There are three major apolipoprotein E (apoE) isoforms. Although APOE-⑀3 is considered a longevity gene, APOE-⑀4 is a dual risk factor to atherosclerosis and Alzheimer disease. We have expressed full-length and N-and C-terminal truncated apoE3 and apoE4 tailored to eliminate helix and domain interactions to unveil structural and functional disturbances. The N-terminal truncated apoE4-(72-299) and C-terminal truncated apoE4-(1-231) showed more complicated or aggregated species than those of the corresponding apoE3 counterparts. This isoformic structural variation did not exist in the presence of dihexanoylphosphatidylcholine. The C-terminal truncated apoE-(1-191) and apoE-(1-231) proteins greatly lost lipid binding ability as illustrated by the dimyristoylphosphatidylcholine turbidity clearance. The low density lipoprotein (LDL) receptor binding ability, determined by a competition binding assay of 3 H-LDL to the LDL receptor of HepG2 cells, showed that apoE4 proteins with N-terminal (apoE4-(72-299)), C-terminal (apoE4-(1-231)), or complete C-terminal truncation (apoE4-(1-191)) maintained greater receptor binding abilities than their apoE3 counterparts. The cholesterol-lowering abilities of apoE3-(72-299) and apoE3-(1-231) in apoE-deficient mice were decreased significantly. The structural preference of apoE4 to remain functional in solution may explain the enhanced opportunity of apoE4 isoform to display its pathophysiologic functions in atherosclerosis and Alzheimer disease. Human apolipoprotein E (apoE)2 is a 299-amino acid protein with a molecular mass of 34 kDa. ApoE is encoded by the three alleles (APOE-⑀2, APOE-⑀3, and APOE-⑀4) of a gene on chromosome 19q13.2 determining the three major isoforms, namely apoE2, apoE3, and apoE4 in six phenotypes (1). The three apoE isoforms differ from each other only by a single amino acid substitution involving cysteine-arginine replacement at residues 112 and 158, i.e. apoE2 (Cys 112 /Cys 158 ), apoE3 (Cys 112 / Arg 158 ), and apoE4 (Arg 112 /Arg 158 ) (Fig. 1A). Genetically, the APOE-⑀4 allele is associated with both familial late-onset and sporadic Alzheimer disease (AD) and atherosclerosis (2-4). AD patients carrying the APOE-⑀4 allele have more profound deposition of ␤-amyloid peptides (A␤) in their brains than those carrying APOE-⑀2 and APOE-⑀3 alleles (5, 6). Considerable evidence supports the view that apoE4 increases the risk of AD by accelerating the plaque formation and by impairing the neurons. ApoE4 appears to modulate amyloid precursor protein processing and A␤ production through both the LDL receptor-related protein pathway and domain interaction (7). Strong correlation of APOE-⑀4 allele with dyslipidemia and atherosclerosis, the major underlying mechanism of coronary heart disease, has been demonstrated (8). Human apoE4 represents a dual risk factor for these two major degenerative diseases.ApoE contains two independently folded domains (N-terminal domain, residues 20 -165, and C-terminal domain, residues 225-299) that are separated by a large nonstructura...

BackgroundsThere are three apolipoprotein E (apoE) isoforms involved in human lipid homeostasis. In the present study, truncated apoE2-, apoE3- and apoE4-(72-166) peptides that are tailored to lack domain interactions are expressed and elucidated the structural and functional consequences.Methods & ResultsCircular dichroism analyses indicated that their secondary structure is still well organized. Analytical ultracentrifugation analyses demonstrated that apoE-(72-166) produces more complicated species in PBS. All three isoforms were significantly dissociated in the presence of dihexanoylphosphatidylcholine. Dimyristoylphosphatidylcholine turbidity clearance assay showed that apoE4-(72-166) maintains the highest lipid-binding capacity. Finally, only apoE4-(72-166) still maintained significant LDL receptor binding ability.ConclusionsOverall, apoE4-(72-166) peptides displayed a higher lipid-binding and comparable receptor-binding ability as to full-length apoE. These findings provide the explanation of diverged functionality of truncated apoE isoforms.

The rebirth of modern analytical ultracentrifugation (AUC) began in 1990s. Since then many advanced AUC detectors have been developed that provide a vast range of versatile choices when characterizing the physical and chemical features of macromolecules. In addition, there have been remarkable advances in software that allow the analysis of AUC data using more sophisticated models, including quaternary structures, conformational changes, and biomolecular interactions. Here we report the application of AUC to protein size-and-shape distribution analysis and structure-and-function analysis in the presence of ligands or lipids. Using band-sedimentation velocity, quaternary structural changes and an enzyme's catalytic activity can be observed simultaneously. This provides direct insights into the correlation between quaternary structure and catalytic activity of the enzyme. On the other hand, also in this study, we have applied size-and-shape distribution analysis to a lipid-binding protein in either an aqueous or lipid environment. The sedimentation velocity data for the protein with or without lipid were evaluated using the c(s,f(r)) two-dimensional distribution model, which provides a precise and quantitative means of analyzing the protein's conformational changes.