Abstract 406: Acrolein, a Component of Tobacco Smoke and Age-Related Oxidative Stress Disrupts Structure and Function of Apolipoprotein E

Abstract

Apolipoprotein E (apoE), an anti-atherogenic apolipoprotein, plays a critical role in regulating plasma cholesterol homeostasis and in lowering cholesterol/triglycerides level. It lowers plasma lipid levels by acting as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins. ApoE mediates this function via essential lysine residues that interact with the LDLr. Our preliminary study shows that rats exposed to second hand smoke displayed oxidative modification of apoE by acrolein, a highly reactive aldehyde, and dissociation of lipoprotein-bound apoE. From the in vivo study, we hypothesize that modification of apoE by acrolein at lysine residues will alter its conformation and disrupt its ability to interact with lipids, heparin and the LDLr. The objective of this project is to study the effect of oxidative stress mediated in vitro modification on recombinant rat apoE. To accomplish our objective, we: (i) over-expressed, isolated and purified recombinant rat apoE; (ii) carried out dose-dependent chemical modification of purified apoE with acrolein followed by mass spectrometry; (iii) performed chemical denaturant-induced unfolding to obtain information on the conformation of acrolein-modified apoE; (iv) performed lipid and heparin binding assays and assessed LDLr binding capability of modified protein. Acrolein modification of apoE was confirmed by Western blot analysis using an antibody specific for acrolein-modified lysines. Mass spectrometric analysis indicates modification by acrolein leading to FDP-lysine formation. Chemical denaturant-induced unfolding studies revealed that the overall fold of acrolein-modified apoE was impaired. Modified apoE also demonstrated a decrease in binding affinity for heparin and decreased lipid binding ability. Lastly, the LDLr binding ability of acrolein-modified apoE was significantly disrupted. 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 (due to environmental factors or aging) mediated modification of apoE in altering lipoprotein metabolism, with direct implications in cardiovascular disease.