Abstracts Category : Other

Structural and Functional Analysis of Apolipoprotein E3/E4 Hybrid

by Kai-Han Tu

Apolipoprotein E (apoE) is a 299 residue, exchangeable apolipoprotein that plays an essential role in cholesterol homeostasis and reverse cholesterol transport. It has anti-atherogenic properties and can exist in lipid-free and lipid-bound states. Lipid-free apoE exists predominantly as tetramers that are self-associated via the C-terminal (CT) domain oligomerization sites. In humans, the <i>APOE</i> gene is polymorphic with three isoforms: apoE2, apoE3 and apoE4. Heterozygous individuals expressing both apoE3 and apoE4 in the body represent the highest population of &epsiv;4 carriers, an allele highly associated with Alzheimers disease. The objective of this study is to determine the ability of apoE3 and apoE4 to exist as heteromeric E3/E4 hybrid complexes at the molecular level. Co-Immunoprecipitation involving His and FLAG tag apoE variants and HRP-His and HRP-FLAG antibody confirmed the presence of both apoE3 and apoE4 on isolated oligomers. Fluorescence resonance energy transfer analysis employing AEDANS, fluorescein-5-maleimide (F5M) and fluorescence quenching studies involving a spin label attached to single-cysteine apoE3 or apoE4 variants at position 291 showed spatial proximity between apoE3 and apoE4. Structural analysis by circular dichroism spectroscopy showed -helical content for E3/E4 hybrid comparable to that of parent proteins. Lastly, E3/E4 hybrid showed a significant decrease in 1,2-Dimyristoyl-<i>sn</i>-glycero-3-phosphocholine (DMPC) binding ability compared to parent proteins. These results confirm the formation of E3/E4 hybrid and lay the ground work for future functional studies as well as the implication of protein-protein oligomerization between two isoforms.