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Biophysical analysis of apolipoprotein E3 variants linked with development of type III hyperlipoproteinemia.

ABSTRACT: Apolipoprotein E (apoE) is a major protein of the lipoprotein transport system that plays important roles in lipid homeostasis and protection from atherosclerosis. ApoE is characterized by structural plasticity and thermodynamic instability and can undergo significant structural rearrangements as part of its biological function. Mutations in the 136-150 region of the N-terminal domain of apoE, reduce its low density lipoprotein (LDL) receptor binding capacity and have been linked with lipoprotein disorders, such as type III hyperlipoproteinemia (HLP) in humans. However, the LDL-receptor binding defects for these apoE variants do not correlate well with the severity of dyslipidemia, indicating that these variants may carry additional properties that contribute to their pathogenic potential.In this study we examined whether three type III HLP predisposing apoE3 variants, namely R136S, R145C and K146E affect the biophysical properties of the protein. Circular dichroism (CD) spectroscopy revealed that these mutations do not significantly alter the secondary structure of the protein. Thermal and chemical unfolding analysis revealed small thermodynamic alterations in each variant compared to wild-type apoE3, as well as effects in the reversibility of the unfolding transition. All variants were able to remodel multillamelar 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles, but R136S and R145C had reduced kinetics. Dynamic light scattering analysis indicated that the variant R136S exists in a higher-order oligomerization state in solution. Finally, 1-anilinonaphthalene-8-sulfonic acid (ANS) binding suggested that the variant R145C exposes a larger amount of hydrophobic surface to the solvent.Overall, our findings suggest that single amino acid changes in the functionally important region 136-150 of apoE3 can affect the molecule's stability and conformation in solution and may underlie functional consequences. However, the magnitude and the non-concerted nature of these changes, make it unlikely that they constitute a distinct unifying mechanism leading to type III HLP pathogenesis.

SUBMITTER: Georgiadou D

PROVIDER: S-EPMC3206067 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Biophysical analysis of apolipoprotein E3 variants linked with development of type III hyperlipoproteinemia.

Georgiadou Dimitra D Chroni Angeliki A Vezeridis Alexander A Zannis Vassilis I VI Stratikos Efstratios E

PloS one 20111101 11

<h4>Background</h4>Apolipoprotein E (apoE) is a major protein of the lipoprotein transport system that plays important roles in lipid homeostasis and protection from atherosclerosis. ApoE is characterized by structural plasticity and thermodynamic instability and can undergo significant structural rearrangements as part of its biological function. Mutations in the 136-150 region of the N-terminal domain of apoE, reduce its low density lipoprotein (LDL) receptor binding capacity and have been lin ...[more]

PMID: 22069485

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Project description:The development of curative glioblastoma treatments and tumour-specific contrast agents that can overcome the blood-brain barrier (BBB) and infiltrative tumour morphology remains a challenge. Apolipoprotein E3 (apoE3) is a high density lipoprotein apolipoprotein that chaperones the transcytosis of nanoparticles across the BBB, and displays high-affinity binding with the low density lipoprotein receptor (LDLR), a cell-surface receptor overexpressed by glioblastoma cells. This LDLR overexpression and apoE3 binding capacity was exploited for the development of glioblastoma-targeted porphyrin-lipid apoE3 lipid nanoparticles (pyE-LNs) with intrinsic theranostic properties. Size-controlled discoidal and cholesteryl oleate (CO)-loaded spherical pyE-LNs were synthesized through the systematic variation of particle composition, which dictated nanoparticle size and morphology. Composition optimization yielded 30 nm pyE-LNs with stable loading of apoE3 and porphyrin-lipid that simultaneously conferred the nanoparticles with glioblastoma targeting and activatable near-infrared fluorescence imaging functionalities. A 4-fold higher uptake of pyE-LNs by LDLR-expressing U87 glioblastomas cells relative to minimally expressing ldlA7 cells was observed in vitro. This uptake was a result of receptor-mediated endocytosis, which could be inhibited through LDL competition and acetylation of particle apoE3 moieties. ApoE3-dependent delivery of pyE-LN to glioblastomas was also demonstrated in orthotopic U87-GFP tumour-bearing animals. Quantification of CO-loaded pyE-LN biodistribution demonstrated successful selective uptake of porphyrin by malignant tissue, with a 4?:?1 tumour?:?healthy tissue particle specificity. This allowed for the detection of strong, tumour-localized porphyrin fluorescence, which was diminished when apoE3-devoid py-LN particles were administered. Furthermore, this selective uptake yielded cell-specific potent PDT sensitization in vitro, resulting in an 83% reduction in glioblastoma cell viability. These results highlight the promising capacity of pyE-LNs to target porphyrin delivery to glioblastoma tumours for theranostic applications.

Dataset Information

Biophysical analysis of apolipoprotein E3 variants linked with development of type III hyperlipoproteinemia.

Publications

Biophysical analysis of apolipoprotein E3 variants linked with development of type III hyperlipoproteinemia.

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