its potential significance to human health.     

Docosahexaenoic acid alters the size and distribution of cell surface microdomains

We recently generated nutritional data suggesting that chemoprotective dietary n-3 polyunsaturated fatty acids (n-3 PUFA) are capable of displacing acylated proteins from lipid raft microdomains in vivo [D.W. Ma, J. Seo, L.A. Davidson, E.S. Callaway, Y.Y. Fan, J.R. Lupton, R.S. Chapkin, n-3 PUFA alter caveolae lipid composition and resident protein localization in mouse colon, FASEB J. 18 (2004) 1040-1042; Y.Y. Fan, L.H. Ly, R. Barhoumi, D.N. McMurray, R.S. Chapkin, Dietary docosahexaenoic acid suppresses T cell protein kinase Cθ lipid raft recruitment and IL-2 recruitment, J. Immunol. 173 (2004) 6151-6160]. A primary source of very long chain n-3 PUFA in the diet is derived from fish enriched with docosahexaenoic acid (DHA, 22:6n-3). In this study, we sought to determine the effect of DHA on cell surface microdomain organization in situ. Using immuno-gold electron microscopy of plasma membrane sheets coupled with spatial point analysis of validated microdomain markers, morphologically featureless microdomains were visualized in HeLa cells at high resolution. Clustering of probes within cholesterol-dependent (GFP-tH) versus cholesterol-independent (GFP-tK) nanoclusters was differentially sensitive to n-3 PUFA treatment of cells. Univariate K-function analysis of GFP-tH (5 nm gold) revealed a significant increase in clustering (p < 0.05) by pre-treatment with DHA and linoleic acid (LA, 18:2^Δ9,12) compared to control fatty acids; whereas LA significantly (p < 0.05) reduced GFP-tK clustering. These novel data suggest that the plasma membrane organization of inner leaflets is fundamentally altered by PUFA-enrichment. We speculate that our findings may help define a new paradigm to better understand the complexity of n-3 PUFA modulation of signaling networks.     

Munc18-2/syntaxin3 complexes are spatially separated from syntaxin3-containing SNARE complexes

Exocytosis of mast cell granules requires a vesicular- and plasma membrane-associated fusion machinery. We examined the distribution of SNARE membrane fusion and Munc18 accessory proteins in lipid rafts of RBL mast cells. SNAREs were found either excluded (syntaxin2), equally distributed between raft and non-raft fractions (syntaxin4, VAMP-8, VAMP-2), or selectively enriched in rafts (syntaxin3, SNAP-23). Syntaxin4-binding Munc18-3 was absent, whereas small amounts of the syntaxin3-interacting partner Munc18-2 consistently distributed into rafts. Cognate SNARE complexes of syntaxin3 with SNAP-23 and VAMP-8 were enriched in rafts, whereas Munc18-2/syntaxin3 complexes were excluded. This demonstrates a spatial separation between these two types of complexes and suggests that Munc18-2 acts in a step different from SNARE complex formation and fusion.