Receptor-Independent Transfer of Low Density Lipoprotein Cargo to Biomembranes.

The fundamental task of lipoprotein particles is extracellular transport of cholesterol, lipids, and fatty acids. Besides, cholesterol-rich apoB-containing lipoprotein particles (i.e., low density lipoprotein LDL) are key players in progression of atherosclerotic cardiovascular disease and are associated with familial hypercholesterolemia (FH). So far, lipoprotein particle binding to the cell membrane and subsequent cargo transfer is directly linked to the lipoprotein receptors on the target cell surface. However, our observations showed that lipoprotein particle cargo transport takes place even in the absence of the receptor. This finding suggests that an alternative mechanism for lipoprotein-particle/membrane interaction, besides the receptor-mediated one, exists. Here, we combined several complementary biophysical techniques to obtain a comprehensive view on the nonreceptor mediated LDL-particle/membrane. We applied a combination of atomic force and single-molecule-sensitive fluorescence microscopy (AFM and SMFM) to investigate the LDL particle interaction with membranes of increasing complexity. We observed direct transfer of fluorescently labeled amphiphilic lipid molecules from LDL particles into the pure lipid bilayer. We further confirmed cargo transfer by fluorescence cross-correlation spectroscopy (FCCS) and spectral imaging of environment-sensitive probes. Moreover, the integration of the LDL particle into the membranes was directly visualized by high-speed atomic force microscopy (HS-AFM) and cryo-electron microscopy (cryo-EM). Overall, our data show that lipoprotein particles are able to incorporate into lipid membranes upon contact to transfer their cargo in the absence of specific receptors.

Effects of enzymes on elastic modulus of low-density lipoproteins were investigated using atomic force microscopy.

Oxidation of low-density lipoproteins (LDLs) induces development of cardiovascular disease. Recently, reports of studies using atomic force microscopy (AFM) have described that the elastic modulus of metal-induced oxidized LDLs is lower than the modulus before oxidation. However, the mechanisms of change of the elastic modulus have not been well investigated. We postulated that disorder of the LDL structure might decrease the elastic modulus. This study measured the elastic modulus of LDLs before and after enzyme treatment with V8 protease, α-chymotrypsin, and phospholipase A2. After LDLs were obtained from serum by ultracentrifugation, LDLs or enzyme-treated LDLs were physically absorbed. They were crowded on a mica surface. Although V8 protease and α-chymotrypsin did not induce the elastic modulus change, treatment with PLA2 decreased the elastic modulus. The LDL particle size did not change during the enzyme treatment. Results suggest that disordering of the lipid structure of the LDL might contribute to the elastic modulus change. Results show that AFM might be a useful tool to evaluate disorders of complex nanoscale particle structures from lipids and proteins such as lipoproteins.

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.

Structural analysis of APOB variants, p.(Arg3527Gln), p.(Arg1164Thr) and p.(Gln4494del), causing Familial Hypercholesterolaemia provides novel insights into variant pathogenicity.

Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.(Arg3527Gln) was the first mutation in APOB being identified and characterized. Recently two novel pathogenic APOB variants have been described: p.(Arg1164Thr) and p.(Gln4494del) showing impaired LDLR binding capacity, and diminished LDL uptake. The objective of this work was to analyse the structure of p.(Arg1164Thr) and p.(Gln4494del) variants to gain insight into their pathogenicity. Secondary structure of the human ApoB100 has been investigated by infrared spectroscopy (IR) and LDL particle size both by dynamic light scattering (DLS) and electron microscopy. The results show differences in secondary structure and/or in particle size of p.(Arg1164Thr) and p.(Gln4494del) variants compared with wild type. We conclude that these changes underlie the defective binding and uptake of p.(Arg1164Thr) and p.(Gln4494del) variants. Our study reveals that structural studies on pathogenic variants of APOB may provide very useful information to understand their role in FH disease.

Study of frozen low density lipoprotein particles by using nanotechnology.

OBJECTIVES: To investigate the impact of freezing in -80°C on the structure of isolated low density lipoproteins (LDLs), using nanotechnology, such as Atomic Force Microscopy (AFM). DESIGN AND METHODS: Blood EDTA plasma was obtained from healthy subject and used immediately to isolate LDL by sequential ultracentrifugation at 10°C in 55,000 rpm for 3h, using a Beckmann XL-90 ultracentrifuge (75Ti rotor), in the presence of KBr in PBS. LDLs were then diluted with PBS until final concentrations of 5 and 15 mg LDL/dl. After initial observation, samples were frozen in -80°C for two weeks and observed again after thawing. Experiments were performed in triplicate on two smooth and clean substrates of different hydrophobicity, glass (HOPG) and Si (c-Si). Statistical significance was set at 0.05. RESULTS: Macroscopically, LDL particles formed aggregations in a dendroid layout. There were no differences between images taken from both substrates (HOPG and c-Si). Frozen samples presented significantly smaller LDL particles, than fresh ones. In specific, mean diameter of LDL particle in the fresh LDL sample was 19.77 nm, ranging from 13.34 to 28.76 nm. The frozen LDL sample had a mean diameter of 5.2 nm, ranging from 2.0 to 8.0 nm, which was significantly different from the unfrozen. CONCLUSIONS: Atomic Force Microscopy showed that freezing of LDL causes alterations in their size.

PCSK9 levels in abdominally obese men: association with cardiometabolic risk profile and effects of a one-year lifestyle modification program.

OBJECTIVES: Studies performed in rodents have suggested a role for proprotein convertase subtilisin/kexin type 9 (PCSK9) in insulin resistance and impaired body fat distribution. Our objective was to examine the relationships between markers of adiposity and insulin resistance and plasma PCSK9 levels in humans. In addition, we explored the effect of a one-year lifestyle modification program on plasma PCSK9 levels in abdominally obese, dyslipidemic men. METHODS: Plasma PCSK9 levels were measured by ELISA in 175 abdominally obese, dyslipidemic sedentary men. Of these abdominally obese men, 117 non-diabetic individuals completed a one-year lifestyle modification program aiming at increasing cardiorespiratory fitness levels and improving nutritional quality. RESULTS: We found no association between plasma PCSK9 levels and body mass index, waist circumference, fat and fat-free mass, or visceral and subcutaneous adipose tissue measured by computed tomography. Compared to men with the lowest PCSK9 levels (bottom tertile), those with the highest PCSK9 levels (top tertile) had the most detrimental lipoprotein-lipid profile including lower LDL particle size (253.6 ± 4.0 vs. 251.6 ± 4.0 Å, p < 0.05) and higher apolipoprotein C-III levels (36.8 ± 10.6 vs. 32.3 ± 32.3, p < 0.05). These men were also characterized by higher HOMA-IR indices (6.78 ± 3.01 vs. 5.54 ± 2.91, p < 0.05). After one year, study participants lost on average 6.7 ± 4.6 kg (p < 0.0001). Plasma PCSK9 decreased by 9.2 ± 53.7 ng/ml (3.8%, p = 0.07). CONCLUSIONS: Plasma PCSK9 levels are not associated with body fat distribution indices, modestly associated with markers of insulin resistance and LDL particle size and are slightly affected by a lifestyle modification program in abdominally obese men.

Misfolding of apoprotein B-100, LDL aggregation and 17-ß -estradiol in atherogenesis.

The long quest for a missing mechanistic rationale accounting for the correlation between plasma cholesterol levels and cardiovascular disease (CVD) has been focused on various possible modifications of low density lipoprotein (LDL), turning this physiological cholesterol carrier into a damaging agent able to trigger atherogenesis and later the onset of the disease. In addition to the debated oxidized LDL (oxLDL), a modified LDL with a misfolded apoprotein B-100, called electronegative LDL(-) for its negative charge due to an increased amount of free fatty acids, is commonly present in plasma. LDL(-) is generated by the action of secretory calcium dependent phospholipase A2. LDL(-) primes LDL aggregation and amyloid formation according to mechanisms very similar to those observed in other misfolding diseases. The LDL particle aggregates recall the structure and size of the subendothelial lipid droplets described in early atherogenesis and elicit a powerful inflammatory response. The use of 17-ß-estradiol (E2) confirmed that the suggested atherogenicity of LDL (-) is mostly dependent on the misfolded character of its apoprotein. E2 binding to the apoprotein of native LDL, through a specific and saturable receptor, inhibits misfolding phenomenon despite an unaffected production of LDL (-) by phospholipase A2, ultimately preventing LDL aggregation. The apoprotein misfolding in LDL(-) emerges as a possible significant trigger mechanism of atherogenesis. Potential implications for the development of novel therapeutic approaches might be hypothesized in perspective. The existing evidence is discussed and reported in this review.

Whole egg consumption improves lipoprotein profiles and insulin sensitivity to a greater extent than yolk-free egg substitute in individuals with metabolic syndrome.

OBJECTIVE: We investigated if daily egg feeding, along with carbohydrate restriction, would alter lipoprotein metabolism and influence atherogenic lipoprotein profiles and insulin resistance in men and women with metabolic syndrome (MetS). METHODS: In a randomized, single-blind, parallel design, participants consumed either 3 whole eggs/day (EGG, n=20) or the equivalent amount of yolk-free egg substitute (SUB, n=17), as part of a moderately carbohydrate-restricted diet (25%-30% energy) for 12 weeks. Plasma lipids, apolipoproteins (apos), oxidized LDL (oxLDL), cholesteryl ester transfer protein (CETP) and lecithin-cholesterol acyltransferase (LCAT) activities were assessed at baseline and week 12. Lipoprotein particle concentrations and sizes were measured by nuclear magnetic resonance spectroscopy. RESULTS: Atherogenic dyslipidemia improved for all individuals as evidenced by reductions in plasma triglycerides, apoC-III, apoE, oxLDL, VLDL particle diameter, large VDL, total IDL, small LDL, and medium LDL particles (P<0.05). Furthermore, there were increases in HDL-cholesterol, large LDL and large HDL particles (P<0.05) for all individuals. However, there were greater increases in HDL-cholesterol and large HDL particles, and reductions in total VLDL and medium VLDL particles for those consuming EGG compared to SUB (P<0.05). Plasma insulin and insulin resistance (HOMA-IR) were reduced, while LCAT activity, and both HDL and LDL diameters increased over time in the EGG group only (P<0.05). CONCLUSIONS: Incorporating daily whole egg intake into a moderately carbohydrate-restricted diet provides further improvements in the atherogenic lipoprotein profile and in insulin resistance in individuals with MetS.

Three-dimensional cryoEM reconstruction of native LDL particles to 16Å resolution at physiological body temperature.

BACKGROUND: Low-density lipoprotein (LDL) particles, the major carriers of cholesterol in the human circulation, have a key role in cholesterol physiology and in the development of atherosclerosis. The most prominent structural components in LDL are the core-forming cholesteryl esters (CE) and the particle-encircling single copy of a huge, non-exchangeable protein, the apolipoprotein B-100 (apoB-100). The shape of native LDL particles and the conformation of native apoB-100 on the particles remain incompletely characterized at the physiological human body temperature (37 °C). METHODOLOGY/PRINCIPAL FINDINGS: To study native LDL particles, we applied cryo-electron microscopy to calculate 3D reconstructions of LDL particles in their hydrated state. Images of the particles vitrified at 6 °C and 37 °C resulted in reconstructions at ~16 Å resolution at both temperatures. 3D variance map analysis revealed rigid and flexible domains of lipids and apoB-100 at both temperatures. The reconstructions showed less variability at 6 °C than at 37 °C, which reflected increased order of the core CE molecules, rather than decreased mobility of the apoB-100. Compact molecular packing of the core and order in a lipid-binding domain of apoB-100 were observed at 6 °C, but not at 37 °C. At 37 °C we were able to highlight features in the LDL particles that are not clearly separable in 3D maps at 6 °C. Segmentation of apoB-100 density, fitting of lipovitellin X-ray structure, and antibody mapping, jointly revealed the approximate locations of the individual domains of apoB-100 on the surface of native LDL particles. CONCLUSIONS/SIGNIFICANCE: Our study provides molecular background for further understanding of the link between structure and function of native LDL particles at physiological body temperature.

Immuno-electron cryo-microscopy imaging reveals a looped topology of apoB at the surface of human LDL.

A single copy of apoB is the sole protein component of human LDL. ApoB is crucial for LDL particle stabilization and is the ligand for LDL receptor, through which cholesterol is delivered to cells. Dysregulation of the pathways of LDL metabolism is well documented in the pathophysiology of atherosclerosis. However, an understanding of the structure of LDL and apoB underlying these biological processes remains limited. In this study, we derived a 22 Å-resolution three-dimensional (3D) density map of LDL using cryo-electron microscopy and image reconstruction, which showed a backbone of high-density regions that encircle the LDL particle. Additional high-density belts complemented this backbone high density to enclose the edge of the LDL particle. Image reconstructions of monoclonal antibody-labeled LDL located six epitopes in five putative domains of apoB in 3D. Epitopes in the LDL receptor binding domain were located on one side of the LDL particle, and epitopes in the N-terminal and C-terminal domains of apoB were in close proximity at the front side of the particle. Such image information revealed a looped topology of apoB on the LDL surface and demonstrated the active role of apoB in maintaining the shape of the LDL particle.

Human LDL core cholesterol ester packing: three-dimensional image reconstruction and SAXS simulation studies.

Human LDL undergoes a reversible thermal order-disorder phase transition associated with the cholesterol ester packing in the lipid core. Structural changes associated with this phase transition have been shown to affect the resistance of LDL to oxidation in vitro studies. Previous electron cryo-microscopy studies have provided image evidence that the cholesterol ester is packed in three flat layers in the core at temperatures below the phase transition. To study changes in lipid packing, overall structure and particle morphology in three dimensions (3D) subsequent to the phase transition, we cryo-preserved human LDL at a temperature above phase transition (53°C) and examined the sample by electron microscopy and image reconstruction. The LDL frozen from 53°C adopted a different morphology. The central density layer was disrupted and the outer two layers formed a "disrupted shell"-shaped density, located concentrically underneath the surface density of the LDL particle. Simulation of the small angle X-ray scattering curves and comparison with published data suggested that this disrupted shell organization represents an intermediate state in the transition from isotropic to layered packing of the lipid. Thus, the results revealed, with 3D images, the lipid packing in the dynamic process of the LDL lipid-core phase transition.

Serum folate and low-density lipoprotein particle size.

AIM: Nutritional parameters, such as B-vitamins, have not been studied for an association with low-density lipoprotein (LDL) particle size. The present study explored whether serum vitamin levels, including folate and vitamin B-12, could be associated with LDL particle size. METHODS: Using a randomly selected population of 255 hospital workers, we collected detailed lipid profiles, including triglyceride (TG), high-density lipoprotein (HDL) cholesterol, and LDL particle sizes. The peak particle size of LDL was measured by density gradient ultracentrifugation and a pore gradient lipoprotein system. Serum folate and vitamin B-12 levels were measured about 1 year later and analyzed. Carotid intima-media thickness (IMT) and hepatic steatosis were diagnosed ultrasonographically, and metabolic syndrome was diagnosed using ATP III criteria. RESULTS: LDL peak particle size was significantly correlated with carotid mean IMT (r=-0.16, p=0.010). Serum folate levels were significantly and positively correlated with HDL cholesterol and negatively with TG, although the latter showed borderline significance. With increasing serum folate levels, the LDL peak particle size showed a gradual independent increase, even when adjusted for age, sex, hepatic steatosis, metabolic syndrome, and the TG/HDL cholesterol ratio. CONCLUSION: Folate may act to enhance LDL particle size. Future clinical and research work should include a study of the safe application and manipulation of folate levels in order to control LDL particle size.

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.

Molecular structure of low density lipoprotein: current status and future challenges.

This review highlights recent advances in structural studies on low density lipoprotein (LDL) with particular emphasis on the apolipoprotein moiety of LDL, apolipoprotein B100 (apoB100). Various molecular aspects of LDL are outlined and obstacles to structure determination are addressed. In this context, the prevailing conceptions of the molecular assembly of LDL and how the synergy of complementary biochemical, biophysical and molecular simulation approaches has lead to the current structural model of LDL are discussed. Evidence is presented that structural heterogeneity and the intrinsic dynamics of LDL are key determinants of the functionality of LDL in both health and disease. Some key research directions, remaining open questions and rapidly emerging new concepts for medical applications of LDL, are furthermore outlined. The article concludes by providing an outlook concerning promising future strategies for the clarification of the molecular details of LDL, in particular of apoB100, combining recent advances in molecular modeling with developments of novel experimental techniques. Although new insights into the molecular organization of LDL are forthcoming, many open questions remain unanswered. The major challenge of the next decade will certainly be the elucidation of the molecular structural and dynamic features of apoB100.

Cytokine-mediated inhibition of fibrillar amyloid-beta peptide degradation by human mononuclear phagocytes.

Vaccination therapy of AD animal models and patients strongly suggests an active role of brain mononuclear phagocytes in immune-mediated clearance of amyloid-beta peptides (Abeta) in brain. Although Abeta uptake by macrophages can be regulated by pro- and anti-inflammatory cytokines, their effects on macrophage-mediated Abeta degradation are poorly understood. To better understand this mechanism of degradation, we examined whether pro- and anti-inflammatory cytokines affect the degradation of Abeta using primary cultured human monocyte-derived macrophages (MDM) and microglia using pulse-chase analysis of fibrillar and oligomer (125)I-Abeta40 and Abeta42. Initial uptake of fibrillar Abeta40 and Abeta42 was 40% and its degradation was saturated by 120 h in both MDM and microglia, compared with an initial uptake of oligomeric Abeta less than 0.5% and saturation of degradation within 24 h. IFN-gamma increased the intracellular retention of fibrillar Abeta40 and Abeta42 by inhibiting degradation, whereas IL-4, IL-10, and TGF-beta1, but not IL-13 and IL-27, enhanced degradation. Fibrillar Abeta degradation in MDM is sensitive to lysosomal and insulin degrading enzyme inhibitors but insensitive to proteasomal and neprilysin inhibitors. IFN-gamma and TNF-alpha directly reduced the expression of insulin degrading enzyme and chaperone molecules (heat shock protein 70 and heat shock cognate protein 70), which are involved in refolding of aggregated proteins. Coculture of MDM with activated, but not naive T cells, suppressed Abeta degradation in MDM, which was partially blocked by a combination of neutralizing Abs against proinflammatory cytokines. These data suggest that proinflammatory cytokines suppress Abeta degradation in MDM, whereas select anti-inflammatory and regulatory cytokines antagonize these effects.

Cationic solid lipid nanoparticles reconstituted from low density lipoprotein components for delivery of siRNA.

Cationic solid lipid nanoparticles (SLN), reconstituted from natural components of protein-free low-density lipoprotein, were used to deliver small interfering RNA (siRNA). The cationic SLN was prepared using a modified solvent-emulsification method. The composition was 45% (w/w) cholesteryl ester, 3% (w/w) triglyceride, 10% (w/w) cholesterol, 14% (w/w) dioleoylphosphatidylethanolamine (DOPE), and 28% (w/w) 3beta-[ N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-chol). The SLN had a mean diameter of 117+/-12 nm and a surface zeta potential value of +41.76+/-2.63 mV. A reducible conjugate of siRNA and polyethylene glycol (PEG) (siRNA-PEG) was anchored onto the surface of SLN via electrostatic interactions, resulting in stable complexes in buffer solution and in even 10% serum. Under an optimal weight ratio of DC-chol of SLN and siRNA-PEG conjugate, the complexes exhibited higher gene silencing efficiency of GFP and VEGF than that of polyethylenimine (PEI) 25K with showing much reduced cell cytotoxicity. Flow cytometry results also showed that siRNA-PEG/SLN complexes were efficiently taken up by cells. Surface-modified and reconstituted protein-free LDL mimicking SLN could be utilized as noncytotoxic, serum-stable, and highly effective carriers for delivery of siRNA.

Method of imaging low density lipoproteins by atomic force microscopy.

This short paper reports a simple method to image low density lipoproteins (LDL) using atomic force microscopy (AFM). This instrument allows imaging of biological samples in liquid and presents the advantage of needing no sample preparation such as staining or fixation that may affect their general structure. Dimensions (diameter and height) of individual LDL particles were successfully measured. AFM imaging revealed that LDL have a quasi-spherical structure on the x and y axis with an oblate spheroid structure in the z axis (i.e., height). LDLs were found to have an average diameter of 23 +/- 3 nm. The obtained mean height was 10 +/- 2 nm.

Low density lipoprotein aged in plasma forms clusters resembling subendothelial droplets: aggregation via surface sites.

In early phases of atherogenesis, droplets and vesicles accumulate in the subendothelial extracellular space of arterial intima. There is much evidence to suggest that these droplets, ranging between 100 and 400 nm, derive from modified low-density lipoprotein (LDL). In investigations of the formation mechanism of these droplets, LDL fusion was previously induced in vitro by proteolysis, lipolysis, oxidation, and vigorous shaking, but all treatments failed to reproduce the size distribution range of in vivo droplets, mostly resulting, instead, in particles with a diameter intermediate between that of one and two LDL. Our approach was meant to mimic LDL aging in plasma. LDL isolated from plasma that was incubated overnight at 37 degrees C is slightly modified in the secondary structure of its protein component and is primed to form very large aggregates according to a reaction-limited mechanism. This mechanism requires interactions between selected surface sites, whereas massive fusion is ruled out. In the frame of the general theory for colloids, the aggregation of LDL aged in plasma fulfills all the requirements of the reaction-limited mechanism, encompassing 1), exponential growth; 2), fractal structure, with the dimension of elementary constituent still consistent with a single LDL; and 3), extreme polydispersity of aggregates, with shape and dimension very close to that of droplets observed in vivo.

Lipid composition influences the shape of human low density lipoprotein in vitreous ice.

Earlier cryo-electron microscopic studies have indicated that the normal low density lipoprotein (N-LDL) has a discoid shape when its core is in the liquid-crystalline state. In the present study, we investigated whether the shape of LDL depends on the physical state and/or the lipid composition of the lipoprotein core. Using a custom-built freezing device, we vitrified NLDL samples from either above or below the phase-transition temperature of the core (42 and 24 degrees C, respectively). Cryo-electron microscopy revealed no differences between these samples and indicated a discoid shape of the N-LDL particle. In contrast, TG-enriched LDL (T-LDL) did not have discoid features and appeared to be quasi-spherical in preparations that were vitrified from either 42 or 24 degrees C. These results suggest that the shape of NLDL is discoid, regardless of the physical state of its core, whereas T-LDL is more spherical. Aspects that may influence the shape of LDL are discussed.

Structural basis for thermal stability of human low-density lipoprotein.

The stability of human low-density lipoprotein (LDL), the major cholesterol carrier in plasma, was analyzed by heating samples of different concentrations at a rate from 11 to 90 K/h. Correlation of the calorimetric, circular dichroism, fluorescence, turbidity, and electron microscopic data shows that thermal disruption of LDL involves irreversible changes in the particle morphology and protein conformation but no global protein unfolding. Heating to 85 degrees C induces LDL conversion into smaller and larger particles and apparent partial dissociation, but not unfolding, of its sole protein, apoB. Further heating leads to partial unfolding of the beta-sheets in apoB and to fusion of the protein-depleted LDL into large aggregated lipid droplets, resulting in a previously unidentified high-temperature calorimetric peak. These lipid droplets resemble in size and morphology the extracellular lipid deposits formed in the arterial wall in early atherosclerosis. The strong concentration dependence of LDL fusion revealed by near-UV/visible CD, turbidity, and calorimetry indicates high reaction order, and the heating rate dependence suggests high activation energy that arises from transient disruption of lipid and/or protein packing interactions in the course of particle fusion and apparent apoB dissociation. Consequently, thermal stability of LDL is modulated by kinetic barriers. Similar barriers may confer structural integrity to LDL subclasses in vivo.