Clustering of α-Synuclein on Supported Lipid Bilayers: Role of Anionic Lipid, Protein, and Divalent Ion Concentration

α-Synuclein is the major component of Lewy body inclusions found in the brains of patients with Parkinson's disease. Several studies indicate that α-synuclein binds to negatively charged phospholipid bilayers. We examined the binding of α-synuclein to membranes containing different amounts of negatively charged lipids using supported lipid bilayers, epifluorescence microscopy, fluorescence recovery after photobleaching, and bulk fluorescence techniques. The membranes contained phosphatidylcholine and phosphatidylglycerol. In the absence of protein, these lipids mix uniformly. Our results show that the propensity of α-synuclein to cluster on the membrane increases as the concentration of anionic lipid and/or protein increases. Regions on the lipid bilayer where α-synuclein is clustered are enriched in phosphatidylglycerol. We also observe divalent metal ions stimulate protein cluster formation, primarily by promoting lipid demixing. The importance of protein structure, lipid demixing, and divalent ions, as well as the physiological implications, will be discussed. Because membrane-bound α-synuclein assemblies may play a role in neurotoxicity, it is of interest to determine how membranes can be used to tune the propensity of α-synuclein to aggregate.     

Lipid Profiling Demonstrates That Suppressing Arabidopsis Phospholipase Dδ Retards ABA-Promoted Leaf Senescence by Attenuating Lipid Degradation

Senescence is the last phase of the plant life cycle and has an important role in plant development. Degradation of membrane lipids is an essential process during leaf senescence. Several studies have reported fundamental changes in membrane lipids and phospholipase D (PLD) activity as leaves senesce. Suppression of phospholipase Dα1 (PLDα1) retards abscisic acid (ABA)-promoted senescence. However, given the absence of studies that have profiled changes in the compositions of membrane lipid molecules during leaf senescence, there is no direct evidence that PLD affects lipid composition during the process. Here, we show that application of n-butanol, an inhibitor of PLD, and N-Acylethanolamine (NAE) 12∶0, a specific inhibitor of PLDα1, retarded ABA-promoted senescence to different extents. Furthermore, phospholipase Dδ (PLDδ) was induced in leaves treated with ABA, and suppression of PLDδ retarded ABA-promoted senescence in Arabidopsis. Lipid profiling revealed that detachment-induced senescence had different effects on plastidic and extraplastidic lipids. The accelerated degradation of plastidic lipids during ABA-induced senescence in wild-type plants was attenuated in PLDδ-knockout (PLDδ-KO) plants. Dramatic increases in phosphatidic acid (PA) and decreases in phosphatidylcholine (PC) during ABA-induced senescence were also suppressed in PLDδ-KO plants. Our results suggest that PLDδ-mediated hydrolysis of PC to PA plays a positive role in ABA-promoted senescence. The attenuation of PA formation resulting from suppression of PLDδ blocks the degradation of membrane lipids, which retards ABA-promoted senescence.     

Lipid trafficking sans vesicles: where, why, how?

Eukaryotic cells possess a remarkable diversity of lipids, which distribute among cellular membranes by well-characterized vesicle trafficking pathways. However, transport of lipids by alternate, or "nonvesicular," routes is also critical for lipid synthesis, metabolism, and proper membrane partitioning. In the past few years, considerable progress has been made in characterizing the mechanisms of nonvesicular lipid transport and how it may go awry in particular diseases, but many fundamental questions remain for this rising field.     

Injectable Lipid Emulsions—Advancements,Opportunities and Challenges

Injectable lipid emulsions, for decades, have been clinically used as an energy source for hospitalized patients by providing essential fatty acids and vitamins. Recent interest in utilizing lipid emulsions for delivering lipid soluble therapeutic agents, intravenously, has been continuously growing due to the biocompatible nature of the lipid-based delivery systems. Advancements in the area of novel lipids (olive oil and fish oil) have opened a new area for future clinical application of lipid-based injectable delivery systems that may provide a better safety profile over traditionally used long- and medium-chain triglycerides to critically ill patients. Formulation components and process parameters play critical role in the success of lipid injectable emulsions as drug delivery vehicles and hence need to be well integrated in the formulation development strategies. Physico-chemical properties of active therapeutic agents significantly impact pharmacokinetics and tissue disposition following intravenous administration of drug-containing lipid emulsion and hence need special attention while selecting such delivery vehicles. In summary, this review provides a broad overview of recent advancements in the field of novel lipids, opportunities for intravenous drug delivery, and challenges associated with injectable lipid emulsions.     

Rhodopsin/Lipid Hydrophobic Matching—Rhodopsin Oligomerization and Function

Lipid composition of the membrane and rhodopsin packing density strongly modulate the early steps of the visual response of photoreceptor membranes. In this study, lipid-order and bovine rhodopsin function in proteoliposomes composed of the sn-1 chain perdeuterated lipids 14:0_d27-14:1-PC, 16:0_d31-16:1-PC, 18:0_d35-18:1-PC, or 20:0_d39-20:1-PC at rhodopsin/lipid molar ratios from 1:70 to 1:1000 (mol/mol) were investigated. Clear evidence for matching of hydrophobic regions on rhodopsin transmembrane helices and hydrophobic thickness of lipid bilayers was observed from ²H nuclear magnetic resonance order parameter measurements at low rhodopsin concentrations. Thin bilayers stretched to match the length of transmembrane helices observed as increase of sn-1 chain order, while thicker bilayers were compressed near the protein. A quantitative analysis of lipid-order parameter changes suggested that the protein adjusts its conformation to bilayer hydrophobic thickness as well, which confirmed our earlier circular-dichroism measurements. Changes in lipid order parameters upon rhodopsin incorporation vanished for bilayers with a hydrophobic thickness of 27 ± 1 Å, suggesting that this is the bilayer thickness at which rhodopsin packs in bilayers at the lowest membrane perturbation. The lipid-order parameter studies also indicated that a hydrophobic mismatch between rhodopsin and lipids triggers rhodopsin oligomerization with increasing rhodopsin concentrations. Both hydrophobic mismatch and rhodopsin oligomerization result in substantial shifts of the equilibrium between the photointermediates metarhodopsin I and metarhodopsin II; increasing bilayer thickness favors formation of metarhodopsin II while oligomerization favors metarhodopsin I. The results highlight the importance of hydrophobic matching for rhodopsin structure, oligomerization, and function.     

Lipid and Peptide Dynamics in Membranes upon Insertion of n-alkyl-β-D-Glucopyranosides

The effect of nonionic detergents of the n-alkyl-β-D-glucopyranoside class on the ordering of lipid bilayers and the dynamics of membrane-embedded peptides were investigated with ²H- and ³¹P-NMR. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was selectively deuterated at methylene segments C-2, C-7, and C-16 of the two fatty acyl chains. Two trans-membrane helices, WALP-19 and glycophorin A_71-98, were synthesized with Ala-d₃ in the central region of the α-helix. n-Alkyl-β-D-glucopyranosides with alkyl chains with 6, 7, 8, and 10 carbon atoms were added at increasing concentrations to the lipid membrane. The bilayer structure is retained up to a detergent/lipid molar ratio of 1:1. The insertion of the detergents leads to a selective disordering of the lipids. The headgroup region remains largely unaffected; the fatty acyl chain segments parallel to the detergent alkyl chain are only modestly disordered (10-20%), whereas lipid segments beyond the methyl terminus of the detergent show a decrease of up to 50%. The change in the bilayer order profile corresponds to an increase in bilayer entropy. Insertion of detergents into the lipid bilayers is completely entropy-driven. The entropy change accompanying lipid disorder is equivalent in magnitude to the hydrophobic effect. Ala-d₃ deuterated WALP-19 and GlycA_71-97 were incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine at a peptide/lipid molar ratio of 1:100 and measured above the 1,2-dimyristoyl-sn-glycero-3-phosphocholine gel/liquid-crystal phase transition. Well-resolved ²H-NMR quadrupole splittings were observed for the two trans-membrane helices, revealing a rapid rotation of the CD₃ methyl rotor superimposed on an additional rotation of the whole peptide around the bilayer normal. The presence of detergent fluidizes the membrane and produces magnetic alignment of bilayer domains but does not produce essential changes in the peptide conformation or dynamics.     

The Interfacial Tension of the Lipid Membrane Formed from Lipid–Amino Acid Systems

The interfacial tension of lipid membranes composed of phosphatidylcholine (lecithin, PC)–valine (Val), phosphatidylcholine–isoleucine (Ile), phosphatidylcholine–tyrosine (Tyr), and phosphatidylcholine–phenylalanine (Phe) has been studied. The membrane components formed 1:1 complexes. The interfacial tension measurements were used to determine the membrane surface concentration A ₃⁻¹, the membrane interfacial tension γ₃, and the stability constant K.     

D1/D2/Cytb-559复合物的共振拉曼光谱

光系统Ⅱ（ＰＳⅡ）反应中心Ｄ１／Ｄ２／Ｃｙｔｂ－５５９ 复合物在４８８ ｎｍ 处激发的共振拉曼光谱显示４ 个主要谱带,其峰位分别在１５３２（υ１）、１１６５（υ２）、１０１０（υ３）和９７０（υ４） ｃｍ － １处,表明ＰＳⅡ反应中心结合的β－胡萝卜素分子是全反式构型。Ｄ１／Ｄ２／Ｃｙｔｂ－５５９ 复合物的色素抽提液的拉曼光谱也显示４ 个主要的拉曼峰,其中υ４ 谱带的强度急剧下降,说明ＰＳⅡ反应中心内部结合的β－胡萝卜素分子与抽提液中自由的β－胡萝卜素分子的构象不同,而与光合细菌反应中心内部的类胡萝卜素分子的构象相似,其共轭多烯链的平面也处于扭曲状态     

D—氨基酸在人体的来龙去脉：人体中D—氨基酸,D—氨基酸氧化酶,D…

本文分析近年,特别是近两年有关人体、食物中Ｄ－氨基酸、Ｄ－氨基酸氧化酶、Ｄ－天冬氨酸氧化酶研究进展,强调：１．必须研究食品中Ｄ－氨基酸水平;２．Ｄ－氨基酸氧化酶、Ｄ．天冬氨酸氧化酶作为药物治疗因Ｄ－氨基酸过量所致疾病。     

Physical Determinants of β-Barrel Membrane Protein Folding in Lipid Vesicles

The spontaneous folding of two Neisseria outer membrane proteins, opacity-associated (Opa)₆₀ and Opa₅₀ into lipid vesicles was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility assays enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states as well as the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged guest lipids, or by the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with more basic pH and low ionic strength; it is hindered by unsaturated or ether-linked lipids. The isolation of the physical determinants of insertion suggests that the membrane surface and dipole potentials are driving forces for outer membrane protein insertion and folding into lipid bilayers.     

Lipid Phase Fatty Acid Flip-Flop, Is It Fast Enough for Cellular Transport?

The mechanism by which fatty acids are transported across cell membranes is controversial. The essence of the controversy is whether transport requires membrane protein mediation or whether the membrane's lipid phase provides a pathway so rapid that a protein is not needed. This review focuses on the mechanisms of fatty acid transport across lipid bilayer membranes. These results for lipid membranes are used to help evaluate transport across cell membranes. Within the context of this analysis, a lipid phase mediated process is consistent with results for the transport of fatty acids across erythrocytes but provides a less adequate explanation for fatty acid transport across more complex cells. Received: 16 June 1999/Revised: 21 January 2000     

Membrane Lipid Co-Aggregation with α-Synuclein Fibrils

Amyloid deposits from several human diseases have been found to contain membrane lipids. Co-aggregation of lipids and amyloid proteins in amyloid aggregates, and the related extraction of lipids from cellular membranes, can influence structure and function in both the membrane and the formed amyloid deposit. Co-aggregation can therefore have important implications for the pathological consequences of amyloid formation. Still, very little is known about the mechanism behind co-aggregation and molecular structure in the formed aggregates. To address this, we study in vitro co-aggregation by incubating phospholipid model membranes with the Parkinson’s disease-associated protein, α-synuclein, in monomeric form. After aggregation, we find spontaneous uptake of phospholipids from anionic model membranes into the amyloid fibrils. Phospholipid quantification, polarization transfer solid-state NMR and cryo-TEM together reveal co-aggregation of phospholipids and α-synuclein in a saturable manner with a strong dependence on lipid composition. At low lipid to protein ratios, there is a close association of phospholipids to the fibril structure, which is apparent from reduced phospholipid mobility and morphological changes in fibril bundling. At higher lipid to protein ratios, additional vesicles adsorb along the fibrils. While interactions between lipids and amyloid-protein are generally discussed within the perspective of different protein species adsorbing to and perturbing the lipid membrane, the current work reveals amyloid formation in the presence of lipids as a co-aggregation process. The interaction leads to the formation of lipid-protein co-aggregates with distinct structure, dynamics and morphology compared to assemblies formed by either lipid or protein alone.     

Lipid rafts: elusive or illusive?

There has been considerable recent interest in the possibility that the plasma membrane contains lipid "rafts," microdomains enriched in cholesterol and sphingolipids. It has been suggested that such rafts could play an important role in many cellular processes including signal transduction, membrane trafficking, cytoskeletal organization, and pathogen entry. However, rafts have proven difficult to visualize in living cells. Most of the evidence for their existence and function relies on indirect methods such as detergent extraction, and a number of recent studies have revealed possible problems with these methods. Direct studies of the distribution of raft components in living cells have not yet reached a consensus on the size or even the presence of these microdomains, and hence it seems that a definitive proof of raft existence has yet to be obtained.     

Specificity of Intramembrane Protein–Lipid Interactions

Our concept of biological membranes has markedly changed, from the fluid mosaic model to the current model that lipids and proteins have the ability to separate into microdomains, differing in their protein and lipid compositions. Since the breakthrough in crystallizing membrane proteins, the most powerful method to define lipid-binding sites on proteins has been X-ray and electron crystallography. More recently, chemical biology approaches have been developed to analyze protein–lipid interactions. Such methods have the advantage of providing highly specific cellular probes. With the advent of novel tools to study functions of individual lipid species in membranes together with structural analysis and simulations at the atomistic resolution, a growing number of specific protein–lipid complexes are defined and their functions explored. In the present article, we discuss the various modes of intramembrane protein–lipid interactions in cellular membranes, including examples for both annular and nonannular bound lipids. Furthermore, we will discuss possible functional roles of such specific protein–lipid interactions as well as roles of lipids as chaperones in protein folding and transport.Our concept of biological membranes has markedly changed in the last two decades, from the fluid mosaic model (Singer and Nicolson 1972), in which the membrane was thought to be formed by a homogenous lipid fluid phase with proteins embedded, to the current model that lipids and proteins are not homogenously distributed, but have the ability to separate into microdomains, differing in their protein and lipid compositions. A well established example of domains are lipid rafts (see Box 1 for definitions). Raft domains are described as dynamic domain structures enriched in cholesterol, sphingolipids, and membrane proteins (Brown and London 1998; Simons and Ikonen 1997) that have an important role in different cellular processes (Lingwood and Simons 2010). Formation of domains within cellular membranes has been extensively investigated over the past years leading to various models that differ in the primary forces involved in the formation and the recruitment of surrounding membrane components into such domains.

BOX 1.

Definitions

Annular Lipids/Lipid Shell

An annular lipid shell is formed when selected lipid classes or molecular species bind preferentially to the hydrophobic and/or hydrophilic surfaces of a membrane protein. Per definition these lipids show markedly reduced residence times at the protein–lipid interface as compared to bulk lipids.

Bulk Lipids

Lipids within the membrane that diffuse rapidly in the bilayer plane and show a low residence time at the protein–lipid interface following random collisions. Typical diffusion coefficients for bulk lipids in a liquid disordered phase are in the range of D_L = 7×10⁻¹² m²/sec (DOPC) (Filippov et al. 2003).

Hydrophobic Mismatch

A term to describe any deviation from the compatibility of the hydrophobic surface of membrane proteins (their TMDs) to the vertically and laterally encountered hydrophobic surfaces of the lipid bilayer in biological membranes. In the case of a hydrophobic mismatch, the resulting energy penalty may cause the recruitment of a suitable local lipid environment, the deformation of the membrane and/or in conformational changes of the protein to achieve a status of hydrophobic match (for advanced reading, see Killian 1998).

Lateral Pressure Field/Profile of Membranes

Biological membranes can be considered as the “solvent” for membrane proteins that are embedded in them. The lateral pressure profile (Ω(z)) describes the force or pressure that is exerted by the membrane on the matter residing inside it. This pressure is modulated by different extents of lipid–lipid interactions and asymmetries across and within the bilayer, which in turn results in varying lateral pressures that may locally correspond to several hundreds of atmospheres.

Lipid Rafts

Sterol and sphingolipid-dependent microdomains that form a network of lipid–lipid, protein–protein, and protein–lipid interactions; involved in the compartmentalization of processes such as signaling within biological membranes.

Liquid-Disordered Phase (I_d)

A predominantly fluid phase of lipids, characterized by a high degree of mobility (cis-gauche flexibility of acyl chains; lateral diffusion) and a high content of short and/or unsaturated fatty acyl chains.

Liquid-Ordered Phase (I_o)

A liquid crystalline phase (that displays physical properties of both liquids and of solid crystals), characterized by a high degree of acyl chain order (“packing”), a reduced lateral mobility of lipid and protein molecules, and a reduction in the elasticity of the membrane as a result of specific interactions between sterols and phospholipids containing long, saturated acyl chains and/or glycosphingolipids.

Microdomains

Membrane compartments of distinct lipid and protein composition that may modulate the enzymatic functions of membrane proteins.

Molecular Lipid Species

Individual members of a lipid class that differ in their fatty acid composition.

Nonannular Lipids

Lipids that specifically interact with membrane proteins are neither bulk lipids, nor do they belong to the shell/annulus of lipids that surround the membrane protein. These nonannular lipids often reside within membrane protein complexes, in which they may fulfill diverse functions ranging from structural building blocks to allosteric effectors of enzymatic activity (see text). Nonannular lipids bind to distinct hydrophobic sites of membrane proteins or membrane protein complexes.According to one model, membrane domains can form by specific protein–protein interactions (Douglass and Vale 2005). This model is based on single-molecule microscopy experiments. In these studies, single fluorophores were chemically attached to specific proteins, and the dynamics of individual proteins was tracked by monitoring the fluorescent probe. In this kind of set up, a dynamic behavior of lipids is not assessed. Here, proteins involved in signaling processes are trapped within interconnected microdomains created by specific protein–protein interactions, probably involving additional scaffolding proteins. The proteins of such domains can exchange with the surrounding membrane area at individual kinetics, some components are immobile over minutes, and others can diffuse rapidly.Another model emphasizes the importance of lipid–lipid interactions, initiating the formation of subdomains of defined lipid compositions. Transmembrane proteins then can be attracted to such subdomains via various specific interactions with lipids. The resulting lipid–protein complexes then eventually coalesce to form larger lipid–protein assemblies (Anderson and Jacobson 2002).The idea of lipid-dependent domain formation is inherent to the biophysical properties and therefore to the complex lipid composition of cellular membranes that include up to a thousand lipids that vary in structure (van Meer et al. 2008). This wide range of lipid species has been proposed to facilitate the “solvation” of membrane proteins. Taken into account the sum of lipid species present in a cellular membrane, it is important to understand the different interactions and affinities within the bilayer between different lipids. Molecular dynamics simulations have been successfully employed to investigate lipid interactions between different lipid species and found specific interactions of various lipid classes and molecular species (Hofsass et al. 2003; Niemela et al. 2004, 2006, 2009; Pandit et al. 2004; Zaraiskaya and Jeffrey 2005; Bhide et al. 2007). These results are supported and expanded by recent data from our group that suggest a specific order of interactions of sphingomyelin species with cholesterol in membranes (A.M. Ernst, F. Wieland, and B. Brügger, unpubl.). At low cholesterol concentrations, some sphingomyelin species preferentially interact with cholesterol, whereas others prefer their kin. At higher cholesterol concentrations, all sphingomyelin species investigated display an increased affinity for the sterol. These findings open the possibility of differentiated pathways of self-assembly of microdomains, dependent on molecular lipid species.In the present article the various modes of intramembrane protein–lipid interactions in cellular membranes (Fig. 1) will be discussed. This includes possible functional roles of such specific protein–lipid interactions.Open in a separate windowFigure 1.Intramembrane protein–lipid interactions within a cell membrane. (A) Bulk lipids; (B) annular lipids; (C) nonannular lipids/lipid ligands. For details see text.     

oxLDL-Induced Lipid Accumulation in Glomerular Podocytes: Role of IFN-γ, CXCL16, and ADAM10

Previous studies have shown that lipid accumulation plays an important role in the pathogenesis and development of glomerular sclerosis. oxLDL caused damage in renal mesangial cells, endothelial cells, and podocytes, and podocytes might be the major victim of oxLDL insult. However, the regulatory mechanism of how oxLDL induces the damage of podocytes remains to be elucidated. In this study, oil red staining was used to investigate the lipid accumulation in podocytes. Moreover, the effects of CXCL16 antibody, IFN-γ, and ADAM10 inhibitor on oxLDL intake and CXCL16 expression were also explored to elucidate the regulatory factors of lipid accumulation in podocytes.