Structural and Functional Analysis of Apolipoprotein E3 Modification by a Lipid Peroxidation Product, 4‐Hydroxynonenal

Abstract

BackgroundPost mortem tissues from brains of Alzheimer’s disease (AD) patients show higher levels of 4‐hydroxynonenal (4‐HNE)‐modified proteins, with 4‐HNE arising as a result of oxidative stress and lipid peroxidation. The overall goal of our study is to understand the effect of 4‐HNE modification on the structure and function of apolipoprotein E3 (apoE3) and apoE4, which are 34 kDa exchangeable apolipoprotein isoforms that play a critical role in brain cholesterol homeostasis. Individuals carrying the APOE ɛ4 allele are at a higher risk of developing AD in a gene‐dose dependent manner. In the present study, we report the biophysical and functional analyses of 4‐HNE modification of apoE3 and apoE4 in terms of protein fold and conformation.MethodsRecombinant apoE3 and apoE4 were modified by 4‐HNE at concentrations typically found in pathological tissues (~20 mM), followed by Western blot and MALDI TOF mass spectrometric analyses to confirm modification. The modified samples were then subjected to circular dichroism (CD), fluorescence (intrinsic and 1‐anilinonaphthalene‐8‐sulfonic acid (ANS) fluorescence) spectroscopic, guanidine hydrochloride (GdnHCl)‐induced unfolding analyses, and lipid binding assessment.ResultsWestern blot with 4‐HNE specific antibody confirmed modification of apoE3 and apoE4, with a major band at ~36 kDa, while mass spectrometric data revealed modification of K72 and K75. 4‐HNE‐modified apoE3 and apoE4 were highly helical (~60%) comparable to that of unmodified proteins (~58%) as revealed by far UV CD spectroscopy. A significant decrease in the intrinsic fluorescence emission was noted for both 4‐HNE‐apoE3 and 4‐HNE‐apoE4, compared to the corresponding unmodified proteins. GdnHCl‐induced denaturation monitored by changes in intrinsic fluorescence revealed a notable difference in terms of increased susceptibility to unfolding for 4‐HNE‐apoE4, but not 4‐HNE‐apoE3. 4‐HNE modification significantly impaired the ability of apoE to transform DMPC vesicles to small discoidal protein/lipid complexes. The calculated t½ (time required for initial absorbance to decrease by 50%) for apoE3 was 165.6 mins, while that for 4‐HNE‐apoE3 was 516.8 mins. Similarly, the t½ for apoE4 was 4.95 min, while that for 4‐HNE‐apoE4 was 16.3 mins. Further, ANS fluorescence emission spectra revealed a 10 nm red shift in the wavelength of maximal fluorescence emission for 4‐HNE‐apoE4 (but not for 4‐HNE‐apoE3) compared to unmodified protein.ConclusionsTaken together, 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 4‐HNE. Assessing the differences in the susceptibility to age‐related oxidative modifications aid in understanding the molecular basis for the role of apoE4 as a risk factor for AD and amyloid pathology.Support or Funding InformationThis project was supported by the National Institutes of Health (NIH) grant award GM105561 (VN), Richard D Green Fellowship (MA), Undergraduate Research Opportunity Program (AA) and Louis Stokes Alliance for Minority Participation (AA).