Biomedical subjects

M C Phillips

Publications and source records attributed to M C Phillips.

At least 55 records · Page 3Linked to original sources

PubMed1997

Interaction of class A amphipathic helical peptides with phospholipid unilamellar vesicles.

The exchangeable apolipoproteins are important in determining the structure/function properties of lipoproteins. These proteins typically contain varying amounts of amphipathic helices. Five model peptides, 18A, Ac-18A-NH2, Ac-18R-NH2, 37pA, and 37aA, have been designed to investigate variations of the amphipathic alpha-helix structural motif on their lipid-binding properties. These include the 18-residue peptides, 18A and Ac-18A-NH2, examples of class A helices, and Ac-18R-NH2, which has the positions of acidic and basic residues interchanged relative to 18A. Three larger peptides were also studied: 36A, a dimer of 18A, 37pA and 37aA, dimers of 18A coupled by Pro (18A-Pro-18A) and Ala (18A-Ala-18A), respectively. We report here the results of a thermodynamic characterization of the binding properties of these peptides to small unilamellar vesicles of POPC. Partition coefficients, Kp, were determined by fluorescence spectroscopy and binding enthalpies, deltaH, by titration calorimetry. These parameters were used to obtain the free energies, deltaG0, and entropies, deltaS0, of binding. The results of this study indicate Kp values on the order of 10(5), with interactions being enthalpically but not entropically favored in all cases. The presence of positively charged residues at the interface (18A and Ac-18A-NH2) enhances binding but has little effect on the extent of bilayer penetration. The presence of tandem repeats decreases lipid affinities for these small, highly curved bilayers. Our results are consistent with the idea that interaction appears to be confined largely to the surface, with some degree of penetration of the hydrophobic face of the helix into the interior of the bilayer.

Calorimetry↗

PubMed1997

Effect of vesicle size on their interaction with class A amphipathic helical peptides.

Throughout the life span of a lipoprotein particle, the type and number of exchangeable apolipoproteins on its surface varies with particle size, suggesting a role of surface curvature on the lipid-binding properties of these proteins. Peptides 18A, Ac-18A-NH2, Ac-18R-NH2, 37pA, and 37aA have been designed to investigate the lipid-binding properties of the amphipathic alpha-helix structural motif that appears to modulate the lipid-binding properties of the exchangeable plasma apolipoproteins. We report here the results of a quantitative thermodynamic characterization of the effects of modifying helix length and of varying both the location of charged residues about the polar face of the peptides and vesicle size on the lipid affinity and depth of bilayer penetration for model amphipathic alpha-helices. Partition coefficients, Kp, were determined by fluorescence spectroscopy, and binding enthalpies, deltaH, by titration calorimetry. The results indicate that Kp values are on the order of 10(5), with similar deltaG(o) values for the interactions of the peptides with vesicles of various sizes. It appears that a class A motif and increased alpha-helical content optimize binding for 18-residue peptides. The interactions of the model peptides with 20 nm SUV are enthalpically driven with small, negative entropy changes; however, interactions for larger vesicles are entropically driven, likely due to disordering of bilayer hydrocarbon chains. Thermodynamic data indicate that 37pA and 37aA induce greater disordering of bilayer hydrocarbon chains than Ac-18A-NH2. The results of this study suggest that the type of interaction, i.e., enthalpically or entropically driven, may be modulated by the lateral compressibility of the bilayer membrane.

Acrylamide↗

PubMed1997

Use of cyclodextrins for manipulating cellular cholesterol content.

Previous studies from this laboratory have demonstrated that exposure of tissue culture cells to cyclodextrins results in rapid cholesterol depletion. In the present study, we have developed experimental systems for using solutions of cyclodextrins, either 2-hydroxypropyl beta-cyclodextrin or methylated beta-cyclodextrin, complexed with varying amounts of free cholesterol to manipulate cell cholesterol content. Cholesterol delivered via the cyclodextrin has been found to be metabolically active, as measured by the acyl-coenzyme A:cholesterol acyltransferase (ACAT)-mediated esterification of [3H]cholesterol in Fu5AH rat hepatoma cells and Chinese hamster ovary cells. The methylated beta-cyclodextrin was found to be a more efficient donor in all cell types studied, with an average cholesterol uptake of at least 100 microg cholesterol/mg protein within 6 h. By modifying the cyclodextrin:cholesterol molar ratio, it is possible to manipulate the cellular cholesterol content of cells, producing conditions ranging from net cholesterol enrichment to depletion. The use of cyclodextrins provides a convenient, precise and reproducible method for modulating the cholesterol content of tissue culture cells.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed1996

Apolipoprotein A-I structural modification and the functionality of reconstituted high density lipoprotein particles in cellular cholesterol efflux.

The role of HDL and its major protein constituent, apolipoprotein (apo) A-I, in promoting the removal of excess cholesterol from cultured cells has been well established; however, the mechanisms by which this occurs are not completely understood. To address the effects of apoA-I modification on cellular unesterified (free) cholesterol (FC) efflux, three recombinant human apoA-I deletion mutants and plasma apoA-I were combined with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and FC to make reconstituted high density lipoprotein (rHDL) discoidal complexes. These particles were characterized structurally and for their efficiency as acceptors of mouse L-cell fibroblast cholesterol. The deletion mutant proteins lacked NH2-terminal (apoA-I (Delta44-126)), central (apoA-I (Delta139-170)), or COOH-terminal (apoA-I (Delta190-243)) domains of apoA-I. The three deletion mutants all displayed lipid-binding abilities and formed discoidal complexes that were similar in major diameter (13.2 +/- 1.5 nm) to those formed by human apoA-I when reconstituted at a 100:5:1 (POPC:FC:protein) mole ratio. Gel filtration profiles indicated unreacted protein in the preparation made with apoA-I (Delta190-243), which is consistent with the COOH terminus portion of apoA-I being an important determinant of lipid binding. Measurements of the percent alpha-helix content of the proteins, as well as the number of protein molecules per rHDL particle, gave an indication of the arrangement of the deletion mutant proteins in the discoidal complexes. The rHDL particles containing the deletion mutants had more molecules of protein present than particles containing intact apoA-I, to the extent that a similar number of helical segments was incorporated into each of the discoidal species. Comparison of the experimentally determined number of helical segments with an estimate of the available space indicated that the deletion mutant proteins are probably more loosely arranged than apoA-I around the edge of the rHDL. The abilities of the complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts. All four discoidal complexes displayed similar abilities to remove FC from the plasma membrane of L-cells when compared at an acceptor concentration of 50 microg of phospholipid/ml. Thus, none of the deletions imposed in this study notably altered the ability of the rHDL particles to participate in cellular FC efflux. These results suggest that efficient apoA-I-mediated FC efflux requires the presence of amphipathic alpha-helical segments but is not dependent on specific helical segments.

Animals↗

PubMed1996

Cellular cholesterol efflux mediated by cyclodextrins. Demonstration Of kinetic pools and mechanism of efflux.

The efflux of cholesterol from cells in culture to cyclodextrin acceptors has been reported to be substantially more rapid than efflux induced by other known acceptors of cholesterol (Kilsdonk, E. P. C., Yancey, P., Stoudt, G., Bangerter, F. W., Johnson, W. J., Phillips, M. C., and Rothblat, G. H. (1995) J. Biol. Chem. 270, 17250-17256). In this study, we compared the kinetics of cholesterol efflux from cells with 2-hydroxypropyl-beta-cyclodextrins and with discoidal high density lipoprotein (HDL) particles to probe the mechanisms governing the remarkably rapid rates of cyclodextrin-mediated efflux. The rate of cholesterol efflux was enhanced by shaking cells growing in a monolayer and further enhanced by placing cells in suspension to achieve maximal efflux rates. The extent of efflux was dependent on cyclodextrin concentration, and maximal efflux was observed at concentrations >50 mM. For several cell types, biexponential kinetics of cellular cholesterol efflux were observed, indicating the existence of two kinetic pools of cholesterol: a fast pool (half-time (t1/2) approximately 19-23 s) and a slow pool with t1/2 of 15-30 min. Two distinct kinetic pools of cholesterol were also observed with model membranes (large unilamellar cholesterol-containing vesicles), implying that the cellular pools are in the plasma membrane. Cellular cholesterol content was altered by incubating cells with solutions of cyclodextrins complexed with increasing levels of cholesterol. The number of kinetic pools was unaffected by raising the cellular cholesterol content, but the size of the fast pool increased. After depleting cells of the fast pool of cholesterol, this pool was completely restored after a 40-min recovery period. The temperature dependence of cyclodextrin-mediated cholesterol efflux from cells and model membranes was compared; the activation energies were 7 kcal/mol and 2 kcal/mol, respectively. The equivalent activation energy observed with apo-HDL-phospholipid acceptor particles was 20 kcal/mol. It seems that cyclodextrin molecules are substantially more efficient than phospholipid acceptors, because cholesterol molecules desorbing from a membrane surface can diffuse directly into the hydrophobic core of a cyclodextrin molecule without having to desorb completely into the aqueous phase before being sequestered by the acceptor.

Animals↗

PubMed1996

Only the two end helixes of eight tandem amphipathic helical domains of human apo A-I have significant lipid affinity. Implications for HDL assembly.

Human apolipoprotein A-I (apo A-I) possesses multiple tandem repeating 22-mer amphipathic alpha-helixes. Computer analysis and studies of model synthetic peptides and recombinant protein-lipid complexes of phospholipids have suggested that apo A-I interacts with HDL surface lipids through cooperation among its individual amphipathic helical domains. To delineate the overall lipid-associating properties of apo A-I, the first step is to understand the lipid-associating properties of individual amphipathic helical domains. To this end, we synthesized and studied each of the eight tandem repeating 22-mer domains of apo A-I: residues 44-65, 66-87, 99-120, 121-142, 143-164, 165-186, 187-208, and 220-241. Among the 22-mers, only the N- and C-terminal peptides (44-65 and 220-241) were effective in clarifying multilamellar vesicles (MLVs) of dimyristoylphosphatidylcholine (DMPC). These two peptides also exhibited the highest partition coefficient into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine liposomes, the highest exclusion pressure for penetration into an egg yolk phosphatidylcholine monolayer, and the greatest reduction in the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. These results suggest that the strong, lipid-associating properties of apo A-I are localized to the N- and C-terminal amphipathic domains. Although each of the eight peptides studied has an amphipathic structure, models based on changes in residual effective amino acid hydrophobicity resulting from differing depths of helix penetration into the lipid are best able to explain the high lipid affinity possessed by the two terminal domains. Differential scanning calorimetry (DSC) studies showed that on a molar basis, apo A-I is about 10 times more effective than the most effective peptide analyzed in reducing the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. Because previous proteolysis experiments coupled with the present DSC results suggest that the lipid-associating domains of apo A-I are distributed throughout the length of the 243 amino acid residues, we propose that the terminal amphipathic helical domains are involved in the initial binding of apo A-I to the lipid surface to form HDL particles, followed by cooperative binding of the middle six amphipathic helical domains, perhaps aided by salt-bridge formation between adjacent helixes arranged in an antiparallel orientation.

Apolipoprotein A-I↗

PubMed1996

Comparison of the intracellular metabolism and trafficking of 25-hydroxycholesterol and cholesterol in macrophages.

Oxysterols arising from the diet or through lipid peroxidation may be important in the modulation of cellular cholesterol metabolism. In this study, the metabolism of one of the oxysterols, 25-hydroxycholesterol (25OHC), was examined in J774 and mouse peritoneal macrophages. Uptake of 25OHC from serum was rapid and substantial. Esterification of the cellular 25OHC was also rapid as was hydrolysis of pre-formed esters. Like cholesterol, 25OHC was removed from cells by an extracellular acceptor such as high density lipoprotein. Unlike cholesterol, 25OHC was also rapidly and extensively removed from cells by serum albumin, but not by ovalbumin. The differential removal of oxysterols and cholesterol from cells by albumin allows separation of cellular effects due to oxysterols and cholesterol. In order to understand more about this differential efflux of sterols, a computer model for sterol mass transport in cells was used to compare intracellular trafficking of cholesterol and 25OHC. The rate constants determined by this model for movement of sterols between cytoplasm and plasma membrane were similar for both cholesterol and 25OHC, whereas those for esterification and ester hydrolysis as well as those for bidirectional movement between plasma membrane and extracellular medium were greater for 25OHC than for cholesterol. For both sterols, the rate-limiting step for removal of cellular esters appeared to be the rate of cytoplasmic ester hydrolysis. As 25OHC and cholesterol differ significantly in aqueous solubility, the similarity in their rate constants for movement between cytoplasm and plasma membrane is consistent with facilitation of transport between these two loci.

Animals↗

PubMed1995

Effects of the neutral lipid content of high density lipoprotein on apolipoprotein A-I structure and particle stability.

Alterations in high density lipoprotein (HDL) composition that occur in dyslipidemic states may modulate a number of events involved in cholesterol homeostasis. To elucidate the details of how HDL-core composition can affect the molecular structure of different kinds of HDL particles, the conformation and stability of apoA-I have been investigated in homogeneous recombinant HDL particles (LpA-I) containing palmitoyloleoyl phosphatidylcholine (POPC), triolein (TG), and/or cholesteryl linoleate (CE). In a discoidal particle containing two molecules of apoA-I and 85 molecules of POPC, apoA-I exhibits an alpha-helix content of 70% and a free energy of stability of its alpha-helical segments (delta G0D) of 2.2 kcal/mol. Inclusion of eight molecules of TG into the complex significantly reduces the alpha-helix content and stability of apoA-I, whereas inclusion of four molecules of CE into the complex has an opposite effect in that the alpha-helix content is significantly reduced and the stability of the remaining alpha-helical structure of apoA-I is increased. Neutral lipids have a different effect on apoA-I conformation in spherical LpA-I particles. In a sonicated-spherical LpA-I particle containing two molecules of apoA-I and 70 molecules of POPC, apoA-I exhibits an alpha-helix content of about 60% and a delta G0D of 1.2 kcal/mol apoA-I. Inclusion of either 10 molecules of TG or six molecules of CE into such a particle increases both the alpha-helix content and stability of apoA-I. Increasing the CE/TG ratio in LpA-I particles that contain both neutral lipids enhances the stability of the alpha-helical segments. ApoA-I molecules tend to dissociate and cause particle instability when delta G0D for the lipid-bound alpha-helices is less than that for helices in the lipid-free state. The stabilities of both discoidal and spherical LpA-I particles are relatively low when the only neutral lipid present is TG but the particle stability is enhanced by the presence of CE molecules. Such dissociation of apoA-I molecules from LpA-I particles that have a low CE/TG ratio would be promoted in the hypertriglyceridemic state in vivo.

Apolipoprotein A-I↗

PubMed1995

Efflux of newly synthesized cholesterol and biosynthetic sterol intermediates from cells. Dependence on acceptor type and on enrichment of cells with cholesterol.

Previous studies suggest that during sterol synthesis in cells, cholesterol and precusor sterols are transported to the plasma membrane and that this transport is stimulated by the binding of high density lipoprotein (HDL) to its putative cell surface receptor, leading to enhanced sterol efflux. Little is known about the identities of synthesized sterols subject to efflux or whether efflux of cholesterol and precursor sterols are stimulated equally by HDL. To address these issues, cells were incubated with [3H]acetate or [3H]mevalonate and sterol acceptors, and then the labeled sterols in cells and efflux media were analyzed by high pressure liquid chromatography methods that resolved cholesterol and precursor sterols. In non-hepatic cells (Chinese hamster ovary (CHO), fibroblasts, and smooth muscle), cholesterol and multiple precursor sterols accumulated. In CHO cells, the major products were cholesterol and desmosterol, which together constituted 50% of labeled nonsaponifiable lipids. When media contained human HDL3 (1 mg of protein/ml), the molar efflux of synthesized desmosterol was four times that of cholesterol, and the 8-h efflux of these sterols, each normalized to its own production, averaged 48 and 16%, respectively. When media contained egg phosphatidylcholine vesicles (1 mg/ml), the efflux of these sterols averaged 18 and 2.4%, respectively. Thus, with both acceptors, desmosterol was the major synthesized sterol released from cells, and its efflux was substantially greater than that of synthesized cholesterol. High relative efflux of desmosterol (or a desmosterol-like sterol) occurred in all cell types and in both cholesterol-enriched and unenriched cells. These results demonstrated qualitatively similar efflux of synthesized sterols in the presence of HDL3 and phospholipid vesicles, arguing against an absolute requirement for acceptors that interact with the HDL receptor. To probe for possible quantitative differences in the capabilities of these two acceptors, the ratios of (efflux to HDL3)/(efflux to phosphatidylcholine vesicles) were calculated for synthesized cholesterol and desmosterol, plasma membrane cholesterol, and lysosomal cholesterol. In comparison to plasma membrane cholesterol, there was little or no HDL selectivity for lysosomal cholesterol or synthesized desmosterol, whereas there was a 2-3-fold selectivity for synthesized cholesterol, suggesting that the ability of HDL to enhance the efflux of synthesized sterols is a modest quantitative effect and confined to cholesterol.

Animals↗

PubMed1995

Effects of acceptor particle size on the efflux of cellular free cholesterol.

Several subspecies of human high density lipoprotein (HDL) have been shown to exist, and particle size is one parameter that can be used to distinguish them. Recently, a small HDL subspecies has been described that may be a particularly efficient acceptor of peripheral cell unesterified (free) cholesterol (FC). To address the effects of particle size on the ability of HDL to remove FC from cells, homogeneous, well defined HDL particles were reconstituted (rHDL) that varied in particle diameter within the size range of human HDL particles (7-13 nm). The abilities of each of these particles to remove cellular FC from mouse L-cells and rat Fu5AH hepatoma cells were compared on the basis of their phospholipid (PL) content as well as on a per particle basis. The effect of particle size was also examined using small unilamellar vesicles (SUV) of 25 nm in diameter and large unilamellar vesicles (LUVs) of 70-180 nm in diameter. The SUV were prepared by sonication, and the LUVs were prepared by extrusion techniques. The FC efflux efficiency of these particles (in order of decreasing efficiency) was: rHDL > SUV > LUV when compared on the basis of acceptor PL content across a range of concentrations (i.e. at a given PL concentration for these three acceptor classes, smaller particles were more efficient). The FC efflux differences between the rHDL and the vesicles were not due to the absence of apolipoprotein in the vesicles. No difference was detected among the rHDL of varying size, nor was a difference detected among the LUVs of varying size when compared on the basis of PL content. When the FC efflux data for rHDL and LUVs were normalized on the basis of the number of acceptor particles present at a given PL concentration, a correlation was found between acceptor particle radius and the ability to accept cellular FC with larger particles being the most efficient. However, the dependence of the rate of FC efflux on acceptor particle size was not quantitatively the same within the rHDL and LUV classes of acceptor particles. The dependence of FC efflux on acceptor particle size may reflect differing abilities of the variously sized acceptor particles to access the region very close to the cell plasma membrane where most of the FC removal is expected to occur.

Animals↗

PubMed1995

Cellular cholesterol efflux mediated by cyclodextrins.

In this study, we compared cholesterol efflux mediated by either high density lipoproteins (HDL3) or beta-cyclodextrins, cyclic oligosaccharides that are able to dissolve lipids in their hydrophobic core. beta-Cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin, and methyl-beta-cyclodextrin at 10 mM induced the release of 50-90% of L-cell [3H]cholesterol after 8 h of incubation, with a major portion of this cholesterol being released in the first 1-2 h of incubation. The cholesterol efflux kinetics are different if cells are incubated with HDL3, which induces a relatively constant rate of release of cholesterol throughout an 8-h incubation. Cholesterol efflux to cyclodextrins was much greater than phospholipid release. To test the hypothesis that maximal efflux rate constants for a particular cell are independent of the type of acceptor, we estimated the maximal rate constants for efflux (Vmax) of cellular cholesterol from L-cells, Fu5AH cells, and GM3468A fibroblasts. The rate constant for HDL3-mediated efflux varied among cell lines in the order Fu5AH > L-cells > fibroblasts. However, these differences were not evident when cyclodextrins were used as cholesterol acceptors. The estimated Vmax values for cyclodextrin-mediated efflux were 3.5-70-fold greater than for HDL3 for the three cell lines. The very high efficiency of cyclodextrins in stimulating cell cholesterol efflux suggests that these compounds can be used in two general ways for studies of atherosclerosis: 1) as research tools to probe mechanisms of cholesterol transport and aspects of membrane structure or 2) as potential pharmacological agents that could modify in vivo cholesterol metabolism and influence the development of the atherosclerotic plaque.

Animals↗

PubMed1995

Microenvironments of basic amino acids in amphipathic alpha-helices bound to phospholipid: 13C NMR studies using selectively labeled peptides.

The lipid-binding properties of serum apolipoproteins are mediated by class A amphipathic alpha-helices in which basic and acidic amino acid residues tend to be localized at the helix polar-nonpolar boundary and the center of the polar face, respectively. To better understand the role of the interfacial basic residues, the microenvironments and titration characteristics of lysine (Lys) residues have been examined using two 18-residue peptides. The parent 18A molecule (AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe) binds phospholipid relatively well because of its class A amphipathic helix, whereas 18R, which has the positions of basic and acidic residues reversed, binds to phospholipid relatively weakly. Lys residues were 13C-dimethylated, and NMR spectra were obtained of the peptides complexed with dimyristoylphosphatidylcholine (1/1 (w/w) DMPC/peptide). The four [13C]-epsilon-dimethyl-Lys in 18A gave four resonances at chemical shifts (delta) of 42-43 ppm, whereas only a single resonance at delta 42.70 ppm was observed from the four Lys in 18R. Measurements of delta as a function of pH gave pKa values. The four Lys in 18R had a common pKa of 10.3, indicating that the Lys in the polar face of the amphipathic helix have the pKa expected for noninteracting, fully hydrated groups. In contrast, the four Lys in Ac-18A-NH2 had pKa's in the range 9.4-11.0, indicating that they exist in various microenvironments at the polar-nonpolar boundary of the 18A helix. To examine individual Lys, 18A was synthesized with various Lys selectively labeled. Lys-4, -9, -13, and -15 in Ac-18A-NH2 associated with DMPC had pKa values of 11.0, 9.4, 9.4, and 10.3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Efflux of cellular cholesterol and phospholipid to lipid-free apolipoproteins and class A amphipathic peptides.

The mechanism(s) by which lipid-free apolipoprotein (apo) AI is able to stimulate efflux of cholesterol and phospholipid from cells in cultures has (have) been examined. This process was found to be enhanced when macrophages were enriched with cholesterol. There were 12- and 4-fold increases in cholesterol and phospholipid efflux, respectively, from cholesterol-enriched mouse macrophages when compared to cells not loaded with cholesterol. This enhancement in cholesterol efflux to lipid-free apo AI from macrophages enriched with cholesterol was found to be controlled by the level of free cholesterol in the cells. When cholesterol-enriched mouse macrophages were exposed to lipid-free apo AI at 20 micrograms/mL (706 nM), there was significant efflux of [14C]cholesterol and [3H]phospholipid (20% +/- 0.5%/24 h and 6% +/- 0.3%/24 h, respectively). In comparison, HDL at equivalent protein concentrations only stimulated 11% and 4% efflux of cholesterol and phospholipid, respectively. Synthetic peptides containing amphipathic helical segments that mimic those present in apo AI were used to examine the structural features of the apoprotein which stimulate lipid efflux. Peptides containing only one (18A) or two (37pA) amphipathic helical segments stimulated as much cholesterol efflux from both mouse macrophages and L-cells as apo AI. The order of efficiency, as assessed by the mass concentration at which half-maximal efflux was reached (EC50), was apo AI > 37pA > 18A, indicating that acceptor efficiency was dependent on the number of amphipathic helical segments per molecule. When the helical content of 18A was increased by neutralizing the charges at the ends of the peptide (Ac-18A-NH2), there was a substantial increase in the efficiency for cholesterol efflux (EC50 18A = 17 micrograms/mL vs Ac-18A-NH2 = 6 micrograms/mL). In contrast, when the amphipathicity of the helix in 18A was decreased by scrambling the amino acid sequence, thereby reducing its lipid affinity, cholesterol and phospholipid efflux were not stimulated. The efficiency with which the peptides stimulated cholesterol efflux was in order of their lipid affinity (37pA > Ac-18A-NH2 > 18A), and this order was similar for phospholipid efflux. The time course of lipid release from mouse macrophages and L-cells indicated that phospholipid appeared in the extracellular medium before cholesterol. These results suggest that the apo AI or peptides first interacted with the cell to form protein/phospholipid complexes, that could then accept cholesterol.

Amino Acid Sequence↗

PubMed1995

Molecular determinants of plasma cholesteryl ester transfer protein binding to high density lipoproteins.

The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids between lipoproteins and is associated with high density lipoproteins (HDL). To understand the mechanism of interaction of CETP with HDL, we studied the binding of pure recombinant CETP to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/apoA-I discoidal particles. Separating bound from free CETP using native gradient gel electrophoresis, complexes of CETP with 10-nm hydrodynamic diameter discoidal particles migrated with a diameter of 12-16 nm, compared with approximately 7.5 nm for CETP. At lower ratios of CETP to discs, CETP bound to discs without displacement of apoA-I. CETP alone was unable to generate discoidal complexes. Cross-linking and fluorescence resonance energy transfer experiments indicated that CETP bound to discs as monomers. Cross-linking of CETP to apoA-I in discs suggested proximity of apoA-I and CETP. By negative-stain electron microscopy, discoidal complexes containing CETP and CETP monoclonal antibody showed localization of antibody molecules to the disc edge, suggesting that CETP was bound to the disc edge. The binding of CETP to discs of different composition or size was studied. Discs (10-nm Stokes diameter) prepared with either apoA-I or apoA-II had a similar Kd (120 nM). Inclusion of 1 mol % cholesteryl oleate, 5 mol % cholesterol, or 6 mol % phosphatidylinositol increased the binding affinity of CETP 3-10 times (20-30 nM). In comparison, plasma HDL3 had a Kd of approximately 450 nM. For POPC/apoA-I discs, 10-nm discs bound CETP with much higher affinity than smaller 7.8-nm discs (Kd = 1-2 microM). 7.7-nm hydrodynamic diameter POPC/apoA-I spherical particles containing either triolein or cholesteryl oleate in their core bound CETP with higher affinity (Kd = 50-100 nM) than 7.8-nm POPC/apoA-I discs. Thus, CETP appears to bind to the perimeter of discoidal particles, possibly in a process in which flexible segments in apoA-I or apoA-II accommodate CETP at the disc edge. The binding of CETP to HDL is markedly influenced by overall particle size and shape and by lipid composition, and the increased binding affinity for cholesterol- and cholesteryl ester-containing discs suggests a higher affinity of CETP for nascent than mature HDL.

Animals↗

PubMed1995

The effect of high density lipoprotein phospholipid acyl chain composition on the efflux of cellular free cholesterol.

High density lipoprotein (HDL) phospholipid (PL) fatty acyl chain composition has been proposed to affect the ability of HDL to participate in the first step of reverse cholesterol transport. To examine the effects of PL fatty acid chain length and degree of unsaturation in this process, reconstituted HDL (rHDL) particles were made with human apolipoprotein (apo) A-I and PL containing fatty acid chains from 14 to 18 carbons in length, which were either fully saturated or unsaturated in one or both chains. These particles were characterized structurally and for their ability to promote free (unesterified) cholesterol (FC) efflux from cells growing in culture. The discoidal rHDL particles were homogeneous and exhibited similar hydrodynamic diameters (10.4 +/- 1.0 nm) indicating that apoA-I forms similarly sized discs with a variety of PL. Measurements of particle surface charge, apoA-I alpha-helix content, and conformational stability indicated that the conformation of apoA-I varies among the particles. These conformational effects on apoA-I are consistent with the PL fluidity influencing the interaction between the amphipathic alpha-helical segments and PL acyl chains. Differential scanning calorimetry demonstrated that the physical state of the rHDL PL at 37 degrees C varied according to acyl chain length and degree of unsaturation; the FC efflux efficiencies for particles with PL in either the gel or liquid crystal states were determined. The ability of the rHDL to accept cellular FC depended on the physical state of the PL in the rHDL. Liquid crystal PL formed the most efficient FC acceptor particles exhibiting a maximal efflux velocity (Vmax) of 12-14% release of total cellular FC per h. Gel-phase PL formed inefficient rHDL acceptors with a Vmax of about 3%/h. A similar hierarchy of FC efflux efficiency was noted when either mouse L-cells or rat Fu5AH hepatoma cells were used as the FC donors. Furthermore, this hierarchy was found to be due to the characteristics of the PL and not due to variable apoA-I conformation because protein-free, small unilamellar vesicles made with the same PL exhibited similar relative efflux capabilities. Generally, the ability of a given rHDL particle to accept cellular FC was related to rHDL PL acyl chain length and degree of unsaturation; decreases in PL acyl chain length and increases in chain unsaturation tended to result in more efficient FC acceptor particles.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Effect of the cholesterol content of reconstituted LpA-I on lecithin:cholesterol acyltransferase activity.

The production of cholesteryl ester (CE) by lecithin: cholesterol acyl transferase (LCAT) is elevated significantly in hyperlipidemic subjects at high risk for coronary artery disease. To elucidate the molecular events involved, the relationship between LCAT activation and apolipoprotein (apo) A-I charge and structure in high density lipoproteins (HDL) has been studied in both native HDL and homogeneous recombinant HDL (Lp2A-I) particles containing apoA-I, palmitoyloleoyl phosphatidylcholine and cholesterol. Increasing the cholesterol content of discoidal Lp2A-I from 4 to 26 molecules/particle raises the maximum rate of cholesterol esterification by LCAT (Vmax) from 3.1 to 9.2 nmol CE/h/unit of LCAT and increases the apparent Km from 0.5 to 3.5 microM cholesterol. Similarly, increasing the cholesterol content in triolein core-containing Lp2A-I (4-18 molecules/particle) and in native HDL3 (12-21 molecules/particle) also significantly increases the Vmax for LCAT (2.8-7.7 and 0.5-3.6 nmol CE/h, respectively) and raises the Km values (7.6-36.9 and 7.3-8.5 microM cholesterol, respectively). In contrast, changes in the cholesterol content of native and recombinant HDL have no significant effect on the apparent Km values when expressed in terms of the concentration of either apoA-I or palmitoyloleoyl phosphatidylcholine. This appears to indicate that interfacial cholesterol content has no effect on the binding affinity of LCAT to different LpA-I particles but directly affects catalysis by modulating the interaction of cholesterol molecules with the active site of LCAT. Increasing the cholesterol content of the different HDL particles progressively increases the particle net negative charge, and these changes in apoA-I charge are strongly correlated with both the Vmax and apparent Km values for LCAT. This suggests that the conformation and charge of apoA-I play a central role in LCAT activation and that these parameters are influenced by the amount of cholesterol in the surface of HDL particles.

Apolipoprotein A-I↗

PubMed1995

Effect of the arrangement of tandem repeating units of class A amphipathic alpha-helixes on lipid interaction.

Exchangeable apolipoproteins possess tandem repeating units of class A amphipathic helical segments and many of them are linked together by proline residues. To understand the optimal arrangement of the amphipathic helixes for lipid association, we have studied the interactions of three model class A amphipathic helical peptides with lipids. The three peptides are: 37pA, a dimer of 18A (DWLKAFYDKVAEKLKEAF) linked together by a Pro (18A-Pro-18A); 37aA, a dimer of 18A linked together by an Ala (18A-Ala-18A); and 36A, a dimer of 18A without any linker residue (18A-18A). Circular dichroism (CD) spectra showed that the peptides are predominantly alpha-helical in aqueous and lipid environments. Temperature dependent CD studies indicated that in buffer helix stability decreases in the order 36A > 37aA > 37pA; however, in the presence of dimyristoyl phosphatidylcholine (DMPC), the above order is reversed. The retention times of the peptides on a C18 reversed-phase high performance liquid chromatography column decreased in the order 36A > 37aA > 37pA, consistent with the lengths of the nonpolar faces of the alpha-helixes being in the same order; the retention time of the parent 18A was shorter than 37pA. While 37pA adsorbed to egg phosphatidylcholine monolayers most strongly, the degree and rate of association of 36A were significantly lower. Differential scanning calorimetry indicated that, while 37pA was most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of DMPC multilamellar vesicles, 36A was least effective; 36A was even less effective than 18A. Fluorescence quenching experiments with iodide and acrylamide indicated that, in the presence of DMPC, Trp residues in 36A are most exposed to the quenchers while in 37pA they are least exposed. In the presence of DMPC, shielding of Trp in 18A from the quenchers was more than that observed with Trp residues in 36A. The results of this study suggest that the arrangement of tandem repeating amphipathic helical units which results in the formation of a class A amphipathic helix with a nonpolar face longer than five or six turns reduces the ability of the helix to associate with phospholipid.

Amino Acid Sequence↗

PubMed1994

The influence of apolipoprotein structure on the efflux of cellular free cholesterol to high density lipoprotein.

The influence of apolipoprotein conformation on the ability of high density lipoprotein (HDL) to remove cellular free cholesterol (FC) has not been studied in detail. To address the effects of amphipathic alpha-helix structure on cellular FC efflux, three class A helical peptides and apolipoprotein (apo) AI were complexed to dimyristoyl phosphatidylcholine (DMPC) to make discoidal complexes that were used as acceptors of cell cholesterol. The peptides consisted of an 18-amino acid, amphipathic, alpha-helical peptide with the sequence DWLKAFYDKVAEKLKEAF (18A), a dimer of 18A covalently linked by a proline residue (37pA), and acetyl-18A-amide (Ac-18A-NH2) that has a higher alpha-helix content than the unblocked 18A molecule. The three peptides strongly mimic the lipid-binding characteristics of the amphipathic segments of apolipoproteins and form discoidal complexes with DMPC that are similar in diameter (11-12 nm) to those formed by human apoAI when reconstituted at a 2.5:1 (w:w) phospholipid to protein ratio. The abilities of these complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts; efflux of FC from both the plasma membrane and the lysosomal pools was examined. For each of the acceptors, the removal of cholesterol from the plasma membrane and lysosomal pools was equally efficient. All four discoidal complexes were equally efficient cell membrane FC acceptors when compared at saturating acceptor concentrations of > 200 micrograms of DMPC/ml of medium. However, at the same lipid concentration, protein-free DMPC small unilamellar vesicles (SUV) were significantly less efficient. The initial rates of FC removal from cells at saturating concentrations of acceptor particles (Vmax) were 12, 10, 10, and 11% per h, respectively, for the complexes containing either 18A, Ac-18A-NH2, 37pA, or apoAI, but only 1% cellular FC per h for the DMPC SUV. The 10-fold higher Vmax for the apoprotein/peptide-containing acceptors was likely due to a reversible interaction of apoprotein or peptide with the plasma membrane that changed the lipid packing characteristics in such a way as to increase the rate of FC desorption from the cell surface. This interaction required amphipathic alpha-helical segments, but it was not affected by the length, number, or lipid-binding affinity of the helices. Furthermore, the efflux efficiency was not dependent on the amino acid sequence of the helical segments which suggests that this interaction is not mediated by a specific cell surface binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗