The Structure of Polyunsaturated Lipid Bilayers Important for Rhodopsin Function: A Neutron Diffraction Study

The structure of oriented 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine bilayers with perdeuterated stearoyl- or docosahexaenoyl hydrocarbon chains was investigated by neutron diffraction. Experiments were conducted at two different relative humidities, 66 and 86%. At both humidities we observed that the polyunsaturated docosahexaenoyl chain has a preference to reside near the lipid water interface. That leaves voids in the bilayer center that are occupied by saturated stearoyl chain segments. This uneven distribution of saturated- and polyunsaturated chain densities is likely to result in membrane elastic stress that modulates function of integral receptor proteins like rhodopsin.     

Exposure to long chain polyunsaturated fatty acids triggers rapid multimerization of synucleins.

Detergent-stable multimers of alpha-synuclein have been found specifically in the brains of patients with Parkinson's disease and other neurodegenerative diseases. Here we show that recombinant alpha-synuclein forms multimers in vitro upon exposure to vesicles containing certain polyunsaturated fatty acid (PUFA) acyl groups, including arachidonoyl and docosahexaenoyl. This process occurs at physiological concentrations and much faster than in aqueous solution. PUFA-induced aggregation involves physical association with the vesicle surface via the large apolipoprotein-like lipid-binding domain that constitutes the majority of the protein. beta- and gamma-synucleins, as well as the Parkinson's disease-associated alpha-synuclein variants A30P and A53T, show similar tendencies to multimerize in the presence of PUFAs. Multimerization does not require the presence of any tyrosine residues in the sequence. The membrane-based interaction of the synucleins with specific long chain polyunsaturated phospholipids may be relevant to the protein family's physiological functions and may also contribute to the aggregation of alpha-synuclein observed in neurodegenerative disease.     

Structure and dynamics of polyunsaturated hydrocarbon chains in lipid bilayers-significance for GPCR function

This review summarizes results of our recent solid-state NMR investigations on polyunsaturated 18:0-22:6n3-PC/PE/PS and 18:0-22:5n6-PC bilayers. Data on structure and dynamics of the polyunsaturated docosahexaenoyl (DHAn3, 22:6n3) and docosapentaenoyl chains (DPAn6, 22:5n6), investigated at physiological conditions, are reported. Lipid–protein interaction was studied on reconstituted bilayers containing the G-protein coupled membrane receptor (GPCR) rhodopsin as well as on rod outer segment (ROS) disk membranes prepared from bovine retinas. Results reveal surprisingly rapid conformational transitions of polyunsaturated chains and existence of weakly specific interactions of DHAn3 with spatially distinct sites on rhodopsin.     

n−3 polyunsaturated N-acylethanolamines are CB2 cannabinoid receptor-preferring endocannabinoids

Anandamide, the first identified endogenous cannabinoid and TRPV1 agonist, is one of a series of endogenous N-acylethanolamines, NAEs. We have generated novel assays to quantify the levels of multiple NAEs in biological tissues and their rates of hydrolysis through fatty acid amide hydrolase. This range of NAEs was also tested in rapid response assays of CB₁, CB₂ cannabinoid and TRPV1 receptors. The data indicate that PEA, SEA and OEA are not endocannabinoids or endovanilloids, and that the higher endogenous levels of these metabolites compared to polyunsaturated analogues are a correlate of their slow rates of hydrolysis. The n?6 NAEs (AEA, docosatetraenoyl and docosapentaenoyl derivatives) activated both CB₁ and CB₂ receptors, as well as TRPV1 channels, suggesting them to be ‘genuine’ endocannabinoids and ‘endovanilloids’. The n?3 NAEs (eicosapentaenoyl, docosapentaenoyl and docosahexaenoyl derivatives) activated CB₂ receptors and some n?3 NAEs (docosapentaenoyl and docosahexaenoyl derivatives) also activated TRPV1 channels, but failed to activate the CB₁ receptor. We hypothesise that the preferential activation of CB₂ receptors by n?3 PUFA NAEs contributes, at least in some part, to their broad anti-inflammatory profile.     

Lipid-protein interactions mediate the photochemical function of rhodopsin

We have investigated the molecular features of recombinant membranes that are necessary for the photochemical function of rhodopsin. The magnitude of the metarhodopsin I to metarhodopsin II phototransient following a 25% +/- 3% bleaching flash was used as a criterion of photochemical activity at 28 degrees C and pH 7.0. Nativelike activity of rhodopsin can be reconstituted with an extract of total lipids from rod outer segment membranes, demonstrating that the protein is minimally perturbed by the reconstitution protocol. Rhodopsin photochemical activity is enhanced by phosphatidylethanolamine head groups and docosahexaenoyl (22:6 omega 3) acyl chains. An equimolar mixture of phosphatidylethanolamine and phosphatidylcholine containing 50 mol% docosahexaenoyl chains results in optimal photochemical function. These results suggest the importance of both the head-group and acyl chain composition of the rod outer segment lipids in the visual process. The extracted rod lipids and those lipid mixtures favoring the conformational change from metarhodopsin I to II can undergo lamellar (L alpha) to inverted hexagonal (HII) phase transitions near physiological temperature. Interaction of rhodopsin with membrane lipids close to a L alpha to HII (or cubic) phase boundary may thus lead to properties which influence the energetics of conformational states of the protein linked to visual function.     

Dehydration induces lateral expansion of polyunsaturated 18:0-22:6 phosphatidylcholine in a new lamellar phase.

To gain a better understanding of the biological role of polyunsaturated phospholipids, infrared (IR) linear dichroism, NMR, and x-ray diffraction studies have been conducted on the lyotropic phase behavior and bilayer dimensions of sn-1 chain perdeuterated 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC-d35), a mixed-chain saturated (18:0)-polyunsaturated (22:6 omega 3) lipid. SDPC films were hydrated at definite values of temperature (T) and relative humidity (RH). In excess water, the lipid forms exclusively lamellar phases in the temperature range 0--50 degrees C. Upon dehydration the lipid undergoes the main phase transition between the liquid-crystalline (L(alpha)) and gel (L(beta)) phase at T < 15 degrees C. Both the saturated and polyunsaturated chains adopt a stretched conformation in the L(beta) phase, presumably the all-trans (stearoyl) and angle iron or helical (docosahexaenoyl) one. A new fluid lamellar phase (L(alpha)') was found in partially hydrated samples at T > 15 degrees C. SDPC membranes expand laterally and contract vertically in the L(alpha)' phase when water was removed. This tendency is in sharp contrast to typical dehydration-induced changes of membrane dimensions. The slope of the phase transition lines in the RH-T phase diagram reveal that the lyotropic L(alpha)'-L(alpha) and L(beta)-L(alpha) transitions are driven by enthalpy and entropy, respectively The possible molecular origin of the phase transitions is discussed. The properties of SDPC are compared with that of membranes of monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d31).     

Evidence for specificity in lipid-rhodopsin interactions

The interaction of bovine rhodopsin with poly- and monounsaturated lipids was studied by (1)H MAS NMR with magnetization transfer from rhodopsin to lipid. Experiments were conducted on bovine rod outer segment (ROS) disks and on recombinant membranes containing lipids with polyunsaturated, docosahexaenoyl (DHA) chains. Poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rates of magnetization transfer from protein to DHA are lipid headgroup-dependent and increased in the sequence PC < PS < PE. Boundary lipids are in fast exchange with the lipid matrix on a time scale of milliseconds or shorter. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for Meta-III rhodopsin. The experiments show clearly that the surface of rhodopsin has sites for specific interaction with lipids. Current theories of lipid-protein interaction do not account for such surface heterogeneity.     

Low-temperature 2H NMR spectroscopy of phospholipid bilayers containing docosahexaenoyl (22:6 omega 3) chains.

Polyunsaturated fatty acids are widely distributed components of biological membranes and are believed to be involved in many biological functions. However, the mechanisms by which they act on a molecular level are not understood. To further investigate the unique properties of omega 3 polyunsaturated phospholipid bilayers, deuterium nuclear magnetic resonance (2H NMR) studies have been made of the liquid-crystalline (L alpha) and gel phases of a homologous series of mixed-chain phosphatidylcholines containing docosahexaenoic acid: (per-2H-n:0)(22:6)PC, where n = 12, 14, 16, and 18. The moments of the 2H NMR lineshapes have been evaluated, and from these the warming and cooling main phase transition temperatures were determined. The transition temperatures of the mixed-chain series were found to be significantly lower than those of the corresponding lipids in the disaturated series, di(per-2H-n:0)PC, with hystereses ranging from 2 to 14 degrees C. Distinct effects of the docosahexaenoyl chain on bilayer order were found, though these effects varied across the mixed-chain series. In evaluating the moment data, an empirical method for normalizing the moments with respect to differences in temperature was applied, in addition to using the reduced temperature method. For the systems studied here, the method of normalization had no significant effect on the interpretation of the moment data.     

Docosahexaenoyl dopamine in freshwater hydra: Effects on regeneration and metabolic changes

The effects of docosahexaenoyl dopamine and docosahexaenoic acid on the regeneration of hydra gastric and basal fragments are studied. Docosahexaenoyl dopamine induced morphogenetic abnormalities such as single ectopic tentacles in the gastric region and projections in the gastric and basal regions. Docosahexaenoic acid had no effect on the morphogenesis except for a mild slowing of the regeneration rate. Since no hydrolysis of docosahexaenoyl dopamine was detected in hydra extract, it was assumed that the morphogenetic effect could be associated with the dopamine component of this complex.     

Recognition of polyunsaturated acyl chains by enzymes acting on membrane lipids

Polyunsaturated acyl chains play an important role in human biology. These lipids cannot be synthesized de novo and they are selectively distributed to certain organs and are found predominantly only in certain lipid classes. Their selective distribution is a consequence of the specificity of the binding of these lipids by certain proteins. Lipoxygenases are a group of well studied enzymes that specifically oxidize polyunsaturated fatty acids. We propose that certain features of the interaction of lipoxygenases with polyunsaturated acyl chains are also found in other unrelated proteins that act on lipids with these moieties. The features common to several of the enzymes that specifically interact with polyunsaturated acyl chains include the fact that the polyunsaturated chain is drawn out of the membrane to bind to a hydrophobic channel within the protein and that a similar pattern of required amino acids residues comprises part of the binding site for the polyunsaturated chain. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Polyunsaturated phospholipids promote the oxidation and fragmentation of gamma-hydroxyalkenals: formation and reactions of oxidatively truncated ether phospholipids

Low density lipoprotein contains traces of biologically active platelet-activating factor (PAF)-like ether phosphatidylcholines (PCs). These oxidatively truncated alkylacylphosphatidylcholines (OxPAFs) are presumably formed through the oxidative truncation of 1-alkyl-2-polyunsaturated fatty acyl PCs. We now report that a diverse structural variety of OxPAFs are generated in small unilamellar vesicles (SUVs) upon myeloperoxidase (MPO)-promoted autoxidation of ether PCs that incorporate linoleoyl, arachidonyl, or docosahexaenoyl groups at the sn-2 position. Total syntheses are reported that confirm the identities of the new OxPAFs and will facilitate the evaluation of their biologically important chemistry and activities. Especially noteworthy is the formation of OxPAFs containing gamma-hydroxyalkenal functionality. Analogous oxidatively truncated diacylphosphatidylcholines are biologically important because they and their more oxidized derivatives are strong ligands for the scavenger receptor CD36. Furthermore, their covalent adduction with proteins can interfere with protein function or generate biologically active carboxyalkylpyrrole derivatives. We now find a profound influence of membrane composition on the stability of OxPAFs. In the presence of a polyunsaturated diacyl PC, the linoleic acid ester of 2-lysophosphatidylcholine, MPO induces the oxidation of aldehydes to carboxylic acids and the further oxidative truncation of gamma-hydroxyalkenals. Remarkably, these reactions do not occur readily with MPO in SUVs composed entirely of saturated diacyl-PCs. A mechanistic rationale is presented that can account for this dichotomy.     

Temperature dependence of conformational properties of natural lipid oligomer chains: computer simulation

The conformational properties of several oligomeric chain molecules at T = 278 and T = 403 K have been studied using Monte Carlo computer simulations. Hydrocarbon oligomers with methylene-interrupted cis double bonds in the main chain were considered. These oligomers are typical constituents of natural lipid molecules. The characteristics of the shape of C-H and C-C bond orientation distribution functions with respect to the principal axis of inertia of the chains and their temperature dependences were studied. It was found that the temperature sensitivity of not only the common geometric characteristics of the polyunsaturated chain is significantly reduced compared with the saturated one, but also that of local characteristics, i. e., the shape of each bond orientation distribution function of the polyunsaturated chain. The relationship between the properties of lipid polyunsaturated hydrocarbon chains and their functions in natural membrane systems, in particular their possible role in the stabilization or optimization of lipid-protein interactions, was discussed.     

“自我剪切”2A肽介导的Δ-12和ω-3脂肪酸脱氢酶以及过氧化氢酶在转基因小鼠肌肉表达研究

哺乳动物因为缺乏Δ-12和ω-3脂肪酸脱氢酶,不能自身合成必需的多不饱和脂肪酸．目前,通过转基因技术在哺乳动物体内表达ω-3脂肪酸脱氢酶,能将长链的n-6多不饱和脂肪酸转化成n-3多不饱和脂肪酸,造成体内长链的n-6多不饱和脂肪酸含量显著减低．本研究通过自我剪切2A肽介导Δ-12和ω-3脂肪酸脱氢酶(FAT-2和FAT-1)以及人过氧化氢酶(human catalase,hCAT)在小鼠的肌肉同时表达．结果表明,转基因小鼠肌肉中长链n-3多不饱和脂肪酸含量提高2.6倍,长链n-6多不饱和脂肪酸含量没有显著变化,而n-6/n-3比例显著降低(P < 0.01)．同时蛋白质印迹检测到人过氧化氢酶hCAT在小鼠的肌肉组织中表达,且过氧化氢酶活性比野生型小鼠显著提高(P < 0.01)．     

On the conformational, physical properties and functions of polyunsaturated acyl chains

The conformational properties of the acyls of biological membranes--hydrocarbon chains with isolated cis double bonds--were studied by computer simulation. The Monte Carlo method was used, with continuous variation of bond rotation angles within the (0, 360 degree) range considered. It has been shown, that if all double bonds of molecules are separated only by one methylene group, and their number in the chain is maximum, the molecule is characterized by the highest equilibrium flexibility (at temperatures only encountered by biological systems) as compared to any similar molecules. It is such a structure which is inherent to docosahexaenoic acid. The above molecule coefficient that characterizes the temperature sensitivity of the molecule sizes is 10-times lower than that of a saturated chain. The polyunsaturated chain segment with high probability assumes the extended (in perfect crystal structures the 'angle iron-shaped') conformation when all the molecules are efficiently packed below the phase-transition temperatures. The annular lipid layer of embedded enzymes is assumed to be enriched with polyunsaturated fatty acid acyls. The above physical properties of polyunsaturated chains are bound to favour the maintenance of the proper conformational mobility of biomembrane enzymes, to relax the negative influence of environmental temperature changes on their activity. When freezing biological membranes they are bound to provide the molecule packing which is free of high tensions.     

Structural determinants of the packing and electrostatic behavior of unsaturated phosphoglycerides

Docosahexaenoic acid-containing phosphoglycerides accumulate preferentially in membranes of the retina, brain, and spermatozoa, but the functional significance of this largely remains to be determined. Previously we compared the physical properties of homogeneous monolayers of these and other phosphoglyceride species to obtain insights into their physiological roles. Particularly noteworthy were the unusually low dipole moments of species having sn-2-docosahexaenoyl chains. In this study, we have investigated the electrostatic and lateral packing properties of related phosphoglycerides and found that: 1), The dipole moment-lowering effect of the docosahexaenoyl group arises from its having a Z double bond at chain position n-3. 2), The large dipole moment-lowering effects at sn-1 of an ether bond to an alkyl or a 1Z alkenyl chain and that of a sn-2-esterified n-3 fatty acid are additive. 3), The 1Z double bond in an alkenyl chain lowers the molecular area of a phosphoglyceride and, concomitantly, makes it less compressible. 4), Ethanolamine-containing phosphoglycerides are generally less compressible than their corresponding choline analogs. Our data showing that relatively small lipid structural changes markedly alter lipid physical properties in fluid phases underscores the need to study the function of peripheral and integral membrane proteins in the presence of appropriate lipid species.     

Cannabinoid receptor antagonists and fatty acids alter endocannabinoid system gene expression and COX activity

Cyclooxygenase (COX) possesses substrate affinity for the endocannabinoids (EC) anandamide (AEA) and 2-arachidonylglycerol (2-AG). We hypothesized that selective antagonism/activation of the cannabinoid receptors will increase COX activity and the availability of EC as substrates will lead to higher COX activity. Since the relationship between EC signaling of the endocannabinoid system (ECS) and the COX pathway in muscle has not been investigated, we examined agonist, antagonists and polyunsaturated fatty acid effects on ECS genes in myoblasts. At 50% confluency, C2C12 myoblasts were pretreated with 5 μM of the cannabinoid receptor (CB)2 inverse agonist AM630 for 2 h and one with both AM630 and 1 μM of the CB1 antagonist NESS0327. Cell cultures pretreated with AM630 were then administered with 25 μM of either arachidonic acid (20:4n6), eicosapentaenoate (EPA) (20:5n3), docosahexaenoate (DHA) (22:6n3), AEA or bovine serum albumin (vehicle control) for 24 h. Quantitative polymerase chain reaction analyses were performed looking at ECS and prostaglandin genes. Total COX activity and COX-1 protein were greater in the AM630+AEA-treated cells compared to all other cell cultures. The mRNA for the AEA synthesis enzyme N-acyl phosphatidylethanolamine phospholipase D and the 2-AG synthesis enzymes diacylglycerol lipase (DAGL)α and DAGLβ were higher in AM630+EPA-treated cells compared to the other groups. The mRNA levels of CB1 and CB2 were both highest in the AM630+EPA group. The mRNA for interleukin-6 and tumor necrosis factor-α was higher with AEA but lower with DHA and docosahexaenoyl ethanolamide (DHEA), supporting previous findings that the EC AEA supports activation of the COX system. These findings suggest that COX activity and protein levels are influenced by the ECS, specifically by the ligand AEA for CB1 and by inverse agonism of CB2.     

Distribution of macular xanthophylls between domains in a model of photoreceptor outer segment membranes

A model of photoreceptor outer segment (POS) membranes has been proposed, consisting of an equimolar ternary mixture of 1-palmitoyl-2-docosahexaenoylphosphatidylcholine/distearoylphosphatidylcholine/cholesterol. It was shown that, as in membranes made from the raft-forming mixture, in the model of POS membranes, two domains are formed: the raft domain (detergent resistant membranes, DRM), and the bulk domain (detergent soluble membranes, DSM). Saturation-recovery EPR discrimination by oxygen transport method also demonstrated the presence of two domains in this model system in situ at a wide range of temperatures (10-55 degrees C), showing additionally that neither lutein nor zeaxanthin at 1 mol% affect the formation of these domains. These membrane domains have been separated using cold Triton X-100 extraction from a model of POS membranes containing 1 mol% of either lutein or zeaxanthin. The results indicated that the macular xanthophylls lutein and zeaxanthin are substantially excluded from DRM and remain concentrated in DSM, a domain enriched in highly unsaturated docosahexaenoyl acid which is abundant in retina membranes. The concentration of xanthophylls in DRM and DSM calculated as the mol ratio of either xanthophyll to total lipid (phospholipid+cholesterol) was 0.0028 and 0.0391, respectively. Thus, xanthophylls are about 14 times more concentrated in DSM than in DRM. No significant difference in the distribution of lutein and zeaxanthin was found. The obtained results suggest that in POS membranes macular xanthophylls should also be concentrated in domains enriched in polyunsaturated chains.     

Lumenal hydrolysis of menhaden and rapeseed oils and their fatty acid methyl and ethyl esters in the rat

Simple alkyl (ethyl) esters of polyunsaturated fish oil fatty acids have been proposed as dietary supplements, but their relative efficiency of digestion and absorption have not been determined. Using stomach tubes, we gave rats menhaden or rapeseed oils, or the corresponding methyl and ethyl esters, and determined by chromatographic methods the lipid classes and molecular species recovered from the lumen of the jejunum during the first 1 to 2.5 h of digestion. Hydrolysis of menhaden oil resulted in a preferential retention of a high proportion of the polyunsaturated long chain acids in the sn-2-monoacylglycerols and in the residual triacyglycerols, while digestion of rapeseed oil led to a preferential release of free long chain monounsaturated fatty acids. In contrast, hydrolysis of the alkyl (methyl and ethyl) esters of the fatty acids of either menhaden or rapeseed oil resulted in a composition of free fatty acids which was much more representative of the original esters. It was therefore concluded that the differential lumenal liberation of the long chain and polyunsaturated (three or more double bonds) fatty acids from fish and rapeseed oil is largely due to their characteristic distribution between the primary and secondary positions in the glycerol molecule, and to a much lesser extent to a chain length discrimination by pancreatic lipase. This study also shows that the methyl and ethyl esters are hydrolyzed about 4 times more slowly than the corresponding triacylglycerols, which is sufficient to maintain a saturated micellar solution of fatty acids in the intestinal lumen during absorption.     

Production of structured lipids by lipase-catalysed interesterification in an ultrafiltration membrane reactor

The application of membranes to the enzymatic production of structured lipids has been investigated using an ultrafiltration membrane reactor for a reaction between medium chain triacylglycerols (MCT) and n-3 polyunsaturated fatty acids from fish oil. Lipozyme IM was used as the biocatalyst. The incorporation of polyunsaturated fatty acids into MCT was increased by about 15% over 80 h by simultaneous separation of the released medium chain fatty acids compared to control.