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.

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.

Reconstituting initial events during the assembly of apolipoprotein B-containing lipoproteins in a cell-free system.

The synthesis of apolipoprotein B (apoB) dictates the formation of chylomicrons and very low-density lipoproteins, two major lipoprotein precursors in the human plasma. Despite its biological significance, the mechanism of the assembly of these apoB-containing lipoproteins remains elusive. An essential obstacle is the lack of systems that allow fine dissection of key components during assembly, including nascent apoB peptide, lipids in defined forms, chaperones, and microsomal triglyceride transfer protein (MTP). In this study, we used a prokaryotic cell-free expression system to reconstitute early events in the assembly of apoB-containing lipoprotein that involve the N-terminal domains of apoB. Our study shows that N-terminal domains larger than 20.5% of apoB (B20.5) have an intrinsic ability to remodel vesicular phospholipid bilayers into discrete protein-lipid complexes. The presence of appropriate lipid substrates during apoB translation plays a pivotal role for successful lipid recruitment, and similar lipid recruitment fails to occur if the lipids are added posttranslationally. Cotranslational presence of MTP can dramatically promote the folding of B6.4-20.5 and B6.4-22. Furthermore, apoB translated in the presence of MTP retains its phospholipid recruitment capability posttranslationally. Our data suggest that during the synthesis of apoB, the N-terminal domain has a short window for intrinsic phospholipid recruitment, the time frame of which is predetermined by the environment where apoB synthesis occurs. The presence of MTP prolongs this window of time by acting as a chaperone. The absence of either proper lipid substrate or MTP may result in the improper folding of apoB and, consequently, its degradation.

Cytoplasmic lipid droplets are sites of convergence of proteasomal and autophagic degradation of apolipoprotein B.

Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make "ApoB-crescents." ApoB-crescents were seen in <10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50-100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12-24 h of proteasomal inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways.

Morphology of sodium deoxycholate-solubilized apolipoprotein B-100 using negative stain and vitreous ice electron microscopy.

The primary and secondary structures of apolipoprotein B-100 (apoB-100) are well established. Previous morphological studies have suggested that apoB is a long, flexible, threadlike molecule that encircles the low density lipoprotein (LDL) particle. Several large domain regions of the protein have been observed in frozen hydrated LDL and may be involved in anchoring of the protein to the lipid surface of LDL. Calorimetric studies of sodium deoxycholate (NaDC)-solubilized apoB indicated a similar number of independently melting domains. We therefore undertook a morphological study of NaDC-solubilized apoB-100 using negative stain and vitreous ice cryoelectron microscopy, a nonperturbing preservation technique. Negative staining experiments were performed in two ways: 1) grids were pulled through NaDC-containing buffer surfaces on which monolayers of apoB had been promoted, or 2) apoB molecules were allowed to diffuse onto carbon surfaces of grids that were floated on sample droplets. Vitrified molecules of apoB were obtained by plunging a thin fluid layer of protein adhered to a holey carbon-coated grid into supercooled ethane and by preserving the molecules in liquid nitrogen. The majority of molecules prepared in negative stain and vitreous ice were curved or arced and had alternating thin and thick regions. In negative stain, the apoB molecules lay on the grid perpendicular to the electron beam and had a mean length of 650 A. In vitreous ice the molecules were randomly oriented and their images ranged from 160 to 650 A in length. Vitrified molecules provided visualization of one or two beaded regions. Similar regions were observed in negative stain but the overall thickness was two to three times greater. Some vitrified molecules contained ribbon-like portions. Our study supports previously obtained data on molecule length but suggests that negative staining overestimates molecule width. These first images of vitrified NaDC-solubilized apoB-100 confirm the long, flexible, beaded thread morphology of the molecule and support the unique potential of this technique when coupled with proper molecule orientation and antibody labeling to correlate the tertiary structure of apoB seen in the intact particle with that of the isolated molecule.

Structural heterogeneity of apoB-containing serum lipoproteins visualized using cryo-electron microscopy.

Cryo-electron microscopy was used to analyze the structure of lipoprotein particles in density gradient subfractions of human very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL). Lipoproteins from a normolipidemic subject with relatively large and buoyant LDL (pattern A) and from a subject with a predominance of small dense LDL (pattern B) were compared. Projections of VLDL in vitreous ice were heterogeneous in size, but all were circular with a relatively even distribution of contrast. Selected projections of LDL, on the other hand, were circular with a high density ring or rectangular with two high density bands. Both circular and rectangular LDL projections decreased in average size with increasing subfraction density, but were found in all of 10 density gradient subfractions, both in pattern A and in pattern B profiles. Preparations of total IDL contained particles with the structural features of VLDL as well as particles resembling LDL. IDL particles resembling LDL were observed in specific density gradient subfractions in the denser region of the VLDL;-IDL density range. Within the group of IDL particles resembling LDL considerable heterogeneity was observed, but no structural features specific for the pattern A or pattern B lipoprotein profile were recognized. The observed structural heterogeneity of the apolipoprotein B-containing serum lipoproteins may reflect differences in the composition of these particles that may also influence their metabolic and pathologic properties.

The effect of caloric restriction on the aortic tissue of aging rats.

Connective tissue shows peculiar and complex age-related modifications, which can be, at least in part, responsible for altered functions and increased susceptibility to diseases. Food restriction has long been known to prolong life in rodents, having antiaging effects on a variety of physiologic and pathologic processes. Therefore, the aorta has been investigated in rats fed normal or hypocaloric diet, from weaning to senescence. Compared with controls, caloric-restricted animals showed less pronounced age-dependent alterations such as elastic fiber degradation, collagen accumulation and cellular modifications. Immunocytochemical analyses revealed that elastic fibers were positively labelled for biglycan, decorin, ApoB100 (LDL), ApoA1 (HDL) and elastase and that the intensity of the reactions was time- and diet-dependent. With age, the major changes affecting aortic elastic fibers were increased positivity for decorin, LDL and elastase. Compared with age-matched normal fed rats, caloric restricted animals revealed lower content of LDL, decorin and elastase and higher positivity for HDL. These data suggest that a caloric restricted diet might influence the aging process of the arterial wall in rats, delaying the appearance of age-related degenerative features, such as structural alterations of cells and matrix and modified interactions of elastin with cells and with other extracellular matrix molecules.

Calnexin and other factors that alter translocation affect the rapid binding of ubiquitin to apoB in the Sec61 complex.

Several secretory proteins, including apolipoprotein B, have been shown to undergo degradation by proteasomes. We found that the rapid degradation of nascent apolipoprotein B in HepG2 cells was diminished but not abolished by the addition of any of three different inhibitors of proteasomes. Ubiquitin is conjugated to apolipoprotein B that is not assembled with sufficient lipids either during or soon after synthesis. In addition, we found that apolipoprotein B that has entered the endoplasmic reticulum sufficiently to become glycosylated can be degraded by proteasomes. Furthermore, we detected ubiquitin-apolipoprotein B that is associated with the Sec61 complex, the major constituent of the translocational channel. Treatment of cells with monomethylethanolamine or dithiothreitol decreased the translocation of apolipoprotein B and increased the proportion of ubiquitin-conjugated molecules associated with Sec61. Conversely, treatment of cells with oleic acid, which increased the proportion of translocated apolipoprotein B, decreased the amount of ubiquitin-apolipoprotein B in the Sec61 complex. Finally, we found that inhibition of the interaction between calnexin and apolipoprotein B decreases the translocation of apolipoprotein B, increases the ubiquitin-apolipoprotein B in the Sec61 complex, and increases the proteasomal degradation of glycosylated apolipoprotein B. Thus, ubiquitin can be attached to unassembled apolipoprotein B in the Sec61 complex, and this process is affected by factors including calnexin that alter the translocation of apolipoprotein B.

Expression, secretion, and lipid-binding characterization of the N-terminal 17% of apolipoprotein B.

The N-terminal 17% of human apolipoprotein B (apoB-17) was expressed in murine C127 cells following transfection with a bovine papilloma virus-based expression vector. A permanent cell line overexpressing the expected 89-kDa protein was selected and characterized. Pulse-chase experiments showed that the depletion of intracellular apoB-17 follows an apparent first-order kinetics with t1/2 = 51 min. Under conditions of continuous labeling, greater than 60% of the total synthesized apoB-17 was secreted in a soluble form, approximately 98% lipid-poor and approximately 2% lipid-bound. Inclusion of 1.2 mM oleate resulted in 5- and 2.5-fold increases in the amount of labeled apoB-17 in the p less than 1.063 g/ml and 1.063 less than p less than 1.21 g/ml fractions, respectively, which was coordinated with increased secretion of radiolabeled core lipids, triacylglycerols, and cholesteryl esters. Thus under conditions in which lipid pools are enriched a greater fraction of apoB-17 may be secreted on lipoprotein-like particles. The lipid-poor apoB-17 present in p greater than 1.21 g/ml readily associates with exogenously added dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles to form discoidal particles. Discs formed with DMPC/apoB-17, 7:1 (wt/wt), are 239 +/- 43 A in diameter and 61 +/- 4 A thick and contain approximately 2 molecules of apoB-17 and 2250 molecules of DMPC per disc. Based on volume calculations we conclude that apoB-17 forms an annulus about one bilayer high and 10 A thick surrounding the DMPC disc. Circular dichroic spectra of apoB-17 on DMPC discs showed apoB-17 to contain 39% alpha-helix, 36% beta-sheet, 9% beta-turn, and 16% random coil. To be consistent with this model greater than 70% of apoB-17 on DMPC discs must bind to lipid. These data suggest that the N-terminal 17% of apoB-100 can bind lipid and may contribute to some extent to the stabilization of triglyceride-rich lipoproteins.

Mapping apolipoprotein B on the low density lipoprotein surface by immunoelectron microscopy.

Apolipoprotein B (apoB) was mapped using electron microscopy to visualize pairs of monoclonal antibodies binding to the low density lipoprotein (LDL) surface. The sites at which these monoclonals bind the apoB polypeptide sequence had already been established. The angular distances between all possible pairs of binding sites except one allowed the relative placement of six epitopes on the LDL sphere. We conclude that apoB extends over at least a hemisphere of the LDL surface since four epitopes are located in the Northern Hemisphere at sites arbitrarily designated as the North Pole, the Aleutian Islands, Bogotá, and in the Atlantic Ocean, while two are found in the Southern Hemisphere at Buenos Aires and at Madagascar. ApoB appears to possess a restricted flexibility, since these relative epitope locations show a substantial standard deviation in latitude and longitude. Mapping of additional epitopes may provide an answer to the question of whether apoB circumnavigates the LDL sphere.

Distribution of lipid-binding regions in human apolipoprotein B-100.

The distribution of lipid-binding regions of human apolipoprotein B-100 has been investigated by recombining proteolytic fragments of B-100 with lipids and characterizing the lipid-bound fragments by peptide mapping, amino acid sequencing, and immunoblotting. Fragments of B-100 were generated by digestion of low-density lipoproteins (LDL) in the presence of sodium decyl sulfate with either Staphylococcus aureus V8 protease, pancreatic elastase, or chymotrypsin. Particles with electron microscopic appearance of native lipoproteins formed spontaneously when detergent was removed by dialysis from enzyme digests containing fragments of B-100 and endogenous lipids, or from incubation mixtures of delipidated B-100 fragments mixed with microemulsions of exogenous lipids (cholesteryl oleate and egg phosphatidylcholine). Fractionation of the recombinant particles by isopycnic or density gradient ultracentrifugation yielded complexes similar to native LDL with respect to shape, diameter, electrophoretic mobility, and surface and core compositions. Circular dichroic spectra of these particles showed helicity similar to LDL but a somewhat decreased content of beta-structure. Most of the fragments of B-100 were capable of binding to lipids; 12 were identified by direct sequence analysis and 14 by reaction with antisera against specific sequences within B-100. Our results indicate that lipid-binding regions of B-100 are widely distributed within the protein molecule and that proteolytic fragments derived from B-100 can reassociate in vitro with lipids to form LDL-like particles.