Apolipoprotein A-I adopts a belt-like orientation in reconstituted high density lipoproteins

Apolipoprotein A-I (apoA-I) is the major protein associated with high density lipoprotein (HDL), and its plasma levels have been correlated with protection against atherosclerosis. Unfortunately, the structural basis of this phenomenon is not fully understood. Over 25 years of study have produced two general models of apoA-I structure in discoidal HDL complexes. The "belt" model states that the amphipathic helices of apoA-I are aligned perpendicular to the acyl chains of the lipid bilayer, whereas the "picket fence" model argues that the helices are aligned parallel with the acyl chains. To distinguish between the two models, various single tryptophan mutants of apoA-I were analyzed in reconstituted, discoidal HDL particles composed of phospholipids containing nitroxide spin labels at various positions along the acyl chain. We have previously used this technique to show that the orientation of helix 4 of apoA-I is most consistent with the belt model. In this study, we performed additional control experiments on helix 4, and we extended the results by performing the same analysis on the remaining 22-mer helices (helices 1, 2, 5, 6, 7, 8, and 10) of human apoA-I. For each helix, two different mutants were produced that each contained a probe Trp occurring two helical turns apart. In the belt model, the two Trp residues in each helix should exhibit maximal quenching at the same nitroxide group position on the lipid acyl chains. For the picket fence model, maximal quenching should occur at two different levels in the bilayer. The results show that the majority of the helices are in an orientation that is consistent with a belt model, because most Trp residues localized to a position about 5 A from the center of the bilayer. This study corroborates a belt hypothesis for the majority of the helices of apoA-I in phospholipid discs.     

The orientation of helix 4 in apolipoprotein A-I-containing reconstituted high density lipoproteins

The three-dimensional structure of the high density lipoprotein (HDL) component apolipoprotein (apo) A-I and the molecular basis for its protection against coronary artery disease are unknown. In terms of discoidal HDL particles, there has been a debate as to the orientation of the apoA-I alpha-helices around the disc edge. The "picket fence" model states that the alpha-helical repeats, separated by turns, are arranged parallel to the phospholipid acyl chains of the enclosed lipid bilayer. On the other hand, the "belt" model states that the helical segments run perpendicular to the acyl chains. To distinguish between these models, we used nitroxide spin labels present at various depths in the bilayer of reconstituted HDL (rHDL) to measure the position of Trp residues in single Trp mutants of human proapoA-I. Two mutants were studied; the first contained a Trp at position 108, which was located near the center of helix 4. The second contained a Trp at position 115, two turns along the same helix. The picket fence model predicts that these Trp residues should be at different depths in the bilayer, whereas the belt model predicts that they should be at similar depths. Different sized rHDL particles were produced that contained 2, 3, and >4 molecules of proapoA-I per complex. In each case, parallax analysis indicated that Trp-108 and Trp-115 were present at similar depths of about 6 A from the center of the bilayer, consistent with helix 4 being oriented perpendicular to the acyl chains (in agreement with the belt model). Similar experiments showed that control transmembrane peptides were oriented parallel to the acyl chains in vesicles, demonstrating that the method was capable of distinguishing between the two models. This study provides one of the first experimental measurements of the location of an apoA-I helix with respect to the bilayer edge.     

Characterization of the discoidal complexes formed between apoA-I-CNBr fragments and phosphatidylcholine.

The structure, composition, and physico-chemical properties of lipid-protein complexes generated between dimyristoylphosphatidylcholine (DPMC) and the CNBr fragments of human apoA-I were studied. The fragments were separated by high performance liquid chromatography and purified on a reversed-phase column. The complexes with DMPC were isolated on a Superose column; their dimensions were obtained by gradient gel electrophoresis and by electron microscopy. The secondary structure of the protein in the complexes was studied both by circular dichroism and by attenuated total reflection infrared spectroscopy. The fragments 1 and 4 of apoA-I, containing, respectively, two and three amphipathic helices, recombined with the phospholipid to generate discoidal particles with sizes similar to that of apoA-I- and apoA-II-DMPC complexes. The infrared measurements indicated that in all complexes the apolipoprotein helical segments were oriented parallel to the phospholipid acyl chains and that the protein was located around the edges of the discs. Computer modelling of the complexes based on energy minimization techniques proposed a model for these particles in agreement with the dimensions measured experimentally. In conclusion, we propose that apoA-I and its longest CNBr fragments are able to generate discoidal particles with DMPC, with apolipoprotein helical segments oriented parallel to the acyl chains of the phospholipids.     

Comparative models for human apolipoprotein A-I bound to lipid in discoidal high-density lipoprotein particles

We have constructed a series of models for apolipoprotein A-I (apo A-I) bound to discoidal high-density lipoprotein (HDL) particles, based upon the molecular belt model [Segrest, J. P., et al. (1999) J. Biol. Chem. 274, 31755-31758] and helical hairpin models [Rogers, D. P., et al. (1998) Biochemistry 37, 11714-11725], and compared these with picket fence models [Phillips, J. C., et al. (1997) Biophys. J. 73, 2337-2346]. Molecular belt models for discoidal HDL particles with differing diameters are presented, illustrating that the belt model can explain the discrete changes in HDL particle size observed experimentally. Hairpin models are discussed for the binding of apo A-I to discoidal HDL particles with diameters identical to those for the molecular belt model. Two models are presented for the binding of three monomers of apo A-I to a 150 A diameter discoidal HDL particle. In one model, two monomers of apo A-I bind to the exterior of the HDL particle in an antiparallel belt, with a third monomer of apo A-I bound to the disk in a hairpin conformation. In the second model, all three monomers of apo A-I are bound to the discoidal HDL particle in a hairpin conformation. Previously published experimental data for each model are reviewed, with FRET favoring either the belt or hairpin models over the picket fence models for HDL particles with diameters of 105 A. Naturally occurring mutations appear to favor the belt model for the 105 A particles, while the 150 A HDL particles favor the presence of at least one hairpin.     

The structure of human lipoprotein A-I. Evidence for the "belt" model.

The two main competing models for the structure of discoidal lipoprotein A-I complexes both presume that the protein component is helical and situated around the perimeter of a lipid bilayer disc. However, the more popular "picket fence" model orients the protein helices perpendicular to the surface of the lipid bilayer, while the alternative "belt" model orients them parallel to the bilayer surface. To distinguish between these models, we have investigated the structure of human lipoprotein A-I using a novel form of polarized internal reflection infrared spectroscopy that can characterize the relative orientation of protein and lipid components in the lipoprotein complexes under native conditions. Our results verify lipid bilayer structure in the complexes and point unambiguously to the belt model.     

The spatial organization of apolipoprotein A-I on the edge of discoidal high density lipoprotein particles: a mass specrometry study

The three-dimensional structure of human apoA-I on nascent, discoidal HDL particles has been debated extensively over the past 25 years. Recent evidence has demonstrated that the alpha-helical domains of apoA-I are arranged in a belt-like orientation with the long axis of the helices perpendicular to the phospholipid acyl chains on the disc edge. However, experimental information on the spatial relationships between apoA-I molecules on the disc is lacking. To address this issue, we have taken advantage of recent advances in mass spectrometry technology combined with cleavable cross-linking chemistry to derive a set of distance constraints suitable for testing apoA-I structural models. We generated highly homogeneous, reconstituted HDL particles containing two molecules of apoA-I. These were treated with a thiol-cleavable cross-linking agent, which covalently joined Lys residues in close proximity within or between molecules of apoA-I in the disc. The cross-linked discs were then exhaustively trypsinized to generate a discrete population of peptides. The resulting peptides were analyzed by liquid chromatography/mass spectrometry before and after cleavage of the cross-links, and resulting peaks were identified based on the theoretical tryptic cleavage of apoA-I. We identified at least 8 intramolecular and 7 intermolecular cross-links in the particle. The distance constraints are used to analyze three current models of apoA-I structure. The results strongly support the presence of the salt-bridge interactions that were predicted to occur in the "double belt" model of apoA-I, but a helical hairpin model containing the same salt-bridge docking interface is also consistent with the data.     

Apolipoprotein A-I: structure-function relationships

The inverse relationship between high density lipoprotein (HDL) plasma levels and coronary heart disease has been attributed to the role that HDL and its major constituent, apolipoprotein A-I (apoA-I), play in reverse cholesterol transport (RCT). The efficiency of RCT depends on the specific ability of apoA-I to promote cellular cholesterol efflux, bind lipids, activate lecithin:cholesterol acyltransferase (LCAT), and form mature HDL that interact with specific receptors and lipid transfer proteins. From the intensive analysis of apoA-I secondary structure has emerged our current understanding of its different classes of amphipathic alpha-helices, which control lipid-binding specificity. The main challenge now is to define apoA-I tertiary structure in its lipid-free and lipid-bound forms. Two models are considered for discoidal lipoproteins formed by association of two apoA-I with phospholipids. In the first or picket fence model, each apoA-I wraps around the disc with antiparallel adjacent alpha-helices and with little intermolecular interactions. In the second or belt model, two antiparallel apoA-I are paired by their C-terminal alpha-helices, wrap around the lipoprotein, and are stabilized by multiple intermolecular interactions. While recent evidence supports the belt model, other models, including hybrid models, cannot be excluded. ApoA-I alpha-helices control lipid binding and association with varying levels of lipids. The N-terminal helix 44-65 and the C-terminal helix 210-241 are recognized as important for the initial association with lipids. In the central domain, helix 100-121 and, to a lesser extent, helix 122-143, are also very important for lipid binding and the formation of mature HDL, whereas helices between residues 144 and 186 contribute little. The LCAT activation domain has now been clearly assigned to helix 144-165 with secondary contribution by helix 166-186. The lower lipid binding affinity of the region 144-186 may be important to the activation mechanism allowing displacement of these apoA-I helices by LCAT and presentation of the lipid substrates. No specific sequence has been found that affects diffusional efflux to lipid-bound apoA-I. In contrast, the C-terminal helices, known to be important for lipid binding and maintenance of HDL in circulation, are also involved in the interaction of lipid-free apoA-I with macrophages and specific lipid efflux. While much progress has been made, other aspects of apoA-I structure-function relationships still need to be studied, particularly its lipoprotein topology and its interaction with other enzymes, lipid transfer proteins and receptors important for HDL metabolism.     

Identification and structural ramifications of a hinge domain in apolipoprotein A-I discoidal high-density lipoproteins of different size

Apolipoprotein (apo) A-I is the major protein constituent of human high-density lipoprotein (HDL) and is likely responsible for many of its anti-atherogenic properties. Since distinct HDL size subspecies may play different roles in interactions critical for these properties, a key question concerns how apoA-I can adjust its conformation in response to changes in HDL particle size. A prominent hypothesis states that apoA-I contains a flexible "hinge domain" that can associate/dissociate from the lipoprotein as its diameter fluctuates. Although flexible domains clearly exist within HDL-bound apoA-I, this hypothesis has not been directly tested by assessing the ability of such domains to modulate their contacts with the lipid surface. In this work, discoidal HDL particles of different size were reconstituted with a series of human apoA-I mutants containing a single reporter tryptophan residue within each of its 22 amino acid amphipathic helical repeats. The particles also contained nitroxide spin labels, potent quenchers of tryptophan fluorescence, attached to the phospholipid acyl chains. We then measured the relative exposure of each tryptophan probe with increasing quencher concentrations. We found that, although there were modest structural changes across much of apoA-I, only helices 5, 6, and 7 exhibited significant differences in terms of exposure to lipid between large (96 A) and small (78 A) HDL particles. From these results, we present a model for a putative hinge domain in the context of recent "belt" and "hairpin" models of apoA-I structure in discoidal HDL particles.     

Mode of assembly of amphipathic helical segments in model high-density lipoproteins

The structure of discoidal apo A-I-phospholipid complexes, representing the metabolic precursors of mature high-density lipoprotein particles, was studied by a combination of both a theoretical and an experimental approach. The secondary structure of the complex was determined by circular dichroic measurements, while the relative orientation of the apo A-I helical segments and of the phospholipid acyl chains was determined by ATR infrared measurements. Fluorescence energy transfer between the tryptophan residues of apo A-I and fluorescent phospholipid probes yielded an estimation of the relative topography of the lipid and apolipoprotein components in discoidal and spherical particles. The theoretical approach consisted of the identification of the helical segments in various apo A-I species. These segments were then oriented at a lipid/water interface by minimization of their hydrophobic and hydrophilic transfer energies. The calculation of the hydrophobicity profiles along the axis of the helices leads to the identification of specific interactions between pairs of helices. The helices were further assembled together with the phospholipids by computer modelling, enabling an estimation of the dimensions of the complex. The combination of the experimental and theoretical results yielded a model for discoidal apolipoprotein-phospholipid complexes, in which the amphipathic helical segments are oriented along the edges of the discs. Such a model can be extended to the conversion of these complexes into mature spherical HDL, through the formation of a cholesteryl ester core.     

Molecular modeling of the amphipathic helices of the plasma apolipoproteins.

In this paper we propose a classification of the amphipathic helical repeats occurring in the plasma apolipoprotein sequences. It is based upon the calculation of the molecular hydrophobicity potential around the helical segments. The repeats were identified using a new autocorrelation matrix, based upon similarities of hydrophobic and hydrophilic properties of the amino acid residues within the apolipoprotein sequences. The helices were constructed by molecular modeling, the molecular hydrophobicity potential was calculated, and isopotential contour lines drawn around the helices yielded a three-dimensional visualization of the hydrophobicity potential. Two classes of apolipoproteins could be differentiated by comparing the hydrophobic angles obtained by projection of the isopotential contour lines on a plane perpendicular to the long axis of the helix. The isopotential contour lines around apo AI, AIV, and E are more hydrophilic than hydrophobic, whereas they are of similar intensity for apo AII, CI, and CIII. In both cases discoidal lipid-protein complexes are generated, with the amphipathic helices around the edge of the lipid core. The long axis of the helices is oriented parallel to the phospholipid acyl chains and the hydrophilic side of the helix toward the aqueous phase. As a result of the differences in hydrophobicity potential, the contact between the hydrophobic side of the helices and the phospholipid acyl chains is larger for apo AII, CI, and CIII than for the other apolipoproteins. This might account for the greater stability of the discoidal complexes generated between phospholipids and these apoproteins.     

Interaction of the alpha-helices of apolipophorin III with the phospholipid acyl chains in discoidal lipoprotein particles: a fluorescence quenching study.

Quenching of tryptophan fluorescence by nitroxide-labeled phospholipids and nitroxide-labeled fatty acids was used to investigate the lipid-binding domains of apolipophorin III. The location of the Trp residues relative to the lipid bilayer was investigated in discoidal lipoprotein particles made with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and five different single-Trp mutants of apoLp-III. A comparison of the quenching efficiencies of phospholipids containing nitroxide groups at the polar head, and at positions 5 and 16 of the sn-2 acyl chain, indicated that the protein is interacting with the acyl chains of the phospholipid along the periphery of the bilayer of the discoidal lipoprotein. N-Bromosuccinimide readily abolished 100% of the fluorescence of all Trp residues in the lipid-bound state. Larger quenching rates were observed for the Trp residues in helices 1, 4, and 5 than for those located in helices 2 and 3, suggesting differences between the interaction of these two groups of helices. However, the extent of Trp fluorescence quenching observed in lipoproteins made with any of the mutants was comparable to that reported for deeply embedded Trp residues, suggesting that all Trp residues interact with the phospholipid acyl chains. This study provides the first experimental evidence of a massive interaction of the alpha-helices of apoLp-III with the phospholipid acyl chains in discoidal lipoproteins. The extent of interaction deduced is consistent with the apolipoprotein adopting a highly extended conformation.     

Assessment of the validity of the double superhelix model for reconstituted high density lipoproteins: a combined computational-experimental approach

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.     

Conformation and lipid binding of a C-terminal (198-243) peptide of human apolipoprotein A-I

Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins that are critically involved in reverse cholesterol transport. The intrinsically flexibility of apoA-I has hindered studies of the structural and functional details of the protein. Our strategy is to study peptide models representing different regions of apoA-I. Our previous report on [1-44]apoA-I demonstrated that this N-terminal region is unstructured and folds into approximately 60% alpha-helix with a moderate lipid binding affinity. We now present details of the conformation and lipid interaction of a C-terminal 46-residue peptide, [198-243]apoA-I, encompassing putative helix repeats 10 and 9 and the second half of repeat 8 from the C-terminus of apoA-I. Far-ultraviolet circular dichroism spectra show that [198-243]apoA-I is also unfolded in aqueous solution. However, self-association induces approximately 50% alpha-helix in the peptide. The self-associated peptide exists mainly as a tetramer, as determined by native electrophoresis, cross-linking with glutaraldehyde, and unfolding data from circular dichroism (CD) and differential scanning calorimetry (DSC). In the presence of a number of lipid-mimicking detergents, above their CMC, approximately 60% alpha-helix was induced in the peptide. In contrast, SDS, an anionic lipid-mimicking detergent, induced helical folding in the peptide at a concentration of approximately 0.003% (approximately 100 microM), approximately 70-fold below its typical CMC (0.17-0.23% or 6-8 mM). Both monomeric and tetrameric peptide can solubilize dimyristoylphosphatidylcholine (DMPC) liposomes and fold into approximately 60% alpha-helix. Fractionation by density gradient ultracentrifugation and visualization by negative staining electromicroscopy demonstrated that the peptide binds to DMPC with a high affinity to form at least two sizes of relatively homogeneous discoidal HDL-like particles depending on the initial lipid:peptide ratio. The characteristics (lipid:peptide weight ratio, diameter, and density) of both complexes are similar to those of plasma A-I/DMPC complexes formed under similar conditions: small discoidal complexes (approximately 3:1 weight ratio, approximately 110 A, and approximately 1.10 g/cm3) formed at an initial 1:1 weight ratio and larger discoidal complexes (approximately 4.6:1 weight ratio, approximately 165 A, and approximately 1.085 g/cm3) formed at initial 4:1 weight ratio. The cross-linking data for the peptide on the complexes of two sizes is consistent with the calculated peptide numbers per particle. Compared to the approximately 100 A disk-like complex formed by the N-terminal peptide in which helical structure was insufficient to cover the disk edge by a single belt, the compositions of these two types of complexes formed by the C-terminal peptide are more consistent with a "double belt" model, similar to that proposed for full-length apoA-I. Thus, our data provide direct evidence that this C-terminal region of apoA-I is responsible for the self-association of apoA-I, and this C-terminal peptide model can mimic the interaction with the phospholipid of plasma apoA-I to form two sizes of homogeneous discoidal complexes and thus may be responsible for apoA-I function in the formation and maintenance of HDL subspecies in plasma.     

Structural determination of lipid-bound ApoA-I using fluorescence resonance energy transfer

Based on the x-ray crystal structure of lipid-free Delta43 apoA-I, two monomers of apoA-I were suggested to bind to a phospholipid bilayer in an antiparallel paired dimer, or "belt orientation." This hypothesis challenges the currently held model in which each of the two apoA-I monomers fold as antiparallel alpha-helices or "picket fence orientation." When apoA-I is bound to a phospholipid disc, the first model predicts that the glutamine at position 132 on one apoA-I molecule lies within 16 A of glutamine 132 in the second monomer, whereas, the second model predicts glutamines at position 132 to be 104 A apart. To distinguish between these models, glutamine at position 132 was mutated to cysteine in wild-type apoA-I to produce Q132C apoA-I, which were labeled with thiol-reactive fluorescent probes. Q132C apoA-I was labeled with either fluorescein (donor probe) or tetramethylrhodamine (acceptor probe) and then used to make recombinant phospholipid discs (recombinant high density lipoprotein (rHDL)). The rHDL containing donor- and acceptor-labeled Q132C apoA-I were of similar size, composition, and lecithin:cholesterol acyltransferase reactivity when compared to rHDL-containing human plasma apoA-I. Analysis of donor probe fluorescence showed highly efficient quenching in rHDL containing one donor- and one acceptor-labeled Q132C apoA-I. rHDL containing only acceptor probe-labeled Q132C apoA-I showed rhodamine self-quenching. Both of these observations demonstrate that position 132 in two lipid-bound apoA-I monomers were in close proximity, supporting the "belt conformation" hypothesis for apoA-I on rHDL.     

Structures of Discoidal High Density Lipoproteins: A COMBINED COMPUTATIONAL-EXPERIMENTAL APPROACH*

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.     

ApoA-I structure on discs and spheres. Variable helix registry and conformational states

Apolipoprotein A-I (apoA-I) readily forms discoidal high density lipoprotein (HDL) particles with phospholipids serving as an ideal transporter of plasma cholesterol. In the lipid-bound conformation, apoA-I activates the enzyme lecithin:cholesterol acyltransferase stimulating the formation of cholesterol esters from free cholesterol. As esterification proceeds cholesterol esters accumulate within the hydrophobic core of the discoidal phospholipid bilayer transforming it into a spherical HDL particle. To investigate the change in apoA-I conformation as it adapts to a spherical surface, fluorescence resonance energy transfer studies were performed. Discoidal rHDL particles containing two lipid-bound apoA-I molecules were prepared with acceptor and donor fluorescent probes attached to cysteine residues located at specific positions. Fluorescence quenching was measured for probe combinations located within repeats 5 and 5 (residue 132), repeats 5 and 6 (residues 132 and 154), and repeats 6 and 6 (residue 154). Results from these experiments indicated that each of the 2 molecules of discoidal bound apoA-I exists in multiple conformations and support the concept of a "variable registry" rather than a "fixed helix-helix registry." Additionally, discoidal rHDL were transformed in vitro to core-containing particles by incubation with lecithin:cholesterol acyltransferase. Compositional analysis showed that core-containing particles contained 11% less phospholipid and 633% more cholesterol ester and a total of 3 apoA-I molecules per particle. Spherical particles showed a lowering of acceptor to donor probe quenching when compared with starting rHDL. Therefore, we conclude that as lipid-bound apoA-I adjusts from a discoidal to a spherical surface its intermolecular interactions are significantly reduced presumably to cover the increased surface area of the particle.     

Linoleic acid-enriched phospholipids act through peroxisome proliferator-activated receptors alpha to stimulate hepatic apolipoprotein A-I secretion

A uniquely formulated soy phospholipid, phosphatidylinositol (PI), is under development as a therapeutic agent for increasing plasma high-density lipoprotein (HDL) levels. Soy PI has been shown to increase plasma HDL and apolipoprotein A-I (apoA-I) levels in phase I human trials. Low micromolar concentrations of PI increase the secretion of apoA-I in model human hepatoma cell lines, through activation of G-protein and mitogen-activated protein (MAP) kinase pathways. Experiments were undertaken to determine the importance of the PI head group and acyl chain composition on hepatic apoA-I secretion. Phospholipids with choline and inositol head groups and one or more linoleic acid (LA) acyl chains were shown to stimulate apoA-I secretion by HepG2 cells and primary human hepatocytes. Phospholipids containing two LA groups (dilinoleoylphosphatidylcholine, DLPC) were twice as active as those with only one LA group and promoted a 4-fold stimulation in apoA-I secretion. Inhibition of cytosolic phospholipase A2 with pyrrolidine 1 (10 microM) resulted in complete attenuation of PI- and DLPC-induced apoA-I secretion. Pretreatment with the peroxisome proliferator-activated receptor alpha (PPARalpha) inhibitor MK886 (10 microM) also completely blocked PI- and DLPC-induced apoA-I secretion. Hepatic PPARalpha expression was significantly increased by both PI and DLPC. However, in contrast to that seen with the fibrate drugs, PI caused minimal inhibition of catalytic activities of cytochrome P450 and UGT1A1 enzymes. These data suggest that LA-enriched phospholipids stimulate hepatic apoA-I secretion through a MAP kinase stimulation of PPARalpha. LA-enriched phospholipids have a greater apoA-I secretory activity than the fibrate drugs and a reduced likelihood to interfere with concomitant drug therapies.     

Apolipoprotein A-II-mediated conformational changes of apolipoprotein A-I in discoidal high density lipoproteins

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.     

Structural requirements for antioxidative and anti-inflammatory properties of apolipoprotein A-I mimetic peptides

Recently, attention has been focused on pharmacological treatments that increase HDL cholesterol to prevent coronary artery disease. Despite three decades of extensive research of human apolipoprotein A-I (apoA-I), the major protein component of HDL, the molecular basis for its antiatherogenic and anti-inflammatory functions remain elusive. Another protein component of HDL, apoA-II, has structural features similar to those of apoA-I but does not possess atheroprotective properties. To understand the molecular basis for the effectiveness of apoA-I, we used model synthetic peptides. We designed analogs of the class A amphipathic helical motif in apoA-I that is responsible for solubilizing phospholipids. None of these analogs has sequence homology to apoA-I, but all are similar in their lipid-associating structural motifs. Although all of these peptide analogs interact with phospholipids to form peptide:lipid complexes, the biological properties of these analogs are different. Physical-chemical and NMR studies of these peptides have enabled the delineation of structural requirements for atheroprotective and anti-inflammatory properties in these peptides. It has been shown that peptides that interact strongly with lipid acyl chains do not have antiatherogenic and anti-inflammatory properties. In contrast, peptides that associate close to the lipid head group (and hence do not interact strongly with the lipid acyl chain) are antiatherogenic and anti-inflammatory. Understanding the structure and function of apoA-I and HDL through studies of the amphipathic helix motif may lead to peptide-based therapies for inhibiting atherosclerosis and other related inflammatory lipid disorders.     

Remodeling of apolipoprotein E-containing spherical reconstituted high density lipoproteins by phospholipid transfer protein

Phospholipid transfer protein (PLTP) transfers phospholipids between HDL and other lipoproteins in plasma. It also remodels spherical, apolipoprotein A-I (apoA-I)-containing HDL into large and small particles in a process involving the dissociation of lipid-free/lipid-poor apoA-I. ApoE is another apolipoprotein that is mostly associated with large, spherical HDL that do not contain apoA-I. Three isoforms of apoE have been identified in human plasma: apoE2, apoE3, and apoE4. This study investigates the remodeling of spherical apoE-containing HDL by PLTP and the ability of PLTP to transfer phospholipids between apoE-containing HDL and phospholipid vesicles. Spherical reconstituted high density lipoproteins (rHDL) containing apoA-I [(A-I)rHDL], apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein were prepared by incubating discoidal rHDL with low density lipoproteins and lecithin:cholesterol acyltransferase. PLTP remodeled the spherical, apoE-containing rHDL into large and small particles without the dissociation of apoE. The PLTP-mediated remodeling of apoE-containing rHDL was more extensive than that of (A-I)rHDL. PLTP transferred phospholipids from small unilamellar vesicles to apoE-containing rHDL in an isoform-dependent manner, but at a rate slower than that for spherical (A-I)rHDL. It is concluded that apoE enhances the capacity of PLTP to remodel HDL but reduces the ability of HDL to participate in PLTP-mediated phospholipid transfers.