Apolipoprotein E3 Containing Nanodiscs as Vehicles for Transport and Targeted Delivery of Flavonoid Luteolin.

Luteolin is a flavonoid that possesses multiple beneficial biological properties, such as anticancer, antioxidant, and anti-inflammatory effects. The objective of this study is to test the hypothesis that luteolin can be transported across a cell via a nanodisc delivery system and delivered to intracellular sites. Luteolin was incorporated into reconstituted high-density lipoprotein complexes made up of apolipoprotein E3 (apoE3) N-terminal domain (apoE3NT) and 1,2-dimystrioyl-sn-glycero-3-phosphocholine. ApoE3NT confers the ability on nanodiscs to traverse the plasma membrane via low-density lipoprotein receptor or scavenger receptor-B1. Physicochemical characterization revealed that the nanodiscs were 17-22 nm in diameter as demonstrated by native polyacrylamide gel electrophoresis and dynamic lightering analysis and ∼660 kDa in size, with a luteolin content of ∼4 luteolin molecules/nanodisc. Luteolin appeared to be embedded in the nonpolar core of nanodiscs, as revealed by fluorescence quenching and polarization analysis and spectroscopic characterization. The presence of luteolin did not affect the ability of apoE3NT to mediate binding and cellular uptake of luteolin containing nanodiscs in macrophages, as inferred from immunofluorescence analysis that revealed apoE- and lipid-related fluorescence as punctate perinuclear vesicles and from flow cytometry studies. Lastly, luteolin appeared to be localized in the nucleus, having escaped the lysosomes following disassembly of the nanodiscs as suggested by fluorescence spectroscopy and microscopy analyses. Taken together, nanodiscs offer the potential to effectively transport luteolin and potentially therapeutic drugs into perinuclear sites in cells, where they can be available to enter the nucleus.

Molecular dynamics simulation of apolipoprotein E3 lipid nanodiscs.

Nanodiscs are binary discoidal complexes of a phospholipid bilayer circumscribed by belt-like helical scaffold proteins. Using coarse-grained and all-atom molecular dynamics simulations, we explore the stability, size, and structure of nanodiscs formed between the N-terminal domain of apolipoprotein E3 (apoE3-NT) and variable number of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) molecules. We study both parallel and antiparallel double-belt configurations, consisting of four proteins per nanodisc. Our simulations predict nanodiscs containing between 240 and 420 DMPC molecules to be stable. The antiparallel configurations exhibit an average of 1.6 times more amino acid interactions between protein chains and 2 times more ionic contacts, compared to the parallel configuration. With one exception, DMPC order parameters are consistently larger in the antiparallel configuration than in the parallel one. In most cases, the root mean square deviation of the positions of the protein backbone atoms is smaller in the antiparallel configuration. We further report nanodisc size, thickness, radius of gyration, and solvent accessible surface area. Combining all investigated parameters, we hypothesize the antiparallel protein configuration leading to more stable and more rigid nanodiscs than the parallel one.

Isoform-specific modification of apolipoprotein E by 4-hydroxynonenal: protective role of apolipoprotein E3 against oxidative species.

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.