莱茵衣藻中脂质代谢过程研究进展

微藻中脂质代谢产生的化合物,可用于生物燃料、营养品和生物药品的生产,因此具有重要的经济价值。脂质代谢贯穿微藻的全部生命过程,对微藻的生长发育和应对外界胁迫都具有重要意义。微藻与研究较清楚的真菌和陆地植物在脂质代谢过程方面具有相似性。当然,随着微藻脂质代谢相关功能基因逐渐被鉴定,人们发现微藻的脂质代谢也具有区别真菌和陆地植物的独特性,因此针对微藻脂质代谢过程的分析具有重要意义。莱茵衣藻是研究脂质代谢过程的模式生物,已经通过基因组、转录组、蛋白质组和代谢组等方法,对其质体、内质网和过氧化物酶体中进行的脂质合成和分解过程进行了研究。本文总结了近年来莱茵衣藻质体、内质网和过氧化物酶体中脂质代谢过程的研究成果,并进行综合阐述。     

FCS Analysis of Protein Mobility on Lipid Monolayers

In vitro membrane model systems are used to dissect complex biological phenomena under controlled unadulterated conditions. In this context, lipid monolayers are a powerful tool to particularly study the influence of lipid packing on the behavior of membrane proteins. Here, monolayers deposited in miniaturized fixed area-chambers, which require only minute amounts of protein, were used and shown to faithfully reproduce the characteristics of Langmuir monolayers. This assay is ideally suited to be combined with single-molecule sensitive fluorescence correlation spectroscopy (FCS) to characterize diffusion dynamics. Our results confirm the influence of lipid packing on lipid mobility and validate the use of FCS as an alternative to conventional surface pressure measurements for characterizing the monolayer. Furthermore, we demonstrate the effect of lipid density on the diffusional behavior of membrane-bound components. We exploit the sensitivity of FCS to characterize protein interactions with the lipid monolayer in a regime in which the monolayer physical properties are not altered. To demonstrate the potential of our approach, we analyzed the diffusion behavior of objects of different nature, ranging from a small peptide to a large DNA-based nanostructure. Moreover, in this work we quantify the surface viscosity of lipid monolayers. We present a detailed strategy for the conduction of point FCS experiments on lipid monolayers, which is the first step toward extensive studies of protein-monolayer interactions.     

Lipid droplets: proteins floating on a pool of fat

Proteins on the surface of lipid storage droplets are crucial to droplet structure and function, but are poorly understood. Recent reports reveal a function for perilipins, major lipid droplet proteins in adipocytes, and show that caveolin proteins can accumulate on lipid droplets.     

Calorimetric and Fourier transform infrared spectroscopic studies on the interaction of glycophorin with phosphatidylserine/dipalmitoylphosphatidylcholine-d62 mixtures

Glycophorin has been isolated in pure form from human erythrocyte membranes and reconstituted into lipid vesicles composed of binary mixtures of bovine brain phosphatidylserine (PS) and acyl-chain perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62). The effect of protein on lipid melting behavior and order has been monitored with differential scanning calorimetry and Fourier transform infrared spectroscopy (FT-IR). The phase diagram for PS/DPPC-d62 is consistent with that previously reported for PS/DPPC (Stewart et al. (1979) Biochim. Biophys. Acta 556, 1-16) and indicates that acyl chain perdeuteration does not greatly alter the lipid mixing characteristics. The use of deuterated lipid allows the examination of lipid order by FT-IR of each lipid component in the binary mixtures as well as in the ternary (lipid/lipid/protein) systems. Addition of glycophorin to a 30:70 PS/DPPC-d62 binary lipid mixture results in a preferential glycophorin/PS interaction leading to bulk lipid enriched in DPPC-d62. This is revealed in two ways: first, through cooperative calorimetric transitions increased in temperature from the binary lipid system and second, through FT-IR melting curves of the DPPC-d62 component which shows transitions increased in both onset and completion temperatures in the presence of protein. In addition, non-cooperative melting events are observed at temperatures below the onset of phase separation. The FT-IR data are used to assign these non-cooperative events to the melting of the PS component. For the 50:50 lipid mixture with protein, two transitions are observed in the DSC experiments. The IR results indicate that both lipid components are involved with the lower temperature event.     

The size dependence of cholate-dialyzed vesicles on phosphatidylcholine concentration.

The size dependence of vesicles prepared by dialysis of cholate from phosphatidylcholine (PC) dispersions has been investigated as a function of lipid concentration (at a constant applied lipid: detergent molar ratio of 0.7). Gel filtration of dialyzed samples produced a symmetrical profile shape, although quasielastic laser light scattering analysis of the fractions revealed an asymmetrical range of sizes about the peak for solutions containing elevated lipid concentrations. Vesicle diameters increased by approximately 20 nm for PC concentrations ranging from 10 to a maximum of 45 mg/ml. This was attributed to mixed micelle sizes being proportional to lipid concentration, since the diameters of vesicles produced from dialysis are determined by mixed micelle sizes. Before commencement of dialysis, mixed micelle sizes are proportional to lipid concentration and, although dialysis causes an increase in mixed micelle sizes, the phase ratios attained are larger for solutions containing elevated lipid concentrations.     

冷却肉糜辐照脂肪氧化影响因素与抑制效果研究

研究了低剂量辐照、抗氧化剂、包装条件以及贮藏时间等影响辐照冷却肉脂肪氧化的主要因素和抑制效果.结果表明,对冷却猪肉的辐照处理会加剧其脂肪的氧化,真空包装或无氧包装可以明显的降低贮藏期间辐照冷却肉的脂肪氧化.辐照处理前在肉糜中添加抗氧化剂并采用真空包装时, 肉糜可以贮藏2周以上而不产生脂肪氧化.比较几种主要的抗氧化剂的效果表明,茶多酚在抑制辐照冷却肉糜脂肪氧化方面效果最好.     

Sorting of streptavidin protein coats on phase-separating model membranes

Heterogeneities in cell membranes due to the ordering of lipids and proteins are thought to play an important role in enabling protein and lipid trafficking throughout the secretory pathway and in maintaining cell polarization. Protein-coated vesicles provide a major mechanism for intracellular transport of select cargo, which may be sorted into lipid microdomains; however, the mechanisms and physical constraints for lipid sorting by protein coats are relatively unexplored. We studied the influence of membrane-tethered protein coats on the sorting, morphology, and phase behavior of liquid-ordered lipid domains in a model system of giant unilamellar vesicles composed of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol. We created protein-coated membranes by forming giant unilamellar vesicles containing a small amount of biotinylated lipid, thereby creating binding sites for streptavidin and avidin proteins in solution. We found that individual tethered proteins colocalize with the liquid-disordered phase, whereas ordered protein domains on the membrane surface colocalize with the liquid-ordered phase. These observations may be explained by considering the thermodynamics of this coupled system, which maximizes its entropy by cosegregating ordered protein and lipid domains. In addition, protein ordering inhibits lipid domain rearrangement and modifies the morphology and miscibility transition temperature of the membrane, most dramatically near the critical point in the membrane phase diagram. This observation suggests that liquid-ordered domains are stabilized by contact with ordered protein domains; it also hints at an approach to the stabilization of lipid microdomains by cross-linked protein clusters or ordered protein coats.     

Lipid microdomain clustering induces a redistribution of antigen recognition and adhesion molecules on human T lymphocytes

The study of lipid microdomains in the plasma membrane is a topic of recent interest in leukocyte biology. Many T cell activation and signaling molecules are found to be associated with lipid microdomains and have been implicated in normal T cell function. It has been proposed that lipid microdomains with their associated molecules move by lateral diffusion to areas of cellular interactions to initiate signaling pathways. Using sucrose density gradients we have found that human T cell beta(1) integrins are not normally associated with lipid microdomains. However, cross-linking of GM1 through cholera toxin B-subunit (CTB) causes an enrichment of beta(1) integrins in microdomain fractions, suggesting that cross-linking lipid microdomains causes a reorganization of molecular associations. Fluorescent microscopy was used to examine the localization of various lymphocyte surface molecules before and after lipid microdomain cross-linking. Lymphocytes treated with FITC-CTB reveal an endocytic vesicle that is enriched in TCR and CD59, while beta(1) integrin, CD43, and LFA-3 were not localized in the vesicle. However, when anti-CTB Abs are used to cross-link lipid microdomains, the microdomains are not internalized but are clustered on the cell surface. In this study, CD59, CD43, and beta(1) integrin are all seen to colocalize in a new lipid microdomain from which LFA-3 remains excluded and the TCR is now dissociated. These findings show that cross-linking lipid microdomains can cause a dynamic rearrangement of the normal order of T lymphocyte microdomains into an organization where novel associations are created and signaling pathways may be initiated.     

Lipid mono- and bilayer supported on polymer films: composite polymer-lipid films on solid substrates.

We report the deposition of lipid monolayers and bilayers on polyacrylamide films deposited by radical chain reaction onto solid substrates in aqueous solutions. Polymer films of various degrees of monomer density and cross-linking are prepared. Lateral diffusion and fluorescent probe permeation measurements yield insight into the continuity of the lipid layers and show that monolayers exposed to air are much less sensitive towards polymer heterogeneities than bilayers below water, which is explained in terms of the wetting laws. The diffusion studies of lipid and lipopeptide probes yield absolute values of the frictional coefficients between the lipid layer and the polymer films and allow one to estimate the surface viscosity of the polymer film. The potential applications of supported membranes on soft thin polymer films for the preparation of biofunctionalized surfaces or biocompatible receptive surfaces for biosensors are discussed.     

Influence of Membrane Lipid Composition on a Transmembrane Bacterial Chemoreceptor

Most bacterial chemoreceptors are transmembrane proteins. Although less than 10% of a transmembrane chemoreceptor is embedded in lipid, separation from the natural membrane environment by detergent solubilization eliminates most receptor activities, presumably because receptor structure is perturbed. Reincorporation into a lipid bilayer can restore these activities and thus functionally native structure. However, the extent to which specific lipid features are important for effective restoration is unknown. Thus we investigated effects of membrane lipid composition on chemoreceptor Tar from Escherichia coli using Nanodiscs, small (∼10-nm) plugs of lipid bilayer rendered water-soluble by an annulus of “membrane scaffold protein.” Disc-enclosed bilayers can be made with different lipids or lipid combinations. Nanodiscs carrying an inserted receptor dimer have high protein-to-lipid ratios approximating native membranes and in this way mimic the natural chemoreceptor environment. To identify features important for functionally native receptor structure, we made Nanodiscs using natural and synthetic lipids, assaying extents and rates of adaptational modification. The proportion of functionally native Tar was highest in bilayers closest in composition to E. coli cytoplasmic membrane. Some other lipid compositions resulted in a significant proportion of functionally native receptor, but simply surrounding the chemoreceptor transmembrane segment with a lipid bilayer was not sufficient. Membranes effective in supporting functionally native Tar contained as the majority lipid phosphatidylethanolamine or a related zwitterionic lipid plus a rather specific proportion of anionic lipids, as well as unsaturated fatty acids. Thus the chemoreceptor is strongly influenced by its lipid environment and is tuned to its natural one.     

Complex biomembrane mimetics on the sub-nanometer scale

Biomimetic lipid vesicles are indispensable tools for gaining insight into the biophysics of cell physiology on the molecular level. The level of complexity of these model systems has steadily increased, and now spans from domain-forming lipid mixtures to asymmetric lipid bilayers. Here, we review recent progress in the development and application of elastic neutron and X-ray scattering techniques for studying these systems in situ and under physiologically relevant conditions on the nanometer to sub-nanometer length scales. In particular, we focus on: (1) structural details of coexisting liquid-ordered and liquid-disordered domains, including their thickness and lipid packing mismatch as a function of a size transition from nanoscopic to macroscopic domains; (2) membrane-mediated protein partitioning into lipid domains; (3) the role of the aqueous medium in tuning interactions between membranes and domains; and (4) leaflet-specific structure in asymmetric bilayers and passive lipid flip-flop.     

Domain formation induced by the adsorption of charged proteins on mixed lipid membranes

Peripheral proteins can trigger the formation of domains in mixed fluid-like lipid membranes. We analyze the mechanism underlying this process for proteins that bind electrostatically onto a flat two-component membrane, composed of charged and neutral lipid species. Of particular interest are membranes in which the hydrocarbon lipid tails tend to segregate owing to nonideal chain mixing, but the (protein-free) lipid membrane is nevertheless stable due to the electrostatic repulsion between the charged lipid headgroups. The adsorption of charged, say basic, proteins onto a membrane containing anionic lipids induces local lipid demixing, whereby charged lipids migrate toward (or away from) the adsorption site, so as to minimize the electrostatic binding free energy. Apart from reducing lipid headgroup repulsion, this process creates a gradient in lipid composition around the adsorption zone, and hence a line energy whose magnitude depends on the protein's size and charge and the extent of lipid chain nonideality. Above a certain critical lipid nonideality, the line energy is large enough to induce domain formation, i.e., protein aggregation and, concomitantly, macroscopic lipid phase separation. We quantitatively analyze the thermodynamic stability of the dressed membrane based on nonlinear Poisson-Boltzmann theory, accounting for both the microscopic characteristics of the proteins and lipid composition modulations at and around the adsorption zone. Spinodal surfaces and critical points of the dressed membranes are calculated for several different model proteins of spherical and disk-like shapes. Among the models studied we find the most substantial protein-induced membrane destabilization for disk-like proteins whose charges are concentrated in the membrane-facing surface. If additional charges reside on the side faces of the proteins, direct protein-protein repulsion diminishes considerably the propensity for domain formation. Generally, a highly charged flat face of a macroion appears most efficient in inducing large compositional gradients, hence a large and unfavorable line energy and consequently lateral macroion aggregation and, concomitantly, macroscopic lipid phase separation.     

Flipping and flopping--lipids on the move

The rapid movement of polar lipids from one membrane leaflet to the other is facilitated by lipid flippases or translocases. Although their activity was first observed over 30 years ago, the structures, physiological roles, and molecular mechanisms of this group of proteins remain enigmatic. Lipid flippases maintain membrane lipid asymmetry, and in eukaryotes they are also intimately involved in membrane budding and vesicle trafficking. The ATP-dependent flippases are members of well-characterized protein families, whose other members transport nonlipid substrates across cell membranes. The P(4)-type ATPases carry out the inward translocation of phospholipids, and various ABC transporters are involved in outward lipid movement. The ATP-independent flippases move lipid substrates in both directions between membrane leaflets. With only a few exceptions, the molecular identity of these proteins is still unknown, despite their involvement in key biosynthetic pathways in both bacteria and eukaryotes. This review provides an overview of the different classes of flippases, and summarizes recent progress in their identification and functional characterization. The possible mechanisms of action of lipid flippases are discussed, and future directions explored.     

Eukaryotic lipid body proteins in oleogenous actinomycetes and their targeting to intracellular triacylglycerol inclusions: Impact on models of lipid body biogenesis

Bacterial neutral lipid inclusions are structurally related to eukaryotic lipid bodies. These lipid inclusions are composed of a matrix of triacylglycerols (TAGs) or wax esters surrounded by a monolayer of phospholipids. Whereas the monolayers of lipid bodies from animal and plant cells harbor specific classes of proteins which are involved in the structure of the inclusions and lipid homoestasis, no such proteins are known to be associated with bacterial lipid inclusions. The present study was undertaken to reveal whether the mammalian lipid body proteins perilipin A, adipose differentiation-related protein, and tail-interacting protein of 47 kDa (TIP47), which comprise the so called PAT family proteins, and the maize (Zea mays L.) oleosin are targeted to prokaryotic TAG bodies in vivo. When fused to enhanced green fluorescent protein, all proteins except the oleosin were mainly located at the surfaces of lipid inclusions when heterologously expressed in the recombinant actinomycetes Rhodococcus opacus PD630 and Mycobacterium smegmatis mc(2)155. A more detailed intracellular distribution analysis of TIP47 in recombinant R. opacus cells by immunocytochemical labeling of ultrathin cryosections and freeze fracture replicas revealed a substantial amount of TIP47 protein also pervading the cores of the inclusions. We discuss the impact of these results on the current model of lipid body biogenesis in prokaryotes.     

Immunocytochemical studies on lipid droplet-surface proteins in adrenal cells

Perilipin and ADRP, located on the surface of intracellular lipid droplets, are proposed to be involved in adipocyte lipid metabolism. The aim of the present study was to investigate the effect of PKA and PKC activities on the distribution of perilipin and ADRP in primary cultured adrenal cells, and the role of ERK in PMA- and calphostin C-induced steroidogenesis. Immunofluorescence staining indicated that in addition to p160, a capsular protein of steroidogenic lipid droplets, perilipin and ADRP were localized on the lipid droplet surface. Stimuli such as activation of PKA by db cAMP or inhibition of PKC by calphostin C, which increase corticosterone synthesis in various magnitudes, caused detachment of p160 and perilipin, but not ADRP, from the lipid droplet surface. Activation of PKC by PMA induced increase in corticosterone synthesis, however, it did not affect the distribution of perilipin, p160, or ADRP on the lipid droplet surface, suggesting the presence of mechanisms for promoting sterodiogensis other than causing detachment of lipid droplet surface proteins. We further demonstrated that ERK pathway was involved in PMA-induced steroidogenesis, since PD98059, specific inhibitor of MEK, blocked the increases in steroidogenesis and phosphorylation of ERK caused by PMA, but not by cAMP-PKA. These data indicate that p160, perilipin, and ADRP were all located on the lipid droplet surface in rat adrenal cells. On the basis of its non-responsiveness to lipolytic stimulation, ADRP may be a structural protein of the lipid droplet surface, whereas their immediate response to lipolytic stimuli suggest that perilipin and p160 are functional proteins. PKC regulates adrenal steroidogenesis through ERK cascade, whereas PKA pathway does not involve ERK.     

Different effects of lipid chain length on the two sides of a membrane and the lipid annulus of MscL

Quenching of the fluorescence of Trp residues in a membrane protein by lipids with bromine-containing fatty acyl chains provides a powerful technique for measuring lipid-protein binding constants. Single Trp residues have been placed on the periplasmic and cytoplasmic sides of the mechanosensitive channel of large conductance MscL from Mycobacterium tuberculosis to measure, separately, lipid binding constants on the two faces of MscL. The chain-length dependence of lipid binding was found to be different on the two sides of MscL, the chain-length dependence being more marked on the cytoplasmic than on the periplasmic side. To determine if lipid binding constants are affected by the properties of the lipid molecules not in direct contact with MscL (the bulk lipid), the amount of bulk lipid present in the system was varied. The binding constant of the short-chain phospholipid didodecylphosphatidylcholine was found to be independent of the molar ratio of lipid/MscL pentamer over the range 500:1-50:1, suggesting that lipid binding constants are determined largely by the properties of the lipid molecules interacting directly with MscL. These results point to a model in which lipid molecules located on the transmembrane surface of a membrane protein (the annular lipid molecules), by playing a dominant role in the interaction between a membrane protein and the surrounding lipid bilayer, could effectively buffer the membrane protein from changes in the properties of the bulk lipid bilayer.     

Immobilization and activity assay of cytochrome P450 on patterned lipid membranes

We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s.     

The organisation of cholesterol and ergosterol in lipid bilayers based on studies using non-perturbing fluorescent sterol probes.

The fluorescence properties of dehydroergosterol and cholesta-5,7,9-trien-3 beta-ol have been studied in organic solution, in aqueous dispersions and incorporated into aqueous lipid dispersions. The absorption spectra of aqueous dispersions of the probes are very different to those in organic solution, and aqueous dispersions are non-fluorescent. This can be attributed to micelle formation with dimerisation and/or aggregation in the micelles. Concentration quenching also occurs when sterols are incorporated into lipid bilayers, but relatively high fluorescence is observed even at a 1 : 1 steroid:lipid molar ratio. Further, the fluorescence is still polarized at these high molar ratios. We attribute this to the formation of ordered arrays of sterol molecules in the lipid bilayers. In these arrays the sterol molecules are organised in an end-to-end fashion, and face-to-face overlap of the sterols is prevented by the lipid molecules. Possible structures for 1 : 1 mixtures are presented.     

Some histochemical observations on Gaucher cells

Plastic-embedded bone marrow biopsies from four patients with Gaucher's disease have been studied histochemically. Concanavalin A (ConA) was found to bind to cytoplasmic inclusions of Gaucher cells; the binding was prevented by lipid extraction or beta-glucosidase digestion. This suggests that glucocerebrosides stored in Gaucher cells are responsible for ConA binding; ConA staining combined with lipid extraction and beta-glucosidase digestion tests may be taken as a tool for the demonstration of Gaucher's cerebrosides of possible practical importance in diagnosis and investigation of Gaucher's disease. An excess of vic-glycol groups with respect to ConA binding-sugar residues and not extractable by lipid solvents are demonstrable in Gaucher cells. Vic-glycols appear to be regularly arranged at the electron microscopy level within Gaucher cell lysosomes along typical Gaucher "tubules", where some kind of interaction between lipid and protein should occur. Acid phosphatase might be one protein species involved in such interaction.     

Ethinyl estradiol: its interaction on blood-lipid

Lipophilicity (log P) of the drug plays an important role when drug reaches in the critical reaction site, i.e., active site cum receptors where the major constituent is lipid moieties. The drug molecule may be responsible for altering the lipid constituents, which is measured in terms of phosphorus content and can be explained by their fatty acid changes that are linked with biological effect of the drug. Having considered the lipophilicity of ethinyl estradiol (log P = 3.67), its interactions with the whole lipid of goat blood have been investigated along with fatty acid changes and lipid peroxidation phenomena. There was significant loss of phosphorus content of phospholipid and change of fatty acid constituents of whole lipid. This may be ascribed to binding affinity of ethinyl estradiol with lipid constituents in blood. Lipid binding potential of the drug may have role in its therapeutic effect. The peroxidation induced by drug has been quantitatively measured along with its suppression by using antioxidant. The results reveal that ethinyl estradiol caused significant extent of lipid peroxidation. Ascorbic acid, a promising antioxidant could significantly reduce drug induced lipid peroxidation.