Phospholipid Transfer Research Articles

Reduction of HDL levels lowers plasma PLTP and affects its distribution among lipoproteins in mice

Phospholipid transfer protein (PLTP) is associated with HDL particles in plasma, where it transfers phospholipids between lipoproteins and remodels HDL particles. Tangier disease patients, with a mutated ABCA1 transporter, have extremely low plasma HDL concentration and reduced PLTP activity levels, a phenotype that is also observed in mice lacking ABCA1. We investigated whether low HDL levels and low PLTP activity are mechanistically related. Firstly, we studied PLTP expression and distribution among lipoproteins in mice lacking ABCA1 (ABCA1 −/−). Parallel to the strong reduction in PLTP activity in plasma of ABCA1 −/− mice, decreased PLTP protein levels were observed. Neither PLTP synthesis in liver or macrophages nor the ability of the macrophages to secrete PLTP were impaired in ABCA1 −/− mice. However, the PLTP activity level in the medium of cultured macrophages was determined by HDL levels in the medium. PLTP was associated with HDL particles in wild type mice, whereas in ABCA1 −/− mice, PLTP was associated with VLDL and LDL particles. Secondly, we treated different mouse models with varying plasma HDL and PLTP levels (wild type, ABCA1 −/−, apoE −/− and PLTPtg mice, overexpressing human PLTP) with a synthetic LXR ligand, and investigated the relationship between LXR-mediated PLTP induction and HDL levels in plasma. Plasma PLTP activity in wild type mice was induced 5.6-fold after LXR activation, whereas in ABCA1 −/−, apoE −/− and PLTPtg mice, all having reduced HDL levels, induction of PLTP activity was 2.4- , 3.2- and 2.0-fold, respectively. The less pronounced PLTP induction in these mice compared to wild type mice was not caused by a decreased PLTP gene expression in the liver or macrophages. Our findings indicate that the extent of LXR-mediated PLTP induction depends on plasma HDL levels. In conclusion, we demonstrate that ABCA1 deficiency in mice affects plasma PLTP level and distribution through an indirect effect on HDL metabolism. In addition, we show that the extent of LXR-mediated PLTP induction is HDL-dependent. These findings indicate that plasma HDL level is an important regulator of plasma PLTP and might play a role in the stabilization of PLTP in plasma.

Avian phospholipid transfer protein causes HDL conversion without affecting cholesterol efflux from macrophages

Circulatory phospholipid transfer protein (PLTP) has two major functions: 1) transfer of phospholipids towards HDL particles; and 2) modulation of HDL size and composition via the HDL conversion process. In the laying hen ( Gallus gallus), the massive oocyte-targeted lipid flow is achieved through the concerted actions of lipases, lipid transfer proteins, and relatives of the LDL receptor family. The aim of the study was to gain insights into the structure and functions of chicken PLTP. The results demonstrate that PLTP is highly conserved from chicken to mammals, as (i) chicken PLTP is associated with plasma HDL; (ii) it clearly possesses phospholipid transfer activity; (iii) it is inactivated at + 58 °C; and (iv) it mediates conversion of avian and human HDL into small preβ-mobile HDL and large fused α-mobile HDL particles. Our data show that HDL from different chicken models is similar in chemical and physical properties to that of man based on PLTP activity, cholesterol efflux, and HDL conversion assays. In contrast to mammals, PLTP-facilitated HDL remodeling did not enhance cholesterol efflux efficiency of chicken HDL particles.

Flip-Flop of Phospholipids in Vesicles: Kinetic Analysis with Time-Resolved Small-Angle Neutron Scattering

We applied a time-resolved small-angle neutron scattering technique to vesicle systems to determine interparticle transfer and flip-flop of phospholipids. Measurements were performed for large unilamellar vesicles, consisting of dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), or 1-palmitoyl-2-oleoylphosphatidic acid (POPA), which differ either in their acyl chains or headgroup. POPC, which is analogous to naturally occurring phosphatidylcholines, exhibited no transbilayer transfer and very slow interbilayer migration. POPC on the inner leaflet of vesicles did not flop even when phospholipase D converted all POPC molecules on the outer leaflet into POPA, which was shown to exhibit fast flip-flop. From these results, together with the observation that the flip-flop of DMPC was entirely inhibited in the presence of cholesterol, it is deduced that the flip-flop of phosphatidylcholines does not take place spontaneously in cellular plasma membranes rich in cholesterol and that it requires enzymatic activities of energy-dependent and/or -independent flippases/floppases.

Relation of baseline plasma phospholipid transfer protein (PLTP) activity to left ventricular systolic dysfunction in patients referred for coronary angiography

Phospholipid transfer protein (PLTP) is an important modulator of phospholipid transfer and exchange among proteins. It also plays a role in inflammation and oxidative stress. Accordingly, PLTP has been implicated in the development of atherosclerosis. Left ventricular (LV) systolic dysfunction is common in patients with atherosclerosis, and both inflammation and oxidative stress have also been implicated in its development and progression. The goal of the present study was to examine the relation between plasma PLTP activity and LV systolic function. Baseline plasma PLTP activity was measured in 389 male patients referred for coronary angiography for a variety of indications. Detailed clinical, angiographic and laboratory characteristics were available for the patients. Compared to those patients with normal LV function (defined as an ejection fraction of ≥55% on ventriculography), patients with any degree of LV dysfunction had elevated PLTP activity (median PLTP 17.8 pmol/μl/h versus 15.9 pmol/μl/h, p = 0.0038). Using multivariate analysis, and adjusting for a variety of confounding variables known to affect both LV function and PLTP activity, PLTP activity was an independent predictor of the presence of any left ventricular systolic dysfunction in the entire population (OR 1.47, 95% CI 1.12–1.93, p = 0.0052). Furthermore, PLTP activity was an independent predictor of the presence of LV dysfunction in both patients with and without myocardial infarction on presentation (OR 2.39, 95% CI 1.18–4.86, p = 0.0161 and OR 1.41, 95% CI 1.05–1.89, p = 0.0206, respectively). In conclusion, PLTP activity may represent a novel marker of LV systolic dysfunction in patients with known or suspected coronary artery disease.

PLTP activity is a risk factor for subsequent cardiovascular events in CAD patients under statin therapy: the AtheroGene Study

Phospholipid transferprotein (PLTP) mediates both net transfer and exchange of phospholipids between different lipoproteins. Although many studies have investigated the role of PLTP in atherogenesis, the role of PLTP in atherosclerotic diseases is unclear. We investigated the association of serum PLTP activity with the incidence of a combined endpoint (myocardial infarction and cardiovascular death) and its relation to other markers of atherosclerosis in 1,085 patients with angiographically documented coronary artery disease (CAD). In the median follow-up of 5.1 years, 156 patients had suffered from the combined endpoint of myocardial infarction or cardiovascular death including 47 of 395 patients who were on statins at baseline. In Kaplan-Meyer analyses serum PLTP activity was not associated with the combined endpoint in all patients. However, in the subgroup of patients receiving statins at baseline, PLTP was shown to be a significant predictor of cardiovascular outcome (P = 0.019), and this also remained stable in univariate (P = 0.027) and multivariate cox regression analyses (P = 0.041) including potential confounders (classical risk factors, HDL cholesterol (HDL-C), and others). We showed in our study that, under statin treatment, high plasma PLTP activity was related to fatal and nonfatal cardiovascular events in CAD patients.

The role of plasma lipid transfer proteins in lipoprotein metabolism and atherogenesis

The plasma lipid transfer proteins promote the exchange of neutral lipids and phospholipids between the plasma lipoproteins. Cholesteryl ester transfer protein (CETP) facilitates the removal of cholesteryl esters from HDL and thus reduces HDL levels, while phospholipid transfer protein (PLTP) promotes the transfer of phospholipids from triglyceride-rich lipoproteins into HDL and increases HDL levels. Studies in transgenic mouse models and in humans with rare genetic deficiencies (CETP) or common genetic variants (CETP and PLTP) highlight the central role of these molecules in regulating HDL levels. Human CETP deficiency is associated with dramatic elevations of HDL cholesterol and apolipoprotein A-I levels, while PLTP variants with increased expression are associated with higher HDL levels. A recent meta-analysis suggests that common CETP alleles causing reduced CETP and increased HDL levels are associated with reduced coronary heart disease. The failure of a clinical trial with the CETP inhibitor torcetrapib may have been related in part to off-target toxicity. Ongoing phase 3 clinical trials with other CETP inhibitors may help to clarify if this strategy can ultimately be successful in the treatment of atherosclerosis.

The Phospholipid Transfer Protein Sec14 is Required for Trafficking from Endosomes and Regulates Distinct trans‐Golgi Export Pathways

Vesicle fission, transport, and fusion are affected by membrane lipid composition. A protein known to regulate both lipid metabolism and vesicular transport is the phosphatidylcholine/phosphatidylinositol transfer protein Sec14 of Saccharomyces cerevisiae. We determined that decreasing Sec14 function affected secretion via the endosome dependent invertase route as well as the endosome independent route followed by Bgl2. Trafficking of a series of other secreted proteins including Hsp150, Cts1, Scw4, Scw10, Exg1, Cis3, and Ygp1 still occurred. Upon diminution of Sec14 function the v‐SNARE Snc1 accumulated in endosomes and the trans‐Golgi but was competent to traffic from the Golgi to the plasma membrane. Sec14 was required for trafficking of Ste3 and the lipophilic dye FM4‐64 from the plasma membrane to the vacuole at the level of endosomes. The results from a synthetic genetic array screen for genes whose inactivation impaired growth of cells with a temperature sensitive SEC14 allele, sec14ts, were consistent with Sec14 regulating endosome function. We go on to determine that lipid ligand occupancy by Sec14 can differentially regulate its functions. (Supported by CIHR and the Canada Research Chairs Fund)

Regulation by unsaturated fatty acids of the expression of key genes involved in HDL metabolism

In humans and animal models, polyunsaturated fatty acids (PUFA) have been suggested to influence the expression and/or activities of proteins involved in the metabolism of high‐density lipoprotein (HDL). The current study determined the effects and the mechanisms of action of different PUFA on genes regulating HDL metabolism in human hepatoma cells (HepG2). HepG2 cells cultured for 24 hr in 200 uM docosahexaenoic acid (DHA), eicosapentaenoic acid or arachidonic acid (AA) had lower mRNA levels of apolipoprotein A‐I (apo A‐I) (30‐40 %, P = .01), ATP‐binding cassette A1 (ABCA1) (35‐50%, P = .05), lecithin: cholesterol acyltransferase (LCAT) (40‐50%, P = .001), and phospholipid transfer protein (PLTP) (25‐40%, P = .05) compared to those exposed to palmitic acid, as determined by real‐time PCR. In transfection assays, 200uM AA or DHA promoted peroxisome proliferator activated receptor (PPAR) and suppressed liver X receptor activities. HepG2 cells transfected with plasmids containing serial deletions of the apo A‐I promoter showed that the element mediating the PUFA‐related transcriptional repression is located in the ‐192 to ‐41 region of the promoter. In conclusion, very long‐chain n‐3 and n‐6 PUFA slowed the formation and the maturation of HDL particles. These FA also downregulated apo A‐I mRNA level and promoter activity via mechanisms other than PPAR activation. Funded by USDA and Unilever Health Institute.Grant Funding SourceUSDA

The HDL proteome: a marker–and perhaps mediator–of coronary artery disease

One important cardioprotective function of HDL is to remove cholesterol from lipid-laden macrophages in the artery wall. HDL also exerts anti-inflammatory effects that might inhibit atherogenesis. However, HDL has been proposed to be dysfunctional in humans with established coronary artery disease (CAD), though the underlying mechanisms are unclear. Therefore, we used mass spectrometry to investigate the roles of HDL proteins in inflammation and cardiovascular disease. Shotgun proteomic analysis identified multiple complement regulatory proteins, protease inhibitors, and acute-phase response proteins in HDL, strongly implicating the lipoprotein in inflammation and the innate immune system. Moreover, mass spectrometry and biochemical analyses demonstrated that HDL3 from subjects with clinically significant CAD was selectively enriched in apolipoprotein E, suggesting that it carries a distinctive protein cargo in humans with atherosclerosis. HDL from CAD subjects also contained markedly elevated levels of chlorotyrosine and nitrotyrosine, two characteristic products of myeloperoxidase, indicating that oxidative damage might generate dysfunctional HDL. Aggressive lipid therapy with a statin and niacin remodeled the HDL proteome to resemble that of apparently healthy subjects. Collectively, our observations indicate that quantifying the HDL proteome by mass spectrometry should help identify novel anti-inflammatory and cardioprotective actions of HDL and provide insights into lipid therapy.

Lipid Transfer between Charged Supported Lipid Bilayers and Oppositely Charged Vesicles

The bidirectional transfer of phospholipids between a charged, supported lipid bilayer (SLB) on SiO(2) and oppositely charged, unilamellar vesicles was studied by means of quartz crystal microbalance with dissipation (QCM-D) and optical reflectometry techniques. SLBs and vesicles were prepared from binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) mixed with different fractions of either 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS) (negatively charged) or 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC) (positively charged). The interaction process consists of an attachment-transfer-detachment (ATD) sequence, where added vesicles first attach to and interact with the SLB, after which they detach, leaving behind a compositionally modified SLB and ditto vesicles. When the process is complete, there is no net addition or reduction of total lipid mass in the SLB, but lipid exchange has occurred. The time scale of the process varies from a few to many tens of minutes depending on the type of charged lipid molecule and the relative concentration of charged lipids in the two membranes. Electrostatically symmetric cases, where only the charge sign (but not the fraction of charged lipid) was reversed between the SLB and the vesicles, produce qualitatively similar but quantitatively different kinetics. The time scale of the interaction varies significantly between the two cases, which is attributed to a combination of the differences in the molecular structure of the lipid headgroup for the positively and the negatively charged lipids used, and to nonsymmetric distribution of charged lipids in the lipid membranes. The maximum amounts of attached vesicles during the ATD process were estimated to be 25-40% of a full monolayer of vesicles, with the precise amount depending on the actual charge fractions in the vesicles and the SLB. Interrupted vesicle exposure experiments, and experiments where the bulk concentration of vesicles was varied, show that vesicles in some cases may be trapped irreversibly on the SLB, when only partial transfer of lipid molecules has occurred. Additional supply of vesicles and further transfer induces detachment, when a sufficient amount of oppositely charged lipids has been transferred to the SLB, so that the latter becomes repulsive to the attached vesicles. Possible mechanistic scenarios, including monomer insertion and hemifusion models, are discussed. The observed phenomena and the actual SLB preparation process form a platform both for studies of various intermembrane molecular transfer processes and for modifying the composition of SLBs in a controlled way, for example, for biosensor and cell culture applications.

Plasma pre β-HDL formation is decreased by atorvastatin treatment in type 2 diabetes mellitus: Role of phospholipid transfer protein

Atorvastatin lowers plasma phospholipid transfer protein (PLTP) activity, which stimulates pre-β-HDL generation in vitro. We determined the effect of atorvastatin on pre-β-HDL formation and its relation with PLTP activity in type 2 diabetes. Methods: Plasma pre-β-HDL formation as well as plasma apo A-I, LpA, LpAI:AII, cholesteryl ester transfer protein (CETP) and PLTP activity were measured before and after 30 weeks treatment in 40 patients randomized to atorvastatin 80 mg daily and 41 placebo receiving patients. Pre-β HDL formation was measured by crossed immunoelectrophoresis under conditions of lecithin:cholesterol acyltransferase (LCAT) inhibition. Results: Plasma pre-β-HDL formation, triglycerides, LDL cholesterol, PLTP activity, and CETP decreased after statin treatment (all P < 0.001 vs placebo), whereas HDL cholesterol increased ( P < 0.005). Plasma apo A-I, LpAI and LpAI:AII remained unchanged compared to placebo. In all patients combined, the changes in pre-β-HDL formation were independently related to the decrease in plasma triglycerides ( β = 0.31; P = 0.006) and PLTP activity ( β = 0.23; P = 0.038), without a contribution of CETP. In the atorvastatin treated patients, the decrease in pre-β-HDL formation tended to be related to the decrease in PLTP activity ( β = 0.30, P = 0.061) after controlling for decreases in triglycerides ( β = 0.22, P = 0.22). Conclusion: High dose atorvastatin decreases the capacity of plasma to generate pre-β-HDL particles in type 2 diabetic patients, probably via lowering of plasma PLTP activity and triglycerides. This could contribute to an improvement in the atherogenic lipoprotein profile.

The COP9 signalosome controls ubiquitinylation of ABCA1

ATP-binding cassette protein A1 (ABCA1) mediates the transfer of cellular free cholesterol and phospholipids to apolipoprotein A-I (apoA-I), an extracellular acceptor in plasma, to form high-density lipoprotein (HDL). ABCA1 has been suggested to be degraded by proteasome in cholesterol-loaded macrophages, however, the mechanism and regulation of proteasomal degradation of ABCA1 remain unclear. In this study, we analyzed the putative interaction between ABCA1 and COP9 signalosome (CSN), a key molecule in controlling protein ubiquitination and deubiquitination. CSN2 and CSN5, subunits of COP9 CSN complex, were coprecipitated with ABCA1 when coexpressed in HEK293 cells and proteasomal degradation was inhibited by MG132. Overexpression of CSN2 increased endogenous CSN7 and CSN8, and decreased ubiquitinylated forms of ABCA1. These results suggest that CSN is a key molecule which controls the ubiquitinylation and deubiquitinylation of ABCA1, and is thus an important target for developing potential drugs to prevent atherosclerosis.

Phospholipid Transfer Protein Sec14 Is Required for Trafficking from Endosomes and Regulates Distinct trans-Golgi Export Pathways

A protein known to regulate both lipid metabolism and vesicular transport is the phosphatidylcholine/phosphatidylinositol transfer protein Sec14 of Saccharomyces cerevisiae. Sec14 is thought to globally affect secretion from the trans-Golgi. The results from a synthetic genetic array screen for genes whose inactivation impaired growth of cells with a temperature-sensitive SEC14 allele implied Sec14 regulates transport into and out of the Golgi. This prompted us to examine the role of Sec14 in various vesicular transport pathways. We determined that Sec14 function was required for the route followed by Bgl2, whereas trafficking of other secreted proteins, including Hsp150, Cts1, Scw4, Scw10, Exg1, Cis3, and Ygp1, still occurred, indicating Sec14 regulates specific trans-Golgi export pathways. Upon diminution of Sec14 function, the v-SNARE Snc1 accumulated in endosomes and the trans-Golgi. Its accumulation in endosomes is consistent with Sec14 being required for transport from endosomes to the trans-Golgi. Sec14 was also required for trafficking of Ste3 and the lipophilic dye FM4-64 from the plasma membrane to the vacuole at the level of the endosome. The combined genetic and cell biology data are consistent with regulation of endosome trafficking being a major role for Sec14. We further determined that lipid ligand occupancy differentially regulates Sec14 functions.

Ataxia with vitamin E deficiency with a mutation in a phospholipid transfer protein gene

Sirs, Ataxia with isolated vitamin E deficiency (AVED) is an autosomal-recessive spinocerebellar degeneration caused by a mutation of the a-tocopherol transfer protein gene (aTTP) [3, 8]. This report presents a case of juvenile spinocerebellar ataxia caused by mutations in the phospholipid transfer protein (PLTP) gene as well as the aTTP gene. This is the first report to indentify a mutation in the PLTP gene associated with vitamin E-mediated spinocerebellar ataxia. The proband was a 54-year-old female who had developed resting tremors in her right hand and ataxia at 14 years of age. At around 30 years of age, her bilateral visual acuity was decreased by retinitis pigmentosa. Her parents were second-degree cousins. On neurological examination she exhibited ataxia, dysarthria, hyporeflexia, decreased proprioceptive and vibratory sensations, and resting tremors of the extremities. The serum vitamin E concentration was observed to be about half normal level, 4.2 mg/l (normal: 10.8 ± 3.3 mg/l). The results of the following laboratory tests were normal: other fat-soluble vitamins, liverand pancreas-function tests, serum lipids, lipoproteins and apoproteins. The gene analysis revealed a homozygous H101Q mutation in the aTTP gene (Fig. 1a). In AVED, the genotype–phenotype correlation is confirmed, thus indicating that the H101Q mutation is associated with retinitis pigmentosa, late onset during the 3rd decade of life and very low serum vitamin E levels (Table 1) [3, 10, 11]. The clinical findings of the current patient characteristic of juvenile onset and a mild decrease in the serum vitamin E level suggested that other genes associated with vitamin E metabolism might affect the phenotype. Genomic analyses in the PLTP gene revealed the patient and her mother were heterozygous for a H154R mutation (Fig. 1b, c). This mutation was not found in 96 healthy subjects and was different from any previously reported polymorphisms. Following intestinal absorption of vitamin E, aTTP incorporates vitamin E into VLDL resulting in secretion from the liver to the circulation. The plasma PLTP transfers vitamin E from VLDL to HDL. HDL is the predominant source of vitamin E for the brain capillary endothelial cells which facilitates uptake of vitamin E to deliver it to the central nervous system [2, 9]. The PLTP is expressed in the brain, and PLTP knock-out studies showed that PLTP deficiency caused a decrease of vitamin E content in the brain and elevated plasma concentration of vitamin E [1, 6]. The proband and mother heterozygous for the H154R mutation showed a normal plasma PLTP concentration measured by ELISA [7]; however, the plasma PLTP activity was decreased to half the normal level measured by a PLTP assay kit (BioVision, Mountain View, CA) (Table 2). His154 is located in the N-terminal domain with conserved lipid-binding pockets associated with biochemical activity [4], and therefore this H154R mutation may induce the biological dysfunction of the PLTP without causing any impairment of intracellular trafficking. PLTP is an important factor regulating the size and composition of HDL and controlling plasma HDL levels [5]. The identification of the PLTP mutation may thus make it S. Kono (&) A. Otsuji K. Shirakawa H. Suzuki H. Miyajima First Department of Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan e-mail: satokono@hama-med.ac.jp

Co-administration of a CpG adjuvant (VaxImmuneTM, CPG 7909) with CETP vaccines increased immunogenicity in rabbits and mice

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that facilitates the transfer of neutral lipids and phospholipids between lipoproteins and contributes to the regulation of the plasma concentration of high density lipoprotein cholesterol (HDL-C). Vaccines have been developed that elicit antibodies that bind to and reduce the lipid transfer function of CETP as a way to increase the plasma concentration of HDL-C and prevent or treat atherosclerosis. This study assessed the immunogenicity of two vaccine peptides. The first, CETi-1, is a dimerized synthetic peptide, including residues 461-476 of human CETP and residues 830-843 of tetanus toxoid, TT(830-843). The second, PADRE-CETP, is a monomeric peptide, in which a PADRE T cell epitope (aK-Cha-VAAWTLKAa) replaces the TT(830-843) T cell epitope of CETi-1. Both peptides were formulated with aluminum-containing adjuvants (Alhydrogel®), and tested in mice and rabbits with or without the co-administration of the investigational TLR9 agonist VaxImmuneTM (CPG 7909). In both mice and rabbits, the vaccine peptide utilizing the PADRE T cell epitope elicited stronger anti-CETP antibody responses than the CETi-1 vaccine. Also, co-administration of VaxImmune enhanced the anti-CETP antibody responses to both vaccines. Isotype analysis of the murine anti-CETP antibody response to both vaccines demonstrated a switch from IgG1 to IgG2a upon co-administration of VaxImmune. We conclude that 1) the PADRE T cell epitope is more potent than the TT(830-843) epitope in providing help for the anti-CETP antibody response; and 2) co-administration of VaxImmune with either vaccine increased immunogenicity as measured by antibody response.

Anionic Phospholipids Lose Their Procoagulant Properties When Incorporated into High Density Lipoproteins

Blood coagulation involves a series of enzymatic protein complexes that assemble on the surface of anionic phospholipid. To investigate whether apolipoproteins affect coagulation reactions, they were included during the preparation of anionic phospholipid vesicles using a detergent solubilization-dialysis method. Apolipoprotein components of high density lipoproteins, especially apolipoprotein A-I, had a pronounced anticoagulant effect. The anionic phospholipids lost their procoagulant effect when the vesicle preparation method was performed in the presence of apolipoprotein A-I. The anionic phospholipid-apolipoprotein A-I particles were 8-10 nm in diameter and contained around 60-80 phospholipid molecules, depending on the phospholipid composition. The phospholipids of these particles were unable to support the activation of prothrombin by factor Xa in the presence of factor Va and unable to support binding of factor Va, whereas binding of prothrombin and factor Xa were efficient. Phospholipid transfer protein was shown to mediate transfer of phospholipids from liposomes to apolipoprotein A-I-containing reconstituted high density lipoprotein. In addition, serum was also shown to neutralize the procoagulant effect of anionic liposomes and to efficiently mediate transfer of phospholipids from liposomes to either apolipoprotein A-I- or apolipoprotein B-containing particles. In conclusion, apolipoprotein A-I was found to neutralize the procoagulant properties of anionic phospholipids by arranging the phospholipids in surface areas that are too small to accommodate the prothrombinase complex. This anionic phospholipid scavenger function may be an important mechanism to control the exposure of such phospholipids to circulating blood and thereby prevent inappropriate stimulation of blood coagulation.

Multiscale Modeling of supported bilayers

Supported Lipid Bilayers are an abundant research platform for understanding the behavior of real cell membranes as they allow for additional mechanical stability. However, in computer simulations these systems have been studied only rarely up to now. Recently, we studied systematically the changes that a support induces on a phospholipid bilayer using coarse-grained molecular modeling on different levels.We characterize the density and pressure profiles as well as the density imbalance inflicted on the membrane by the support. We also determine the diffusion coefficients and characterize the influence of different corrugations of the support. We then determine the free energy of transfer of phospholipids between the proximal (close to the surface) and distal leaflet of a supported membrane using the coarse-grained Martini model. It turns out that there is at equilibrium about a 2-3% higher density in the proximal leaflet.These results are in favorable agreement with recent data obtained by very large scale modeling using a water free model where flip-flop can be observed directly. We compare results of the free energy of transfer obtained by pulling the lipid across the membrane in different ways. There are small quantitative differences but the overall picture is consistent. We are additionally characterizing the intermediate states which determine the barrier height and therefore the rate of translocation. Simulations in atomistic detail are performed for selected systems in order to confirm the findings. Calculations on unsupported bilayers are used to validate the approach and to determine the barrier to flip-flop in a free membrane.

PLTP is present in the nucleus, and its nuclear export is CRM1-dependent

Phospholipid transfer protein (PLTP), one of the key lipid transfer proteins in plasma and cerebrospinal fluid, is nearly ubiquitously expressed in cells and tissues. Functions of secreted PLTP have been extensively studied. However, very little is known about potential intracellular PLTP functions. In the current study, we provide evidence for PLTP localization in the nucleus of cells that constitutively express PLTP (human neuroblastoma cells, SK-N-SH; and human cortical neurons, HCN2) and in cells transfected with human PLTP (Chinese hamster ovary and baby hamster kidney cells). Furthermore, we have shown that incubation of these cells with leptomycin B (LMB), a specific inhibitor of nuclear export mediated by chromosome region maintenance 1 (CRM1), leads to intranuclear accumulation of PLTP, suggesting that PLTP nuclear export is CRM1-dependent. We also provide evidence for entry of secreted PLTP into the cell and its translocation to the nucleus, and show that intranuclear PLTP is active in phospholipid transfer. These findings suggest that PLTP is involved in novel intracellular functions.

Retroendocytosis pathway of ABCA1/apoA‐I contributes to HDL formation

ATP-binding cassette protein A1 (ABCA1) mediates transfer of cellular free cholesterol and phospholipids to apolipoprotein A-I (apoA-I), an extracellular acceptor in plasma, to form high-density lipoprotein (HDL). It is currently unknown to what extent ABCA1 endocytosis and recycling contribute to the HDL formation. To address this issue, we expressed human ABCA1 constructs with either an extracellular HA tag or an intracellular GFP tag in cells, and used this system to characterize endocytosis and recycling of ABCA1 and apoA-I. Under basal conditions, ABCA1 and apoA-I are endocytosed via a clathrin- and Rab5-mediated pathway and recycled rapidly back to the cell surface, at least in part via a Rab4-mediated route; approximately 30% of the endocytosed ABCA1 is recycled back to the cell surface. When receptor-mediated endocytosis is inhibited, the level of ABCA1 at the cell surface increases and apoA-I internalization is blocked. Under these conditions, apoA-I mediated cholesterol efflux from cells that have accumulated lipoprotein-derived cholesterol is decreased, whereas efflux from cells without excess cholesterol is increased. These results suggest that the retroendocytosis pathway of ABCA1/apoA-I contributes to HDL formation when excess lipoprotein-derived cholesterol has accumulated in cells.

Human plasma phospholipid transfer protein specific activity is correlated with HDL size: Implications for lipoprotein physiology

To gain further insights into the relationship between plasma phospholipid transfer protein (PLTP) and lipoprotein particles, PLTP mass and phospholipid transfer activity were measured, and their associations with the level and size of lipoprotein particles examined in 39 healthy adult subjects. No bivariate correlation was observed between PLTP activity and mass. PLTP activity was positively associated with cholesterol, triglyceride, apo B and VLDL particle level ( r s = 0.40–0.56, p ≤ 0.01) while PLTP mass was positively associated with HDL-C, large HDL particles, and mean LDL and HDL particle sizes ( r s = 0.44–0.52, p < 0.01). Importantly, plasma PLTP specific activity (SA) was significantly associated with specific lipoprotein classes, positively with VLDL, IDL, and small LDL particles ( r s = 0.42–0.62, p ≤ 0.01) and inversely with large LDL, large HDL, and mean LDL and HDL particle size ( r s = − 0.42 to − 0.70, p ≤ 0.01). After controlling for triglyceride levels, the correlation between PLTP mass or SA and HDL size remained significant. In linear models, HDL size explained 45% of the variability of plasma PLTP SA while triglyceride explained 34% of the PLTP activity. Thus, in healthy adults a significant relationship exists between HDL size and plasma PLTP SA ( r s = − 0.70), implying that HDL particle size may modulate PLTP SA in the vascular compartment.