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.     

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.     

Molecular packing of high-density and low-density lipoprotein surface lipids and apolipoprotein A-I binding

The surface pressure (pi)-molecular area (A) isotherms for monolayers of human high-density lipoprotein (HDL3) and low-density lipoprotein (LDL) phospholipids and of mixed monolayers of these phospholipids with cholesterol spread at the air-water interface were used to deduce the likely molecular packing at the surfaces of HDL3 and LDL particles. LDL phospholipids form more condensed monolayers than HDL3 phospholipids; for example, the molecular areas of LDL and HDL3 phospholipids at pi = 10 dyn/cm are 88 and 75 A2/molecule, respectively. The closer packing in the LDL phospholipids monolayer can be attributed to the higher contents of saturated phosphatidylcholines and sphingomyelin relative to HDL3. Cholesterol condenses both HDL3 and LDL phospholipid monolayers but has a greater condensing effect on the LDL phospholipid monolayer. The pi-A isotherms for mixed monolayer of HDL3 phospholipid/cholesterol and LDL phospholipid/cholesterol at stoichiometries similar to those at the surfaces of lipoprotein particles suggest that the monolayer at the surface of the LDL particle is significantly more condensed than that at the surface of the HDL3 particle. The closer lateral packing in LDL is due to at least three factors: (1) the difference in phospholipid composition; (2) the higher unesterified cholesterol content in LDL; and (3) a stronger interaction between cholesterol and LDL phospholipids relative to HDL3 phospholipids. The influence of lipid molecular packing on the affinity of human apolipoprotein A-I (apo A-I) for HDL3 and LDL surface lipids was evaluated by monitoring the adsorption of 14C-methylated apo A-I to monolayers of these lipids spread at various initial surface pressures (pi i).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cholesterol feeding on primate serum lipoproteins. III. The change in high density lipoprotein components

Sooty mangabey (Cercocebus atys) monkeys had a lower serum HDL cholesterol concentration than any other Old World monkey species reported. In addition, they had a higher serum Lp(a) concentration than other species. The mangabeys were fed a cholesterol-fat diet for 5 weeks. HDL2 and HDL3 amounts were determined from the two peaks apparent upon analytical ultracentrifugation. In the first 1-3 weeks, 13 of the 14 mangabeys increased 30% (mean) in total HDL, this increase occurring only in the HDL2 fraction. After 5 weeks, HDL and HDL2 decreased markedly. During the cholesterol feeding, HDL3 continually decreased in flotation rate, indicating it was either smaller and/or denser. HDL2 and HDL3 separated well on molecular sieving agarose columns during the diet period, whereas a single symmetrical elution peak was found for chow-fed HDL. Thus on a cholesterol-fat diet, HDL2 and HDL3 increased in difference in molecular size.     

High density lipoprotein structural changes and drug response in lipidomic profiles following the long-term fenofibrate therapy in the FIELD substudy

In a recent FIELD study the fenofibrate therapy surprisingly failed to achieve significant benefit over placebo in the primary endpoint of coronary heart disease events. Increased levels of atherogenic homocysteine were observed in some patients assigned to fenofibrate therapy but the molecular mechanisms behind this are poorly understood. Herein we investigated HDL lipidomic profiles associated with fenofibrate treatment and the drug-induced Hcy levels in the FIELD substudy. We found that fenofibrate leads to complex HDL compositional changes including increased apoA-II, diminishment of lysophosphatidylcholines and increase of sphingomyelins. Ethanolamine plasmalogens were diminished only in a subgroup of fenofibrate-treated patients with elevated homocysteine levels. Finally we performed molecular dynamics simulations to qualitatively reconstitute HDL particles in silico. We found that increased number of apoA-II excludes neutral lipids from HDL surface and apoA-II is more deeply buried in the lipid matrix than apoA-I. In conclusion, a detailed molecular characterization of HDL may provide surrogates for predictors of drug response and thus help identify the patients who might benefit from fenofibrate treatment.     

The refined structure of nascent HDL reveals a key functional domain for particle maturation and dysfunction

The cardioprotective function of high-density lipoprotein (HDL) is largely attributed to its ability to facilitate transport of cholesterol from peripheral tissues to the liver. However, HDL may become dysfunctional through oxidative modification, impairing cellular cholesterol efflux. Here we report a refined molecular model of nascent discoidal HDL, determined using hydrogen-deuterium exchange mass spectrometry. The model reveals two apolipoprotein A1 (apoA1) molecules arranged in an antiparallel double-belt structure, with residues 159-180 of each apoA1 forming a protruding solvent-exposed loop. We further show that this loop, including Tyr166, a preferred target for site-specific oxidative modification within atheroma, directly interacts with and activates lecithin cholesterol acyl transferase. These studies identify previously uncharacterized structural features of apoA1 in discoidal HDL that are crucial for particle maturation, and elucidate a structural and molecular mechanism for generating a dysfunctional form of HDL in atherosclerosis.     

"Sticky" and "promiscuous", the yin and yang of apolipoprotein A-I termini in discoidal high-density lipoproteins: a combined computational-experimental approach

Apolipoprotein (apo) A-I-containing lipoproteins in the form of high-density lipoproteins (HDL) are inversely correlated with atherosclerosis. Because HDL is a soft form of condensed matter easily deformable by thermal fluctuations, the molecular mechanisms for HDL remodeling are not well understood. A promising approach to understanding HDL structure and dynamics is molecular dynamics (MD). In the present study, two computational strategies, MD simulated annealing (MDSA) and MD temperature jump, were combined with experimental particle reconstitution to explore molecular mechanisms for phospholipid- (PL-) rich HDL particle remodeling. The N-terminal domains of full-length apoA-I were shown to be "sticky", acting as a molecular latch largely driven by salt bridges, until, at a critical threshold of particle size, the associated domains released to expose extensive hydrocarbon regions of the PL to solvent. The "sticky" N-termini also associate with other apoA-I domains, perhaps being involved in N-terminal loops suggested by other laboratories. Alternatively, the overlapping helix 10 C-terminal domains of apoA-I were observed to be extremely mobile or "promiscuous", transiently exposing limited hydrocarbon regions of PL. Based upon these models and reconstitution studies, we propose that separation of the N-terminal domains, as particles exceed a critical size, triggers fusion between particles or between particles and membranes, while the C-terminal domains of apoA-I drive the exchange of polar lipids down concentration gradients between particles. This hypothesis has significant biological relevance since lipid exchange and particle remodeling are critically important processes during metabolism of HDL particles at every step in the antiatherogenic process of reverse cholesterol transport.     

Isolation and identification of HDL particles of low molecular weight

Small particles of high density lipoproteins (HDL) were isolated from fresh, fasting human plasma and from the ultracentrifugally isolated high density lipoprotein fraction by means of ultrafiltration through membranes of molecular weight cutoff of 70,000. These particles were found to contain cholesterol, phospholipids, and apolipoproteins A-I and A-II; moreover, they floated at a density of 1.21 kg/l. They contained 67.5% of their mass as protein and the rest as lipid. Two populations of small HDL particles were identified: one containing apolipoprotein A-I alone [(A-I)HDL] and the other containing both apolipoproteins A-I and A-II [A-I + A-II)HDL]. The molar ratio of apoA-I to apoA-II in the latter subclass isolated from plasma or HDL was 1:1. The molecular weights of these subpopulations were determined by nondenaturing gradient polyacrylamide gel electrophoresis and found to be 70,000; 1.5% of the plasma apoA-I was recovered in the plasma ultrafiltrate.     

Isolation and characterization of a dog serum lipoprotein having apolipoprotein A-I as its predominant protein constituent.

The serum high density lipoproteins (HDL) of normolipemic dogs (beagles) were isolated in the density range of p 1.063 to 1.21 g/ml, and characterized in terms of composition and physical properties (flotation and diffusion coefficients, partial specific volume, molecular weight, electrophoretic mobility, ultraviolet absorption, and circular dichroism). The results indicated that canine HDL is a relatively homogeneous class with a molecular weight of about 230 000 and general properties similar to those reported for human HDL. After delipidation, the resulting apolipoprotein, apo-HDL, was fractionated by Sephadex G-200 column chromatography in urea or guanidine hydrochloride solutions. About 90% of the apo-HDL consisted of a protein with a molecular weight of about 28 000, similar in amino acid composition to human apolipoprotein A-I and having the same NH2 terminus (aspartic acid) and COOH terminus (glutamine) and no carbohydrates. Two other proteins were isolated, one having an apparent mol wt of 55 000 and representing, at least in part, an aggregate of apolipoprotein A-I and the other component with a mol wt of 8000, not yet characterized. The results indicate that canine HDL, as an intact complex, has general physical properties that lie between those reported for human HDL2 and HDL3, and that it differs compositionally from the human products mainly in its predominant content of apo-A-I. These findings together with evidence for the relatively homogeneous nature of the canine HDL provide new prospects for unraveling the relationship between polypeptide composition and HDL structure.     

Apolipoprotein AI tertiary structures determine stability and phospholipid‐binding activity of discoidal high‐density lipoprotein particles of different sizes

Human high‐density lipoprotein (HDL) plays a key role in the reverse cholesterol transport pathway that delivers excess cholesterol back to the liver for clearance. In vivo, HDL particles vary in size, shape and biological function. The discoidal HDL is a 140–240 kDa, disk‐shaped intermediate of mature HDL. During mature spherical HDL formation, discoidal HDLs play a key role in loading cholesterol ester onto the HDL particles by activating the enzyme, lecithin:cholesterol acyltransferase (LCAT). One of the major problems for high‐resolution structural studies of discoidal HDL is the difficulty in obtaining pure and, foremost, homogenous sample. We demonstrate here that the commonly used cholate dialysis method for discoidal HDL preparation usually contains 5–10% lipid‐poor apoAI that significantly interferes with the high‐resolution structural analysis of discoidal HDL using biophysical methods. Using an ultracentrifugation method, we quickly removed lipid‐poor apoAI. We also purified discoidal reconstituted HDL (rHDL) into two pure discoidal HDL species of different sizes that are amendable for high‐resolution structural studies. A small rHDL has a diameter of 7.6 nm, and a large rHDL has a diameter of 9.8 nm. We show that these two different sizes of discoidal HDL particles display different stability and phospholipid‐binding activity. Interestingly, these property/functional differences are independent from the apoAI α‐helical secondary structure, but are determined by the tertiary structural difference of apoAI on different discoidal rHDL particles, as evidenced by two‐dimensional NMR and negative stain electron microscopy data. Our result further provides the first high‐resolution NMR data, demonstrating a promise of structural determination of discoidal HDL at atomic resolution using a combination of NMR and other biophysical techniques.     

A model structure for the heterodimer apoA-IMilano-apoA-II supports its peculiar susceptibility to proteolysis

In this study, we propose a structure for the heterodimer between apolipoprotein A-I(Milano) and apolipoprotein A-II (apoA-I(M)-apoA-II) in a synthetic high-density lipoprotein (HDL) containing L-alpha-palmitoyloleoyl phosphatidylcholine. We applied bioinformatics/computational tools and procedures, such as molecular docking, molecular and essential dynamics, starting from published crystal structures for apolipoprotein A-I and apolipoprotein A-II. Structural and energetic analyses onto the simulated system showed that the molecular dynamics produced a stabilized synthetic HDL. The essential dynamic analysis showed a deviation from the starting belt structure. Our structural results were validated by limited proteolysis experiments on HDL from apoA-I(M) carriers in comparison with control HDL. The high sensitivity of apoA-I(M)-apoA-II to proteases was in agreement with the high root mean-square fluctuation values and the reduction in secondary structure content from molecular dynamics data. Circular dichroism on synthetic HDL containing apoA-I(M)-apoA-II was consistent with the alpha-helix content computed on the proposed model.     

Mass spectrometric determination of apolipoprotein molecular stoichiometry in reconstituted high density lipoprotein particles

Plasma HDL-cholesterol and apolipoprotein A-I (apoA-I) levels are strongly inversely associated with cardiovascular disease. However, the structure and protein composition of HDL particles is complex, as native and synthetic discoidal and spherical HDL particles can have from two to five apoA-I molecules per particle. To fully understand structure-function relationships of HDL, a method is required that is capable of directly determining the number of apolipoprotein molecules in heterogeneous HDL particles. Chemical cross-linking followed by SDS polyacrylamide gradient gel electrophoresis has been previously used to determine apolipoprotein stoichiometry in HDL particles. However, this method yields ambiguous results due to effects of cross-linking on protein conformation and, subsequently, its migration pattern on the gel. Here, we describe a new method based on cross-linking chemistry followed by MALDI mass spectrometry that determines the absolute mass of the cross-linked complex, thereby correctly determining the number of apolipoprotein molecules in a given HDL particle. Using well-defined, homogeneous, reconstituted apoA-I-containing HDL, apoA-IV-containing HDL, as well as apoA-I/apoA-II-containing HDL, we have validated this method. The method has the capability to determine the molecular ratio and molecular composition of apolipoprotein molecules in complex reconstituted HDL particles.     

Radiation inactivation of binding sites for high density lipoproteins in human fibroblast membranes

Radiation inactivation and target analysis were used to determine the molecular mass of the binding sites for high density lipoproteins (HDL) on membranes prepared from human fibroblasts. These membrane binding sites shared characteristics with the previously described HDL binding sites on whole fibroblasts in tissue culture. They exhibited the same affinity for HDL, the same ligand specificity, and the same sensitivity to proteolytic agents. They were also up-regulated by cholesterol loading of the cells. Kinetics of HDL dissociation from membrane binding sites could not be described by a single exponential function, indicating that HDL probably bind to multiple classes of sites on fibroblast membranes. After exposure to ionizing radiation, these sites decreased in number as an apparent single exponential function of radiation dose, corresponding to an average molecular mass of 16,000 +/- 1,000 Da, which is smaller than any known cell-surface receptor protein. These data indicate that HDL binding sites on fibroblast membranes are not "classical" receptors in that they are kinetically heterogeneous and small in molecular mass.     

Characterization of the apolipoproteins of rat plasma lipoproteins.

Purified fractions of three major rat high-density lipoproteins (HDL) and one rat very low-density lipoprotein (VLDL) were isolated by Sephadex gel chromatography or preparative sodium dodecyl sulfate gel electrophoresis. These proteins were characterized by amino acid analysis, end-group analysis, molecular-weight determination, polyacrylamide gel electrophoresis, and circular dichroism. One of these rat proteins, of molecular weight 27 000, appears to be homologous with the human A-I protein. However, rat HDL possesses two additional major components not reported in human HDL - an arginine-rich protein of molecular weight 35 000 and a protein of molecular weight 46 000. The arginine-rich protein of the rat is similar in size and amino acid analysis to the arginine-rich protein reported in human VLDL. A major component of rat VLDL of 35 000 molecular weight appears similar or identical to the arginine-rich protein in rat HDL by every criterion employed for their characterization.     

Synthesis and secretion of apoE in thioglycolate-elicited mouse peritoneal macrophages: effect of cholesterol efflux

ApoE synthesis and secretion, as a function of cellular cholesterol content and cholesterol efflux, was studied in thioglycolate-elicited mouse peritoneal macrophages. As expected, loading elicited macrophages with cholesterol induced a 5-fold increase in apoE secretion and a 2.5-fold increase in cellular apoE content over a 5-h period. Treatment of cholesterol-loaded cells with HDL3 further increased apoE secretion 1.7-fold and decreased cellular cholesterol by 20%. Treatment of cholesterol-loaded cells with HDL3 and SAH 58.035 (an ACAT inhibitor) increased apoE secretion 2.4-fold and decreased cellular cholesterol content by 35%. Treatment of the cells with the ACAT inhibitor alone suppressed apoE secretion by 40% but did not change cellular cholesterol content. Northern blot analysis of RNA indicated that cholesterol loading increased apoE mRNA 2-fold. ApoE mRNA levels were not further affected by treatment with HDL3 and/or the ACAT inhibitor. Cholesterol-loaded cells, in the absence of HDL3, secreted apoE into the media in two fractions as determined by column chromatography: a large molecular weight complex, (larger than HDL), and an essentially lipid-free protein. In the presence of HDL3, the cells secreted apoE in three fractions: a large molecular weight complex, an essentially lipid-free protein, and over 50% of apoE associated with HDL. In the process, HDL3 became larger and eluted in a position identical to that of HDL2. A small amount of HDL3-derived material was also transformed to an LDL-size particle. Incubation of HDL3 in the absence of cholesterol-loaded cells did not produce these changes. It is concluded that cholesterol-loading increases apoE mRNA content and apoE synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radiation inactivation of binding sites for high-density lipoproteins in human liver membranes

High-density lipoproteins (HDL) are involved in 'reverse cholesterol transport'. Whether or not cell-surface receptors for HDL exist and participate in this process remains controversial, and part of this controversy has centered on the nature of the HDL binding sites. We therefore used radiation inactivation to determine the molecular mass of the HDL binding sites in human liver membranes in situ. These binding sites, which shared all the characteristics of previously described putative HDL receptors, had a molecular mass of less than 10 kDa, indicating that they are probably not proteins. In addition, the binding of HDL to protein-free liposomes was characterized and was found to display the same affinity (KD = 5 micrograms protein/ml approximately 5.10(-8) M) as that to cell membranes, indicating that HDL binding to cell membranes may not require membrane proteins. These observations highlight an important application of radiation inactivation: the ability to demonstrate that something - in this case, a high-molecular-weight protein that accounts for the majority of the HDL binding activity in human liver membranes - is absent.     

Heterogeneity of serum lipoproteins during the fetal and neonatal development of the pig

Serum lipoproteins from fetal, neonatal and adult pigs were characterized with the use of lipid analysis, polyacrylamide gradient gel electrophoresis, two-dimensional immunoelectrophoresis and zonal ultracentrifugation. Almost all serum cholesterol was found in LDL during the early stages of fetal development, while low but increasing levels appeared in the fetal pig HDL by the end of the gestation period. In the fetal pig, most of the serum triglycerides could be found in the HDL fraction. After the start of suckling, the levels of serum triglycerides and cholesterol increased. Most of these exogenous lipids were found in the chylomicrons + VLDL + LDL fraction of the newborn pig serum. The molecular weights of the native serum lipoproteins were calculated as being 2.0-2.4 X 10(5) daltons for newborn pig HDL and 1.4-1.7 X 10(6) daltons for newborn pig LDL. Minor changes in the molecular weight distributions were detected within these ranges for both HDL and LDL during fetal and neonatal development of the pig. Zonal ultracentrifugation of neonatal pig serum partly separated the LDL into three subfractions, whereas neonatal HDL appeared as one broad fraction.     

A high density lipoprotein from Piaractus mesopotamicus, pacu, (Osteichthyes, Characidae), is associated with paraoxonase activity.

We have characterized the serum lipoprotein profile and localized the serum paraoxonase activity of pacu, Piaractus mesopotamicus, a tropical fish species. The total lipoprotein profile of pacu serum obtained after KBr density ultracentrifugation shows the predominance of HDL (1.1267 g/mL). SDS-PAGE electrophoresis revealed a negligible amount of LDL. Pacu HDL was purified by gel filtration column on HPLC, and its molecular mass was estimated to be 246 kDa. Protein composition was 35%, and comprised four protein components with molecular masses of 45, 38, 25 and 12.5 kDa. Lipids represent 58% of total HDL, comprising 40% neutral lipids and 18% phospholipids by weight. The HDL contains 7% of carbohydrates, and mannose was the only sugar detected by paper chromatography in HDL hydrolysates. HDL-containing fractions showed the major paraoxonase activity. Purification of HDL resulted in a 23-fold enrichment of this activity. This is the first experimental evidence demonstrating the association of paraoxonase activity with a HDL in fish.     

HDL: structure, function and metabolism.

The major advances in our knowledge of the structure, function and metabolism of the plasma lipoproteins have occurred as a result of the rapid increase in our knowledge of the structure and function of the apolipoproteins, lipoproteins, and the heterogeneity of the individual classes of lipoproteins. Over the last decade, there has been a tremendous increase in our knowledge of the structure and molecular properties of ApoA-I and ApoA-II which has permitted an analysis of the functions of these apolipoproteins in lipid and lipoprotein metabolism and the initiation of kinetic studies of HDL metabolism. The elucidation of the structures of the ApoA-I and ApoA-II genes has permitted the determination of genetic defects resulting in decreased levels of HDL and premature cardiovascular disease, as well as the identification of new diseases (e.g. hereditary systemic amyloidosis). The future focus of research on HDL will be the analysis of the individual lipoprotein particles within HDL which have different physiological functions and important roles in reverse cholesterol transport. An improved understanding of the role of HDL in the transport of cellular cholesterol to the liver and the exchange of cholesterol between plasma lipoproteins will provide critical information on cholesterol metabolism in normal subjects and permit the elucidation of the molecular defects of new genetic diseases which may be associated with the development of premature cardiovascular disease.