MD simulations suggest important surface differences between reconstituted and circulating spherical HDL.
ABSTRACT: Since spheroidal HDL particles (sHDL) are highly dynamic, molecular dynamics (MD) simulations are useful for obtaining structural models. Here we use MD to simulate sHDL with stoichiometries of reconstituted and circulating particles. The hydrophobic effect during simulations rapidly remodels discoidal HDL containing mixed lipids to sHDL containing a cholesteryl ester/triglyceride (CE/TG) core. We compare the results of simulations of previously characterized reconstituted sHDL particles containing two or three apoA-I created in the absence of phospholipid transfer protein (PLTP) with simulations of circulating human HDL containing two or three apoA-I without apoA-II. We find that circulating sHDL compared with reconstituted sHDL with the same number of apoA-I per particle contain approximately equal volumes of core lipid but significantly less surface lipid monolayers. We conclude that in vitro reconstituted sHDL particles contain kinetically trapped excess phospholipid and are less than ideal models for circulating sHDL particles. In the circulation, phospholipid transfer via PLTP decreases the ratio of phospholipid to apolipoprotein for all sHDL particles. Further, sHDL containing two or three apoA-I adapt to changes in surface area by condensation of common conformational motifs. These results represent an important step toward resolving the complicated issue of the protein and lipid stoichiometry of circulating HDL.
Project description:1. Phospholipid transfer protein (PLTP) mediates conversion of high-density lipoprotein (HDL3) to large particles, with concomitant release of apolipoprotein A-I (apoA-I). To study the mechanisms involved in this conversion, reconstituted HDL (rHDL) particles containing either fluorescent pyrenylacyl cholesterol ester (PyrCE) in their core (PyrCE-rHDL) or pyrenylacyl phosphatidylcholine (PysPC) in their surface lipid layer (PyrPC-rHDL) were prepared. Upon incubation with PLTP they behaved as native HDL3, in that their size increased considerably. 2. When PyrPC-rHDL was incubated with HDL3 in the presence of PLTP, a rapid decline of the pyrene excimer/monomer fluorescence ratio (E/M) occurred, demonstrating that PLTP induced mixing of the surface lipids of PyrPC-rHDL and HDL3. As this mixing was almost complete before any significant increase in HDL particle size was observed, it represents PLTP-mediated phospholipid transfer or exchange that is not directly coupled to the formation of large HDL particles. 3. When core-labelled PyrCE-rHDL was incubated in the presence of PLTP, a much slower, time-dependent decrease of E/M was observed, demonstrating that PLTP also promotes mixing of the core lipids. The rate and extent of mixing of core lipids correlated with the amount of PLTP added and with the increase in particle size. The enlarged particles formed could be visualized as discrete, non-aggregated particles by electron microscopy. Concomitantly with the appearance of enlarged particles, lipid-poor apoA-I molecules were released. These data, together with the fact that PLTP has been shown not to mediate transfer of cholesterol esters, strongly suggest that particle fusion rather than (net) lipid transfer or particle aggregation is responsible for the enlargement of HDL particles observed upon incubation with PLTP.4.ApoA-I rHDL, but not apoA-II rHDL, were converted into large particles, suggesting that the presence of apoA-I is required for PLTP-mediated HDL fusion. A model for PLTP-mediated enlargement of HDL particles is presented.
Project description:Phospholipid transfer protein (PLTP), which binds phospholipids and facilitates their transfer between lipoproteins in plasma, plays a key role in lipoprotein remodeling, but its influence on nascent high-density lipoprotein (HDL) formation is not known. The effect of PLTP overexpression on apolipoprotein A-I (apoA-I) lipidation by primary mouse hepatocytes was investigated.Overexpression of PLTP through an adenoviral vector markedly affected the amount and size of lipidated apoA-I species that were produced in hepatocytes in a dose-dependent manner, ultimately generating particles that were <7.1 nm but larger than lipid-free apoA-I. These <7.1-nm small particles generated in the presence of overexpressed PLTP were incorporated into mature HDL particles more rapidly than apoA-I both in vivo and in vitro and were less rapidly cleared from mouse plasma than lipid-free apoA-I. The <7.1-nm particles promoted both cellular cholesterol and phospholipid efflux in an ATP-binding cassette transporter A1-dependent manner, similar to apoA-I in the presence of PLTP. Lipid-free apoA-I had a greater efflux capacity in the presence of PLTP than in the absence of PLTP, suggesting that PLTP may promote ATP-binding cassette transporter A1-mediated cholesterol and phospholipid efflux. These results indicate that PLTP alters nascent HDL formation by modulating the lipidated species and by promoting the initial process of apoA-I lipidation.Our findings suggest that PLTP exerts significant effects on apoA-I lipidation and nascent HDL biogenesis in hepatocytes by promoting ATP-binding cassette transporter A1-mediated lipid efflux and the remodeling of nascent HDL particles.
Project description:apoA-I, apoA-I mimetic peptides, and their lipid complexes or reconstituted high-density lipoprotein (HDL) have been studied as treatments for various pathologies. However, consensus is lacking about the best method for administration, by intravenous (IV) or intraperitoneal (IP) routes, and formulation, as an HDL particle or in a lipid-free form. The objective of this study was to systematically examine peptide plasma levels, cholesterol mobilization, and lipoprotein remodeling in vivo following administration of lipid-free apoA-I peptide (22A) or phospholipid reconstituted 22A-sHDL by IV and IP routes. The mean circulation half-life was longer for 22A-sHDL (T<sub>1/2</sub> = 6.27 h) than for free 22A (T<sub>1/2</sub> = 3.81 h). The percentage of 22A absorbed by the vascular compartment after the IP dosing was ?50% for both 22A and 22A-sHDL. The strongest pharmacologic response came from IV injection of 22A-sHDL, specifically a 5.3-fold transient increase in plasma-free cholesterol (FC) level compared with 1.3- and 1.8-fold FC increases for 22A-IV and 22A-sHDL-IP groups. Addition of either 22A or 22A-sHDL to rat plasma caused lipoprotein remodeling and appearance of a lipid-poor apoA-I. Hence, both the route of administration and the formulation of apoA-I peptide significantly affect its pharmacokinetics and pharmacodynamics.
Project description:Targeting at enhancing reverse cholesterol transport (RCT) is apromising strategy for treating atherosclerosis via infusion of reconstitute high density lipoprotein (HDL) as cholesterol acceptors or increase of cholesterol efflux by activation of macrophage liver X receptors (LXRs). However, systemic activation of LXRs triggers excessive lipogenesis in the liver and infusion of HDL downregulates cholesterol efflux from macrophages. Here we describe an enlightened strategy using phospholipid reconstituted apoA-I peptide (22A)-derived synthetic HDL (sHDL) to deliver LXR agonists to the atheroma and examine their effect on atherosclerosis regression in vivo. A synthetic LXR agonist, T0901317 (T1317) was encapsulated in sHDL nanoparticles (sHDL-T1317). Similar to the T1317 compound, the sHDL-T1317 nanoparticles upregulated the expression of ATP-binding cassette transporters and increased cholesterol efflux in macrophages in vitro and in vivo. The sHDL nanoparticles accumulated in the atherosclerotic plaques of ApoE-deficient mice. Moreover, a 6-week low-dose LXR agonist-sHDL treatment induced atherosclerosis regression while avoiding lipid accumulation in the liver. These findings identify LXR agonist loaded sHDL nanoparticles as a promising therapeutic approach to treat atherosclerosis by targeting RCT in a multifaceted manner: sHDL itself serving as both a drug carrier and cholesterol acceptor and the LXR agonist mediating upregulation of ABC transporters in the aorta.
Project description:Reconstituted high-density lipoprotein particles (rHDL) are powerful platforms used as a model phospholipid bilayer system to study membrane proteins. They consist of a discoidal-shaped planar bilayer of phospholipids that is surrounded by a dimer of apolipoprotein A-I (apoA-I). The amphipathic nature of apoA-1 shields the hydrophobic acyl chains of the lipids from solvent and keeps the particles soluble in aqueous environments. These monodispersed, nanoscale discoidal HDL particles are approximately 10-11 nm in diameter with a thickness that is dependent on the length of the phospholipid acyl chain. Reconstituted HDL particles can be assembled in vitro using purified apoA-1 and purified lipids. Investigators have utilized this model bilayer system to co-reconstitute membrane proteins, and take advantage of the small size and its monodispersion. Our laboratory and others have utilized the rHDL approach to study the behavior of G protein-coupled receptors. In this chapter, we describe strategies for the preparation of rHDL particles containing GPCRs in their monomeric form and discuss various methodologies used to analyze the reconstituted receptor function.
Project description:For several decades, the standard model for high density lipoprotein (HDL) particles reconstituted from apolipoprotein A-I (apoA-I) and phospholipid (apoA-I/HDL) has been a discoidal particle ?100 Å in diameter and the thickness of a phospholipid bilayer. Recently, Wu et al. (Wu, Z., Gogonea, V., Lee, X., Wagner, M. A., Li, X. M., Huang, Y., Undurti, A., May, R. P., Haertlein, M., Moulin, M., Gutsche, I., Zaccai, G., Didonato, J. A., and Hazen, S. L. (2009) J. Biol. Chem. 284, 36605-36619) used small angle neutron scattering to develop a new model they termed double superhelix (DSH) apoA-I that is dramatically different from the standard model. Their model possesses an open helical shape that wraps around a prolate ellipsoidal type I hexagonal lyotropic liquid crystalline phase. Here, we used three independent approaches, molecular dynamics, EM tomography, and fluorescence resonance energy transfer spectroscopy (FRET) to assess the validity of the DSH model. (i) By using molecular dynamics, two different approaches, all-atom simulated annealing and coarse-grained simulation, show that initial ellipsoidal DSH particles rapidly collapse to discoidal bilayer structures. These results suggest that, compatible with current knowledge of lipid phase diagrams, apoA-I cannot stabilize hexagonal I phase particles of phospholipid. (ii) By using EM, two different approaches, negative stain and cryo-EM tomography, show that reconstituted apoA-I/HDL particles are discoidal in shape. (iii) By using FRET, reconstituted apoA-I/HDL particles show a 28-34-Å intermolecular separation between terminal domain residues 40 and 240, a distance that is incompatible with the dimensions of the DSH model. Therefore, we suggest that, although novel, the DSH model is energetically unfavorable and not likely to be correct. Rather, we conclude that all evidence supports the likelihood that reconstituted apoA-I/HDL particles, in general, are discoidal in shape.
Project description:Conversion of discoidal phospholipid (PL)-rich high density lipoprotein (HDL) to spheroidal cholesteryl ester-rich HDL is a central step in reverse cholesterol transport. A detailed understanding of this process and the atheroprotective role of apolipoprotein A-I (apoA-I) requires knowledge of the structure and dynamics of these various particles. This study, combining computation with experimentation, illuminates structural features of apoA-I allowing it to incorporate varying amounts of PL. Molecular dynamics simulated annealing of PL-rich HDL models containing unesterified cholesterol results in double belt structures with the same general saddle-shaped conformation of both our previous molecular dynamics simulations at 310 K and the x-ray structure of lipid-free apoA-I. Conversion from a discoidal to a saddle-shaped particle involves loss of helicity and formation of loops in opposing antiparallel parts of the double belt. During surface expansion caused by the temperature-jump step, the curved palmitoyloleoylphosphatidylcholine bilayer surfaces approach planarity. Relaxation back into saddle-shaped structures after cool down and equilibration further supports the saddle-shaped particle model. Our kinetic analyses of reconstituted particles demonstrate that PL-rich particles exist in discrete sizes corresponding to local energetic minima. Agreement of experimental and computational determinations of particle size/shape and apoA-I helicity provide additional support for the saddle-shaped particle model. Truncation experiments combined with simulations suggest that the N-terminal proline-rich domain of apoA-I influences the stability of PL-rich HDL particles. We propose that apoA-I incorporates increasing PL in the form of minimal surface bilayers through the incremental unwinding of an initially twisted saddle-shaped apoA-I double belt structure.
Project description:Although HDL is inversely correlated with coronary heart disease, elevated HDL-cholesterol is not always protective. Additionally, HDL has biological functions that transcend any antiatherogenic role: shotgun proteomics show that HDL particles contain 84 proteins (latest count), many correlating with antioxidant and anti-inflammatory properties of HDL. ApoA-I has been suggested to serve as a platform for the assembly of these protein components on HDL with specific functions - the HDL proteome. However, the stoichiometry of apoA-I in HDL subspecies is poorly understood. Here we use a combination of immunoaffinity chromatography data and volumetric analysis to evaluate the size and stoichiometry of LpA-I and LpA-I,A-II particles. We conclude that there are three major LpA-I subspecies: two major particles, HDL in the HDL3 size range (d = 85.0 ± 1.2 Å) and HDL in the HDL2 size range (d = 108.5 ± 3.8 Å) with apoA-I stoichiometries of 3 and 4, respectively, and a small minor particle, HDL (d = 73.8 ± 2.1Å) with an apoA-I stoichiometry of 2. Additionally, we conclude that the molar ratio of apolipoprotein to surface lipid is significantly higher in circulating HDL subspecies than in reconstituted spherical HDL particles, presumably reflecting a lack of phospholipid transfer protein in reconstitution protocols.
Project description:HDL-Cholesterol (HDL-C) is not an accurate surrogate marker to measure the cardioprotective functions of HDL in coronary artery diseases (CAD) patients. Hence, measurement of other HDL-related parameters may have prognostic superiority over HDL-C. In this work, we examined the predictive value of HDL particles profile for long-term mortality in CAD patients and to compare its informative value to that of HDL-C and apoA-I. HDL particles profiles were measured by nuclear magnetic resonance (NMR) spectroscopy in 214 male participants with stable CAD (45-74 years). Median follow up was 12.5 years with a 36.4% mortality rate. Cardiovascular mortality accounted for 64.5%. Mean concentrations of total HDL particles (HDL-P), small-sized HDL (SHDL-P) and apoA-I were lower in deceased than in surviving patients whereas no difference was observed according to HDL-C and large HDL particles. All NMR-HDL measures were correlated between themselves and with other HDL markers (HDL-C, apoA-I and LpA-I). In a multivariate model adjusted for cardiovascular risk factors and bioclinical variables, HDL-P and SHDL-P displayed the strongest inverse association with all-cause and cardiovascular mortality. Weaker associations were recorded for apoA-I. Based on our results, we conclude that HDL particle profile measured by NMR spectroscopy should be considered to better stratify risk in population at high risk or in the setting of pharmacotherapy.
Project description:Phospholipid transfer protein (PLTP) is an important modulator of lipoprotein metabolism, including interparticle phospholipid transfer, remodeling of HDL, cholesterol and phospholipid efflux from peripheral tissues, and the production of hepatic VLDL. PLTP also plays an important role in inflammation and oxidative stress. Accordingly, PLTP has been implicated in the development of atherosclerosis. In this study, we evaluated the association between PLTP activity and lipoprotein metabolism in a Chinese patients cohort with or without coronary heart disease (CHD group n = 407, control group n = 215), the PLTP activity was measured and PLTP genotyping was screened for sequence anomalies by PCR. We found that human plasma PLTP activity was negatively associated with plasma HDL and apoA-I levels, and positively associated with plasma TG, apoB and apoE levels. We also found that PLTP rs2294213 polymorphism was tended to be associated with increased plasma PLTP activity.