S-nitrosylation of ApoE in Alzheimer's disease

Abstract

The mechanism by which apolipoprotein E (ApoE) isoforms functionally influence the risk and progression of late-onset Alzheimer's disease (LOAD) remains hitherto unknown. Herein, we present evidence that all ApoE isoforms bind to nitric oxide synthase 1 (NOS1) and that such protein-protein interaction results in S-nitrosylation of ApoE2 and ApoE3 but not ApoE4. Our structural analysis at the atomic level reveals that S-nitrosylation of ApoE2 and ApoE3 proteins may lead to conformational changes resulting in the loss of binding to low-density lipoprotein (LDL) receptors. Collectively, our data suggest that S-nitrosylation of ApoE proteins may play an important role in regulating lipid metabolism and in the pathogenesis of LOAD.

Figure 1

Protein interaction between ApoE isoforms and NOS1. (A) Co-IP of overexpressed ApoE isoforms and NOS1 in HEK-293 cells. (B) Co-localization of ApoE3 and NOS1 in human hippocampus by immunofluorescence.

Figure 2

S-nitrosylation of ApoE proteins. (A) Biotin-switch assay of recombinant human ApoE isoforms. (B) Biotin-switch assay of human hippocampus lysate.

Figure 3

Effect of S-nitrosylation on the 3D structure of human ApoE3. (A) Fully processed ApoE3, without the N-terminal signal peptide sequence (18 residues), is comprised of an N-terminal LDL receptor binding (RB) domain and a C-terminal lipid binding (LB) domain. Note that all the amino acid numbering referred to herein is based on the amino acid sequence of the fully processed ApoE (residues 1–299). (B) 3D atomic model of the wildtype (WT) RB domain of ApoE. (C) 3D atomic model of the S-nitrosothiol derivative (C112SNO) of the RB domain of ApoE. Note that in both (b) and (c), the RB domains are colored brown, while the sidechain moieties of R61, E109 and C112/C112SNO are respectively colored blue, red and green. Insets show close-up views of intramolecular interactions of C112/C112SNO with R61 and E109. (D) A schematic showing the S-nitrosylation of C112 within the RB domain of ApoE. Note that the resulting C112SNO S-nitrosothiol derivative may undergo resonance arrangement to form a zwitterion with an internal dipole characterized by the separation of a positive charge and a negative charge on sulfur and oxygen atoms, respectively. (E) A schematic showing a plausible hydrogen bonding and/or ion pairing network between the polarized S-nitrosothiol moiety of C112SNO, the guanidino group of R61 and the sidechain carboxylate of E109. The double-headed red arrows indicate potential hydrogen bonding and/or ion pairing contacts.