Presentation Title

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

Faculty Mentor

Vasanthy Narayanaswami

Start Date

23-11-2019 9:15 AM

End Date

23-11-2019 9:30 AM

Location

Markstein 102

Session

oral 1

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

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. 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 will test the hypothesis that 4-HNE modification alters the structure and function of apoE in an isoform-specific manner.

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.

Western blot with 4-HNE specific antibody confirmed modification of apoE3 and apoE4, 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. 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, tertiary fold and functional ability as a consequence of modification of apoE by 4-HNE

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Nov 23rd, 9:15 AM Nov 23rd, 9:30 AM

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

Markstein 102

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 will test the hypothesis that 4-HNE modification alters the structure and function of apoE in an isoform-specific manner.

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.

Western blot with 4-HNE specific antibody confirmed modification of apoE3 and apoE4, 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. 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, tertiary fold and functional ability as a consequence of modification of apoE by 4-HNE