57 research outputs found

    Pathophysiology of Lipoprotein Oxidation

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    Molecular Dynamics Simulation of Apolipoprotein E3 Lipid Nanodiscs

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    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

    Inter-molecular coiled-coil formation in human apolipoprotein E C-terminal domain.

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    Human apolipoprotein E (apoE) is composed of an N-terminal (NT) domain (residues 1-191) that bears low-density lipoprotein receptor-binding sites, and a C-terminal (CT) domain (residues 210-299), which houses lipoprotein binding and apoE self-association sites. The NT domain is comprised of a four-helix bundle, while the structural organization of the CT domain is not known. Secondary structural algorithms predict that the apoE CT domain adopts an amphipathic alpha-helical conformation. On the basis of further sequence predictions, we identified a segment (residues 218-266) in the apoE CT domain that bears a high propensity to form a coiled-coil helix, which coincides with the putative lipoprotein-binding surface. An apoE construct bearing residues 201-299 that encompasses the entire CT domain was designed, expressed in Escherichia coli and purified by affinity chromatography. Circular dichroism (CD) spectroscopy of the apoE CT domain reveals spectra characteristic of coiled-coil helices, with the ratio of molar ellipticities at 222 nm and 208 nm ([theta](222)/[theta](208)) of 1.03. Trifluoroethanol (TFE) stabilized the secondary structure of the apoE CT domain and disrupted coiled-coil helix formation as determined by CD and tryptophan fluorescence analysis. Analytical ultracentrifugation and lysine-specific cross-linking analysis of the apoE CT domain revealed predominant formation of dimeric and tetrameric species in aqueous buffers, and monomeric forms in 50% TFE. Guanidine hydrochloride-induced denaturation studies reveal that, at low concentrations of denaturant, the apoE CT domain maintains the [theta](222)/[theta](208) ratio at approximately 1.0 and elicits an altered tertiary environment with a shift in oligomeric state towards a dimer, indicative of the role of coiled-coil helix formation in inter molecular interactions. Further, coiled-coil formation is disrupted by protonation below pH 6.0, with a corresponding decrease in Trp fluorescence emission intensity, demonstrating that salt-bridge interactions play a critical role in maintaining the structural integrity of the apoE CT domain. The data support the concept that inter molecular coiled-coil helix formation is an essential structural feature of the apoE CT domain, which likely plays a role in clustering heparin-binding sites and/or sequestering the lipid-binding surface in lipid-free states.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

    Apolipoprotein E LDL receptor-binding domain-containing high-density lipoprotein: A nanovehicle to transport curcumin, an antioxidant and anti-amyloid bioflavonoid

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    AbstractCurcumin is an antioxidant and anti-inflammatory bioflavonoid that has been recently identified as an anti-amyloid agent as well. To make it more available in its potent form as a potential amyloid disaggregation agent, we employed high-density lipoproteins (HDL), which are lipid–protein complexes that transport plasma cholesterol, to transport curcumin. The objective of this study was to employ reconstituted HDL containing human apoE3 N-terminal (NT) domain, as a vehicle to transport curcumin. The NT domain serves as a ligand to mediate binding and uptake of lipoprotein complexes via the low-density lipoprotein receptor (LDLr) family of proteins located at the cell surface. Reconstituted HDL was prepared with phospholipids and recombinant apoE3-NT domain in the absence or presence of curcumin. Non-denaturing polyacrylamide gel electrophoresis indicated that the molecular mass and Stokes' diameter of HDL bearing curcumin were ~670kDa and ~17nm, respectively, while electron microscopy revealed the presence of discoidal particles. Fluorescence emission spectra of HDL bearing (the intrinsically fluorescent) curcumin indicated that the wavelength of maximal fluorescence emission (λmax) of curcumin was ~495nm, which is highly blue-shifted compared to λmax of curcumin in solvents of varying polarity (λmax ranging from 515–575nm) or in aqueous buffers. In addition, an enormous enhancement in fluorescence emission intensity was noted in curcumin-containing HDL compared to curcumin in aqueous buffers. Curcumin fluorescence emission was quenched to a significant extent by lipid-based quenchers but not by aqueous quenchers. These observations indicate that curcumin has partitioned efficiently into the hydrophobic milieu of the phospholipid bilayer of HDL. Functional assays indicated that the LDLr-binding ability of curcumin-containing HDL with apoE3-NT is similar to that of HDL without curcumin. Taken together, we report that apoE-containing HDL has a tremendous potential as a ‘nanovehicle’ with a homing device to transport curcumin to target sites
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