ApoE4-specific Misfolded Intermediate Identified by Molecular Dynamics Simulations.

The increased risk of developing Alzheimer's disease (AD) is associated with the APOE gene, which encodes for three variants of Apolipoprotein E, namely E2, E3, E4, differing only by two amino acids at positions 112 and 158. ApoE4 is known to be the strongest risk factor for AD onset, while ApoE3 and ApoE2 are considered to be the AD-neutral and AD-protective isoforms, respectively. It has been hypothesized that the ApoE isoforms may contribute to the development of AD by modifying the homeostasis of ApoE physiological partners and AD-related proteins in an isoform-specific fashion. Here we find that, despite the high sequence similarity among the three ApoE variants, only ApoE4 exhibits a misfolded intermediate state characterized by isoform-specific domain-domain interactions in molecular dynamics simulations. The existence of an ApoE4-specific intermediate state can contribute to the onset of AD by altering multiple cellular pathways involved in ApoE-dependent lipid transport efficiency or in AD-related protein aggregation and clearance. We present what we believe to be the first structural model of an ApoE4 misfolded intermediate state, which may serve to elucidate the molecular mechanism underlying the role of ApoE4 in AD pathogenesis. The knowledge of the structure for the ApoE4 folding intermediate provides a new platform for the rational design of alternative therapeutic strategies to fight AD.

Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy.

Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.

The Molecular Basis for Apolipoprotein E4 as the Major Risk Factor for Late-Onset Alzheimer's Disease.

Apolipoprotein E4 (ApoE4) is one of three (E2, E3 and E4) human isoforms of an α-helical, 299-amino-acid protein. Homozygosity for the ε4 allele is the major genetic risk factor for developing late-onset Alzheimer's disease (AD). ApoE2, ApoE3 and ApoE4 differ at amino acid positions 112 and 158, and these sequence variations may confer conformational differences that underlie their participation in the risk of developing AD. Here, we compared the shape, oligomerization state, conformation and stability of ApoE isoforms using a range of complementary biophysical methods including small-angle x-ray scattering, analytical ultracentrifugation, circular dichroism, x-ray fiber diffraction and transmission electron microscopy We provide an in-depth and definitive study demonstrating that all three proteins are similar in stability and conformation. However, we show that ApoE4 has a propensity to polymerize to form wavy filaments, which do not share the characteristics of cross-ß amyloid fibrils. Moreover, we provide evidence for the inhibition of ApoE4 fibril formation by ApoE3. This study shows that recombinant ApoE isoforms show no significant differences at the structural or conformational level. However, self-assembly of the ApoE4 isoform may play a role in pathogenesis, and these results open opportunities for uncovering new triggers for AD onset.

Prevention and promotion effects of apolipoprotein E4 on amylin aggregation.

The misfolding of islet amyloid polypeptide (IAPP, amylin) results in the formation of islet amyloid, which is one of the most common pathological features of type 2 diabetes (T2D). Amylin, a 37-amino-acid peptide co-secreted with insulin and apolipoprotein E (ApoE) from the beta-cells of pancreatic islets, is thought to be responsible for the reduced mass of insulin-producing beta-cells. However, neither the relationship between amylin and ApoE nor the biological consequence of amylin misfolding is known. Here we have characterized the interaction between ApoE4 and amylin in vitro. We found that ApoE4 can strongly bind to amylin, and insulin can hardly inhibit amylin-ApoE binding. We further found that amylin fibrillization can be prevented by low concentration of ApoE4 and promoted by high concentration of ApoE4. Taken together, we propose that under physiological conditions ApoE4 efficiently binds and sequesters amylin, preventing its aggregation, and in T2D the enhanced ApoE4-amylin binding leads to the critical accumulation of amylin, facilitating islet amyloid formation.