Properties Of apoE Research Articles

Characterization of a novel apolipophorin‐III/apolipoprotein E C‐terminal domain chimera

Human apolipoprotein E (apoE) is a two‐domain anti‐atherogenic lipid transport protein that mediates plasma cholesterol homeostasis by serving as a ligand for the low density lipoprotein (LDL) receptor. Of its three isoforms E3, E2, E4, the latter two have been associated with increased risk of cardiovascular and Alzheimer's disease respectively, yet all three contain an identical C‐terminal (CT) domain. The CT domain (residues 201–299) is responsible for tetramerization and has been found to initiate lipid binding that influences critical functional features of apoE. The N‐terminal (NT) domain of apoE has four α‐helices arranged in a bundle similar to apolipophorin III (apoLp‐III), a model insect apolipoprotein containing five α‐helices in one domain. To better understand the role apolipoprotein domains play in structure and function, a novel chimeric apolipoprotein was designed by attaching apoE‐CT to apoLp‐III. A disulfide bond was introduced to apoLp‐III to lock the helix bundle and eliminate its lipid binding. Recombinant apoLp‐III/apoE‐CT, apoE3, apoE‐CT, and apoLp‐III were expressed in bacterial cells, purified by affinity chromatography, and the purity verified by SDS‐PAGE. Western blot analysis using monoclonal apoE‐CT specific antibody confirmed the presence of apoE‐CT in the chimera. Crosslinking studies using dimethylsuberimidate revealed that the apoLp‐III/apoE‐CT chimera formed oligomers similar to apoE, while apoLp‐III was monomeric. Far UV circular dichroism showed increased α‐helical content, and incubation with 1‐anilinonaphthalene‐8‐sulfonic acid showed increased fluorescence intensity when apoE‐CT was added to apoLp‐III, consistent with the presence of water‐shielded dye binding exposed hydrophobic regions. Our results suggest that the chimeric protein has structural similarities to the parent proteins, and that self‐association properties of apoE can be transferred to apoLp‐III through the addition of the CT domain. Future studies will include measuring the ability of the chimera to solubilize phospholipid bilayers and binding to low density lipoproteins. The chimeric approach offers the potential to obtain insight into domain interactions and the structure‐function relationships in apolipoproteins.Support or Funding InformationThis research was supported by the National Institute of General Medical Sciences of the NIH (8UL1GM118979‐02, 8TL4GM118980‐02, 8RL5GM118978‐02, GM089564 and GM105561).

Targeted In Situ Gene Correction of DysfunctionalAPOEAlleles to Produce Atheroprotective Plasma ApoE3 Protein

Cardiovascular disease is the leading worldwide cause of death. Apolipoprotein E (ApoE) is a 34-kDa circulating glycoprotein, secreted by the liver and macrophages with pleiotropic antiatherogenic functions and hence a candidate to treat hypercholesterolaemia and atherosclerosis. Here, we describe atheroprotective properties of ApoE, though also potential proatherogenic actions, and the prevalence of dysfunctional isoforms, outline conventional gene transfer strategies, and then focus on gene correction therapeutics that can repair defective APOE alleles. In particular, we discuss the possibility and potential benefit of applying in combination two technical advances to repair aberrant APOE genes: (i) an engineered endonuclease to introduce a double-strand break (DSB) in exon 4, which contains the common, but dysfunctional, ε2 and ε4 alleles; (ii) an efficient and selectable template for homologous recombination (HR) repair, namely, an adeno-associated viral (AAV) vector, which harbours wild-type APOE sequence. This technology is applicable ex vivo, for example to target haematopoietic or induced pluripotent stem cells, and also for in vivo hepatic gene targeting. It is to be hoped that such emerging technology will eventually translate to patient therapy to reduce CVD risk.

Impact of apoE genotype on oxidative stress, inflammation and disease risk

Although in developing countries an apolipoprotein E4 (apoE4) genotype may offer an evolutionary advantage, as it has been shown to offer protection against certain infectious disease, in Westernised societies it is associated with increased morbidity and mortality, and represents a significant risk factor for cardiovascular disease, late-onset Alzheimer's disease and other chronic disorders. ApoE is an important modulator of many stages of lipoprotein metabolism and traditionally the increased risk was attributed to higher lipid levels in E4 carriers. However, more recent evidence demonstrates the multifunctional nature of the apoE protein and the fact that the impact of genotype on disease risk may be in large part due to an impact on oxidative status or the immunomodulatory/anti-inflammatory properties of apoE. An increasing number of studies in cell lines, targeted replacement rodents and human volunteers indicate higher oxidative stress and a more pro-inflammatory state associated with the epsilon4 allele. The impact of genotype on the antioxidant and immunomodulatory/anti-inflammatory properties of apoE is the focus of the current review. Furthermore, current information on the impact of environment (diet, exercise, smoking status, alcohol) on apoE genotype-phenotype associations are discussed with a view to identifying particular lifestyle strategies that could be adapted to counteract the 'at-risk' E4 genotype.

Apolipoprotein E: a cholesterol transport protein with lipid transport-independent cell signaling properties.

Apolipoprotein (apo) E is a 34-kDa glycoprotein associated with plasma lipoproteins. In contrast to other apolipoproteins, apoE is expressed ubiquitously in all tissues and appears to have a wide variety of functions in addition to lipid transport. Although the lipid transport properties of apoE are well characterized, the appreciation apoE functions other than lipid transport is still in an infancy stage. Nevertheless, there is increasing evidence that apoE regulates cell functions in a variety of tissues and organs. This review article summarizes briefly the current status of our understanding on the lipid transport-independent properties of this apolipoprotein.

Recent insights into the pathogenesis of type III hyperlipoproteinemia

Type III hyperlipoproteinemia is a genetic disorder of lipoprotein metabolism characterized by high plasma cholesterol and triglycerides, the presence of a characteristic lipoprotein termed β-very low density lipoprotein (gb-VLDL), xanthomatosis, and premature atherosclerosis. Defects in the gene for apolipoprotein (apo) E represent the molecular cause of this disorder. ApoE serves as a ligand for the receptor-mediated clearance of the triglyceride-rich lipoproteins from the circulation. All defects in apoE cause impaired lipoprotein clearance through the same mechanism, that is, the disruption, either direct or indirect, of the receptor-binding region of the apoE molecule. However, the receptor-binding defect alone does not fully explain the pathogenesis of β-VLDL formation, the development of type III hyperlipoproteinemia, and its mode of inheritance, which can be either recessive or dominant, depending on the apoE mutation. Recent studies suggest that other functional properties of apoE might play a role in the formation of β-VLDL. Modulation of the receptor-binding defect, association with VLDL, heparin-binding affinity, and interaction with lipolytic enzymes could all contribute to the expression of the disease phenotype and determine its mode of inheritance.