Biophysical and Biochemical Characterization of Acrolein-Modified Apolipoprotein E

Abstract

Apolipoprotein E (apoE), an anti-atherogenic apolipoprotein, plays a significant role in the metabolism of lipoproteins. It lowers plasma lipid levels by acting as a ligand for low-density lipoprotein receptors (LDLr). ApoE mediates this function via essential lysine residues that interact with the LDLr. Our preliminary study shows that rats exposed to environmental tobacco smoke displayed oxidative modification of apoE and dissociation of lipoprotein-bound apoE. The objective of this project is to study the effect of oxidative stress (specifically acrolein) mediated in vitro modification on the structure and function of recombinant rat apoE. SDS-PAGE and RP-HPLC confirmed that the protein was purified to homogeneity with no signs of degradation. Acrolein modification of apoE was confirmed by Western blot analysis. Circular dichroism and fluorescence spectroscopy revealed that the secondary and tertiary structures of acrolein-modified apoE were affected with significant difference in the overall fold of the modified protein. Modified apoE also demonstrated a decrease in binding affinity for heparin and lipid binding ability. Lastly, the LDLr binding ability of acrolein-modified apoE was significantly impaired. Overall, we conclude that acrolein disrupts the structural and functional integrity of apoE, which is likely to affect its role in maintaining plasma cholesterol homeostasis. Our data provide a molecular basis for the potential role of oxidative stress mediated Modification of apoE in altering lipoprotein metabolism, with direct implications in cardiovascular disease.Funded by CSULB, TRDRP 17RT-0165 (VN), NIH HL096365 (VN), McNair Scholar Program, CSU Long Beach (TT), NSF HRD-0802628 (TT), and CSULB Women and Philanthropy scholarship (TT).