Lipid headgroup superlattice modulates the activity of surface-acting cholesterol oxidase in ternary phospholipid/cholesterol bilayers

The relationship between the molecular organization of lipid headgroups and the activity of surface-acting enzyme was examined using a bacterial cholesterol oxidase (COD) as a model. The initial rate of cholesterol oxidation by COD in fluid state 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPE/POPC/CHOL) bilayers was measured as a function of POPE-to-phospholipid mole ratio (X(PE)) and cholesterol-to-lipid mole ratio (X(CHOL)) at 37 degrees C. At X(PE) = 0, the COD activity changed abruptly at X(CHOL) approximately 0.40, whereas major activity peaks were detected at X(PE) approximately 0.18, 0.32, 0.50, 0.64, and 0.73 when X(CHOL) was fixed to 0.33 or 0.40. At a fixed X(CHOL) of 0.50, the COD activity increased progressively with PE content and exhibited small peaks or kinks at X(PE) approximately 0.40, 0.50, 0.58, 0.69, and 0.81. When X(PE) and X(CHOL) were systematically varied within a narrow 2-D lipid composition window, an onset of COD activity at X(CHOL) approximately 0.40 and the elimination of the activity peak at X(PE) approximately 0.64 for X(CHOL) >0.40 were clearly observed. Except for X(PE) approximately 0.40 and 0.58, the observed critical PE mole ratios agree closely (+/-0.03) with those predicted by a headgroup superlattice model (Virtanen, J.A., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4964-4969; Cannon, B., et al. (2006) J. Phys. Chem. B 110, 6339-6350), which proposes that lipids with headgroups of different sizes tend to adopt regular, superlattice-like distributions at discrete and predictable compositions in fluid lipid bilayers. Our results indicate that headgroup superlattice domains exist in lipid bilayers and that they may play a crucial role in modulating the activity of enzymes acting on the cell membrane surface.     

Cholesterol modulates alkaline phosphatase activity of rat intestinal microvillus membranes

Experiments were conducted, using a nonspecific lipid transfer protein, to vary the cholesterol/phospholipid molar ratio of rat proximal small intestinal microvillus membranes in order to assess the possible role of cholesterol in modulating enzymatic activities of this plasma membrane. Cholesterol/phospholipid molar ratios from 0.71 to 1.30 were produced from a normal value of 1.05 by incubation with the transfer protein and an excess of either phosphatidylcholine or cholesterol/phosphatidylcholine liposomes for 60 min at 37 degrees C. Cholesterol loading or depletion of the membranes was accompanied by a decrease or increase, respectively, in their lipid fluidity, as assessed by steady-state fluorescence polarization techniques using the lipid-soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene. Increasing the cholesterol/phospholipid molar ratio also decreased alkaline phosphatase specific activity by approximately 20-30%, whereas decreasing this ratio increased this enzymatic activity by 20-30%. Sucrase, maltase, and lactase specific activities were not affected in these same preparations. Since the changes in alkaline phosphatase activity could be secondary to alterations in fluidity, cholesterol, or both, additional experiments were performed using benzyl alcohol, a known fluidizer. Benzyl alcohol (25 mM) restored the fluidity of cholesterol-enriched preparations to control levels, did not change the cholesterol/phospholipid molar ratio, and failed to alter alkaline phosphatase activity. These findings, therefore, indicate that alterations in the cholesterol content and cholesterol/phospholipid molar ratio of microvillus membranes can modulate alkaline phosphatase but not sucrase, maltase, or lactase activities. Moreover, membrane fluidity does not appear to be an important physiological regulator of these enzymatic activities.     

Cholesterol oxidase catalyzed oxidation of cholesterol in mixed lipid monolayers: effects of surface pressure and phospholipid composition on catalytic activity

The catalytic activity of cholesterol oxidase from Streptomyces sp. in mixed monolayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), N-oleoylsphingomyelin (O-SPM), and cholesterol (CHL) has been determined at lateral surface pressures between 10 and 30 mN/m. The highest cholesterol oxidase activity (determined at 37 degrees C) was observed at surface pressures around 20 mN/m in a POPC/CHL monolayer (50:50 mol %). Above and below this surface pressure, the enzyme activity decreased markedly. A similar optimal activity vs surface pressure relationship was observed also for an O-SPM/CHL monolayer (50:50 mol %). The activity of cholesterol oxidase toward cholesterol in the O-SPM/CHL monolayer was, however, less than in the corresponding POPC mixed monolayer. The surface activity of cholesterol oxidase decreased markedly when the temperature was lowered to 20 degrees C, and hardly any enzyme activity was observed in an O-SPM/CHL monolayer at 25 mN/m or above. With a monolayer containing POPC/O-SPM/CHL (42:18:40 mol %), maximal cholesterol oxidase activity was observed at the lowest surface pressure tested (i.e., 10 mN/m), and the catalytic activity decreased markedly with increasing lateral surface pressures in the monolayer. The results of this study show (i) that the activity of cholesterol oxidase in general is highly dependent on the lateral surface pressure in the substrate membranes and (ii) that sphingomyelin, by interacting tightly with cholesterol, can prevent or restrain the accessibility of cholesterol for oxidation by cholesterol oxidase.     

Cholesterol effect on the dipole potential of lipid membranes

The effect of cholesterol removal by methyl-beta-cyclodextrin on the dipole potential, psi(d), of membrane vesicles composed of natural membrane lipids extracted from the kidney and brain of eight vertebrate species was investigated using the voltage-sensitive fluorescent probe di-8-ANEPPS. Cyclodextrin treatment reduced cholesterol levels by on average 80% and this was associated with an average reduction in psi(d) of 50 mV. Measurements of the effect of a range of cholesterol derivatives on the psi(d) of DMPC lipid vesicles showed that the magnitude of the effect correlated with the component of the sterol's dipole moment perpendicular to the membrane surface. The changes in psi(d) observed could not be accounted for solely by the electric field originating from the sterols' dipole moments. Additional factors must arise from sterol-induced changes in lipid packing, which changes the density of dipoles in the membrane, and changes in water penetration into the membrane, which changes the effective dielectric constant of the interfacial region. In DMPC membranes, the cholesterol-induced change in psi(d) was biphasic, i.e., a maximum in psi(d) was observed at approximately 35-45 mol %, after which psi(d) started to decrease. We suggest that this could be associated with a maximum in the strength of DMPC-cholesterol intermolecular forces at this composition.     

Cholesterol modulates the interaction of the islet amyloid polypeptide with membranes

The deposition of insoluble amyloid fibrils resulting from the aggregation of the human islet amyloid polypeptide (hIAPP) within the islet of Langerhans is a pathological feature of type 2 diabetes mellitus (T2DM). Increasing evidence indicates that biological membranes play a key role in amyloid aggregation, modulating among others the kinetics of amyloid formation, and being the target of toxic species generated during amyloid formation. In T2DM patients, elevated levels of cholesterol, an important determinant of the physical state of biological membranes, are observed in β-cells and are thought to directly impair β-cell function and insulin secretion. However, it is not known whether cholesterol enhances membrane-interaction or membrane-insertion of hIAPP. In this study, we investigated the effect of cholesterol incorporated in zwitterionic and anionic membranes. Our circular dichroism and liquid state NMR data reveal that 10–30% of cholesterol slightly affects the aggregational and conformational behaviour of hIAPP. Additional fluorescence results indicate that 10 and 20% of cholesterol slightly slow down the kinetics of oligomer and fibril formation while anionic lipids accelerate this kinetics. This behavior might be caused by differences in membrane insertion and therefore in membrane binding of hIAPP. The membrane binding affinity was evaluated using ¹H NMR experiments and our results show that the affinity of hIAPP for membranes containing cholesterol is significantly smaller than that for membranes containing anionic lipids. Furthermore, we found that hIAPP-induced membrane damage is synchronized to fibril formation in the absence and in the presence of cholesterol.     

Cholesterol oxidase senses subtle changes in lipid bilayer structure

We investigated the dependence of cholesterol oxidase catalytic activity and membrane affinity on lipid structure in model membrane bilayers. The binding affinities of cholesterol oxidase to 100-nm unilamellar vesicles composed of mixtures of DOPC or DPPC and cholesterol are not sensitive to cholesterol mole fraction if the phase of the membrane is in a fluid state. When the membrane is in a solid-ordered state, the binding affinity of cholesterol oxidase increases approximately 10-fold. The second-order rate constants (kcat*/Km*) for different lipid mixtures show a 2-fold substrate specificity for cholesterol in the l(d) phase of high cholesterol chemical activity over cholesterol in the l(o) phase. Moreover, the enzyme is 2-fold more specific for cholesterol in the l(o) phase than in the s(o) phase. Likewise, there is 2-fold substrate specificity for the high cholesterol chemical activity l(d) phase over the low chemical activity l(d) phase. The specificities for the l(d) phase of low cholesterol chemical activity and the l(o) phase are the same. These data indicate that the more ordered the lipid cholesterol structure in the bilayer, the lower the catalytic rate. However, under all of the conditions investigated, the enzyme is never saturated with substrate. The enzymatic activity directly reflects the facility with which cholesterol can move out of the membrane, whether changes in cholesterol transfer facility are due to phase changes or more localized changes in packing. We conclude that the activity of cholesterol oxidase is directly and sensitively dependent on the physical properties of the membrane in which its substrate is bound.     

Desmosterol may replace cholesterol in lipid membranes

Recently, knockout mice entirely lacking cholesterol have been described as showing only a mild phenotype. For these animals, synthesis of cholesterol was interrupted at the level of its immediate precursor, desmosterol. Since cholesterol is a major and essential constituent of mammalian cellular membranes, we asked whether cholesterol with its specific impact on membrane properties might be replaced by desmosterol. By employing various approaches of NMR, fluorescence, and EPR spectroscopy, we found that the properties of phospholipid membranes like lipid packing in the presence of cholesterol or desmosterol are very similar. However, for lanosterol, a more distant precursor of cholesterol synthesis, we found significant differences in comparison with cholesterol and desmosterol. Our results show that, from the point of view of membrane biophysics, cholesterol and desmosterol behave identically and, therefore, replacement of cholesterol by desmosterol may not impact organism homeostasis.     

Cholesterol modulates glycolipid conformation and receptor activity

We document a new dimension of surface recognition in which communication is controlled through the collective behavior of lipids. Membrane cholesterol induces a tilt in glycolipid receptor headgroup, resulting in loss of access for ligand binding. This property appears to organize erythrocyte blood group presentation and glycolipid receptor function during the activation of sperm fertility, suggesting that lipid 'allostery' is a means to regulate membrane recognition processes.     

Role of sterol superlattice in free radical-induced sterol oxidation in lipid membranes

We developed a new fluorescence assay for sterol oxidation and used it to study the relationship between free radical-induced sterol oxidation and membrane sterol lateral organization. This assay used dehydroergosterol (DHE) as both a membrane probe and a membrane component. Sterol oxidation was induced by a free radical generator, AAPH (2,2'-azobis(2-amidinopropane)dihydrochloride). Using this new assay, we found that, in unilamellar vesicles composed of DHE and 1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC), the initial rate of DHE oxidation induced by AAPH changed with membrane sterol content in an alternating manner, exhibiting a local maximum at 20.3, 22.2, 25.0, 32.3, and 40.0 mol % DHE. These mole fractions correspond to the critical sterol mole fractions C(r) predicted for maximal sterol superlattice formation. In three-component bilayers composed of POPC, cholesterol, and DHE (fixed at 1 and 5 mol %), the initial rate of AAPH-induced DHE oxidation exhibited a biphasic change whenever the total sterol mole fraction, irrespective of the DHE content, was near C(r), indicating that the correlation between sterol oxidation and sterol superlattice formation revealed in this study is not an artifact due to the use of the fluorescent cholesterol analogue DHE. The alternating variation of AAPH-induced sterol oxidation with sterol content also appeared in multicomponent unilamellar vesicles containing bovine brain sphingomyelins (bbSPM), POPC, and DHE. The present work and our previous study on cholesterol oxidase-induced sterol oxidation [Wang et al. (2004) Biochemistry 43, 2159-2166] suggest that sterol oxidation in general, either by reactive oxygen species or by enzymes, may be regulated by the extent of sterol superlattice in the membrane and thus regulated by the membrane sterol content in a fine-tuning manner.     

Chemical activity of cholesterol in membranes

Measurements are reported for the rate constants for the release of cholesterol (and dihydrocholesterol) to beta-cyclodextrin from mixtures with phospholipids in homogeneous monolayers at constant pressure at the air-water interface. In each mixture, it is found that the release rate shows a sharp decrease as the cholesterol concentration in the monolayer decreases through a composition corresponding to the stoichiometry of a cholesterol-phospholipid complex. The stoichiometry of the complex was established previously by the position of a sharp cusp in the thermodynamic phase diagram of each mixture and also by a minimum in average molecular area versus composition measurements. A theoretical model used earlier to account for the phase diagrams predicts the chemical potential and chemical activity of cholesterol in these mixtures. The calculated chemical activity also shows a sharp change at the complex stoichiometry in homogeneous monolayers. The similarities in change of observed release rate and calculated chemical activity are expected from reaction rate theory where the release rate is proportional to the cholesterol chemical activity. The chemical activity of cholesterol as determined by complex formation between some phospholipids and cholesterol in the plasma membrane of cells may serve a regulatory function with respect to intracellular cholesterol transport and biosynthesis.     

Induction of lipid peroxidation in the erythrocytes during cholesterol oxidation catalysed by cholesterol oxidase

Using the chemiluminescence technique to assay the activity of cholesterol oxidase it has been shown that enzymic oxidation of cholesterol to cholest-4-en-3-one red cell membranes is accompanied by accumulation of lipid peroxidation products--malonyl dialdehyde (MDA). The amount of MDA formed was dependent on the amount of cholesterol oxidized. The free radical scavenger 4-methyl-2,6-ditretbutylphenol, the transition metal chelator EDTA and catalase inhibited lipid peroxidation in red blood cells. The participation of OH radicals in the initiation of lipid peroxidation in red cell membranes in the course of cholesterol oxidation is discussed.     

Membrane cholesterol modulates serotonin transporter activity

The synaptic actions of the neurotransmitter serotonin are terminated by a selective high-affinity reuptake mediated by the serotonin transporter (SERT). To gain insight into the modulation of the functional properties of this integral membrane protein by cholesterol, a main component of the lipid bilayer, we stably expressed the rat SERT in human embryonic kidney 293 cells and, upon altering the cholesterol content of these cells by different means, analyzed SERT activity. Depletion of the level of membrane cholesterol by treatment with either the cholesterol chelating agent methyl-beta-cyclodextrin (MbetaCD), cholesterol oxidase, or the cholesterol-binding fluorochrome filipin resulted in a decrease in SERT activity due to both a loss of affinity of substrate and ligand binding and a concomitant reduction of the maximal transport rate. In cholesterol-depleted membranes, cholesterol levels could be restored to those found in untreated membranes by incubation of the membranes with an MbetaCD-cholesterol complex, which correlated with a reversal of the cholesterol depletion-mediated decrease in the level of high-affinity binding. This was not the case when other steroids, such as ergosterol, 5-cholestene, or pregnenolone, were substituted into cholesterol-depleted membranes. These results suggest that membrane cholesterol modulates the functional properties of the SERT by specific molecular interactions which are needed to stabilize the transporter in its optimally active form.     

Cholesterol modulates P-glycoprotein activity in human peripheral blood mononuclear cells

P-glycoprotein (P-gp) is expressed in a wide range of cell types including peripheral blood mononuclear cells (PBMCs) where it may restrict intracellular accumulation of substrates like antineoplastic agents, HIV protease inhibitors, or rhodamine123. P-gp is known to be located in membrane microdomains, whose structure and function are susceptible to cholesterol alterations. This study evaluated the effect of cholesterol alteration in human PBMCs on P-gp activity. Whereas cholesterol depletion had no effect, cholesterol repletion of depleted cells significantly decreased intracellular rhodamine123 concentrations in lymphocytes to 32.2%+/-2.7 (p<0.001) and to 41.9%+/-3.5 (p<0.001) in monocytes. After cholesterol saturation of native cells intracellular rhodamine123 fluorescence decreased to 12.4%+/-1.6 (p<0.001) in lymphocytes and 12.9%+/-3.5 (p<0.001) in monocytes. These data demonstrate that elevated cellular cholesterol levels can markedly increase P-gp activity in human PBMCs.     

Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes

Aspirin and other non-steroidal anti-inflammatory drugs have a high affinity for phospholipid membranes, altering their structure and biophysical properties. Aspirin has been shown to partition into the lipid head groups, thereby increasing membrane fluidity. Cholesterol is another well known mediator of membrane fluidity, in turn increasing membrane stiffness. As well, cholesterol is believed to distribute unevenly within lipid membranes leading to the formation of lipid rafts or plaques. In many studies, aspirin has increased positive outcomes for patients with high cholesterol. We are interested if these effects may be, at least partially, the result of a non-specific interaction between aspirin and cholesterol in lipid membranes.We have studied the effect of aspirin on the organization of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) membranes containing cholesterol. Through Langmuir–Blodgett experiments we show that aspirin increases the area per lipid and decreases compressibility at 32.5 mol% cholesterol, leading to a significant increase of fluidity of the membranes. Differential scanning calorimetry provides evidence for the formation of meta-stable structures in the presence of aspirin. The molecular organization of lipids, cholesterol and aspirin was studied using neutron diffraction. While the formation of rafts has been reported in binary DPPC/cholesterol membranes, aspirin was found to locally disrupt membrane organization and lead to the frustration of raft formation. Our results suggest that aspirin is able to directly oppose the formation of cholesterol structures through non-specific interactions with lipid membranes.     

Cholesterol modulates the exposure and orientation of pulmonary surfactant protein SP-C in model surfactant membranes

Cholesterol is the major neutral lipid in lung surfactant, accounting for up to 8-10% of surfactant mass, while surfactant protein SP-C (∼ 4.2 kDa) accounts for no more than 1-1.5% of total surfactant weight but plays critical roles in formation and stabilization of pulmonary surfactant films. It has been reported that surfactant protein SP-C interacts with cholesterol in lipid/protein interfacial films and this interaction could have a potential role on modulating surfactant function. In the present study, we have analyzed the effect of cholesterol on the structure, orientation and dynamic properties of SP-C embedded in physiologically relevant model membranes. The presence of cholesterol does not induce substantial changes in the secondary structure of SP-C, as analyzed by Attenuated Reflection Fourier Transformed Infrared spectroscopy (ATR-FTIR). However, the presence of cholesterol modifies the orientation of the transmembrane helix and the dynamic properties of the protein, as demonstrated by hydrogen/deuterium exchange kinetics. The effect of cholesterol on SP-C reconstituted in zwitterionic, entirely fluid, membranes made of POPC (palmitoyloleoylphospatidylcholine) or in anionic membranes with coexistence of ordered and disordered phases, such as those made of dipalmitoylphosphatidylcholine (DPPC):POPC:Palmitoyloleoylphosphatidylglycerol (POPG) (50:25:15) is dual. Cholesterol decreases the exposure of the protein to the aqueous environment and the tilt of its transmembrane helical segment up to a ratio Cholesterol:SP-C of 4.8 and 2.4 (mol/mol) in the two lipid systems tested, respectively, and it increases the exposure and tilt at higher cholesterol proportions. The results presented here suggest the existence of an interaction between SP-C and cholesterol-enriched phases, with consequences on the behavior of the protein, which could be of relevance for cholesterol-dependent structure-function relationships in pulmonary surfactant membranes and films.     

Investigating the competitive effects of cholesterol and melatonin in model lipid membranes

We have studied the impact of cholesterol and/or melatonin on the static and dynamical properties of bilayers made of DPPC or DOPC utilizing neutron scattering techniques, Raman spectroscopy and molecular dynamics simulations. While differing in the amplitude of the effect due to cholesterol or melatonin when comparing their interactions with the two lipids, their addition ensued recognizable changes to both types of bilayers. As expected, based on the two-component systems of lipid/cholesterol or lipid/melatonin studied previously, we show the impact of cholesterol and melatonin being opposite and competitive in the case of three-component systems of lipid/cholesterol/melatonin. The effect of cholesterol appears to prevail over that of melatonin in the case of structural properties of DPPC-based bilayers, which can be explained by its interactions targeting primarily the saturated lipid chains. The dynamics of hydrocarbon chains represented by the ratio of trans/gauche conformers reveals the competitive effect of cholesterol and melatonin being somewhat more balanced. The additive yet opposing effects of cholesterol and melatonin have been observed also in the case of structural properties of DOPC-based bilayers. We report that cholesterol induced an increase in bilayer thickness, while melatonin induced a decrease in bilayer thickness in the three-component systems of DOPC/cholesterol/melatonin. Commensurately, by evaluating the projected area of DOPC, we demonstrate a lipid area decrease with an increasing concentration of cholesterol, and a lipid area increase with an increasing concentration of melatonin. The demonstrated condensing effect of cholesterol and the fluidizing effect of melatonin appear in an additive manner upon their mutual presence.     

How cholesterol is distributed between monolayers in asymmetric lipid membranes

The distribution of cholesterol in asymmetric lipid bilayers was studied by extensive coarse-grained molecular dynamics simulations. The effects of the lipid head group charge, acyl chain saturation, spontaneous membrane curvature and surface tension of the membrane were investigated. Four asymmetric bilayers containing DOPC, DOPS, DSPC or DSPS lipids were simulated on a time scale extended to tens of microseconds. We show that cholesterol strongly prefers anionic lipids to neutral and saturated lipid tails to unsaturated with a distribution ratio of ~0.7 in neutral/anionic bilayers and of ~0.4 in unsaturated/saturated bilayers. Multiple flip-flop transitions of cholesterol were observed directly, and their mean times ranged from 80 to 250?ns. It was shown that the distribution of cholesterol in the asymmetric membrane depends not only on the type of lipid, but also on the local membrane curvature and the surface tension. The membrane curvature enhances the influence of the lipid head groups on cholesterol distribution, while non-optimal surface tension caused by different areas per lipid in different monolayers increases the effect of the lipid tail saturation. It was clearly seen that the monolayers of asymmetric bilayers are interdependent. Mean distances from the bilayer center to cholesterol molecules depend not only on the type of the lipid in the considered monolayer but also on the composition of the opposite monolayer.     

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.     

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.