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.     

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.     

Polyunsaturated fatty acid supplements modulate mast cell membrane microdomain composition

In the present study, the lipid raft composition of a canine mastocytoma cell line (C2) was analyzed. Lipid rafts were well separated from non-raft plasma membranes using a detergent-free isolation technique. To study the influence of n-3 and n-6 polyunsaturated fatty acids (PUFA) on raft fatty acid composition in comparison to non-raft cell membrane, C2 were supplemented with one of the following: α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid or arachidonic acid. Enrichment of the culture medium with a specific PUFA resulted in an increase in the content of this fatty acid both in rafts and non-raft membranes. Contents of cholesterol and protein were found not to be affected by the changes in the fatty acid profiles. In conclusion, our data provide strong evidence that PUFA modulate lipid composition and physiological properties of membrane micro domains of mast cells which in turn may have effects on mast cell function.     

脂筏与精子膜信号传导

脂筏是细胞膜内由特殊脂质与蛋白质构成的微域。小窝是脂筏的一种形式,小窝标记蛋白有小窝蛋白和小窝舟蛋白。脂筏或小窝与生物信号传导、细胞蛋白转运和胆固醇平衡有关。最近实验证实哺乳动物精子膜具有脂筏结构,脂筏与膜胆固醇外逸对于启动受精的信号传导具有重要作用。     

Severe alterations in lipid composition of frontal cortex lipid rafts from Parkinson's disease and incidental Parkinson's disease

Lipid rafts are cholesterol- and sphingomyelin-enriched microdomains that provide a highly saturated and viscous physicochemical microenvironment to promote protein-lipid and protein-protein interactions. We purified lipid rafts from human frontal cortex from normal, early motor stages of Parkinson's disease (PD) and incidental Parkinson's disease (iPD) subjects and analyzed their lipid composition. We observed that lipid rafts from PD and iPD cortices exhibit dramatic reductions in their contents of n-3 and n-6 long-chain polyunsaturated fatty acids, especially docosahexaenoic acid (22:6-n3) and arachidonic acid (20:4n-6). Also, saturated fatty acids (16:0 and 18:0) were significantly higher than in control brains. Paralleling these findings, unsaturation and peroxidability indices were considerably reduced in PD and iPD lipid rafts. Lipid classes were also affected in PD and iPD lipid rafts. Thus, phosphatidylserine and phosphatidylinositol were increased in PD and iPD, whereas cerebrosides and sulfatides and plasmalogen levels were considerably diminished. Our data pinpoint a dramatic increase in lipid raft order due to the aberrant biochemical structure in PD and iPD and indicate that these abnormalities of lipid rafts in the frontal cortex occur at early stages of PD pathology. The findings correlate with abnormal lipid raft signaling and cognitive decline observed during the development of these neurodegenerative disorders.     

Effect of docosahexaenoic acid on interleukin-2 receptor signaling pathway in lipid rafts

Recent studies have shown that polyunsaturated fatty acids (PUFA) regulated the functions of membrane receptors in T cells and suppressed T cell-mediated immune responses. But the molecular mechanisms of immune regulation are not yet elucidated. Lipid rafts are plasma membrane microdomains, in which many receptors localized. The purpose of this study was to investigate the effect of DHA on IL-2R signaling pathway in lipid rafts. We isolated lipid rafts by discontinuous sucrose density gradient ultracentrifugation, and found that DHA could change the composition of lipid rafts and alter the distribution of key molecules of IL-2R signaling pathway, which transferred from lipid rafts to detergent-soluble membrane fractions. These results revealed that DHA treatment increased the proportion of polyunsaturated fatty acids especially n−3 polyunsaturated fatty acids in lipid rafts and changed the lipid environment of membrane microdomains in T cells. Compared with controls, DHA changed the localization of IL-2R, STAT5a and STAT5b in lipid rafts and suppressed the expression of JAK1, JAK3 and tyrosine phosphotyrosine in soluble membrane fractions. Summarily, this study concluded the effects of DHA on IL-2R signaling pathway in lipid rafts and explained the regulation of PUFAs in T cell-mediated immune responses.     

Effect of docosahexaenoic acid on interleukin-2 receptor signaling pathway in lipid rafts

PUFAs have been shown to mediate immune re-sponse especially the functions of T cells[1]. Recent researches have demonstrated that PUFAs can in-crease membrane fluidity and modify the functions of membrane receptors and enzymes in T cell membra-ne[2,3]. M…     

生物膜结构研究的一些进展

膜蛋白三维结构的解析存在很多困难.最近几年由于一些通道(如K⁺通道,Cl^-通道,水通道Aquaporin 1等)和泵（如Ca^2＋泵）的结晶获得成功,这些膜蛋白具有原子分辨率三维结构的解析才得以完成,从而基本阐明一些极性分子和离子选择性通过生物膜的分子机理.在膜脂结构方面,动物细胞质膜膜脂的分布是不均匀的.近年来已多方面证明,质膜具有一些被命名为“脂筏（lipid rafts）”和“质膜微囊（Caveolae）”的微区.它们富含鞘脂和胆固醇。简单介绍了这些脂质微区的大小、组分以及动态变化.根据研究结果,这类脂质微区含有大量信号分子,很可能具有信号传递中心的作用.此外,对脂筏在膜运送过程中的作用也进行一些评述.     

Critical role for lipid raft-associated Src kinases in activation of PI3K-Akt signalling

Lipid rafts are membrane microdomains distinct from caveolae, whose functions in polypeptide growth factor signalling remain unclear. Here we show that in small cell lung cancer (SCLC) cells, specific growth factor receptors such as c-Kit associate with lipid rafts and that these domains play a critical role in the activation of phosphoinositide 3-kinase (PI3K) signalling. The class IA p85/p110alpha associated with Src in lipid rafts and was activated by Src in vitro. Lipid raft integrity was essential for Src activation in response to stem cell factor (SCF) and raft disruption selectively inhibited activation of protein kinase B (PKB)/Akt in response to SCF stimulation. Moreover, inhibition of Src kinases blocked PKB/Akt activation and SCLC cell growth. The use of fibroblasts with targeted deletion of the Src family kinase genes confirmed the role of Src kinases in PKB/Akt activation by growth factor receptors. Moreover a constitutively activated mutant of Src also stimulated PI3K/Akt in lipid rafts, indicating that these microdomains play a role in oncogenic signalling. Together our data demonstrate that lipid rafts play a key role in the activation of PI3K signalling by facilitating the interaction of Src with specific PI3K isoforms.     

Plasma membrane microdomains: Organization,function and trafficking (Review)

The plasma membrane consists of a mosaic of functional microdomains facilitating a variety of physiological processes associated with the cell surface. In most cells, the majority of the cell surface is morphologically featureless, leading to difficulties in characterizing its organization and microdomain composition. The reliance on indirect and perturbing techniques has led to vigorous debate concerning the nature and even existence of some microdomains. Recently, increasing technical sophistication has been applied to study cell surface compartmentalization providing evidence for small, short-lived clusters that may be much less than 50 nm in diameter. Lipid rafts and caveolae are cholesterol-dependent, highly ordered microdomains that have received most attention in recent years, yet their precise roles in regulating functions such as cell signalling remain to be determined. Endocytosis of lipid rafts/caveolae follows a clathrin-independent route to both early endosomes and non-classical caveosomes. The observation that a variety of cellular pathogens localize to and internalize with these microdomains provides an additional incentive to characterize the organization, dynamics and functions of these domains.     

Plasma membrane microdomains: organization, function and trafficking

The plasma membrane consists of a mosaic of functional microdomains facilitating a variety of physiological processes associated with the cell surface. In most cells, the majority of the cell surface is morphologically featureless, leading to difficulties in characterizing its organization and microdomain composition. The reliance on indirect and perturbing techniques has led to vigorous debate concerning the nature and even existence of some microdomains. Recently, increasing technical sophistication has been applied to study cell surface compartmentalization providing evidence for small, short-lived clusters that may be much less than 50 nm in diameter. Lipid rafts and caveolae are cholesterol-dependent, highly ordered microdomains that have received most attention in recent years, yet their precise roles in regulating functions such as cell signalling remain to be determined. Endocytosis of lipid rafts/caveolae follows a clathrin-independent route to both early endosomes and non-classical caveosomes. The observation that a variety of cellular pathogens localize to and internalize with these microdomains provides an additional incentive to characterize the organization, dynamics and functions of these domains.     

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.     

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.     

Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids

Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid–protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease.     

Production of phosphatidylinositol 3,4,5-trisphosphate and phosphatidic acid in platelet rafts: evidence for a critical role of cholesterol-enriched domains in human platelet activation.

Glycosphingolipid- and cholesterol-enriched membrane microdomains, called rafts, can be isolated from several mammalian cells, including platelets. These microdomains appear to play a critical role in signal transduction in several hematopoietic cells, but their function in blood platelets remains unknown. Herein, we first characterized the lipid composition, including the fatty acid composition of phospholipids, of human platelet rafts. Then their role in platelet activation process was investigated. Interestingly, thrombin stimulation led to morphological changes of rafts correlating with the production of lipid second messengers in these microdomains. Indeed, we could demonstrate for the first time that a large part of the stimulation-dependent production of phosphatidic acid and phosphoinositide 3-kinase products was concentrated in rafts. Moreover, cholesterol depletion with methyl-beta-cyclodextrin disrupted platelet rafts, dramatically decreased the agonist-dependent production of these lipid signaling molecules, and impaired platelet secretion and aggregation. Cholesterol repletion restored the physiological platelet responses. Altogether our data indicate that rafts are highly dynamic platelet membrane structures involved in critical signaling mechanisms linked to the production of lipid second messengers. The demonstration of phosphatidylinositol 3,4,5-trisphosphate production in rafts may have general implications for the understanding of the role of this key second messenger found ubiquitously in higher eucaryotic cells.     

Lipid rafts and little caves. Compartmentalized signalling in membrane microdomains.

Lipid rafts are liquid-ordered membrane microdomains with a unique protein and lipid composition found on the plasma membrane of most, if not all, mammalian cells. A large number of signalling molecules are concentrated within rafts, which have been proposed to function as signalling centres capable of facilitating efficient and specific signal transduction. This review summarizes current knowledge regarding the composition, structure, and dynamic nature of lipid rafts, as well as a number of different signalling pathways that are compartmentalized within these microdomains. Potential mechanisms through which lipid rafts carry out their specialized role in signalling are discussed in light of recent experimental evidence.     

Docosahexaenoic acid affects endothelial nitric oxide synthase in caveolae

n-3 Polyunsaturated fatty acids are assumed to play an important role in the prevention and treatment of atherosclerosis. Endothelial nitric-oxide synthase (eNOS) is responsible for cardiovascular homeostasis involving in regulation of vascular function, and the subcellular localization is critical for its activation. Here we determined the effect of docosahexaenoic acid (DHA, 22:6 n-3) on distribution of eNOS and its activity. DHA treatment markedly altered lipid environment of caveolae microdomains, which was coincided with selective displacement of caveolin-1 and eNOS from caveolae. Akt was not detected in caveolae fractions and CaM was distributed in both of caveolin-1-enriched membranes and non-caveolar fractions, whose distribution was unaffected by DHA. These data demonstrated for the first time that DHA altered caveolae microenvironment not only by modifying membrane lipid composition, but also by changing distribution of major structural proteins. DHA-induced alterations in caveolae lipid/protein environment may be an important mechanism in the development of pathogenesis of atherosclerosis.