P4‐515: Biophysical Analysis of Apolipoprotein E3 and E4 Modification by 4‐hydroxynonenal, a Lipid Peroxidation Product
Year: 2019
Type: article
Abstract: Post 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. Individual carrying the APOE e4 allele are at a higher risk of developing AD in a gene-dose dependent manner. In the present study, we report the biophysical analysis of 4-HNE modification of apoE3 and apoE4 in terms of protein fold and conformation. Recombinant 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 and guanidine hydrochloride (GdnHCl)-induced unfolding analyses. Western 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. 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. Taken together, our data indicate that there are isoform-specific differences in protein conformation and tertiary fold as a consequence of modification of apoE by 4-HNE. These 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.
Source: Alzheimer s & Dementia
Authors: Muhammad I. Abeer, Vasanthy Narayanaswami
Institution: California State University, Long Beach
Language: English
Subfield: Physiology
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W2980416674