Structure-function analysis of naturally occurring apolipoprotein A-I L144R, A164S and L178P mutants provides insight on their role on HDL levels and cardiovascular risk.

Naturally occurring point mutations in apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), may affect plasma HDL-cholesterol levels and cardiovascular risk. Here, we evaluated the effect of human apoA-I mutations L144R (associated with low HDL-cholesterol), L178P (associated with low HDL-cholesterol and increased cardiovascular risk) and A164S (associated with increased cardiovascular risk and mortality without low HDL-cholesterol) on the structural integrity and functions of lipid-free and lipoprotein-associated apoA-I in an effort to explain the phenotypes of subjects carrying these mutations. All three mutants, in lipid-free form, presented structural and thermodynamic aberrations, with apoA-I[L178P] presenting the greatest thermodynamic destabilization. Additionally, apoA-I[L178P] displayed reduced ABCA1-mediated cholesterol efflux capacity. When in reconstituted HDL (rHDL), apoA-I[L144R] and apoA-I[L178P] were more thermodynamically destabilized compared to wild-type apoA-I, both displayed reduced SR-BI-mediated cholesterol efflux capacity and apoA-I[L144R] showed severe LCAT activation defect. ApoA-I[A164S] was thermodynamically unaffected when in rHDL, but exhibited a series of functional defects. Specifically, it had reduced ABCG1-mediated cholesterol and 7-ketocholesterol efflux capacity, failed to reduce ROS formation in endothelial cells and had reduced capacity to induce endothelial cell migration. Mechanistically, the latter was due to decreased capacity of rHDL-apoA-I[A164S] to activate Akt kinase possibly by interacting with endothelial LOX-1 receptor. The impaired capacity of rHDL-apoA-I[A164S] to preserve endothelial function may be related to the increased cardiovascular risk for this mutation. Overall, our structure-function analysis of L144R, A164S and L178P apoA-I mutants provides insights on how HDL-cholesterol levels and/or atheroprotective properties of apoA-I/HDL are impaired in carriers of these mutations.

Macrophages release plasma membrane-derived particles rich in accessible cholesterol.

Macrophages are generally assumed to unload surplus cholesterol through direct interactions between ABC transporters on the plasma membrane and HDLs, but they have also been reported to release cholesterol-containing particles. How macrophage-derived particles are formed and released has not been clear. To understand the genesis of macrophage-derived particles, we imaged mouse macrophages by EM and nanoscale secondary ion mass spectrometry (nanoSIMS). By scanning EM, we found that large numbers of 20- to 120-nm particles are released from the fingerlike projections (filopodia) of macrophages. These particles attach to the substrate, forming a "lawn" of particles surrounding macrophages. By nanoSIMS imaging we showed that these particles are enriched in the mobile and metabolically active accessible pool of cholesterol (detectable by ALO-D4, a modified version of a cholesterol-binding cytolysin). The cholesterol content of macrophage-derived particles was increased by loading the cells with cholesterol or by adding LXR and RXR agonists to the cell-culture medium. Incubating macrophages with HDL reduced the cholesterol content of macrophage-derived particles. We propose that release of accessible cholesterol-rich particles from the macrophage plasma membrane could assist in disposing of surplus cholesterol and increase the efficiency of cholesterol movement to HDL.

Structures of apolipoprotein A-I in high density lipoprotein generated by electron microscopy and biased simulations.

BACKGROUND: Apolipoprotein A-I (apoA-I) in high-density lipoprotein (HDL) is a key protein for the transport of cholesterol from the vascular wall to the liver. The formation and structure of nascent HDL, composed of apoA-I and phospholipids, is critical to this process. METHODS: The HDL was assembled in vitro from apoA-I, cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at a 1:4:50 molar ratio. The structure of HDL was investigated in vitreous samples, frozen at cryogenic temperatures, as well as in negatively stained samples by transmission electron microscopy. Low resolution electron density maps were next used as restraints in biased Monte Carlo simulations of apolipoprotein A-I dimers, with an initial structure derived from atomic resolution X-ray structures. RESULTS: Two final apoA-I structure models for the full-length structure of apoA-I dimer in the lipid bound conformation were generated, showing a nearly circular, flat particle with an uneven particle thickness. CONCLUSIONS: The generated structures provide evidence for the discoidal, antiparallel arrangement of apoA-I in nascent HDL, and propose two preferred conformations of the flexible N-termini. GENERAL SIGNIFICANCE: The novel full-length structures of apoA-I dimers deepens the understanding to the structure-function relationship of nascent HDL with significance for the prevention of lipoprotein-related disease. The biased simulation method used in this study provides a powerful and convenient modelling tool with applicability for structural studies and modelling of other proteins and protein complexes.

Patients with Rheumatoid Arthritis Show Altered Lipoprotein Profiles with Dysfunctional High-Density Lipoproteins that Can Exacerbate Inflammatory and Atherogenic Process.

OBJECTIVE: In order to identify putative biomarkers in lipoprotein, we compared lipid and lipoprotein properties between rheumatoid arthritis (RA) patients and control with similar age. METHODS: We analyzed four classes of lipoproteins (VLDL, LDL, HDL2, HDL3) from both male (n = 8, 69±4 year-old) and female (n = 25, 53±7 year-old) rheumatoid arthritis (RA) patients as well as controls with similar age (n = 13). RESULTS: Although RA group showed normal levels of total cholesterol (TC), low-density lipoprotein (LDL)-cholesterol, and glucose, however, the RA group showed significantly reduced high-density lipoprotein (HDL)-C level and ratio of HDL-C/TC. The RA group showed significantly elevated levels of blood triglyceride (TG), uric acid, and cholesteryl ester transfer protein (CETP) activity. The RA group also showed elevated levels of advanced glycated end (AGE) products in all lipoproteins and severe aggregation of apoA-I in HDL. As CETP activity and TG contents were 2-fold increased in HDL from RA group, paraoxonase activity was reduced upto 20%. Electron microscopy revealed that RA group showed much less HDL2 particle number than control. LDL from the RA group was severely oxidized and glycated with greater fragmentation of apo-B, especially in female group, it was more atherogenic via phagocytosis. CONCLUSION: Lipoproteins from the RA patients showed severely altered structure with impaired functionality, which is very similar to that observed in coronary heart patients. These dysfunctional properties in lipoproteins from the RA patients might be associated with high incidence of cardiovascular events in RA patients.

Cholesteryl ester transfer between lipoproteins does not require a ternary tunnel complex with CETP.

The cholesteryl ester transfer protein (CETP) enables the transfer of cholesteryl ester (CE) from high-density lipoproteins (HDL) to low-density lipoproteins (LDL) in the plasma compartment. CETP inhibition raises plasma levels of HDL cholesterol; a ternary tunnel complex with CETP bridging HDL and LDL was suggested as a mechanism. Here, we test whether the inhibition of CETP tunnel complex formation is a promising approach to suppress CE transfer from HDL to LDL, for potential treatment of cardio-vascular disease (CVD). Three monoclonal antibodies against different epitopes of CETP are assayed for their potential to interfere with CE transfer between HDL and/or LDL. Surprisingly, antibodies that target the tips of the elongated CETP molecule, interaction sites sterically required to form the suggested transfer complexes, do not interfere with CETP activity, but an antibody binding to the central region does. We show that CETP interacts with HDL, but not with LDL. Our findings demonstrate that a ternary tunnel complex is not the mechanistic prerequisite to transfer CE among lipoproteins.

HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation.

Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobic environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL.

Hyaluronic acid-decorated reconstituted high density lipoprotein targeting atherosclerotic lesions.

The primary aim of our current study was to utilize hyaluronic acid (HA) to decorate reconstituted high density lipoprotein (rHDL) loaded with lovastatin (LT), termed as HA-LT-rHDL, in order to investigate whether coating HA could efficiently evade from the undesired uptake of LT-rHDL in liver mediated by scavenger receptor class B type I (SR-BI) and then greatly accumulate LT-rHDL in atherosclerotic lesions via strong HA affinity to CD44 up-regulated at inflammatory sites such as atherosclerotic lesions, thus exerting enhanced atheroprotective efficacy. In vitro characterizations indicated the successful HA decoration onto the surface of LT-rHDL, which could be indirectly verified by the increased particle size, enhanced negative surface charge and reduced in vitro drug release rate after HA decoration. Compared with rHDL without HA, HA decoration endowed rHDL with better atherosclerotic lesions targeting efficiency and lower liver accumulation, proved by results from ex vivo imaging and tissue distribution. Furthermore, atheroprotective efficacy in model animal showed that HA-LT-rHDL had the best potent efficacy than other LT preparations, which was demonstrated by the fewest atherosclerotic lesions sizes, the most minimum mean intima-media thickness (MIT), the lowest macrophage infiltration and expression of matrix metalloproteinase-9 (MMP-9), respectively. Above results demonstrated that the newly designed HA-LT-rHDL could decrease the non-targeted uptake by liver and deliver a large amount of drug into atherosclerotic lesions so as to efficiently suppress the advancement of atherosclerosis.

Pharmacokinetics and atherosclerotic lesions targeting effects of tanshinone IIA discoidal and spherical biomimetic high density lipoproteins.

High density lipoproteins (HDL) have been successfully reconstructed to deliver a large number of lipophilic drugs. Here, discoidal and spherical recombinant HDL loaded with cardiovascular drug tanshinone IIA (TA) were constructed (TA-d-rHDL and TA-s-rHDL), respectively. And next their in vitro physiochemical and biomimetic properties were characterized. Furthermore, pharmacokinetics, atherosclerotic lesions targeting effects and antiatherogenic efficacies were elaborately performed and compared in atherosclerotic New Zealand White (NZW) rabbits. In vitro characterizations results showed that both TA-d-rHDL and TA-s-rHDL had nano-size diameter, high entrapment efficiency (EE) and drug-loading capacity (DL). Additionally, similar to their native counterparts, TA-d-rHDL maintained remodeling behaviors induced by lecithin cholesterol acyltransferase (LCAT), and TA leaked during remodeling behaviors. Pharmacokinetic studies manifested that both TA-d-rHDL and TA-s-rHDL markedly improved pharmacokinetic behaviors of TA in vivo. Ex vivo imaging demonstrated that both d-rHDL and s-rHDL bound more avidly to atherosclerotic lesions than to normal vessel walls, and s-rHDL had better targeting effect than d-rHDL. Pharmacodynamic tests illustrated that both TA-d-rHDL and TA-s-rHDL had much stronger antiatherogenic efficacies than conventional TA nanostructured lipid carriers (TA-NLC), TA liposomes (TA-L) and commercially available preparation Sulfotanshinone Sodium Injection (SSI). Moreover, TA-s-rHDL had more potent antiatherogenic efficacies than TA-d-rHDL. Collectively our studies indicated that rHDL could be exploited as potential delivery vehicles of TA targeting atherosclerotic lesions as well as synergistically improving efficacies, especially for s-rHDL.

Qualitative characteristics of HDL in young patients of an acute myocardial infarction.

AIM: Recently, the concept that high density lipoprotein (HDL) quality is an important parameter for atheroprotection is gaining ground, though little data exists so far to support it. In an attempt to identify measurable qualitative parameters of HDL associated with increased risk for premature myocardial infarction (MI), we studied the structural characteristics of HDL from patients who survived an MI at a young age (≤35 years). METHODS AND RESULTS: We studied 20 MI patients and 20 healthy control subjects. HDL of patients had reduced apolipoprotein A-I (apoA-I), apolipoprotein M, and paraoxonase 1 levels and significantly elevated apolipoprotein C-III (apoCIII) levels (all p<0.05). Specifically, the HDL apoA-I/apoC-III ratio was 0.24±0.01 in patients versus 4.88±0.90 in controls (p<0.001). These structural alterations correlated with increased oxidation potential of HDL of the MI group compared to controls (2.5-fold, p=0.026). Electron microscopy showed no significant difference in average HDL particle diameter between the two groups though a significant difference existed in HDL diameter distribution, suggesting the presence of different HDL subpopulations in MI and control subjects. Indeed, non-denaturing two-dimensional electrophoresis revealed that MI patients had reduced pre-ß1(α), pre-ß1(b) and α(2), and elevated α(1), α(3), and pre-α(4) HDL. CONCLUSIONS: Reduction in the HDL apoA-I/apoC-III ratio, changes in the HDL subpopulation distribution and an increase in HDL oxidation potential correlated with the development of MI in young patients. The possibility that such changes may serve as markers for the early identification of young individuals at high risk for an acute coronary event should be further explored.

Combined light and electron microscopy using diaminobenzidine photooxidation to monitor trafficking of lipids derived from lipoprotein particles.

Diaminobenzidine (DAB) photooxidation is a method for conversion of fluorescent signals into electron-dense precipitates that are visible in the electron microscope. Recently, we have applied this method to analyze organelles involved in holo-high density lipoprotein (HDL) particle uptake at the ultrastructural level. In the present work we extended the spectrum of molecules visualized via photooxidation to monitor the uptake of HDL-derived lipids in HepG2 cells. By the combined light-electron microscopic method and with the aid of the DAB photooxidation technique, it became possible for the first time to visualize different intracellular pathways of lipoprotein particle-derived lipids and analyze the compartments involved at the ultrastructural level. HDL-Alexa 568 was used to visualize holo-HDL particle uptake. Reconstituted HDL particles containing the fluorescent cholesterol analogues Bodipy-cholesterol, Bodipy-cholesteryl oleate, or cholesteryl Bodipy-ester were used to visualize uptake of the HDL-associated sterol. In Bodipy-cholesteryl oleate and cholesteryl Bodipy-ester, the cholesterol moiety or the fatty acid moiety is fluorescently labeled, respectively; in contrast, Bodipy-cholesterol is an analogue of free cholesterol. The cellular compartments involved in their intracellular routes after uptake were analyzed in the fluorescence and electron microscope after DAB photooxidation. Bodipy-cholesterol was found to be localized in tubular endosomes and multivesicular bodies (MVBs), in the trans-Golgi network, and in stacked Golgi cisternae. In contrast, HepG2 cells incubated with HDL containing Bodipy-cholesteryl oleate or cholesteryl Bodipyester gave an uptake pattern comparable to that of holo-HDL particles, with MVBs being involved. Bodipy-cholesteryl oleate was also found in lysosomes. These results indicate that HDL-derived cholesterol and cholesteryl ester are transported by different intracellular pathways in HepG2 cells. Thus, the DAB photooxidation method enables the analysis of intracellular transport of lipoprotein particle-derived lipids at the light and at the ultrastructural level.

Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein.

Dengue virus (DENV) causes the major arboviral disease of the tropics, characterized in its severe forms by signs of hemorrhage and plasma leakage. DENV encodes a nonstructural glycoprotein, NS1, that associates with intracellular membranes and the cell surface. NS1 is eventually secreted as a soluble hexamer from DENV-infected cells and circulates in the bloodstream of infected patients. Extracellular NS1 has been shown to modulate the complement system and to enhance DENV infection, yet its structure and function remain essentially unknown. By combining cryoelectron microscopy analysis with a characterization of NS1 amphipathic properties, we show that the secreted NS1 hexamer forms a lipoprotein particle with an open-barrel protein shell and a prominent central channel rich in lipids. Biochemical and NMR analyses of the NS1 lipid cargo reveal the presence of triglycerides, bound at an equimolar ratio to the NS1 protomer, as well as cholesteryl esters and phospholipids, a composition evocative of the plasma lipoproteins involved in vascular homeostasis. This study suggests that DENV NS1, by mimicking or hijacking lipid metabolic pathways, contributes to endothelium dysfunction, a key feature of severe dengue disease.

Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy.

Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.

Responses of LDL and HDL particle size and distribution to omega-3 fatty acid supplementation and aerobic exercise.

The purpose of this investigation was to determine the independent and combined effects of aerobic exercise and omega-3 fatty acid (n-3fa) supplementation on lipid and lipoproteins. Sedentary, normoglycemic, nonsmoking men (n = 11) were assigned to perform rest and exercise before and during n-3fa supplementation. Exercise consisted of 3 consecutive days of treadmill walking at 65% maximum O(2) consumption for 60 min. Supplementation consisted of 42 days of 4.55 g/day of n-3fa. A two-way factorial ANOVA with repeated measures revealed significant reductions in total cholesterol (P = 0.001, -9.2%) and triglyceride (P = 0.007, -32.4%) concentrations postexercise. In addition, exercise increased LDL peak particle size (P = 0.001) from 26.2 to 26.4 nm, but not HDL size. The n-3fa supplementation resulted in a significant shift in the distribution of HDL-cholesterol (HDL-C) carried by HDL(2b+2a) (P = 0.001, 14.2%) and HDL(3a+3b) (P = 0.001, -22.8%), despite no significant changes in lipid and lipoprotein-cholesterol concentrations. The majority of the shift in HDL-C was noted in HDL(2b) (P = 0.001, 20.9%) and HDL(3a) (P < 0.001, -31.0%) particles. There were no combined effects of exercise and n-3fa supplementation on lipids and lipoproteins. Three consecutive days of aerobic exercise reduced triglyceride and total cholesterol concentrations with a concomitant increase in LDL peak particle size. In contrast, n-3fa supplementation shifted HDL-C from HDL(3) particles to HDL(2) particles, despite no significant changes in HDL(2)-C and HDL(3)-C concentrations. Exercise and n-3fa supplementation do not synergistically improve serum lipids and lipoproteins, but rather independently affect the metabolism of lipids and lipoproteins.

Smaller discoidal high-density lipoprotein particles form saddle surfaces, but not planar bilayers.

Discoidal high-density lipoprotein (HDL) particles are known to be fractionalized into several discrete populations in plasma and to differ in behavior according to size; however, their structural differences and the factors regulating their size are less understood. In this study, we prepared several reconstituted HDLs (rHDLs) for structural evaluation by gel filtration chromatography and fluorometric analyses. With initial ratios of phospholipid (PL) to apolipoprotein A-I (apoA-I) between 25:1 and 100:1, unsaturated PLs constructed rHDLs with diameters of 9.5-9.6, 8.8-9.0, and 7.8-7.9 nm. Conversely, saturated PLs formed only the largest type of rHDLs (9.5-9.9 nm). While the largest rHDL comprised 23% cholesterol (Chol), the smallest rHDL contained only 13% Chol, which approximates liquid-ordered phase composition. As the size of rHDLs decreased, both the lateral pressure in the lipid bilayer, as determined from the excimer fluorescence of dipyrenylphosphatidylcholine, and the degree of hydration of the membrane surface, which was examined using the mean fluorescence lifetime of dansyl phosphatidylethanolamine, decreased well below the values obtained for large unilamellar vesicles. These results demonstrated that smaller rHDLs form a saddle surface, distinct from the planar bilayer produced by the largest forms.

Maturation of high-density lipoproteins.

Human high-density lipoproteins (HDLs) are involved in the transport of cholesterol. The mechanism by which HDL assembles and functions is not well understood owing to a lack of structural information on circulating spherical HDL. Here, we report a series of molecular dynamics simulations that describe the maturation of discoidal HDL into spherical HDL upon incorporation of cholesterol ester as well as the resulting atomic level structure of a mature circulating spherical HDL particle. Sixty cholesterol ester molecules were added in a stepwise fashion to a discoidal HDL particle containing two apolipoproteins wrapped around a 160 dipalmitoylphosphatidylcholine lipid bilayer. The resulting matured particle, captured in a coarse-grained description, was then described in a consistent all-atom representation and analysed in chemical detail. The simulations show that maturation results from the formation of a highly dynamic hydrophobic core comprised of cholesterol ester surrounded by phospholipid and protein; the two apolipoprotein strands remain in a belt-like conformation as seen in the discoidal HDL particle, but with flexible N- and C-terminal helices and a central region stabilized by salt bridges. In the otherwise flexible lipoproteins, a less mobile central region provides an ideal location to bind lecithin cholesterol acyltransferase, the key enzyme that converts cholesterol to cholesterol ester during HDL maturation.

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 alpha-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.

[Lipid transfer to HDl in women with type 1 diabetes]. / Transferência de lípides para a lipoproteína de alta densidade (HDl) em mulheres com diabetes melito tipo 1.

INTRODUCTION: People with type 1 diabetes mellitus (T1DM) have an increased risk of cardiovascular disease and may still have a normal lipid profile. In order to clarify whether normal HDL cholesterol levels may conceal defects in HDL function, we have studied the transfer of lipids to HDL in T1DM. METHODS: Twenty-one young women with T1DM were compared with 21 non-diabetic women. Nanoemulsion preparations were used as lipid donor to HDL: one labeled with (3)H-triglycerides and 14C-free cholesterol and the other with (3)H-cholesteryl esters and 14C-phospholipids. These preparations were incubated with plasma samples for 1h. After chemical precipitation, the supernatant containing HDL was counted for radioactivity. RESULTS: No difference in transfer was observed to nanoemulsion HDL from cholesteryl esters, triglycerides, free cholesterol and phospholipids. CONCLUSION: Simultaneous lipid transfer to HDL was not affected in T1DM patients. This suggests that the disease does not alter lipoprotein composition and transfer protein action in such way as to disturb HDL metabolism.

Structural and functional consequences of the Milano mutation (R173C) in human apolipoprotein A-I.

Carriers of the apolipoprotein A-I(Milano) (apoA-I(M)) variant, R173C, have reduced levels of plasma HDL but no increase in cardiovascular disease. Despite intensive study, it is not clear whether the removal of the arginine or the introduction of the cysteine is responsible for this altered functionality. We investigated this question using two engineered variations of the apoA-I(M) mutation: R173S apoA-I, similar to apoA-I(M) but incapable of forming a disulfide bond, and R173K apoA-I, a conservative mutation. Characterization of the lipid-free proteins showed that the order of stability was wild type approximately R173K>R173S>R173C. Compared with wild-type apoA-I, apoA-I(M) had a lower affinity for lipids, while R173S apoA-I displayed intermediate affinity. The in vivo effects of the apoA-I variants were measured by injecting apoA-I-expressing adeno-associated virus into apoA-I-null mice. Mice that expressed the R173S variant again showed an intermediate phenotype. Thus, both the loss of the arginine and its replacement by a cysteine contribute to the altered properties of apoA-I(M). The arginine is potentially involved in an intrahelical salt bridge with E169 that is disrupted by the loss of the positively charged arginine and repelled by the cysteine, destabilizing the helix bundle domain in the apoA-I molecule and modifying its lipid binding characteristics.

Transferência de lípides para a lipoproteína de alta densidade (HDl) em mulheres com diabetes melito tipo 1 / Lipid transfer to HDl in women with type 1 diabetes

INTRODUÇÃO: Os portadores de diabetes melito tipo 1 (DM1) possuem aumentado risco de doença cardiovascular e, ainda assim, podem apresentar perfil lipídico normal. Para esclarecer se os níveis normais de HDL podem ocultar defeitos na função, foram estudados a transferência de lípides para a HDL em DM1. MÉTODOS: Vinte e uma mulheres jovens portadoras de DM1 foram comparadas com 21 mulheres não-diabéticas. Nanoemulsões foram usadas como doadoras de lípides para HDL: uma marcada com ³H-triglicérides e 14C-colesterol livre e outra com ³H-éster de colesterol e 14C-fosfolípides. Após 1 hora de incubação com amostras de plasma, seguida por precipitação química, o sobrenadante, contendo HDL, teve a radioatividade contada. RESULTADOS: Nenhuma diferença foi encontrada nas transferências dos ésteres de colesterol, triglicérides, colesterol livre e fosfolípides para as HDL. CONCLUSÃO: A transferência de lípides para a HDL não está afetada em portadoras de DM1. Isso sugere que a doença não altera a composição de lipoproteínas e a ação de proteínas de transferência.

INTRODUCTION: People with type 1 diabetes mellitus (T1DM) have an increased risk of cardiovascular disease and may still have a normal lipid profile. In order to clarify whether normal HDL cholesterol levels may conceal defects in HDL function, we have studied the transfer of lipids to HDL in T1DM. METHODS: Twenty-one young women with T1DM were compared with 21 non-diabetic women. Nanoemulsion preparations were used as lipid donor to HDL: one labeled with ³H-triglycerides and 14C-free cholesterol and the other with ³H-cholesteryl esters and 14C-phospholipids. These preparations were incubated with plasma samples for 1h. After chemical precipitation, the supernatant containing HDL was counted for radioactivity. RESULTS: No difference in transfer was observed to nanoemulsion HDL from cholesteryl esters, triglycerides, free cholesterol and phospholipids. CONCLUSION: Simultaneous lipid transfer to HDL was not affected in T1DM patients. This suggests that the disease does not alter lipoprotein composition and transfer protein action in such way as to disturb HDL metabolism.

Nanocrystal core high-density lipoproteins: a multimodality contrast agent platform.

High density lipoprotein (HDL) is an important natural nanoparticle that may be modified for biomedical imaging purposes. Here we developed a novel technique to create unique multimodality HDL mimicking nanoparticles by incorporation of gold, iron oxide, or quantum dot nanocrystals for computed tomography, magnetic resonance, and fluorescence imaging, respectively. By including additional labels in the corona of the particles, they were made multifunctional. The characteristics of these nanoparticles, as well as their in vitro and in vivo behavior, revealed that they closely mimic native HDL.