High levels of 4-hydroxynonenal (HNE), arising from lipid peroxidation, and HNE-modified proteins have been identified in postmortem brains of ageing and Alzheimer's disease (AD) patients. The goal of this study is to understand the effect of HNE modification on the structure and function of recombinant apolipoprotein E3 (apoE3) and apolipoprotein E4 (apoE4), which play a critical role in brain cholesterol homeostasis. The two isoforms differ in a single amino acid at position 112: Cys in apoE3 and Arg in apoE4. Immunoblot with HNE-specific antibody indicates HNE modification of apoE3 and apoE4 with a major band at ~36 kDa, while LC-MS/MS revealed Michael addition at His140 (60-70% abundance) and His299 (3-5% abundance) in apoE3 and apoE4, and Cys112 adduct in apoE3 (75% abundance). Circular dichroism spectroscopy revealed no major differences in the overall secondary structure or helical content between unmodified and HNE-modified apoE. HNE modification did not affect their ability to promote cholesterol efflux from J774.1 macrophages. However, it led to a 3-fold decrease in their ability to bind lipids and 25-50% decrease in the ability of cerebral cortex endothelial cells to uptake lipoproteins bearing HNE-modified HNE-apoE3 or HNE-apoE4 as noted by fluorescence microscopy and flow cytometry. Taken together, the data indicate that HNE modification impairs lipid binding and cellular uptake of both isoforms, and that apoE3, bearing a Cys, offers a protective role by sequestering lipid peroxidation products that would otherwise cause indiscriminate damage to biomolecules. ApoE4, lacking Cys, is unable to protect against oxidative damage that is commensurate with ageing.
Background 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. 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. Methods 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, guanidine hydrochloride (GdnHCl)‐induced unfolding analyses, and lipid binding assessment. Results 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. 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. Conclusions Taken 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 Information This project was supported by the National Institutes of Health (NIH) grant award GM105561 (VN), Richard D Green Fellowship (MA), Undergraduate Research Oppor...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.