Volumetric determination of apolipoprotein stoichiometry of circulating HDL subspecies

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[4] in the HDL₃ size range (d = 85.0 ± 1.2 Å) and HDL[7] 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[1] (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.     

Molecular dynamics simulations on discoidal HDL particles suggest a mechanism for rotation in the apo A-I belt model

Apolipoprotein A-I (apo A-I) is the major protein component of high-density lipoprotein (HDL) particles. Elevated levels of HDL in the bloodstream have been shown to correlate strongly with a reduced risk factor for atherosclerosis. Molecular dynamics simulations have been carried out on three separate model discoidal high-density lipoprotein particles (HDL) containing two monomers of apo A-I and 160 molecules of palmitoyloleoylphosphatidylcholine (POPC), to a time-scale of 1ns. The starting structures were on the basis of previously published molecular belt models of HDL consisting of the lipid-binding C-terminal domain (residues 44-243) wrapped around the circumference of a discoidal HDL particle. Subtle changes between two of the starting structures resulted in significantly different behavior during the course of the simulation. The results provide support for the hypothesis of Segrest et al. that helical registration in the molecular belt model of apo A-I is modulated by intermolecular salt bridges. In addition, we propose an explanation for the presence of proline punctuation in the molecular belt model, and for the presence of two 11-mer helical repeats interrupting the otherwise regular pattern of 22-mer helical repeats in the lipid-binding domain of apo A-I.     

Effects of cholesteryl ester transfer protein inhibition on apolipoprotein A-II-containing HDL subspecies and apolipoprotein A-II metabolism

This study was designed to establish the mechanism responsible for the increased apolipoprotein (apo) A-II levels caused by the cholesteryl ester transfer protein inhibitor torcetrapib. Nineteen subjects with low HDL cholesterol (<40 mg/dl), nine of whom were also treated with 20 mg of atorvastatin daily, received placebo for 4 weeks, followed by 120 mg of torcetrapib daily for the next 4 weeks. Six subjects in the nonatorvastatin cohort participated in a third phase, in which they received 120 mg of torcetrapib twice daily for 4 weeks. At the end of each phase, subjects underwent a primed-constant infusion of [5,5,5-²H₃]l-leucine to determine the kinetics of HDL apoA-II. Relative to placebo, torcetrapib significantly increased apoA-II concentrations by reducing HDL apoA-II catabolism in the atorvastatin (−9.4%, P < 0.003) and nonatorvastatin once- (−9.9%, P = 0.02) and twice- (−13.2%, P = 0.02) daily cohorts. Torcetrapib significantly increased the amount of apoA-II in the α-2-migrating subpopulation of HDL when given as monotherapy (27%, P < 0.02; 57%, P < 0.003) or on a background of atorvastatin (28%, P < 0.01). In contrast, torcetrapib reduced concentrations of apoA-II in α-3-migrating HDL, with mean reductions of −14% (P = 0.23), −18% (P < 0.02), and −18% (P < 0.01) noted during the atorvastatin and nonatorvastatin 120 mg once- and twice-daily phases, respectively. Our findings indicate that CETP inhibition increases plasma concentrations of apoA-II by delaying HDL apoA-II catabolism and significantly alters the remodeling of apoA-II-containing HDL subpopulations.     

Enhancement by LDL of transfer of L-4F and oxidized lipids to HDL in C57BL/6J mice and human plasma

The apoA-I mimetic peptide L-4F [(Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2) synthesized from all L-amino acids] has shown potential for the treatment of a variety of diseases. Here, we demonstrate that LDL promotes association between L-4F and HDL. A 2- to 3-fold greater association of L-4F with human HDL was observed in the presence of human LDL as compared with HDL by itself. This association further increased when LDL was supplemented with the oxidized lipid 15S-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid (15HETE). Additionally, L-4F significantly (P = 0.02) promoted the transfer of 15HETE from LDL to HDL. The transfer of L-4F from LDL to HDL was demonstrated both in vitro and in C57BL/6J mice. L-4F, injected into C57BL/6J mice, associated rapidly with HDL and was then cleared quickly from the circulation. Similarly, L-4F loaded onto human HDL and injected into C57BL/6J mice was cleared quickly with T(1/2) = 23.6 min. This was accompanied by a decline in human apoA-I with little or no effect on the mouse apoA-I. Based on these results, we propose that i) LDL promotes the association of L-4F with HDL and ii) in the presence of L-4F, oxidized lipids in LDL are rapidly transferred to HDL allowing these oxidized lipids to be acted upon by HDL-associated enzymes and/or cleared from the circulation.     

The effect of phospholipid composition of reconstituted HDL on its cholesterol efflux and anti-inflammatory properties

The goal of this study was to understand how the reconstituted HDL (rHDL) phospholipid (PL) composition affects its cholesterol efflux and anti-inflammatory properties. An ApoA-I mimetic peptide, 5A, was combined with either SM or POPC. Both lipid formulations exhibited similar in vitro cholesterol efflux by ABCA1, but 5A-SM exhibited higher ABCG1- and SR-BI-mediated efflux relative to 5A-POPC (P < 0.05). Injection of both rHDLs in rats resulted in mobilization of plasma cholesterol, although the relative potency was 3-fold higher for the same doses of 5A-SM than for 5A-POPC. Formation of preβ HDL was observed following incubation of rHDLs with both human and rat plasma in vitro, with 5A-SM inducing a higher extent of preβ formation relative to 5A-POPC. Both rHDLs exhibited anti-inflammatory properties, but 5A-SM showed higher inhibition of TNF-α, IL-6, and IL-1β release than did 5A-POPC (P < 0.05). Both 5A-SM and 5A-POPC showed reduction in total plaque area in ApoE^−/− mice, but only 5A-SM showed a statistically significant reduction over placebo control and baseline (P < 0.01). The type of PL used to reconstitute peptide has significant influence on rHDL’s anti-inflammatory and anti-atherosclerosis properties.     

Formation of high density lipoproteins containing both apolipoprotein A-I and A-II in the rabbit

Human plasma HDLs are classified on the basis of apolipoprotein composition into those that contain apolipoprotein A-I (apoA-I) without apoA-II [(A-I)HDL] and those containing apoA-I and apoA-II [(A-I/A-II)HDL]. ApoA-I enters the plasma as a component of discoidal particles, which are remodeled into spherical (A-I)HDL by LCAT. ApoA-II is secreted into the plasma either in the lipid-free form or as a component of discoidal high density lipoproteins containing apoA-II without apoA-I [(A-II)HDL]. As discoidal (A-II)HDL are poor substrates for LCAT, they are not converted into spherical (A-II)HDL. This study investigates the fate of apoA-II when it enters the plasma. Lipid-free apoA-II and apoA-II-containing discoidal reconstituted HDL [(A-II)rHDL] were injected intravenously into New Zealand White rabbits, a species that is deficient in apoA-II. In both cases, the apoA-II was rapidly and quantitatively incorporated into spherical (A-I)HDL to form spherical (A-I/A-II)HDL. These particles were comparable in size and composition to the (A-I/A-II)HDL in human plasma. Injection of lipid-free apoA-II and discoidal (A-II)rHDL was also accompanied by triglyceride enrichment of the endogenous (A-I)HDL and VLDL as well as the newly formed (A-I/A-II)HDL. We conclude that, irrespective of the form in which apoA-II enters the plasma, it is rapidly incorporated into spherical HDLs that also contain apoA-I to form (A-I/A-II)HDL.     

Effect of rosiglitazone on HDL metabolism in subjects with metabolic syndrome and low HDL

Treatment with the peroxisome proliferator-activated receptor γ agonist rosiglitazone has been reported to increase HDL-cholesterol (HDL-C) levels, although the mechanism responsible for this is unknown. We sought to determine the effect of rosiglitazone on HDL apolipoprotein A-I (apoA-I) and apoA-II metabolism in subjects with metabolic syndrome and low HDL-C. Subjects were treated with placebo followed by rosiglitazone (8 mg) once daily. At the end of each 8 week treatment, subjects (n = 15) underwent a kinetic study to measure apoA-I and apoA-II production rate (PR) and fractional catabolic rate. Rosiglitazone significantly reduced fasting insulin and high-sensitivity C-reactive protein (hsCRP) and increased apoA-II levels. Mean apoA-I and HDL-C levels were unchanged following rosiglitazone treatment, although there was considerable individual variability in the HDL-C response. Rosiglitazone had no effect on apoA-I metabolism, whereas the apoA-II PR was increased by 23%. The change in HDL-C in response to rosiglitazone was significantly correlated with the change in apoA-II concentration but not to changes in apoA-I, measures of glucose homeostasis, or hsCRP. Treatment with rosiglitazone significantly increased apoA-II production in subjects with metabolic syndrome and low HDL-C but had no effect on apoA-I metabolism. The change in HDL-C in response to rosiglitazone treatment was unrelated to effects on apoA-I, instead being related to the change in the metabolism of apoA-II.     

Validation of previous computer models and MD simulations of discoidal HDL by a recent crystal structure of apoA-I

HDL is a population of apoA-I-containing particles inversely correlated with heart disease. Because HDL is a soft form of matter deformable by thermal fluctuations, structure determination has been difficult. Here, we compare the recently published crystal structure of lipid-free (Δ185-243)apoA-I with apoA-I structure from models and molecular dynamics (MD) simulations of discoidal HDL. These analyses validate four of our previous structural findings for apoA-I: i) a baseline double belt diameter of 105 Å ii) central α helixes with an 11/3 pitch; iii) a “presentation tunnel” gap between pairwise helix 5 repeats hypothesized to move acyl chains and unesterified cholesterol from the lipid bilayer to the active sites of LCAT; and iv) interchain salt bridges hypothesized to stabilize the LL5/5 chain registry. These analyses are also consistent with our finding that multiple salt bridge-forming residues in the N-terminus of apoA-I render that conserved domain “sticky.” Additionally, our crystal MD comparisons led to two new hypotheses: i) the interchain leucine-zippers previously reported between the pair-wise helix 5 repeats drive lipid-free apoA-I registration; ii) lipidation induces rotations of helix 5 to allow formation of interchain salt bridges, creating the LCAT presentation tunnel and “zip-locking” apoA-I into its full LL5/5 registration.     

Defective functionality of HDL particles in familial apoA-I deficiency: relevance of alterations in HDL lipidome and proteome

To evaluate functional and compositional properties of HDL in subjects from a kindred of genetic apoA-I deficiency, two homozygotes and six heterozygotes, with a nonsense mutation at APOA1 codon -2, Q[-2]X, were recruited together with age- and sex-matched healthy controls (n = 11). Homozygotes displayed undetectable plasma levels of apoA-I and reduced levels of HDL-cholesterol (HDL-C) and apoC-III (5.4% and 42.6% of controls, respectively). Heterozygotes displayed low HDL-C (21 ± 9 mg/dl), low apoA-I (79 ± 24 mg/dl), normal LDL-cholesterol (132 ± 25 mg/dl), and elevated TG (130 ± 45 mg/dl) levels. Cholesterol efflux capacity of ultracentrifugally isolated HDL subpopulations was reduced (up to −25%, P < 0.01, on a glycerophospholipid [GP] basis) in heterozygotes versus controls. Small, dense HDL3 and total HDL from heterozygotes exhibited diminished antioxidative activity (up to −48%, P < 0.001 on a total mass basis) versus controls. HDL subpopulations from both homozygotes and heterozygotes displayed altered chemical composition, with depletion in apoA-I, GP, and cholesteryl ester; enrichment in apoA-II, free cholesterol, and TG; and altered phosphosphingolipidome. The defective atheroprotective activities of HDL were correlated with altered lipid and apo composition. These data reveal that atheroprotective activities of HDL particles are impaired in homozygous and heterozygous apoA-I deficiency and are intimately related to marked alterations in protein and lipid composition.     

Atomistic MD simulation reveals the mechanism by which CETP penetrates into HDL enabling lipid transfer from HDL to CETP

Inhibition of cholesterol ester transfer protein (CETP), a protein mediating transfer of neutral lipids between lipoproteins, has been proposed as a means to elevate atheroprotective HDL subpopulations and thereby reduce atherosclerosis. However, off-target and adverse effects of the inhibition have raised doubts about the molecular mechanism of CETP-HDL interaction. Recent experimental findings have demonstrated the penetration of CETP into HDL. However, atomic level resolution of CETP penetration into HDL, a prerequisite for a better understanding of CETP functionality and HDL atheroprotection, is missing. We constructed an HDL particle that mimics the actual human HDL mass composition and investigated for the first time, by large-scale atomistic molecular dynamics, the interaction of an upright CETP with a human HDL-mimicking model. The results demonstrated how CETP can penetrate the HDL particle surface, with the formation of an opening in the N barrel domain end of CETP, put in evidence the major anchoring role of a tryptophan-rich region of this domain, and unveiled the presence of a phenylalanine barrier controlling further access of HDL-derived lipids to the tunnel of CETP. The findings reveal novel atomistic details of the CETP-HDL interaction mechanism and can provide new insight into therapeutic strategies.     

Disruption of the murine procollagen C-proteinase enhancer 2 gene causes accumulation of pro-apoA-I and increased HDL levels

Given the increased prevalence of cardiovascular disease in the world, the search for genetic variations that impact risk factors associated with the development of this disease continues. Multiple genetic association studies demonstrate that procollagen C-proteinase enhancer 2 (PCPE2) modulates HDL levels. Recent studies revealed an unexpected role for this protein in the proteolytic processing of pro-apolipoprotein (apo) A-I by enhancing the cleavage of the hexapeptide extension present at the N-terminus of apoA-I. To investigate the role of the PCPE2 protein in an in vivo model, PCPE2-deficient (PCPE2 KO) mice were examined, and a detailed characterization of plasma lipid profiles, apoA-I, HDL speciation, and function was done. Results of isoelectric focusing (IEF) electrophoresis together with the identification of the amino terminal peptides DEPQSQWDK and WHVWQQDEPQSQWDVK, representing mature apoA-I and pro-apoA-I, respectively, in serum from PCPE2 KO mice confirmed that PCPE2 has a role in apoA-I maturation. Lipid profiles showed a marked increase in plasma apoA-I and HDL-cholesterol (HDL-C) levels in PCPE2 KO mice compared with wild-type littermates, regardless of gender or diet. Changes in HDL particle size and electrophoretic mobility observed in PCPE2 KO mice suggest that the presence of pro-apoA-I impairs the maturation of HDL. ABCA1-dependent cholesterol efflux is defective in PCPE2 KO mice, suggesting that the functionality of HDL is altered.     

An apoA-I mimetic peptide containing a proline residue has greater in vivo HDL binding and anti-inflammatory ability than the 4F peptide

Modifying apolipoprotein (apo) A-I mimetic peptides to include a proline-punctuated α-helical repeat increases their anti-inflammatory properties as well as allows better mimicry of full-length apoA-I function. This study compares the following mimetics, either acetylated or biotinylated (b): 4F (18mer) and 4F-proline-4F (37mer, Pro). b4F interacts with both mouse HDL (moHDL) and LDL in vitro. b4F in vivo plasma clearance kinetics are not affected by mouse HDL level. Administration of biotinylated peptides to mice demonstrates that b4F does not associate with lipoproteins smaller than LDL in vivo, though it does associate with fractions containing free hemoglobin (Hb). In contrast, bPro specifically interacts with HDL. b4F and bPro show opposite binding responses to HDL by surface plasmon resonance. Administration of acetylated Pro to apoE^−/− mice significantly decreases plasma serum amyloid A levels, while acetylated 4F does not have this ability. In contrast to previous reports that inferred that 4F associates with HDL in vivo, we systematically examined this potential interaction and demonstrated that b4F does not interact with HDL in vivo but rather elutes with Hb-containing plasma fractions. bPro, however, specifically binds to moHDL in vivo. In addition, the number of amphipathic α-helices and their linker influences the anti-inflammatory effects of apoA-I mimetic peptides in vivo.     

Atherogenic, enlarged, and dysfunctional HDL in human PLTP/apoA-I double transgenic mice

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.     

SR-BI-mediated selective lipid uptake segregates apoA-I and apoA-II catabolism

The HDL receptor scavenger receptor class B type I (SR-BI) binds HDL and mediates the selective uptake of cholesteryl ester. We previously showed that remnants, produced when human HDL(2) is catabolized in mice overexpressing SR-BI, become incrementally smaller, ultimately consisting of small alpha-migrating particles, distinct from pre-beta HDL. When mixed with mouse plasma, some remnant particles rapidly increase in size by associating with HDL without the mediation of cholesteryl ester transfer protein, LCAT, or phospholipid transfer protein. Here, we show that processing of HDL(2) by SR-BI-overexpressing mice resulted in the preferential loss of apolipoprotein A-II (apoA-II). Short-term processing generated two distinct, small alpha-migrating particles. One particle (8.0 nm diameter) contained apoA-I and apoA-II; the other particle (7.7 nm diameter) contained only apoA-I. With extensive SR-BI processing, only the 7.7 nm particle remained. Only the 8.0 nm remnants were able to associate with HDL. Compared with HDL(2), this remnant was more readily taken up by the liver than by the kidney. We conclude that SR-BI-generated HDL remnants consist of particles with or without apoA-II and that only those containing apoA-II associate with HDL in an enzyme-independent manner. Extensive SR-BI processing generates small apoA-II-depleted particles unable to reassociate with HDL and readily taken up by the liver. This represents a pathway by which apoA-I and apoA-II catabolism are segregated.     

Accuracy and precision of NMR relaxation experiments and MD simulations for characterizing protein dynamics

Model-free parameters obtained from nuclear magnetic resonance (NMR) relaxation experiments and molecular dynamics (MD) simulations commonly are used to describe the intramolecular dynamical properties of proteins. To assess the relative accuracy and precision of experimental and simulated model-free parameters, three independent data sets derived from backbone ¹⁵N NMR relaxation experiments and two independent data sets derived from MD simulations of Escherichia coli ribonuclease HI are compared. The widths of the distributions of the differences between the order parameters for pairs of NMR data sets are congruent with the uncertainties derived from statistical analyses of individual data sets; thus, current protocols for analyzing NMR data encapsulate random uncertainties appropriately. Large differences in order parameters for certain residues are attributed to systematic differences between samples for intralaboratory comparisons and unknown, possibly magnetic field-dependent, experimental effects for interlaboratory comparisons. The widths of distributions of the differences between the order parameters for two NMR sets are similar to widths of distributions for an NMR and an MD set or for two MD sets. The linear correlations between the order parameters for an MD set and an NMR set are within the range of correlations observed between pairs of NMR sets. These comparisons suggest that the NMR and MD generalized order parameters for the backbone amide N—H bond vectors are of comparable accuracy for residues exhibiting motions on a fast time scale (<100 ps). Large discrepancies between NMR and MD order parameters for certain residues are attributed to the occurrence of “rare” motional events over the simulation trajectories, the disruption of an element of secondary structure in one of the simulations, and lack of consensus among the experimental data sets. Consequently, (easily detectable) severe distortions of local protein structure and infrequent motional events in MD simulations appear to be the most serious artifacts affecting the accuracy and precision, respectively, of MD order parameters relative to NMR values. In addition, MD order parameters for motions on a fast (<100 ps) timescale are more precisely determined than their NMR counterparts, thereby permitting more detailed dynamic characterization of biologically important residues by MD simulation than is sometimes possible by experimental methods. Proteins 28:481–493, 1997. © 1997 Wiley-Liss, Inc.     

Composition and lipid spatial distribution of HDL particles in subjects with low and high HDL-cholesterol

A low level of high density lipoprotein cholesterol (HDL-C) is a powerful risk factor for cardiovascular disease. However, despite the reported key role of apolipo-proteins, specifically, apoA-I, in HDL metabolism, lipid molecular composition of HDL particles in subjects with high and low HDL-C levels is currently unknown. Here lipidomics was used to study HDL derived from well-characterized high and low HDL-C subjects. Low HDL-C subjects had elevated triacylglycerols and diminished lysophosphatidylcholines and sphingomyelins. Using information about the lipid composition of HDL particles in these two groups, we reconstituted HDL particles in silico by performing large-scale molecular dynamics simulations. In addition to confirming the measured change in particle size, we found that the changes in lipid composition also induced specific spatial distributions of lipids within the HDL particles, including a higher amount of triacylglycerols at the surface of HDL particles in low HDL-C subjects. Our findings have important implications for understanding HDL metabolism and function. For the first time we demonstrate the power of combining molecular profiling of lipoproteins with dynamic modeling of lipoprotein structure.     

Differential effects of HDL subpopulations on cellular ABCA1- and SR-BI-mediated cholesterol efflux

Our objective was to evaluate the associations of individual apolipoprotein A-I (apoA-I)-containing HDL subpopulation levels with ABCA1- and scavenger receptor class B type I (SR-BI)-mediated cellular cholesterol efflux. HDL subpopulations were measured by nondenaturing two-dimensional gel electrophoresis from 105 male subjects selected with various levels of apoA-I in pre-beta-1, alpha-1, and alpha-3 HDL particles. ApoB-containing lipoprotein-depleted serum was incubated with [(3)H]cholesterol-labeled cells to measure efflux. The difference in efflux between control and ABCA1-upregulated J774 macrophages was taken as a measure of ABCA1-mediated efflux. SR-BI-mediated efflux was determined using cholesterol-labeled Fu5AH hepatoma cells. Fractional efflux values obtained from these two cell systems were correlated with the levels of individual HDL subpopulations. A multivariate analysis showed that two HDL subspecies correlated significantly with ABCA1-mediated efflux: small, lipid-poor pre-beta-1 particles (P=0.0022) and intermediate-sized alpha-2 particles (P=0.0477). With regard to SR-BI-mediated efflux, multivariate analysis revealed significant correlations with alpha-2 (P=0.0004), alpha-1 (P=0.0030), pre-beta-1 (P=0.0056), and alpha-3 (P=0.0127) HDL particles. These data demonstrate that the small, lipid-poor pre-beta-1 HDL has the strongest association with ABCA1-mediated cholesterol even in the presence of all other HDL subpopulations. Cholesterol efflux via the SR-BI pathway is associated with several HDL subpopulations with different apolipoprotein composition, lipid content, and size.