apoA-I Molecules Research Articles

Nascent HDL (High-Density Lipoprotein) Discs Carry Cholesterol to HDL Spheres: Effects of HDL Particle Remodeling on Cholesterol Efflux.

To characterize the fate of protein and lipid in nascent HDL (high-density lipoprotein) in plasma and explore the role of interaction between nascent HDL and mature HDL in promoting ABCA1 (ATP-binding cassette transporter 1)-dependent cholesterol efflux. Approach and Results: Two discoidal species, nascent HDL produced by RAW264.7 cells expressing ABCA1 (LpA-I [apo AI containing particles formed by incubating ABCA1-expressing cells with apo AI]), and CSL112, human apo AI (apolipoprotein AI) reconstituted with phospholipids, were used for in vitro incubations with human plasma or purified spherical plasma HDL. Fluorescent labeling and biotinylation of HDL were employed to follow the redistribution of cholesterol and apo AI, cholesterol efflux was measured using cholesterol-loaded cells. We show that both nascent LpA-I and CSL112 can rapidly fuse with spherical HDL. Redistribution of the apo AI molecules and cholesterol after particle fusion leads to the formation of (1) enlarged, remodeled, lipid-rich HDL particles carrying lipid and apo AI from LpA-I and (2) lipid-poor apo AI particles carrying apo AI from both discs and spheres. The interaction of discs and spheres led to a greater than additive elevation of ABCA1-dependent cholesterol efflux. These data demonstrate that although newly formed discs are relatively poor substrates for ABCA1, they can interact with spheres to produce lipid-poor apo AI, a much better substrate for ABCA1. Because the lipid-poor apo AI generated in this interaction can itself become discoid by the action of ABCA1, cycles of cholesterol efflux and disc-sphere fusion may result in net ABCA1-dependent transfer of cholesterol from cells to HDL spheres. This process may be of particular importance in atherosclerotic plaque where cholesterol acceptors may be limiting.

The structure of apoA‐II on HDL reveals novel insights into its regulation of lipoprotein composition and function

High‐density lipoproteins (HDL) are compositionally and functionally diverse. HDL can host at least 95 different proteins and studies suggest many of these interactions are governed by specific conformations of HDL's primary scaffold protein, apolipoprotein (apo)A‐I. Previously, we showed that the presence of HDL's second most abundant scaffold protein, apoA‐II, impacts particle composition and function; presumably through interactions with apoA‐I. The goal of this study was to determine the structural conformation of apoA‐II on HDL to understand how its presence impacts HDL metabolism.We used chemical cross‐linking and mass spectrometry to derive spatial information on homogenous populations of recombinant HDL (rHDL) generated in vitro. The rHDL was created using a mixture of wild‐type and isotopically‐labeled apoA‐I to unambiguously identify the molecular span of the cross‐linked peptides; i.e. within a molecule of apoA‐I (intramolecular) or between two molecules of apoA‐I (intermolecular). Two rHDL populations were studied: (i) rHDL that contained only apoA‐I (LpA‐I) and (ii) rHDL containing both apoA‐I and apoA‐II (LpA‐I/A‐II).Cross‐linking and SDS‐PAGE showed both populations contained two molecules of apoA‐I while the LpA‐I/A‐II rHDL contained a single dimer of apoA‐II. Analysis by negative stain electron microscopy revealed both populations were discoidal in shape with no differences in size. The intermolecular cross‐linking pattern between apoA‐I molecules were consistent with the “double‐belt” model of apoA‐I on HDL and, surprisingly, the presence of apoA‐II didn't appear to perturb the pattern. However, we observed strong cross‐links between apoA‐II and the C‐terminus of apoA‐I.In a parallel approach, native LpA‐I and LpA‐I/A‐II HDL was isolated from human plasma using immunoaffinity and size exclusion chromatography. Shotgun proteomics and cross‐linking revealed accessory proteins were mostly associated with LpA‐I HDL and interactions were primarily localized to the N‐ and C‐termini of apoA‐I. As with the rHDL, we found apoA‐II specifically interacts with the C‐terminus of apoA‐I in the LpA‐I/A‐II particles. Taken together, this suggests that apoA‐II's compositional and functional effects on HDL are exerted via its interaction with the C‐terminus of apoA‐I possibly limiting its availability to interact with the accessory proteins. We have gone on to generate preliminary all‐atoms models of apoA‐II on HDL to illustrate this structural mechanism.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Disease-Causing Mutations on the Conformation of Human Apolipoprotein A-I in Model Lipoproteins

Plasma high-density lipoproteins (HDLs) are protein-lipid nanoparticles that transport lipids and protect against atherosclerosis. Human apolipoprotein A-I (apoA-I) is the principal HDL protein whose mutations can cause either aberrant lipid metabolism or amyloid disease. Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) was used to study the apoA-I conformation in model discoidal lipoproteins similar in size to large plasma HDL. We examined how point mutations associated with hereditary amyloidosis (F71Y and L170P) or atherosclerosis (L159R) influence the local apoA-I conformation in model lipoproteins. Unlike other apoA-I forms, the large particles showed minimal conformational heterogeneity, suggesting a fully extended protein conformation. Mutation-induced structural perturbations in lipid-bound protein were attenuated compared to the free protein and indicated close coupling between the two belt-forming apoA-I molecules. These perturbations propagated to distant lipoprotein sites, either increasing or decreasing their protection. This HDX MS study of large model HDL, compared with previous studies of smaller particles, ascertained that apoA-I's central region helps accommodate the protein conformation to lipoproteins of various sizes. This study also reveals that the effects of mutations on lipoprotein conformational dynamics are much weaker than those in a lipid-free protein. Interestingly, the mutation-induced perturbations propagate to distant sites nearly 10 nm away and alter their protection in ways that cannot be predicted from the lipoprotein structure and stability. We propose that long-range mutational effects are mediated by both protein and lipid and can influence lipoprotein functionality.

ABCA1, apoA-I, and BTN3A1: A Legitimate Ménage à Trois in Dendritic Cells.

Human Vγ9Vδ2 T cells have the capacity to detect supra-physiological concentrations of phosphoantigens (pAgs) generated by the mevalonate (Mev) pathway of mammalian cells under specific circumstances. Isopentenyl pyrophosphate (IPP) is the prototypic pAg recognized by Vγ9Vδ2 T cells. B-cell derived tumor cells (i.e., lymphoma and myeloma cells) and dendritic cells (DCs) are privileged targets of Vγ9Vδ2 T cells because they generate significant amounts of IPP which can be boosted with zoledronic acid (ZA). ZA is the most potent aminobisphosphonate (NBP) clinically available to inhibit osteoclast activation and a very potent inhibitor of farnesyl pyrophosphate synthase in the Mev pathway. ZA-treated DCs generate and release in the supernatants picomolar IPP concentrations which are sufficient to induce the activation of Vγ9Vδ2 T cells. We have recently shown that the ATP-binding cassette transporter A1 (ABCA1) plays a major role in the extracellular release of IPP from ZA-treated DCs. This novel ABCA1 function is fine-tuned by physical interactions with IPP, apolipoprotein A-I (apoA-I), and butyrophilin-3A1 (BTN3A1). The mechanisms by which soluble IPP induces Vγ9Vδ2 T-cell activation remain to be elucidated. It is possible that soluble IPP binds to BTN3A1, apoA-I, or other unknown molecules on the cell surface of bystander cells like monocytes, NK cells, Vγ9Vδ2 T cells, or any other cell locally present. Investigating this scenario may represent a unique opportunity to further characterize the role of BTN3A1 and other molecules in the recognition of soluble IPP by Vγ9Vδ2 T cells.

Abstract 560: Protein and Lipid Composition of HDL Particles

Objective: HDL cell-cholesterol efflux capacity is influenced by both the concentration and the functionality (efflux capacity per particle) of HDL particles. It is assumed that the lipid and protein composition of HDL particles significantly influence their functionality; however there is little data to support that. Our aim was to determine the protein and lipid composition of individual HDL particles to better understand the large variability observed in HDL functionality studies. Approach: ApoA-I-containing HDL particles (preβ-1, α-4, α-3, α-2, and α-1) were separated by non-denaturing 2d-PAGE, electro-eluted from the gel, and purified by immuno-affinity chromatography using anti-human apoA-I IgG. The protein and lipid composition of the particles were assessed by mass-spectrometry. The number of apoA-I molecules in each particle was determined by cross-linking experiments. Results: The number of protein species, associated with HDL particles, increased with particle size: the largest (α-1) particles carried more than 50, while the smallest (preβ-1) particles carried only a few different proteins. ApoE was present in trace amount, if any, in these HDL particles. The apoA-II/apoA-I ratio was very low in the α-1, α-4, and preβ-1 particles but substantially higher in the α-2 and α-3 particles. The maximum number of apoA-I molecules was 4 in α-1 and 3 in α-2, α-3, and α-4 particles. The largest (α-1) particles contained significantly more lipid molecules, especially triglycerides, for each apoA-I molecule compared to smaller particles. The lipid composition of the intermediate-size α-2 and α-3 particles was similar. In contrast to larger HDL particles, the small α-4 particles were enriched in sphingomyelin. Interestingly, the very-small preβ-1 particles contained about equal amounts of polar (phospholipids and free cholesterol) and non-polar (cholesteryl ester and triglycerides) lipids. Phospholipids were represented only by sphingomyelin. These findings contradict previous models that indicated that each preβ-1 particle contained one or two apoA-I and a few polar lipid molecules. Conclusions: Both the protein and the lipid composition of HDL particles are size dependent. Further studies are needed to determine how the protein and lipid composition of HDL particles influence HDL functions.

Arginine 123 of apolipoprotein A-I is essential for lecithin:cholesterol acyltransferase activity

ApoA-I activates LCAT that converts lipoprotein cholesterol to cholesteryl ester (CE). Molecular dynamic simulations suggested earlier that helices 5 of two antiparallel apoA-I molecules on discoidal HDL form an amphipathic tunnel for migration of acyl chains and unesterified cholesterol to the active sites of LCAT. Our recent crystal structure of Δ(185-243)apoA-I showed the tunnel formed by helices 5/5, with two positively charged residues arginine 123 positioned at the edge of the hydrophobic tunnel. We hypothesized that these uniquely positioned residues Arg123 are poised for interaction with fatty acids produced by LCAT hydrolysis of the sn-2 chains of phosphatidylcholine, thus positioning the fatty acids for esterification to cholesterol. To test the importance of Arg123 for LCAT phospholipid hydrolysis and CE formation, we generated apoA-I[R123A] and apoA-I[R123E] mutants and made discoidal HDL with the mutants and WT apoA-I. Neither mutation of Arg123 changed the particle composition or size, or the protein conformation or stability. However, both mutations of Arg123 significantly reduced LCAT catalytic efficiency and the apparent Vmax for CE formation without affecting LCAT phospholipid hydrolysis. A control mutation, apoA-I[R131A], did not affect LCAT phospholipid hydrolysis or CE formation. These data suggest that Arg123 of apoA-I on discoidal HDL participates in LCAT-mediated cholesterol esterification.

ApoA-I/A-II-HDL positively associates with apoB-lipoproteins as a potential atherogenic indicator

BackgroundWe recently reported distinct nature of high-density lipoproteins (HDL) subgroup particles with apolipoprotein (apo) A-I but not apoA-II (LpAI) and HDL having both (LpAI:AII) based on the data from 314 Japanese. While plasma HDL level almost exclusively depends on concentration of LpAI having 3 to 4 apoA-I molecules, LpAI:AII appeared with almost constant concentration regardless of plasma HDL levels having stable structure with two apoA-I and one disulfide-dimeric apoA-II molecules (Sci. Rep. 6; 31,532, 2016). The aim of this study is further characterization of LpAI:AII with respect to its role in atherogenesis.MethodsAssociation of LpAI, LpAI:AII and other HDL parameters with apoB-lipoprotein parameters was analyzed among the cohort data above.ResultsApoA-I in LpAI negatively correlated with the apoB-lipoprotein parameters such as apoB, triglyceride, nonHDL-cholesterol, and nonHDL-cholesterol + triglyceride, which are apparently reflected in the relations of the total HDL parameters to apoB-lipoproteins. In contrast, apoA-I in LpAI:AII and apoA-II positively correlated to the apoB-lipoprotein parameters even within their small range of variation. These relationships are independent of sex, but may slightly be influenced by the activity-related CETP mutations.ConclusionsThe study suggested that LpAI:AII is an atherogenic indicator rather than antiatherogenic. These sub-fractions of HDL are to be evaluated separately for estimating atherogenic risk of the patients.

Significance of Lipid-Free and Lipid-Associated ApoA-I in Cellular Cho-lesterol Efflux.

The structure and stability of apolipoprotein (apo)A-I, the major apolipoprotein of human plasma high-density lipoproteins (HDL), determine the efficiency of the protein in the process of HDL generation and affect HDL properties in binding and exchanging its constituents, thus playing an essential role in reverse cholesterol transport. The equilibrium stability of an apoA-I molecule at the lipid interface (12.7 kcal/mol) predicted by a thermodynamic cycle for apolipoprotein folding-unfolding in water and at interface, largely exceeds apoA-I helix stability in HDL against chemical denaturation (3-5 kcal/mol). An ensemble of structures of lipid-bound apoA-I with different stabilities is assumed to exist. The conformational transitions between apoA-I conformers in water and lipid phases correspond to Lumry-Eyring model OL ⇔ CL ⇒ MW, where OL and CL are open and closed structures of HDLbound apoA-I, and MW is the molten globule in water. The model includes the reversible foldingunfolding transitions of N- and C-domains at HDL interface and apolipoprotein irreversible dissociation. We gathered published data on cholesterol efflux for apoA-I proteins with missense mutations in C-domain and calculated the stability of these mutants as a change of free energy relative to a wild type protein. Significant negative correlation was found between this stability and the efficiency of cAMP-stimulated cholesterol efflux. Thus, besides the known role of C-domain hydrophobicity, structure-destabilizing changes may significantly contribute to ABCA1-mediated cholesterol efflux by free apolipoprotein.

Bioinformatic Analysis of Plasma Apolipoproteins A-I and A-II Revealed Unique Features of A-I/A-II HDL Particles in Human Plasma.

Plasma concentration of apoA-I, apoA-II and apoA-II-unassociated apoA-I was analyzed in 314 Japanese subjects (177 males and 137 females), including one (male) homozygote and 37 (20 males and 17 females) heterozygotes of genetic CETP deficiency. ApoA-I unassociated with apoA-II markedly and linearly increased with HDL-cholesterol, while apoA-II increased only very slightly and the ratio of apoA-II-associated apoA-I to apoA-II stayed constant at 2 in molar ratio throughout the increase of HDL-cholesterol, among the wild type and heterozygous CETP deficiency. Thus, overall HDL concentration almost exclusively depends on HDL with apoA-I without apoA-II (LpAI) while concentration of HDL containing apoA-I and apoA-II (LpAI:AII) is constant having a fixed molar ratio of 2 : 1 regardless of total HDL and apoA-I concentration. Distribution of apoA-I between LpAI and LpAI:AII is consistent with a model of statistical partitioning regardless of sex and CETP genotype. The analysis also indicated that LpA-I accommodates on average 4 apoA-I molecules and has a clearance rate indistinguishable from LpAI:AII. Independent evidence indicated LpAI:A-II has a diameter 20% smaller than LpAI, consistent with a model having two apoA-I and one apoA-II. The functional contribution of these particles is to be investigated.

Abstract 541: The Molecular Interaction of Apolipoprotein A-I Containing High Density Lipoproteins with Lecithin: Cholesterol Acyl Transferase

The structure of apolipoprotein (apo)A-I on a high density lipoprotein (HDL) particle can elucidate the protein interactions and cardioprotective functions of HDL. This includes critical interactions with HDL modifying proteins like lecithin:cholesterol acyl transferase (LCAT) which performs a key function in reverse cholesterol transport by using apoA-I as a cofactor to esterify cholesterol. Data from our lab and others demonstrate that apoA-I molecules dimerize into an antiparallel stacked ring structure that encapsulates lipid in reconstituted (r)HDL particles. Cross-linking analysis of rHDL particles imply two possible registries: one with the fifth helical repeat of an apoA-I molecule adjacent to the fifth helical repeat of its antiparallel partner (5/5), and the other with the fifth helical repeat adjacent to the second helical repeat of its antiparallel partner (5/2). We hypothesized that apoA-I registry on rHDL can modulate LCAT activity. Site-directed cysteine mutagenesis was used to lock two apoA-I molecules into each of the aforementioned helical registries, and an intermediate registry created as a negative control. rHDL particles were generated using these dimerized mutants, and their ability to activate LCAT was determined. The 5/5 mutant demonstrated higher LCAT activity than wild-type rHDL particles while the 5/2 and intermediate mutants showed dramatically lower LCAT activity (p<0.001). To determine where LCAT interacts with apoA-I, rHDL containing wild-type apoA-I was cross-linked to LCAT. The majority of the cross-links were concentrated between S240 of LCAT and a region encompassing helices 4-6 of apoA-I. We propose that LCAT binds to a discontinuous epitope comprised of two apoA-I molecules in rHDL and that changes in this registry can alter LCAT function. These studies provide a basis for understanding how apoA-I structure may modulate the association and activity of a multitude of different HDL protein partners. This implicates apoA-I conformation as a target for altering HDL function in order to enhance the cardioprotective properties of HDL.

Abstract 393: Covalently Connected Apolipoprotein A-I Molecules Provide Support for the Double Belt Model in Reconstituted HDL

Because of its critical role in high density lipoprotein (HDL) metabolism, significant work has been devoted to studying apolipoprotein (apo)A-I structure in its lipid-bound state, particularly reconstituted forms of HDL (rHDL). Unfortunately, high-resolution structural studies have not been successful because of HDL heterogeneity and compositional dynamics. There is general agreement that apoA-I adopts an antiparallel arrangement on rHDL with the majority of the molecule conforming to the “double belt” model. However, less is understood about the locations of the N- and C-termini in these particles. The classic double belt predicts that, in a particle containing two molecules of apoA-I, all four termini are localized to one area of the particle. However, other models predict that they are far apart. To address this issue, we generated a series of covalently connected apoA-I dimers and tested their ability to generate rHDL particles. The dimeric (d)-apoA-I C-N mutant, locked the C-terminus of one apoA-I molecule to the N-terminus of a second molecule with three intervening Ala residues. Linked by strategically introduced cysteine residues, the d-apoA-I C-C , and d-apoA-I N-N mutants lock the C-termini and the N-termini of two apoA-I molecules together, respectively. When all three of these mutants were reconstituted into rHDL particles using sodium cholate and synthetic phospholipids, the resulting particles closely resembled those generated with wild-type (WT) apoA-I. This indicates a ring-like structure predicted by the double belt. However, when testing the ability of these mutants to spontaneously clear lipid, we found that both the N- and C- termini required the ability to separate and move independently to form normal particles. Native gel analysis showed the dimerized lipid-free mutants present as a tight homogenous band compared to wild-type which runs as a mixture of oligomers. We are currently evaluating if the homogeneity of these mutants can allow for more facile crystallization of full-length apoA-I. Further studies with these stabilized mutants should help us understand the structural basis underlying HDL’s diverse functions, which is essential for developing HDL-based therapies for cardiovascular disease.

Abstract 27: Structure of Lipid-free and Lipid-bound Apolipoprotein A-I Determined by Stable Isotope-assisted Cross-linking and Small Angle X-ray Scattering

Apolipoprotein (apo)A-I, the major protein constituent of high density lipoproteins (HDL), acts as a conformationally dynamic scaffold on the surface of the particle. Evidence suggests apoA-I conformation dictates HDL functionality and capacity to interface with other HDL proteins; however, the details of apoA-I structure and HDL formation are not completely understood. X-ray crystal structures of truncated forms of apoA-I and its cousin, apoA-IV, show reciprocal domain swapping structures in the lipid-free state. Recently, we showed the truncated mutant of apoA-I is also highly dynamic in solution with a flexible N-terminus. The purpose of this study was to a) derive a structural model of wild-type (WT) apoA-I using the new in-solution truncation model as a template and b) determine the relationship between monomeric, and oligomeric forms of lipid-free WT apoA-I. Using a novel isotope labeling strategy and chemical cross-linking, we defined spatial relationships within and between different molecules of WT apoA-I. The cross-linking patterns revealed a high degree of structural similarity between WT apoA-I oligomers. We found that monomeric and dimeric WT apoA-I structures were similar, but not identical, to those predicted by the crystal and solution structures of truncated apoA-I. Using cross-linking data, we derived two interconverting structures each for monomeric WT apoA-I, both containing dynamic N- and C-terminal regions. Similar structures were generated for WT dimeric and trimeric species. Small angle X-ray scattering was used to derive molecular envelopes of the molecules and refine the arrangements to propose the most detailed molecular models of lipid-free WT apoA-I to date. The models were used in conjunction with the reported double-belt and trefoil structures reconstituted HDL to reveal evidence of a parsimonious relationship between lipid-free and lipid-bound WT apoA-I. We propose a structural framework of WT apoA-I self-association and lipidation, centric to the C-terminal region of the molecule, that forms a basis for understanding HDL particle formation at the cell surface.

Apolipoprotein A-I mimetic peptide reverses impaired arterial healing after injury by reducing oxidative stress.

Endothelial cell (EC) migration is essential for healing of arterial injuries caused by angioplasty, but a high cholesterol diet inhibits endothelial repair. Invivo studies suggest that apolipoprotein A-I (apoA-I), the major protein constituent of HDL, is essential for normal healing of arterial injuries. ApoA-I mimetics, including 4F, have been designed to mimic the amphipathic portion of the apoA-I molecule. This study was undertaken to determine if 4F improves endothelial migration and healing. A razor scrape assay was used to analyze the effect of 4F on EC migration invitro. Endothelial healing invivo was assessed following electrical injury of carotid arteries in mice. Markers of oxidative stress were also examined. Lipid oxidation products inhibited EC migration invitro, but preincubation with L-4F preserved EC migration. Endothelial healing of carotid arterial injuries in mice on a high cholesterol diet was delayed compared with mice on a chow diet with 27.8% vs. 48.2% healing, respectively, at 5 days. Administration of D-4F improved endothelial healing in mice on a high cholesterol diet to 43.4%. D-4F administration had no effect on lipid levels but decreased markers of oxidation. Invivo, there was a significant inverse correlation between endothelial healing and plasma markers of oxidative stress. These studies suggested that an apoA-I mimetic can improve endothelial healing of arterial injuries by decreasing oxidative stress.

Serum Amyloid A 1.1 Forms a Conformational Temperature Sensor at the Lipid Surface

Serum amyloid A (SAA) is a natively unfolded acute-phase protein. Normally SAA circulates on plasma high-density lipoproteins (HDL) whose major protein is apoA-I. In inflammation SAA levels can increase 1000-fold and it becomes the major plasma protein that displaces up to 50% of apoA-I from HDL, forming SAA-HDL with altered metabolic properties. To determine whether SAA alters structural stability of HDL, human HDL were incubated with murine SAA 1.1 to obtain SAA-HDL. Calorimetric, spectroscopic and biochemical studies showed that HDL fusion and rupture, which were characterized in our previous work, occur at similar temperatures in normal HDL and in SAA-HDL. Therefore, the exchangeable fraction of apoA-I has little effect on HDL stability which, we propose, is determined by the apoA-I fraction that cannot be displaced. This apoA-I likely forms an antiparallel double-belt dimer around HDL. Our results argue against domain swapping among apoA-I molecules on HDL, which is important for HDL structure and function. Surprisingly, our results reveal helical unfolding in SAA-HDL circa 38°C. This novel transition was observed only in samples containing excess SAA and the lipid surface, but neither in lipid-free SAA nor in isolated SAA-HDL containing lipoproteins alone. The transition was invariant in a wide range of solvent conditions (pH 5.5-8.5, 10-150 mMol Na salt), upon lipolysis by PLA2, or upon lipoprotein rupture. In sum, SAA near the lipid surface undergoes folding/unfolding at physiologic temperatures. We propose that excess SAA forms a local conformational temperature sensor and suggest a new function for SAA in acute phase response. Funded by NIH GM067260.

Cholesterol and lipoprotein metabolism: Early Career Committee contribution.

Cholesterol and lipoprotein metabolism: Early Career Committee contribution.

Abstract 223: The Structural Organization of Apolipoprotein A-I in Reconstituted High-Density Lipoprotein Discs Using Stable Isotope-Assisted Cross-Linking

X-ray crystallography studies of lipid-free apolipoprotein A-I (apoA-I) and related apoA-IV have shown that the proteins form a reciprocating dimer which could be directly related to the structural organization of lipid-bound apoA-I. Previous work has demonstrated the success of chemical cross-linking combined with mass spectrometry to derive distance constraints for molecular modeling of apoA-I in reconstituted high density lipoprotein (HDL) particles. However, these studies have been limited by i) the relative paucity of distance constraint data, and ii) ambiguities in determining the molecular span of the cross-links. Here, we have addressed the first issue by using an affinity-tagged cross-linker to purify cross-linked peptides away from non-modified peptides prior to sampling with mass spectrometry. This essentially boosts the signal from cross-linked peptides of interest that would have otherwise been suppressed by detection of non-modified peptides. Using this method, we identified >25 cross-linked peptides; the most reported to date for these particles. To address the issue of molecular span, we used a stable isotopic-labeling approach in which wild-type human recombinant apoA-I ( 14 N) was mixed 1:1 with an isotopically labeled ( 15 N) apoA-I. Analysis of the isotopic patterns of the resulting mass spectra allowed the unambiguous determination of the molecular span of the cross-linked peptides; i.e. whether the links were within the same molecule of apoA-I (intramolecular) or between the dimers of apoA-I (intermolecular). This information will be used to derive a detailed novel model of lipid-bound apoA-I in reconstituted HDL, an important step in our overall understanding of how native HDL species are organized and how they interact with plasma remodeling factors and cell surface proteins.

An In-Silico Model of Lipoprotein Metabolism and Kinetics for the Evaluation of Targets and Biomarkers in the Reverse Cholesterol Transport Pathway

High-density lipoprotein (HDL) is believed to play an important role in lowering cardiovascular disease (CVD) risk by mediating the process of reverse cholesterol transport (RCT). Via RCT, excess cholesterol from peripheral tissues is carried back to the liver and hence should lead to the reduction of atherosclerotic plaques. The recent failures of HDL-cholesterol (HDL-C) raising therapies have initiated a re-examination of the link between CVD risk and the rate of RCT, and have brought into question whether all target modulations that raise HDL-C would be atheroprotective. To help address these issues, a novel in-silico model has been built to incorporate modern concepts of HDL biology, including: the geometric structure of HDL linking the core radius with the number of ApoA-I molecules on it, and the regeneration of lipid-poor ApoA-I from spherical HDL due to remodeling processes. The ODE model has been calibrated using data from the literature and validated by simulating additional experiments not used in the calibration. Using a virtual population, we show that the model provides possible explanations for a number of well-known relationships in cholesterol metabolism, including the epidemiological relationship between HDL-C and CVD risk and the correlations between some HDL-related lipoprotein markers. In particular, the model has been used to explore two HDL-C raising target modulations, Cholesteryl Ester Transfer Protein (CETP) inhibition and ATP-binding cassette transporter member 1 (ABCA1) up-regulation. It predicts that while CETP inhibition would not result in an increased RCT rate, ABCA1 up-regulation should increase both HDL-C and RCT rate. Furthermore, the model predicts the two target modulations result in distinct changes in the lipoprotein measures. Finally, the model also allows for an evaluation of two candidate biomarkers for in-vivo whole-body ABCA1 activity: the absolute concentration and the % lipid-poor ApoA-I. These findings illustrate the potential utility of the model in drug development.

Structural basis for distinct functions of the naturally occurring Cys mutants of human apolipoprotein A-I

HDL removes cell cholesterol and protects against atherosclerosis. ApoA-I provides a flexible structural scaffold and an important functional ligand on the HDL surface. We propose structural models for apoA-I(Milano) (R173C) and apoA-I(Paris) (R151C) mutants that show high cardioprotection despite low HDL levels. Previous studies established that two apoA-I molecules encircle HDL in an antiparallel, helical double-belt conformation. Recently, we solved the atomic structure of lipid-free Δ(185-243)apoA-I and proposed a conformational ensemble for apoA-I(WT) on HDL. Here we modify this ensemble to understand how intermolecular disulfides involving C173 or C151 influence protein conformation. The double-belt conformations are modified by belt rotation, main-chain unhinging around Gly, and Pro-induced helical bending, and they are verified by comparison with previous experimental studies and by molecular dynamics simulations of apoA-I(Milano) homodimer. In our models, the molecular termini repack on various-sized HDL, while packing around helix-5 in apoA-I(WT), helix-6 in apoA-I(Paris), or helix-7 in apoA-I(Milano) homodimer is largely conserved. We propose how the disulfide-induced constraints alter the protein conformation and facilitate dissociation of the C-terminal segment from HDL to recruit additional lipid. Our models unify previous studies of apoA-I(Milano) and demonstrate how the mutational effects propagate to the molecular termini, altering their conformations, dynamics, and function.

New insights into the determination of HDL structure by apolipoproteins : Thematic Review Series: High Density Lipoprotein Structure, Function, and Metabolism

Apolipoprotein (apo)A-I is the principal protein component of HDL, and because of its conformational adaptability, it can stabilize all HDL subclasses. The amphipathic α-helix is the structural motif that enables apoA-I to achieve this functionality. In the lipid-free state, the helical segments unfold and refold in seconds and are located in the N-terminal two thirds of the molecule where they are loosely packed as a dynamic, four-helix bundle. The C-terminal third of the protein forms an intrinsically disordered domain that mediates initial binding to phospholipid surfaces, which occurs with coupled α-helix formation. The lipid affinity of apoA-I confers detergent-like properties; it can solubilize vesicular phospholipids to create discoidal HDL particles with diameters of approximately 10 nm. Such particles contain a segment of phospholipid bilayer and are stabilized by two apoA-I molecules that are arranged in an anti-parallel, double-belt conformation around the edge of the disc, shielding the hydrophobic phospholipid acyl chains from exposure to water. The apoA-I molecules are in a highly dynamic state, and they stabilize discoidal particles of different sizes by certain segments forming loops that detach reversibly from the particle surface. The flexible apoA-I molecule adapts to the surface of spherical HDL particles by bending and forming a stabilizing trefoil scaffold structure. The above characteristics of apoA-I enable it to partner with ABCA1 in mediating efflux of cellular phospholipid and cholesterol and formation of a heterogeneous population of nascent HDL particles. Novel insights into the structure-function relationships of apoA-I should help reveal mechanisms by which HDL subclass distribution can be manipulated.

Comparison of apoA-I helical structure and stability in discoidal and spherical HDL particles by HX and mass spectrometry

Elucidation of apoA-I secondary structure in spherical plasma HDL particles is essential for understanding HDL structure and function at the molecular level. To provide this information, we have applied hydrogen exchange (HX) and mass spectrometry methods to compare apoA-I secondary structure in discoidal (two apoA-I molecules/particle) and spherical (five apoA-I molecules/particle) HDL particles. The HX kinetics indicate that the locations of helical segments within the apoA-I molecules are the same in both discoidal and spherical HDL particles (approximately 10 nm hydrodynamic diameter). Helix stabilities in both types of particles are 3–5 kcal/mol, consistent with the apoA-I molecules being in a highly dynamic state with helical segments unfolding and refolding in seconds. For the spherical HDL, apoA-I fragments corresponding to residues 115–158 exhibit bimodal HX kinetics consistent with this segment adopting an inter-converting (on the timescale of tens of minutes) helix-loop configuration. The segment adopting this configuration in the 10 nm disc is shorter because the surface area available to each apoA-I molecule is apparently larger. Loop formation in the central region of the apoA-I molecule contributes to the ability of the protein to adapt to changes in available space on the HDL particle surface. Overall, apoA-I secondary structure is largely unaffected by a change in HDL particle shape from disc to sphere.