Apolipoprotein (apo) E is an exchangeable apolipoprotein that plays an integral role in cholesterol transport in the plasma and the brain. It is also associated with protein misfolding or amyloid proteopathy of the beta amyloid peptide (Abeta) in Alzheimer's disease (AD) and cerebral amyloid angiopathy. The C-terminal domain (CT) of apoE encompasses two types of amphipathic alpha helices: a class A helix (residues 216-266) and a class G* helix (residues 273-299). This domain also harbors high-affinity lipoprotein binding and apoE self-association sites that possibly overlap. The objective of this study is to examine if the neurotoxic oligomeric Abeta interacts with apoE CT and if this association affects the lipoprotein binding function of recombinant human apoE CT. Site-specific fluorescence labeling of single cysteine-containing apoE CT variants with donor probes were employed to identify the binding of Abeta bearing an acceptor probe by intermolecular fluorescence resonance energy-transfer analysis. A higher efficiency of energy transfer was noted with probes located in the class A helix than with those located in the class G* helix of apoE CT. In addition, incubation of apoE CT with Abeta severely impaired the lipid binding ability and the overall amount of lipid-associated apoE CT. However, when apoE CT is present in a lipid-bound state, Abeta appears to be localized within the lipid milieu of the lipoprotein particle and not associated with any specific segments of the protein. When our data are taken together, they suggest that Abeta association compromises the fundamental lipoprotein binding function of apoE, which may have implications not only in terms of amyloid buildup but also in terms of the accumulation of cholesterol at extracellular sites.

Apolipoprotein E (apoE) is a 34-kDa resident of lipoproteins that plays a key role in cholesterol homeostasis in plasma and in brain. It is composed of an N-terminal (NT) domain (residues 1-191) and a C-terminal (CT) domain (residues 201-299). Of the three major isoforms (apoE2, -E3, and -E4), apoE4 is considered a risk factor for both cardiovascular and Alzheimer disease. Compared with apoE3, domain interaction between NT and CT domains is believed to direct the lipoprotein distribution preference of apoE4 for very low density lipoprotein-sized particles. We examined the relative disposition of apoE4 NT and CT domains in lipid-free and lipid-bound forms by monitoring pyrene excimer fluorescence emission as a direct indicator of spatial proximity. Site-specific labeling of apoE4 by N-(1-pyrene)maleimide was accomplished after substitution of Cys residues for Arg-61 in NT domain and Glu-255 in CT domain. Pyrene labeling did not alter the lipoprotein distribution pattern of apoE4 in plasma. Pyrene excimer fluorescence was noted in lipid-free pyrene-R61C/E255C/apoE4 in mixtures containing excess wildtype apoE4, which was attributed to intramolecular spatial proximity between these specified sites. Upon disruption of tertiary interaction, a large decrease in excimer fluorescence emission was noted in pyrene-R61C/ E255C/apoE4. In dimyristoylphosphatidylcholine/pyrene-R61C/E255C/apoE4 discoidal complexes, pyrene excimer fluorescence emission was retained. Taken together with fluorescence quenching and cross-linking analysis, a looped-back model of apoE4 is proposed in lipid-bound state, including spherical lipoprotein particles, wherein residues Arg-61 and Glu-255 are proximal to one another. Apolipoprotein E (apoE)1 belongs to the super family of exchangeable apolipoproteins that are major constituents of lipoproteins (1). It plays a crucial role in lipid homeostasis, not only in the plasma but also in the brain. ApoE exerts a protective effect against development of atherosclerosis (2, 3) by promoting cellular uptake (4) and clearance of triglyceride-rich lipoproteins from blood (5, 6). In addition, apoE in macrophages has a direct antiatherogenic effect independent of alterations in plasma lipoproteins (7) by promoting cholesterol efflux (8) from cell lining of arteries by a process termed reverse cholesterol transport (9 -11). The role of apoE in cholesterol homeostasis in the brain is emerging with evidence of its protective role against neuronal injury and neurodegeneration (12).In humans, apoE displays polymorphism with three major genetic variants identified in the population, apo⑀2, -⑀3, and -⑀4, with allelic frequencies of 8%, 77%, and 14%, respectively, determining six apoE phenotypes, apoE2/E2, apoE3/E3, apoE4/ E4, apoE2/E3, apoE2/E4, and apoE3/E4 (13). The apoE isoforms determine the atherogenic fate of the lipoproteins on which they reside. Although the apoE2 isoform is associated with type III hyperlipoproteinemia, peripheral atherosclerosis, and accumulation of remnant lipoproteins, apoE4 is consistent...

ApoE (apolipoprotein E) is an anti-atherogenic lipid transport protein that plays an integral role in lipoprotein metabolism and cholesterol homoeostasis. Lipid association educes critical functional features of apoE, mediating reduction in plasma and cellular cholesterol levels. The 10-kDa CT (C-terminal) domain of apoE facilitates helix-helix interactions in lipid-free state to promote apoE self-association and helix-lipid interactions during binding with lipoproteins, although the mode of lipid-binding interaction is not well understood. We investigated the mode of lipid-binding interaction and orientation of apoE CT domain on reconstituted lipoproteins. Isolated recombinant human apoE CT domain (residues 201-299) possesses a strong ability to interact with phospholipid vesicles, yielding lipoprotein particles with an apparent molecular mass of approximately 600 kDa, while retaining the overall alpha-helical content. Electron microscopy and non-denaturing PAGE analysis of DMPC (dimyristoylphosphatidylcholine)--apoE CT domain lipoprotein complexes revealed discoidal complexes with a diameter of approx. 17 nm. Cross-linking apoE CT domain on discoidal particles yielded dimeric species as the major product. Attenuated total reflectance Fourier transform IR spectroscopy of phospholipid-apoE CT domain complexes reveals that the helical axis is oriented perpendicular to fatty acyl chains of the phospholipid. Fluorescence quenching analysis of DMPC-apoE CT domain discoidal complexes by spin-labelled stearic acid indicated a relatively superficial location of the native tryptophan residues with respect to the plane of the phospholipid bilayer. Taken together, we propose that apoE CT domain interacts with phospholipid vesicles, forming a long extended helix that circumscribes the discoidal bilayer lipoprotein complex.

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