Structural and Functional Analysis of Apolipoprotein E Modification by a Lipid Peroxidation Product, 4-Hydroxynonenal

Abstract

Postmortem tissues from brains of Alzheimer's disease (AD) patients show higher levels of 4-hydroxynonenal (HNE)-modified proteins, with HNE arising as a result of oxidative stress and lipid peroxidation. The overall goal of our study is to understand the effect of HNE modification on the structure and function of apolipoprotein E3 (apoE3) and apoE4, which are cholesterol transport proteins. Individuals carrying the APOE ɛ4 allele are at a higher risk of developing AD in a gene-dose dependent manner. Recombinant apoE3 and apoE4 were modified by HNE at typical pathological concentrations (about 20 μM). Western blot with HNE-specific antibody confirmed modification of apoE3 and apoE4, with a major band at 36 kDa, while LC-MS/MS data revealed Michael adduct formation at C112 for HNE-apoE3, and H140 and H299 for both HNE-apoE3 and HNE-apoE4. Far UV-CD spectroscopy indicated that HNE-modified apoE3 and apoE4 were highly helical (60%) comparable to that of unmodified proteins (58%). A significant decrease in the intrinsic fluorescence emission was noted for both HNE-apoE3 and HNE-apoE4. Guanidine HCl-induced denaturation revealed an increased susceptibility to unfolding for HNE-apoE4, but not HNE-apoE3. HNE modification greatly reduced the lipid-binding ability of apoE4, but only modestly decreased the ability of apoE3. 1-Anilinonaphthalene-8-sulfonic acid fluorescence emission spectra showed a 10 nm red shift in the wavelength of maximal fluorescence emission for HNE-apoE4 (but not for HNE-apoE3), indicative of significant changes in exposure of hydrophobic patches upon modification. Overall, our data indicate that there are isoform-specific differences in protein conformation, tertiary fold, and functional ability as a consequence of modification of apoE by HNE. These findings are significant since they offer a potential to understand the structure-function relationship of oxidatively-modified apoE and determine its role in modulating cholesterol metabolism in AD.