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 APOE 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 ϵ4 carriers, an allele highly associated with Alzheimer’s 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 α-helical content for E3/E4 hybrid comparable to that of parent proteins. Lastly, E3/E4 hybrid showed a significant decrease in 1,2-Dimyristoyl-sn-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.
|Commitee:||McAbee, Douglas D., Schwans, Jason|
|School:||California State University, Long Beach|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- California|
|Source:||MAI 56/06M(E), Masters Abstracts International|
|Keywords:||Apolipoprotein E3, Apolipoprotein E3/E4, Apolipoprotein E4, E3/E4 heterotetramer, E3/E4 hybrid, Heterozygote E3/E4|
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