Cellular Uptake and Clearance of Oxidatively-modified Apolipoprotein E3 by Cerebral Cortex Endothelial Cells

Apolipoprotein E3 (apoE3) plays a critical role in the metabolism of lipoproteins and lowers plasma lipid levels by serving as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins and by promoting macrophage cholesterol efflux. The current study examines the effect of acrolein (an endogenously generated metabolite and an environmental pollutant) modification on the structure and function of apoE3. Acrolein modification was confirmed in Western blots by reactivity with acrolein–lysine-specific antibody and by the presence of oligomeric species due to cross-linking. LC-MS/MS analysis revealed modification of 10 out of 12 lysines in apoE3, with Nε-(3-methylpyridinium)-lysine being the predominant form of modification, and Lys75 being a 'hot spot' in terms of susceptibility to oxidation. Circular dichroism spectroscopy showed no major change in overall secondary structure compared to unmodified apoE3. Reconstituted high density lipoprotein (HDL) bearing acrolein modified apoE3 showed loss of binding to soluble LDLr; however, incubation with mouse endothelioma bEnd.3 cells showed that it was internalized. Incubation with excess LDL did not abolish cellular uptake of acrolein modified apoE3, suggesting alternative mechanism(s) not involving LDLr. Incubation with anti-CD36 antibody did not show a decrease in internalization while incubation with anti- lectin-like oxidized LDL receptor 1 (LOX1) showed partial internalization. However, incubation with anti-scavenger receptor class B type I (SRB1) antibody abolished internalization of acrolein modified apoE3. Taken together, our studies suggest that acrolein modification of apoE3 at lysine residues leads to increase in net negative charge, and as a consequence, results in clearance by LOX1 and SRB1 on endothelial cells. Overall, oxidative modification of apoE3 likely impairs its role in regulating plasma cholesterol homeostasis, eventually leading to lipid disorders.https://doi.org/10.3390/ijms201845821422-006

Cellular Uptake and Clearance of Oxidatively-modified Apolipoprotein E3 by Cerebral Cortex Endothelial Cells

Apolipoprotein E3 (apoE3) plays a critical role in the metabolism of lipoproteins and lowers plasma lipid levels by serving as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins and by promoting macrophage cholesterol efflux. The current study examines the effect of acrolein (an endogenously generated metabolite and an environmental pollutant) modification on the structure and function of apoE3. Acrolein modification was confirmed in Western blots by reactivity with acrolein–lysine-specific antibody and by the presence of oligomeric species due to cross-linking. LC-MS/MS analysis revealed modification of 10 out of 12 lysines in apoE3, with Nε-(3-methylpyridinium)-lysine being the predominant form of modification, and Lys75 being a ‘hot spot’ in terms of susceptibility to oxidation. Circular dichroism spectroscopy showed no major change in overall secondary structure compared to unmodified apoE3. Reconstituted high density lipoprotein (HDL) bearing acrolein modified apoE3 showed loss of binding to soluble LDLr; however, incubation with mouse endothelioma bEnd.3 cells showed that it was internalized. Incubation with excess LDL did not abolish cellular uptake of acrolein modified apoE3, suggesting alternative mechanism(s) not involving LDLr. Incubation with anti-CD36 antibody did not show a decrease in internalization while incubation with anti- lectin-like oxidized LDL receptor 1 (LOX1) showed partial internalization. However, incubation with anti-scavenger receptor class B type I (SRB1) antibody abolished internalization of acrolein modified apoE3. Taken together, our studies suggest that acrolein modification of apoE3 at lysine residues leads to increase in net negative charge, and as a consequence, results in clearance by LOX1 and SRB1 on endothelial cells. Overall, oxidative modification of apoE3 likely impairs its role in regulating plasma cholesterol homeostasis, eventually leading to lipid disorders

Cellular Uptake and Clearance of Oxidatively-modified Apolipoprotein E3 by Cerebral Cortex Endothelial Cells

Apolipoprotein E3 (apoE3) plays a critical role in the metabolism of lipoproteins and lowers plasma lipid levels by serving as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins and by promoting macrophage cholesterol efflux. The current study examines the effect of acrolein (an endogenously generated metabolite and an environmental pollutant) modification on the structure and function of apoE3. Acrolein modification was confirmed in Western blots by reactivity with acrolein–lysine-specific antibody and by the presence of oligomeric species due to cross-linking. LC-MS/MS analysis revealed modification of 10 out of 12 lysines in apoE3, with Nε-(3-methylpyridinium)-lysine being the predominant form of modification, and Lys75 being a ‘hot spot’ in terms of susceptibility to oxidation. Circular dichroism spectroscopy showed no major change in overall secondary structure compared to unmodified apoE3. Reconstituted high density lipoprotein (HDL) bearing acrolein modified apoE3 showed loss of binding to soluble LDLr; however, incubation with mouse endothelioma bEnd.3 cells showed that it was internalized. Incubation with excess LDL did not abolish cellular uptake of acrolein modified apoE3, suggesting alternative mechanism(s) not involving LDLr. Incubation with anti-CD36 antibody did not show a decrease in internalization while incubation with anti- lectin-like oxidized LDL receptor 1 (LOX1) showed partial internalization. However, incubation with anti-scavenger receptor class B type I (SRB1) antibody abolished internalization of acrolein modified apoE3. Taken together, our studies suggest that acrolein modification of apoE3 at lysine residues leads to increase in net negative charge, and as a consequence, results in clearance by LOX1 and SRB1 on endothelial cells. Overall, oxidative modification of apoE3 likely impairs its role in regulating plasma cholesterol homeostasis, eventually leading to lipid disorders.</jats:p

Reconfiguring Nature’s Cholesterol Accepting Lipoproteins as Nanoparticle Platforms for Transport and Delivery of Therapeutic and Imaging Agents

Apolipoproteins are critical structural and functional components of lipoproteins, which are large supramolecular assemblies composed predominantly of lipids and proteins, and other biomolecules such as nucleic acids. A signature feature of apolipoproteins is the preponderance of amphipathic &alpha;-helical motifs that dictate their ability to make extensive non-covalent inter- or intra-molecular helix&ndash;helix interactions in lipid-free states or helix&ndash;lipid interactions with hydrophobic biomolecules in lipid-associated states. This review focuses on the latter ability of apolipoproteins, which has been capitalized on to reconstitute synthetic nanoscale binary/ternary lipoprotein complexes composed of apolipoproteins/peptides and lipids that mimic native high-density lipoproteins (HDLs) with the goal to transport drugs. It traces the historical development of our understanding of these nanostructures and how the cholesterol accepting property of HDL has been reconfigured to develop them as drug-loading platforms. The review provides the structural perspective of these platforms with different types of apolipoproteins and an overview of their synthesis. It also examines the cargo that have been loaded into the core for therapeutic and imaging purposes. Finally, it lays out the merits and challenges associated with apolipoprotein-based nanostructures with a future perspective calling for a need to develop &ldquo;zip-code&rdquo;-based delivery for therapeutic and diagnostic applications

Reconfiguring Nature’s Cholesterol Accepting Lipoproteins as Nanoparticle Platforms for Transport and Delivery of Therapeutic and Imaging Agents

Apolipoproteins are critical structural and functional components of lipoproteins, which are large supramolecular assemblies composed predominantly of lipids and proteins, and other biomolecules such as nucleic acids. A signature feature of apolipoproteins is the preponderance of amphipathic α-helical motifs that dictate their ability to make extensive non-covalent inter- or intra-molecular helix–helix interactions in lipid-free states or helix–lipid interactions with hydrophobic biomolecules in lipid-associated states. This review focuses on the latter ability of apolipoproteins, which has been capitalized on to reconstitute synthetic nanoscale binary/ternary lipoprotein complexes composed of apolipoproteins/peptides and lipids that mimic native high-density lipoproteins (HDLs) with the goal to transport drugs. It traces the historical development of our understanding of these nanostructures and how the cholesterol accepting property of HDL has been reconfigured to develop them as drug-loading platforms. The review provides the structural perspective of these platforms with different types of apolipoproteins and an overview of their synthesis. It also examines the cargo that have been loaded into the core for therapeutic and imaging purposes. Finally, it lays out the merits and challenges associated with apolipoprotein-based nanostructures with a future perspective calling for a need to develop “zip-code”-based delivery for therapeutic and diagnostic applications.</jats:p

Abstract 388: Transcellular Transport of HDL Bearing Gold Nanoparticles Across the Blood Brain Barrier

The overall objective of this study was to develop a HDL-based multifunctional platform for transport and delivery of highly hydrophobic gold nanoparticles (AuNP) bearing photothermic properties across the blood brain barrier (BBB). We exploited the ability of apolipoprotein E3 (apoE3) to act as a high affinity ligand for the low-density lipoprotein receptor to gain entry into endothelial and glioblastoma cells. The issue of poor aqueous solubility of AuNP of varying diameters (3, 10, or 10 nm) was overcome by integrating them with phospholipids and apoE3, yielding reconstituted rHDL bearing AuNP (rHDL-AuNP). Transmission electron microscopy (TEM) revealed the presence of AuNP embedded in spherical particles. Incubation of human brain microvasculature endothelial cells or glioblastoma cells with rHDL-AuNP bearing unlabeled or FITC-labeled apoE3 revealed robust uptake of particles that were localized in endocytic/lysosomal vesicles. The transport of rHDL-AuNP across an in vitro BBB model developed from primary porcine endothelial cells was examined. The addition of rHDL-AuNP to the luminal side of the cells did not affect the integrity of the BBB as assessed by the localization of key tight junction markers such as occludin, claudins and ZO-1 by immunofluorescence, and, by continual measurement of the transepithelial electrical resistance by impedance spectroscopy under physiological conditions. Lastly, the appearance of fluorescein fluorescence and AuNP in the abluminal side suggested transport of rHDL-AuNP across the neurovascular junction. These findings demonstrate that rHDL bearing apoE3 acts as a detergent in solubilizing and dramatically improving the aqueous solubility of AuNP, facilitates cellular uptake and transcellular transport of rHDL-AuNP across endothelial cells. They are significant since they present rHDL bearing apoE3 as an effective platform for delivering AuNP across the BBB. </jats:p

Swapping the N- and C-terminal domains of human apolipoprotein E3 and AI reveals insights into their structure/activity relationship.

Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma lipoprotein metabolism. ApoE3 (299 residues) is composed of an N-terminal (NT) domain bearing a 4-helix bundle and a C-terminal (CT) domain bearing a series of amphipathic α-helices. ApoAI (243 residues) also comprises a highly helical NT domain and a less structured CT tail. The objective of this study was to understand their structural and functional role by generating domain swapped chimeras: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. The bacterially overexpressed chimeras were purified by affinity chromatography and their identity confirmed by immunoblotting and mass spectrometry. Their α-helical content was comparable to that of the parent proteins. ApoE3-NT/apoAI-CT retained the denaturation profile of apoE3 NT domain, with apoAI CT tail eliciting a relatively unstructured state; its lipid binding ability improved dramatically compared to apoE3 indicative of a significant role of apoAI CT tail in lipid binding interaction. The LDL receptor interaction and ability to promote ABCA1-mediated cholesterol efflux of apoE3-NT/apoAI-CT was comparable to that of apoE3. In contrast, apoAI-NT/apoE-CT elicited an unfolding pattern and lipid binding ability that were similar to that of apoAI. As expected, DMPC/apoAI-NT/apoE-CT discoidal particles did not elicit LDLr binding ability, and promoted SR-B1 mediated cellular uptake of lipids to a limited extent. However, apoAI-NT/apoE-CT displayed an enhanced ability to promote cholesterol efflux compared to apoAI, indicative of a significant role for apoE CT domain in mediating this function. Together, these results indicate that the functional attributes of apoAI and apoE3 can be conferred on each other and that NT-CT domain interactions significantly modulate their structure and function

Swapping the N- and C-terminal domains of human apolipoprotein E3 and AI reveals insights into their structure/activity relationship.

Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma lipoprotein metabolism. ApoE3 (299 residues) is composed of an N-terminal (NT) domain bearing a 4-helix bundle and a C-terminal (CT) domain bearing a series of amphipathic α-helices. ApoAI (243 residues) also comprises a highly helical NT domain and a less structured CT tail. The objective of this study was to understand their structural and functional role by generating domain swapped chimeras: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. The bacterially overexpressed chimeras were purified by affinity chromatography and their identity confirmed by immunoblotting and mass spectrometry. Their α-helical content was comparable to that of the parent proteins. ApoE3-NT/apoAI-CT retained the denaturation profile of apoE3 NT domain, with apoAI CT tail eliciting a relatively unstructured state; its lipid binding ability improved dramatically compared to apoE3 indicative of a significant role of apoAI CT tail in lipid binding interaction. The LDL receptor interaction and ability to promote ABCA1-mediated cholesterol efflux of apoE3-NT/apoAI-CT was comparable to that of apoE3. In contrast, apoAI-NT/apoE-CT elicited an unfolding pattern and lipid binding ability that were similar to that of apoAI. As expected, DMPC/apoAI-NT/apoE-CT discoidal particles did not elicit LDLr binding ability, and promoted SR-B1 mediated cellular uptake of lipids to a limited extent. However, apoAI-NT/apoE-CT displayed an enhanced ability to promote cholesterol efflux compared to apoAI, indicative of a significant role for apoE CT domain in mediating this function. Together, these results indicate that the functional attributes of apoAI and apoE3 can be conferred on each other and that NT-CT domain interactions significantly modulate their structure and function

Spectra characteristics of rHDL and rHDL/res.

<p>(<b>A</b>) The absorbance spectra of pooled fractions of rHDL (a), rHDL/res (b), resveratrol in DMSO (c) and res/NBD in DMSO (d) were recorded. (<b>B</b>) Fluorescence emission spectra of resveratrol were recorded in: water (<b>a</b>), DMSO (<b>b</b>), 95% ethanol (<b>c</b>), isopropanol (<b>d</b>), and ethyl acetate (<b>e</b>) with dielectric constants of 80.4, 33.0, 24.3, 7.5 and 6.0, respectively, following excitation at 310 nm. (<b>C</b>). Fluorescence emission spectra of rHDL (<b>a</b>) and rHDL/res (<b>b</b>) were recorded with 10 μg protein in PBS. For comparison, emission spectrum of resveratrol in DMSO is shown (<b>c</b>).</p