Circular RNAs Variously Participate in Coronary Atherogenesis.

Over the past decade, numerous studies have shown that circular RNAs (circRNAs) play a significant role in coronary artery atherogenesis and other cardiovascular diseases. They belong to the class of non-coding RNAs and arise as a result of non-canonical splicing of premature RNA, which results in the formation of closed single-stranded circRNA molecules that lack 5'-end caps and 3'-end poly(A) tails. circRNAs have broad post-transcriptional regulatory activity. Acting as a sponge for miRNAs, circRNAs compete with mRNAs for binding to miRNAs, acting as competing endogenous RNAs. Numerous circRNAs are involved in the circRNA-miRNA-mRNA regulatory axes associated with the pathogenesis of cardiomyopathy, chronic heart failure, hypertension, atherosclerosis, and coronary artery disease. Recent studies have shown that сirc_0001445, circ_0000345, circ_0093887, сircSmoc1-2, and circ_0003423 are involved in the pathogenesis of coronary artery disease (CAD) with an atheroprotective effect, while circ_0002984, circ_0029589, circ_0124644, circ_0091822, and circ_0050486 possess a proatherogenic effect. With their high resistance to endonucleases, circRNAs are promising diagnostic biomarkers and therapeutic targets. This review aims to provide updated information on the involvement of atherogenesis-related circRNAs in the pathogenesis of CAD. We also discuss the main modern approaches to detecting and studying circRNA-miRNA-mRNA interactions, as well as the prospects for using circRNAs as biomarkers and therapeutic targets for the treatment of cardiovascular diseases.

Differential Expression of Subsets of Genes Related to HDL Metabolism and Atherogenesis in the Peripheral Blood in Coronary Artery Disease.

Differential expression of genes (DEGs) in coronary artery disease (CAD) and the association between transcript level and high-density lipoprotein cholesterol (HDL-C) were studied with 76 male patients with CAD and 63 control patients. The transcript level of genes related to HDL metabolism (24 genes) and atherosclerosis-prone (41 genes) in RNA isolated from peripheral blood mononuclear cells was measured by real-time RT-PCR. Twenty-eight DEGs were identified. The expression of cholesterol transporters, ALB, APOA1, and LCAT was down-regulated, while the expression of AMN, APOE, LDLR, LPL, PLTP, PRKACA, and CETP was up-regulated. The systemic inflammation in CAD is evidenced by the up-regulation of IL1B, TLR8, CXCL5, and TNFRSF1A. For the controls, TLR8 and SOAT1 were negative predictors of the HDL-C level. For CAD patients, PRKACG, PRKCQ, and SREBF1 were positive predictors, while PRKACB, LCAT, and S100A8 were negative predictors. For CAD patients, the efficiency of reverse cholesterol transport is 73-79%, and intracellular free cholesterol seems to accumulate at hyperalphalipoproteinemia. Both atheroprotective (via S100A8) and proatherogenic (via SREBF1, LCAT, PRKACG, PRKACB, and PRKCQ) associations of gene expression with HDL-C determine HDL functionality in CAD patients. The selected key genes and involved pathways may represent HDL-specific targets for the diagnosis and treatment of CAD and atherosclerosis.

Different Pathways of Cellular Cholesterol Efflux.

Cholesterol efflux is the first and rate-limiting step of reverse cholesterol transport (RCT) from peripheric cells to the liver. The involvement of high-density lipoprotein (HDL) in RCT determines the atheroprotective properties of HDL. Cholesterol efflux from different membrane pools includes both passive and energy-dependent processes. The first type of route consists of cholesterol desorption from the cell membrane into the unstirred layer adjacent to the cell surface and diffusion in the water phase. Moreover, the selective uptake and facilitated diffusion of cholesterol and cholesteryl ester molecules through the hydrophobic tunnel in the scavenger receptor BI molecule does not require energy consumption. The second type of route includes active cholesterol export by the ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1). Several cholesterol acceptors specifically bind cholesterol and phospholipid molecules, and cholesterol binding to the albumin molecule, which acts as a shuttle, significantly increases cholesterol movement between acceptors and red blood cells, thus functioning as a sink for cholesterol. Cholesterol and phospholipid molecules effluxed from macrophages by ABCA1 are accepted exclusively by the lipid-free apolipoprotein apoA-I, which is the major protein moiety of HDL, whereas those effluxed by ABCG1 are accepted by HDL. ABCA1- and ABCG1-mediated cholesterol transport, together with cholesterol diffusion, largely determine cholesterol turnover at the physiological level of intracellular cholesterol. However, at cholesterol overload, ABCA1-mediated efflux prevails over other routes. The exchange of apoA-I between lipid-free and lipid-associated states and the synergism of nascent and mature HDL contribute to cholesterol efflux efficiency. Moreover, extracellular cholesterol deposits and microvesicles may be involved in RCT.

Genomic Variants and Multilevel Regulation of ABCA1, ABCG1, and SCARB1 Expression in Atherogenesis.

Atheroprotective properties of human plasma high-density lipoproteins (HDLs) are determined by their involvement in reverse cholesterol transport (RCT) from the macrophage to the liver. ABCA1, ABCG1, and SR-BI cholesterol transporters are involved in cholesterol efflux from macrophages to lipid-free ApoA-I and HDL as a first RCT step. Molecular determinants of RCT efficiency that may possess diagnostic and therapeutic meaning remain largely unknown. This review summarizes the progress in studying the genomic variants of ABCA1, ABCG1, and SCARB1, and the regulation of their function at transcriptional and post-transcriptional levels in atherosclerosis. Defects in the structure and function of ABCA1, ABCG1, and SR-BI are caused by changes in the gene sequence, such as single nucleotide polymorphism or various mutations. In the transcription initiation of transporter genes, in addition to transcription factors, long noncoding RNA (lncRNA), transcription activators, and repressors are also involved. Furthermore, transcription is substantially influenced by the methylation of gene promoter regions. Post-transcriptional regulation involves microRNAs and lncRNAs, including circular RNAs. The potential biomarkers and targets for atheroprotection, based on molecular mechanisms of expression regulation for three transporter genes, are also discussed in this review.

Interaction of lipid-free apolipoprotein A-I with cholesterol revealed by molecular modeling.

We report the modeling of the interaction of differently self-associated lipid-free apoA-I with cholesterol monomer and tail-to-tail (TT) or face-to-face (FF) cholesterol dimer. Cholesterol dimerization is exploited to reconcile the existing experimental data on cholesterol binding to apoA-I with extremely low critical micelle concentration of cholesterol. Two crystal structures of 1-43 N-truncated apolipoprotein Δ(1-43)A-I tetramer (PDB ID: 1AV1, structure B), 185-243 C-truncated apolipoprotein Δ(185-243)A-I dimer (PDB ID: 3R2P, structure M) were analyzed. Cholesterol monomers bind to multiple binding sites in apoA-I monomer, dimer and tetramer with low, moderate and high energy (-10 to -28 kJ/mol with Schrödinger package), still insufficient to overcome the thermodynamic restriction by cholesterol micellization (-52.8 kJ/mol). The binding sites partially coincide with the putative cholesterol-binding motifs. However, apoA-I monomer and dimer existing in structure B, that contain nonoverlapping and non-interacting pairs of binding sites with high affinity for TT and FF cholesterol dimers, can bind in common 14 cholesterol molecules that correspond to existing values. ApoA-I monomer and dimer in structure M can bind in common 6 cholesterol molecules. The values of respective total energy of cholesterol binding up to 64.5 and 67.0 kJ/mol for both B and M structures exceed the free energy of cholesterol micellization. We hypothesize that cholesterol dimers may simultaneously interact with extracellular monomer and dimer of lipid-free apoA-I, that accumulate at acid pH in atheroma. The thermodynamically allowed apolipoprotein-cholesterol interaction outside the macrophage may represent a new mechanism of cholesterol transport by apoA-I from atheroma, in addition to ABCA1-mediated cholesterol efflux.

Denaturation of human plasma high-density lipoproteins by urea studied by apolipoprotein A-I dissociation.

We studied the mechanism of HDL denaturation with concomitant apoA-I dissociation with HDL preparations from 48 patients with a wide range of plasma HDL-C and evaluated the contribution of lipid-free apoA-I into cholesterol efflux from macrophage, in particular, mediated by cholesterol transporter ABCA1. We prepared HDL by precipitation of apoB-containing lipoproteins by polyethylene glycol and used the chaotropic agent urea to denature HDL preparations. Apo-I dissociation from urea-treated HDL was assessed by the increase of preß-band fraction with agarose gel electrophoresis followed by electro transfer and immunodetection and by the increase of ABCA1-mediated efflux of fluorescent analogue BODIPY-Cholesterol from RAW 264.7 macrophages. The HDL denaturation is governed by a single transition to fully dissociated apoA-I and the transition cooperativity decreases with increasing HDL-C. The apoA-I release depends on phospholipid concentration of HDL preparation and HDL compositional and structural heterogeneity and is well described by apolipoprotein partition between aqueous and lipid phases. Dissociated apoA-I determines the increase of ABCA1-mediated efflux of BODIPY-Cholesterol from RAW 264.7 macrophages to patient HDL. The increase in apoA-I dissociation is associated with the increase of ABCA1 gene transcript in peripheral blood mononuclear cells from patients. The low level of plasma HDL particles may be compensated by their increased potency for apoA-I release, thus suggesting apoA-I dissociation as a new HDL functional property.

HDL cholesterol is associated with PBMC expression of genes involved in HDL metabolism and atherogenesis.

BACKGROUND: To reveal the association of plasma level of high density lipoprotein cholesterol (HDL-C) level with the transcript level of annotated genes in peripheral blood mononuclear cells (PBMC) and involved in HDL metabolism and atherogenesis at the absence of morphologically evident coronary stenosis. METHODS: Transcript levels of 63 genes in PBMC from 38 male patients 40-60 years without coronary atherosclerosis with widely varied HDL-C level were measured. The protein interactions were analyzed with STRING database. RESULTS: Among 22 HDL-related genes, the transcript levels for 10 genes (ABCA1, BMP1, CUBN, HDLBP, LCAT, LDLR, PRKACB, PRKACG, SCARB1 and ZDHHC8) negatively correlated with HDL-C, while positively for APOA1 gene. Among 41 atherosclerosis-prone genes, the transcript levels for 11 genes (CSF1R, CSF2RB, IL18R1, ITGAM, ITGB3, PRKCQ, SREBF1, TLR5, TLR8, TNFRSF1A and TNFRSF1B) negatively correlated with HDL-C only, not with LDL-C and plasma TG. The protein products efficiently interacted within each cluster while only two intersection nodes existed between clusters. CONCLUSIONS: Coordinate regulation of cholesterol influx and efflux in PBMC in atherosclerosis-free subjects with widely varied HDL-C level is suggested. The decreased synthesis and transport of cholesteryl ester to the liver may contribute to hyperalphalipoproteinemia. HDL-C increase is associated with the decrease of expression of innate immunity and inflammation genes. Visualization of 22 responder genes is suggested to be useful in the validation of HDL functionality and atherogenesis even at the absence of morphologically evident coronary stenosis.

Analysis of Low Molecular Weight Substances and Related Processes Influencing Cellular Cholesterol Efflux.

Cholesterol efflux is the key process protecting the vascular system from the development of atherosclerotic lesions. Various extracellular and intracellular events affect the ability of the cell to efflux excess cholesterol. To explore the possible pathways and processes that promote or inhibit cholesterol efflux, we applied a combined cheminformatic and bioinformatic approach. We performed a comprehensive analysis of published data on the various substances influencing cholesterol efflux and found 153 low molecular weight substances that are included in the Chemical Entities of Biological Interest (ChEBI) database. Pathway enrichment was performed for substances identified within the Reactome database, and 45 substances were selected in 93 significant pathways. The most common pathways included the energy-dependent processes related to active cholesterol transport from the cell, lipoprotein metabolism and lipid transport, and signaling pathways. The activators and inhibitors of cholesterol efflux were non-uniformly distributed among the different pathways: the substances influencing 'biological oxidations' activate cholesterol efflux and the substances influencing 'Signaling by GPCR and PTK6' inhibit efflux. This analysis may be used in the search and design of efflux effectors for therapies targeting structural and functional high-density lipoprotein deficiency.

Significance of Cholesterol-Binding Motifs in ABCA1, ABCG1, and SR-B1 Structure.

ABCA1, ABCG1 transporters, and SR-B1 receptor are the major proteins involved in cholesterol efflux from cells. We superposed in silico the location of putative cholesterol (Chol)-binding motifs CRAC/CARC and CCM in human ABCA1, ABCG1, and SR-B1 with (1) transmembrane protein topology, (2) a profile of structural order of protein, and (3) with an influence of single amino acid substitutions on protein structure and function. ABCA1, ABCG1, and SR-B1 molecules contain 50, 19, and 13 Chol-binding motifs, respectively, that are localized either in membrane helices, or at membrane-water interface, or in water-exposed protein regions. Arginine residues in motifs that coincide with molecular recognition features within intrinsically disordered regions of the transporters are suggested to be important in cholesterol binding; cholesterol-arginine interaction may result in the induction of local order in protein structure. Chol-binding motifs in membrane helices may immobilize cholesterol, while motifs at membrane-water interface may be involved into the efflux of "active" cholesterol. Cholesterol may interfere with ATP binding in both nucleotide-binding domains of ABCA1 structure. For ABCA1 and ABCG1, but not for SR-B1, the presence of mirror code as a CARC-CRAC vector couple in the C-terminal helices controlling protein-cholesterol interactions in the outer and inner membrane leaflets was evidenced. We propose the role of Chol-binding motifs with different immersion in membrane in transport of different cholesterol pools by ABCA1 and ABCG1.

Relation of High-Density Lipoprotein Charge Heterogeneity, Cholesterol Efflux Capacity, and the Expression of High-Density Lipoprotein-Related Genes in Mononuclear Cells to the HDL-Cholesterol Level.

The heterogeneity and content of human plasma high-density lipoprotein (HDL) related to their atheroprotective properties determined by various molecular and cellular mechanisms still remain to be completely clarified. For 29 atherosclerosis-free male subjects, we studied the relationship of plasma lipid levels and the content of apolipoprotein A-I (apoA-I)-containing HDL with preß-electrophoretic mobility, the efficiency of BODIPY-cholesterol efflux from RAW 264.7 macrophages to apolipoprotein B (apoB)-deficient plasma, and the expression level of 22 genes related to HDL metabolism in mononuclear cells. A significant decrease in the absolute content of apoA-I in preß-HDL was found in subjects with hypoalphalipoproteinemia compared with the subjects with hyperalphalipoproteinemia. The preß-to-α-ratio of the apoA-I content was constant within the HDL-cholesterol (HDL-C) range 0.59 to 2.24 mM. However, this ratio was significantly increased with an increase in the plasma triacylglycerol (TAG) content from 0.59 to 3.42 mM. A correlation of the level of preß-HDL with the basal and ABCA1-mediated efflux of cholesterol is shown. The transcript levels for six HDL-metabolizing genes (LDLR, LCAT, ABCA1, SCARB1, ZDHHC8, and BMP1) were decreased, while the transcript level of APOA1 gene was increased in mononuclear cells of subjects with hyperalphalipoproteinemia as compared with subjects with hypoalphalipoproteinemia. A reduction of the intracellular cholesterol level and inhibition of the expression of cholesterol transporters by nascent HDL in mononuclear cells from subjects with hyperalphalipoproteinemia are suggested. Hyperalphalipoproteinemia can be a driving force of the decreased flux of cholesteryl ester to the liver and the increased TAG hydrolysis. The atheroprotective effect of preß-HDL in hypertriglyceridemia is proposed.

Intracellular and Plasma Membrane Events in Cholesterol Transport and Homeostasis.

Cholesterol transport between intracellular compartments proceeds by both energy- and non-energy-dependent processes. Energy-dependent vesicular traffic partly contributes to cholesterol flux between endoplasmic reticulum, plasma membrane, and endocytic vesicles. Membrane contact sites and lipid transfer proteins are involved in nonvesicular lipid traffic. Only "active" cholesterol molecules outside of cholesterol-rich regions and partially exposed in water phase are able to fast transfer. The dissociation of partially exposed cholesterol molecules in water determines the rate of passive aqueous diffusion of cholesterol out of plasma membrane. ATP hydrolysis with concomitant conformational transition is required to cholesterol efflux by ABCA1 and ABCG1 transporters. Besides, scavenger receptor SR-B1 is involved also in cholesterol efflux by facilitated diffusion via hydrophobic tunnel within the molecule. Direct interaction of ABCA1 with apolipoprotein A-I (apoA-I) or apoA-I binding to high capacity binding sites in plasma membrane is important in cholesterol escape to free apoA-I. ABCG1-mediated efflux to fully lipidated apoA-I within high density lipoprotein particle proceeds more likely through the increase of "active" cholesterol level. Putative cholesterol-binding linear motifs within the structure of all three proteins ABCA1, ABCG1, and SR-B1 are suggested to contribute to the binding and transfer of cholesterol molecules from cytoplasmic to outer leaflets of lipid bilayer. Together, plasma membrane events and intracellular cholesterol metabolism and traffic determine the capacity of the cell for cholesterol efflux.

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.

Cholesterol Efflux and Reverse Cholesterol Transport: Experimental Approaches.

BACKGROUND: Cholesterol efflux as a key event in reverse cholesterol transport (RCT) is considered now as both diagnostic tool and a promising target for the treatment of atherosclerosis. Radioactive in vitro cholesterol efflux assay (CEA) is the gold standard for determination of efflux at cellular level. Fluorescent tracers and stable isotope-labeled cholesterol gradually come into use as convenient tools for non-radioactive CEAs. RESULTS: We review the use of various tracer-based and tracer-free methods for CEAs and for measuring RCT with focus on macrophage-specific cholesterol efflux. CEA utilizing stable isotope-labeled cholesterol is equally reliable with radioactive assay and especially well suited for the determination of both cholesterol efflux and net cholesterol flux. Fluorescent tracers cannot fully mimic cholesterol; however, they are successfully applied in CEA in specific well-defined conditions. Fluorescent CEAs can be high throughput and can provide unique information on efflux from fast cholesterol pools or with single cell resolution. Enzymatic and chromatographic CEAs are net cholesterol flux assays, and they can be applied as efflux assays when used with specific acceptors only. In vivo tests are suited for studies of cholesterol efflux and RCT at the level of the organism. They include injection of tracer-loaded macrophages, a method suitable at present for animal models only, and recently invented modification of whole body tracer kinetics with multicompartment modeling that is capable to determine cholesterol efflux from macrophages. CONCLUSION: Despite the decisive role of in vitro assays in our understanding of cholesterol efflux mechanism, the in vivo assays are highly desired to study cholesterol efflux in atherosclerotic lesions and RCT in whole body.

Cholesteryl ester diffusion, location and self-association constraints determine CETP activity with discoidal HDL: excimer probe study.

The transfer of cholesteryl ester by recombinant cholesteryl ester transfer protein (CETP) between reconstituted discoidal high-density lipoprotein (rHDL) was studied. Particles contained apolipoprotein A-I, unsaturated POPC or saturated DPPC and cholesteryl ester as cholesteryl 1-pyrenedecanoate (CPD) or cholesteryl laurate (CL) in donor and acceptor rHDL, respectively. Probe dynamics fulfilled the quenching sphere-of-action model. The cholesteryl ester exchange between donor and acceptor particles was characterized by a heterogeneous kinetics; the fast exchanging CPD pool was much higher in a case of POPC compared to DPPC complexes. Probe fraction accessible to CETP increased with temperature, suggesting a more homogeneous probe distribution. Noncompetitive inhibition of probe transfer by acceptor particles was observed. The values of Vmax (0.063µMmin(-1)) and catalytic rate constant kcat (0.42s(-1)) together with a similarity of Km (0.9µM CPD) and KI (2.8µM CL) values for POPC-containing rHDL suggest the efficient cholesteryl ester transfer between nascent HDL with unsaturated phosphatidylcholine in vivo. The phospholipid matrix in discoidal HDL may underlie CETP activity through the self-association, diffusivity and location of cholesteryl ester in the bilayer, the accessibility of cholesteryl ester to cholesterol-binding site in apoA-I structure and the binding of cholesteryl ester, positionable by apoA-I, to CETP.

Prediction of the influences of missense mutations on cholesteryl ester transfer protein structure.

The structure of human plasma cholesteryl ester transfer protein (CETP) was mapped in silico by a search of the structural effects of missense mutations in the CETP gene. Sixteen deleterious substitutions were chosen among 54 known missense mutations and further ranked by stability change score into six structural and ten functional mutations with large and small stability changes, respectively. A cluster of eight mutations in a central region spanning residues 184-296 with exclusively destabilizing effects was evident. Moreover, the mutations were differently distributed between ordered and highly fluctuating regions. Putative cholesterol-binding regions, mostly unique for CETP in a whole CETP-including protein family, were identified. Three of six structural mutations influence cholesteryl ester and phosphatidylcholine binding by CETP. The local partially disordered structure of some putative cholesterol-binding regions is suggested to be differently influenced by cholesterol binding. This may underlie the impairment of the local ordering effect of cholesterol by the L261R substitution. Also, cholesterol may competitively inhibit cholesteryl ester binding to the CETP molecule, with triglyceride binding being largely undisturbed. This analysis may contribute to the ongoing design and mechanistic studies of new CETP inhibitors.

Mutation mapping of apolipoprotein A-I structure assisted with the putative cholesterol recognition regions.

Twenty-nine from 52 missense mutations in apoA-I gene are predicted to be deleterious by both SIFT and PolyPhen-2 algorithms. Among those, eight mutations with a prominent change in structure stability as modeled by the SDM tool for both lipid-free (Mei and Atkinson (2011) PDB ID: 3R2P) and HDL-bound (Wu et al. (2009) PDB ID: 3K2S) apoA-I, are referred as structural. The remaining mutations with a preferential location in a long intrinsically disordered region, predicted by the SPINE-D and DNdisorder tools, may influence the functional sites. Among structural mutations, five amyloidosis-only-related mutations, significant in a lipid-free structure, are located in 1-90 region. Six amyloidosis- and hypoalphalipoproteinemia-associated mutations, differently significant in two chains of lipid-bound apoA-I, are distributed among the N-domain. Six cholesterol recognition putative motifs (5 CRAC/1 CCM) in apoA-I structure are suggested to interact with cholesterol. Among those, the K40-W50 partially conserved CCM sequence with a putative recognition feature, predicted by the MoRF tool, may underlie cholesterol binding to lipid-free apoA-I, the binding triggering the disorder-to-order transition within MoRF. Thus, the impairment of helix formation and accelerated protein aggregation may underlie the amyloidogenic effect of W50R substitution. Also, D102H substitution in conserved CRAC2 V97-K106 sequence may be harmful in reverse cholesterol transport. With PDBe Motifs and Sites algorithm, cholesterol is a ligand for L101, F104 and W108 residues in HDL-bound apoA-I. The influence of specific mutation on apoA-I structure and mutated apolipoprotein switch between different pathologies is suggested to depend on the surrounding phase properties.

Sequence-specific apolipoprotein A-I effects on lecithin:cholesterol acyltransferase activity.

Existing kinetic data of cholesteryl ester formation by lecithin:cholesterol acyltransferase in discoidal high-density lipoproteins with 34 mutations of apoA-I that involved all putative helices were grouped by cluster analysis into four noncoincident regions with mutations both without any functional impairment and with profound isolated (V- and K-mutations) or common (VK-mutations) effect on V(max)(app) and K(m)(app). Data were analyzed with a new kinetic model of LCAT activity at interface that exploits the efficiency of LCAT binding to the particle, particle dimensions, and surface concentrations of phosphatidylcholine and cholesterol. V-mutations with major location in the central part and C-domain affected the second-order rate constant of cholesteryl ester formation at the solvolysis of acyl-enzyme intermediate by cholesterol as nucleophile. The central region in apoA-I sequence is suggested to influence the proper positioning of cholesterol molecule toward LCAT active center with major contribution of arginine residue(s). K-mutations with major location in N-domain may affect binding and stability of enzyme-phosphatidylcholine complex. VK-mutations may possess mixed effects; the independent binding measurement may segregate individual steps.

A mechanistic model of lecithin:cholesterol acyltransferase activity exploits discoidal HDL composition and structure.

Lecithin:cholesterol acyltransferase (LCAT) activity towards discoidal HDL with apoA-I was analyzed in conjunction with re-evaluation of conformational stability of apoA-I (Sparks et al., 1993). The reaction at water-lipid interface involves the formation of acyl-enzyme and cholesterol (Chol) as a nucleophilic agent can compete with water at deacylation step. Raw data on apparent kinetic parameters for LCAT activity toward discoidal HDL with fixed (Sparks et al., 1995) or varying (Sparks et al., 1998) palmitoyloleoylphosphatidylcholine (POPC) content fit the kinetic equation derived. At the increase of Chol content in complexes with fixed POPC, interfacial dissociation constant K(d)(∗) for LCAT penetration decreased and interfacial Michaelis constant K(m)(∗) did not change. Also, differences in stability and unfolding cooperativity between two domains in apoA-I molecule increased. At the increase of surface area of the complexes with varying POPC, K(d)(∗) increased, while K(m)(∗) decreased. For both lipidation states the rate constant of acyl-LCAT formation did not vary and any changes in K(m)(∗) are postulated to originate from the change(s) in association/dissociation rate constants of enzyme-substrate complex. Then, at the increase of POPC, the LCAT-POPC complex becomes more stable. ApoA-I seems to "activate" substrate by increasing the exposure of POPC ester bond to active center of LCAT.

Kinetic analysis of lecithin:cholesterol acyltransferase activity toward discoidal HDL.

The kinetics of lecithin:cholesterol acyltransferase(LCAT, EC 2.3.1.43)-catalyzed generation of cholesteryl ester in discoidal high density lipoproteins (HDL) was analyzed in terms of initial binding of LCAT to the disc surface followed by a three-state reaction of the hydrolysis of phosphatidylcholine sn-2 ester bond and acyl-enzyme formation. Cholesterol was considered as alcoholic nucleophile that increases the solvolysis rate of acyl-LCAT. The raw kinetic data of Sparks et al. (J Biol Chem 270:5151-5157, 1995) for four preparations of reconstituted discoidal HDL with a constant level of apolipoprotein A-I and palmitoyloleoylphosphatidylcholine per disc but with cholesterol in a lipid phase continuously increasing from 2.1 to 12.5 mol%, were analyzed in terms of the kinetic equation and a complete set of rate constants was obtained. Data at high cholesterol content do not indicate a saturation phenomenon, thus giving no evidence for a binding of cholesterol to the enzyme. This analysis may be used in the study of LCAT activation by exchangeable apolipoproteins and contribution of the HDL structure.

Local/bulk determinants of conformational stability of exchangeable apolipoproteins.

GuHCl-induced denaturation of human plasma apoA-I, apoA-II, apoA-IV, apoE3 and three recombinant apoE isoforms in solution and discoidal complexes with phosphatidylcholine (only plasma proteins) was studied. The protein conformational stability (ΔG(H(2)O)) and a slope of linear dependence of free energy of unfolding on GuHCl concentration (m-value) were estimated with the three equilibrium schemes. The data for all proteins, except apoA-II, fit with the three-state model, thus evidencing two-domain structure. The predicted folding rate of the four apoE in solution correlated with conformational stability. The dependence disappeared at the inclusion of apoA-I and apoA-IV into analysis and the m-values, adjusted for residue number in helices (m(rh)), differed between those for apoE and apoA-I/apoA-IV. However, the m(rh)-values for six proteins correlated positively with the fractional change in accessible surface area at unfolding for Phe, Lys and Asn, while negatively for Arg, Ala and Gly residues. The difference between the adjusted ΔG(rh)(H(2)O) values for apolipoproteins in complexes and in solution decreased at the increase of reduced temperature (T(obs)-T(t))/T(t). The induction of intrinsic disorder by arginine residues may be of primary importance in metabolism and function of exchangeable apolipoproteins, while their stability in nascent discoidal HDL is controlled by the physical state of phosphatidylcholine.