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Apolipoprotein E (apoE) is a 299 residue, exchangeable apolipoprotein that has essential roles in cholesterol homeostasis and reverse cholesterol transport. It is a two-domain protein with the C-terminal (CT) domain mediating protein self-association via helix-helix interactions. In humans, the APOE gene is polymorphic with three common alleles, ε2, ε3, and ε4, occurring in frequencies of ~ 5%, 77%, and 18%, respectively. Heterozygotes expressing apoE3 and apoE4 isoforms, which differ in residue at position 112 in the N-terminal domain (C112 in apoE3 and R112 in apoE4), represent the highest population of ε4 carriers, an allele highly associated with Alzheimer's disease. The objective of this study was to determine if apoE3 and apoE4 have the ability to hybridize to form a heteromer in lipid-free state. Refolding an equimolar mixture of His-apoE3 and FLAG-apoE4 (or vice versa) followed by pull-down and immunoblotting indicated formation of apoE3/apoE4 heteromers. Förster resonance energy transfer between donor fluorophore on one isoform and acceptor on the other, both located in the respective CT domains, revealed a distance of separation of ~ 46 Å between the donor/acceptor pair. Similarly, a quencher placed on one was able to mediate significant quenching of fluorescence emission on the other, indicative of spatial proximity within collisional distance between the two. ApoE3/apoE4 heteromer association was also noted in lipid-associated state in reconstituted lipoprotein particles. The possibility of heteromerization of apoE3/apoE4 bears implications in the potential mitigating role of apoE3 on the folding and physiological behavior of apoE4 and its role in maintaining cholesterol homeostasis.

Apolipoprotein E (apoE) (299 residues) is a highly helical protein that plays a critical role in cholesterol homeostasis. It comprises a four-helix bundle N-terminal (NT) and a C-terminal (CT) domain that can exist in lipid-free and lipid-associated states. In humans, there are two major apoE isoforms, apoE3 and apoE4, which differ in a single residue in the NT domain, with apoE4 strongly increasing risk of Alzheimer's disease (AD) and cardiovascular diseases (CVD). It has been proposed that the CT domain initiates rapid lipid binding, followed by a slower NT domain helix bundle opening and lipid binding to yield discoidal reconstituted high density lipoprotein (rHDL). However, the contribution of the NT domain on the CT domain organization in HDL remains poorly understood. To understand this, we employed Cys-specific cross-linking and spatially-sensitive fluorophores in the NT and CT domains of apoE3 and apoE4, and in isolated CT domain. We noted that the helices in isolated CT domain are oriented parallel to those in the neighboring molecule in rHDL, whereas full length apoE3 and apoE4 adopt either an anti-parallel or hairpin-like organization. It appears that the bulky NT domain determines the spatial organization of its CT domain in rHDL, a finding that has significance for apoE4, which is more susceptible to proteolytic cleavage in AD brains, showing increased accumulation of neurotoxic NT and CT fragments. We envisage that the structural organization of HDL apoE would have profound functional consequences in its ability to regulate cholesterol homeostasis in AD and CVD.

Apolipophorin III (apoLp-III) is an insect apolipoprotein (18 kDa) that comprises a single five-helix bundle domain. In contrast, human apolipoprotein A-I (apoA-I) is a 28 kDa two-domain protein: an α-helical N-terminal domain (residues 1-189) and a less structured C-terminal domain (residues 190-243). To better understand the apolipoprotein domain organization, a novel chimeric protein was engineered by attaching residues 179 to 243 of apoA-I to the C-terminal end of apoLp-III. The apoLp-III/apoA-I chimera was successfully expressed and purified in E. coli. Western blot analysis and mass spectrometry confirmed the presence of the C-terminal domain of apoA-I within the chimera. While parent apoLp-III did not self-associate, the chimera formed oligomers similar to apoA-I. The chimera displayed a lower α-helical content, but the stability remained similar compared to apoLp-III, consistent with the addition of a less structured domain. The chimera was able to solubilize phospholipid vesicles at a significantly higher rate compared to apoLp-III, approaching that of apoA-I. The chimera was more effective in protecting phospholipase C-treated low density lipoprotein from aggregation compared to apoLp-III. In addition, binding interaction of the chimera with phosphatidylglycerol vesicles and lipopolysaccharides was considerably improved compared to apoLp-III. Thus, addition of the C-terminal domain of apoA-I to apoLp-III created a two-domain protein, with self-association, lipid and lipopolysaccharide binding properties similar to apoA-I. The apoA-I like behavior of the chimera indicate that these properties are independent from residues residing in the N-terminal domain of apoA-I, and that they can be transferred from apoA-I to apoLp-III.

Context In end-stage renal disease (ESRD), serum high-density lipoprotein cholesterol (HDL-C) level is not an accurate predictor of mortality, partly because it does not necessarily correlate with indices of HDL function. Paraoxonase (PON) is a major enzyme constituent of HDL and a key component of HDL antioxidant activity. Apolipoprotein A-I (Apo A-1) is the core HDL structural protein that plays a major role in various aspects of HDL function. Objective We sought to examine PON activity and Apo A-I levels in patients with ESRD vs healthy controls. Design and Setting PON/arylesterase activity was measured in 499 patients with maintenance hemodialysis (MHD) and 24 healthy controls with similar distributions of age, sex, and race/ethnicity. Serum acrolein-modified Apo A-I was measured in 30 patients with MHD and 10 healthy controls. Main Outcome Measures Multilevel Cox models were used to assess associations among PON activity, Apo A-I, and HDL-C levels with 12-month all-cause mortality. Results PON activity was significantly lower in patients with MHD vs controls. Furthermore, acrolein-modified Apo A-I levels were higher in patients with MHD vs controls. In fully adjusted models, high PON activity was associated with lower 12-month mortality, whereas no difference of mortality risk was observed across HDL-C levels. The combination of high PON and low Apo A-I compared with low PON and low Apo A-I was associated with lower mortality risk. Conclusions In patients with MHD, PON activity had a stronger association with 12-month mortality than HDL-C. Future studies are needed to examine the role of these markers as potential diagnostic and therapeutic tools in ESRD.

The LDL family of receptors and its member low-density lipoprotein receptor-related protein 1 (LRP1) have classically been associated with a modulation of lipoprotein metabolism. Current studies, however, indicate diverse functions for this receptor in various aspects of cellular activities, including cell proliferation, migration, differentiation, and survival. LRP1 is essential for normal neuronal function in the adult CNS, whereas the role of LRP1 in development remained unclear. Previously, we have observed an upregulation of LewisX (LeX) glycosylated LRP1 in the stem cells of the developing cortex and demonstrated its importance for oligodendrocyte differentiation. In the current study, we show that LeX-glycosylated LRP1 is also expressed in the stem cell compartment of the developing spinal cord and has broader functions in the developing CNS. We have investigated the basic properties of LRP1 conditional knockout on the neural stem/progenitor cells (NSPCs) from the cortex and the spinal cord, created by means of Cre-loxp-mediated recombination in vitro. The functional status of LRP1-deficient cells has been studied using proliferation, differentiation, and apoptosis assays. LRP1 deficient NSPCs from both CNS regions demonstrated altered differentiation profiles. Their differentiation capacity toward oligodendrocyte progenitor cells (OPCs), mature oligodendrocytes and neurons was reduced. In contrast, astrocyte differentiation was promoted. Moreover, LRP1 deletion had a negative effect on NSPCs proliferation and survival. Our observations suggest that LRP1 facilitates NSPCs differentiation via interaction with apolipoprotein E (ApoE). Upon ApoE4 stimulation wild type NSPCs generated more oligodendrocytes, but LRP1 knockout cells showed no response. The effect of ApoE seems to be independent of cholesterol uptake, but is rather mediated by downstream MAPK and Akt activation. GLIA 2016 GLIA 2016;64:1363-1380.

Background Human apolipoprotein E3 (apoE3) is an exchangeable apolipoprotein that plays a critical role in maintaining plasma cholesterol/triglyceride homeostasis. The C-terminal (CT) domain of apoE3 (residues 201-299) is composed of amphipathic α-helices C1: W210-S223, C2: V236-E266, and C3: D271-W276, which play a dominant role in mediating high-affinity lipid binding. Objective The objective is to understand the accessibility of the CT domain at the sub-domain level and the mechanistic details regarding lipid-binding interaction. Methods Hydrogen-deuterium exchange coupled to mass spectrometry (HDX/MS) of recombinant wild type (WT) apoE(201-299), chemical-induced unfolding monitored as changes in fluorescence polarization (FP) of labeled apoE(201-299) bearing a probe at specified sites, and lipid binding studies were carried out. Results HDX/MS revealed that residues towards the C-terminal end of the domain display significantly lower %D uptake compared to those towards the center, suggesting extensive protein-protein interaction in this segment. Functional assays showed that locking apoE(201-299) in an inter-molecular disulfide-bonded state at position 209, 223, 255, or 277 significantly decreases its ability to interact with lipids, especially when tethered towards the ends; this could be restored by reduction. Unfolding studies indicate that the C-terminal end offers less resistance to unfolding compared to the central portion of the domain. Conclusion Taken together, our data suggest that two dimers of CT domain are juxtaposed around helix C3 leading to apoE3 tetramerization, and that dissociation to monomeric units is a required step in lipid binding, with helix C3 likely seeking stability via lipid interaction prior to helices C1 or C2.

Aim: Cotinine is known as a stable metabolite of nicotine, the plasma half-life is over 20-30 hrs. Here, we evaluated its characteristic properties on the apoA-I mediated HDL generation. Method: Cotinine (5.6 to 11.2μM), or nicotine (5 to 150μM) were supplemented to medium and co-incubated with apoA-I (10μg/mL) for 18 hrs with THP-1 cells to determine their effect on efflux activity. Cellular ACAT accessible cholesterol pool size was determined by [ 14 C]oleic acid labeling of cells for 60 min immediately after removal of the culture medium. The culture medium and the cellular lipids were extracted by organic solvents and cholesterol and phospholipids content were determined. Nicotine was administered orally as an aqueous solution (10 μM) to C57BL/6 mice for 10 days and the plasma lipoprotein profiles were measured by gel-HPLC (tandem TSK-lipopropak XL ) with online enzymatic lipid assay system (Skylight biotech, Inc.). Results: Supplementation of nicotine into the medium showed no effect on the apoA-I mediated cellular cholesterol release from THP-1 cells. On the other hand, cotinine increased apoA-I mediated cellular cholesterol efflux 2.8 fold. ACAT accessible cholesterol pool size was dramatically decreased to 80% and 58% of control with 5 and 10 μM cotinine treatment, respectively, indicating that cotinine effectively increased apoA-I/ABCA1 mediated cellular cholesterol efflux. Mice with oral nicotine administration showed increased plasma HDL by 11.4% (50.1±4.4 and 55.8±3.9 mg/dL for control and nicotine treatment, respectively). Conclusions: Cotinine increased apoA-I mediated cellular cholesterol efflux in THP-1 cells. The cellular ACAT accessible cholesterol pool size reduction is one of the early reactions of apoA-I/ABCA1 mediated cellular cholesterol efflux. Cotinine seemingly modifies cellular factor/factors to induce a change in cholesterol distribution. Mice administrated oral nicotine may convert the compound to cotinine by CYP2A6 and aldehyde oxidase activities in their liver, and which likely increased HDL generation. Further experiments are necessary to examine the direct effect of cotinine on the ABCA1 transporter for increasing HDL assembly system.

Apolipoprotein E3 (apoE3) is an exchangeable apolipoprotein that plays a critical role in cholesterol homeostasis. The N-terminal (NT) domain of apoE3 (residues 1–191) is folded into a helix bundle comprised of 4 amphipathic α-helices: H1, H2, H3 and H4, flanked by flexible helices N1 and N2, and Hinge Helix 1 (Hinge H1), at the N-and C-terminal sides of the helix bundle, respectively. The NT domain plays a critical role in binding to the low density lipoprotein receptor (LDLR), which eventually leads to lowering of plasma cholesterol levels. In order to be recognized by the LDLR, the helix bundle has to open and undergo a conformational change. The objective of the study was to understand the mechanism of opening of the helix bundle. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) revealed that apoE3 NT domain adopts several disordered and unfolded regions, with H2 exhibiting relatively little protection against exchange-in compared to H1, H3, and H4. Site-directed fluorescence labeling indicated that H2 not only has the highest degree of solvent exposure but also the most flexibility in the helix bundle. It also indicated that the lipoprotein behavior of H1 was significnatly different from that of H2, H3 and H4. These results suggest that the opening of the helix bundle is likely initiated at the flexible end of H2 and the loop linking H2/H3, and involves movement of H2/H3 away from H1/H4. Together, these observations offer mechanistic insight suggesting a regulated helix bundle opening of apoE3 NT domain can be triggered by lipid binding.

Skylar T Chuang, Young-Seok Shon, Vasanthy Narayanaswami Department of Chemistry and Biochemistry, California State University Long Beach, Long Beach, CA, USA Abstract: We have developed a high-density lipoprotein (HDL)-based platform for transport and delivery of hydrophobic gold nanoparticles (AuNPs). The ability of apolipoprotein E3 (apoE3) to act as a high-affinity ligand for the low-density lipoprotein receptor (LDLr) was exploited to gain entry of HDL with AuNPs into glioblastoma cells. AuNPs of 3, 10, and 17nm diameter, the latter two synthesized by phase transfer process, were solubilized by integration with phospholipids and apoE3, yielding reconstituted HDL (rHDL) bearing AuNPs. Ultraviolet–visible spectra of rHDL-AuNP indicated the presence of stable particles with surface plasmon band at~530 nm. Transmission electron microscopy (TEM) of rHDL-AuNP revealed roughly spherical particles with AuNPs embedded in the core. The rHDL-AuNP particles displayed robust binding to the LDLr and were internalized by receptor-mediated endocytosis in glioblastoma cells. Confocal microscopy confirmed cellular uptake of AuNPs in the endosomal–lysosomal compartments, while TEM revealed intracellular aggregated AuNPs. Cell viability assay demonstrated that>85% of cells were viable with rHDL-AuNP treatment of 0.1–100 µg/mL for 24hours. These findings are significant since they offer an effective means of delivering AuNPs across the cell membrane, which is particularly relevant in tumor cells that overexpress LDLr. Keywords: apolipoprotein E, gold nanoparticles, lipoproteins, HDL, cancer

Apolipoprotein E3 (apoE3) is a 34 kDa exchangeable apolipoprotein critical for lipid transport and associates with chylomicrons, VLDL, and a subclass of HDL. The protein contains two independently folded domains: a 22 kDa N-terminal domain and a 10 kDa C-terminal domain. The N-terminal domain contains LDL receptor binding sites and is tightly folded into a 4-helix bundle. The smaller C-terminal (CT) domain contains helical segments that: (i) promote protein-protein interaction and apoE3 self-association, (ii) bear high lipid binding affinity, and, (iii) facilitate cholesterol efflux from macrophages. In contrast, insect apolipophorin III (apoLp-III) is a one-domain protein bearing a 5-helix bundle, does not self-associate and binds to diacylglycerol enriched lipoproteins. To better understand the domain organization of apolipoproteins, a chimeric protein was constructed of apoLp-III and the CT domain of apoE3 (residues 201-299). The structure and function of the 28 kDa apoLp-III/CT-apoE3 chimera was determined and compared to that of parent proteins apoLp-III and apoE3. The novel chimeric construct was expressed in E. coli and purified using an N-terminal poly-histidine tag. Electrophoresis and Western blot analysis showed that the apoLp-III/CT-apoE3 chimera was of the expected size and reacted with apoE3 specific antibodies. Circular dichroism demonstrated that the chimera was α-helical, similar to both parent proteins. While apoLp-III was monomeric, the chimera formed tetramers similar to apoE3. Probing for hydrophobic pockets on the protein surface with anilinonaphthalene-8-sulfonic acid demonstrated that the CT domain provided increased binding sites for the probe. Addition of the CT domain to apoLp-III conferred an increased ability to promote cholesterol efflux from J774 macrophages, comparable to that of parent apoE3. Thus, the fold of apoLp-III/CT-apoE3 chimera was similar to that of apoE3, promoted self-association and cholesterol efflux. This demonstrated that functional characteristics of CT domain of apoE3 can be transferred independently to another apolipoprotein, allowing us to further increase our understanding of apolipoprotein domains.

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.

Apolipoprotein E3 (apoE3) is an anti-atherogenic apolipoprotein with the ability to exist in lipid-free and lipoprotein-associated states. During atherosclerosis, its function in promoting cholesterol efflux from macrophages via the ATP-binding cassette transporter A1 (ABCA1) takes a prominent role, leading to generation of nascent high density lipoprotein (nHDL) particles. The objective of this study is to understand the conformation adopted by apoE3 in macrophage-generated nHDL using a fluorescence spectroscopic approach involving pyrene. Pyrene-labeled recombinant human apoE3 displayed a robust ability to stimulate ABCA1-mediated cholesterol efflux from cholesterol-loaded J774 macrophages (which do not express apoE), comparable to that elicited by unlabeled apoE3. The nHDL recovered from the conditioned medium revealed the presence of apoE3 by immunoblot analysis. A heterogeneous population of nHDL bearing exogenously added apoE3 was generated with particle size varying from ~ 12 to ~19 nm in diameter, corresponding to molecular mass of ~ 450 to ~700 kDa. The lipid: apoE3 ratio varied from ~60:1 to 10:1. A significant extent of pyrene excimer emission was noted in nHDL, indicative of spatial proximity between Cys112 on neighboring apoE3 molecules similar to that noted in reconstituted HDL. Cross-linking analysis using Cys-specific cross-linkers revealed the predominant presence of dimers. Taken together the data indicate a double belt arrangement of apoE molecules on nHDL. A similar organization of the C-terminal tail of apoE on nHDL was noted when pyrene-apoEA277C(201-299) was used as the cholesterol acceptor. These studies open up the possibility of using exogenously labeled apoE3 to generate nHDL for structural and conformational analysis.

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&ndash, 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 N&epsilon, (3-methylpyridinium)-lysine being the predominant form of modification, and Lys75 being a &lsquo, hot spot&rsquo, 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.

ATI-5261 is a novel, single-helix peptide that stimulates cellular cholesterol efflux with high potency similar to native apolipoproteins on a molar basis. Presently we investigated structural features of the peptide that conferred cholesterol efflux activity. Analogs of ATI-5261 with amino acids arranged in reverse order or with individual arginine (R) to glutamine (Q) substitutions (i.e. R3Q, R14Q, or R23Q) stimulated ABCA1 dependent cholesterol efflux similar to ATI-5261. Consequently, neither the presence of specific positively charged residues nor their specific arrangement along the length of the peptide was necessary for mediating cholesterol efflux. Similarly, peptides composed of all d-amino acids stimulated cholesterol efflux efficiently, indicating a stereospecific component was not required for promotion of cholesterol efflux from macrophages. Removal of two or more positively charged residues (R3, 14→Q and R3, 14, 23→Q) however, greatly reduced the ability of ATI-5261 to mediate cellular cholesterol efflux. This was accompanied by a loss of α-helical structure upon dilution, indicating the secondary structure of individual peptide strands was important for stimulating cholesterol efflux. Surprisingly, peptides with removal of two or more positively charged residues retained the ability to bind phospholipid and adopt an α-helical structure. These data indicate that the propensity of a hydrophobic peptide to form an amphipathic α-helix is not sufficient to mediate cellular cholesterol efflux. Efficient stimulation of cholesterol efflux requires that ATI-5261 retain α-helical structure upon dilution.

© 2017 Lek et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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.

We have developed a novel HDL-based multifunctional platform for transport and delivery of highly hydrophobic gold nanoparticles (AuNP) bearing photothermic properties. We exploit the ability of apolipoprotein E3 (apoE3) to act as a high affinity ligand for the low-density lipoprotein receptor (LDLr) to gain entry into glioblastoma cells. The issue of poor aqueous solubility of AuNP was overcome by integrating them with phospholipids and apoE3, yielding reconstituted rHDL bearing 3, 10, or 10 nm AuNP. UV-Vis spectra of rHDL-AuNP indicated the presence of stable particles with surface plasmon band at ~530 nm, a signature feature of AuNP. Transmission electron microscopy (TEM) revealed discoidal geometry for rHDL with 3, 10 and 17 nm particles. Co-immunoprecipitation assay using a soluble form of the LDLr indicated robust binding of rHDL-AuNP to the receptor. Immunofluorescence analysis indicated that all 3 rHDL particles were internalized by glioblastoma cells, as revealed by the presence of punctate, peri-nuclear endocytic/lysosomal vesicles; this suggests cellular uptake of rHDL-AuNP by receptor-mediated endocytosis. Cellular uptake was further confirmed by TEM, in which aggregated AuNP were found in the endosomal-lysosomal compartments. Finally, cytotoxicity studies demonstrated that more than 50% of cells were viable with rHDL-AuNP treatment of up to 0.1 mg/ml for 24 h. The findings that apoE3: (i) acts as a detergent in solubilizing and dramatically improving the aqueous solubility of AuNP, and, (ii) facilitates cellular uptake of rHDL-AuNP by the LDLr pathway, are significant since they offer an effective means of delivering AuNP across the cell membrane. This is particularly relevant in tumor cells since they overexpress LDLr to meet the high demand for cholesterol that is required for rapid proliferation and membrane biogenesis.

Apolipoproteins (apo) A-I and E are exchangeable apolipoproteins that play a dominant role in regulating lipoprotein metabolism. They are composed of a series of amphipathic α-helices, which are organized into an N-terminal (NT) helix bundle domain and a smaller C-terminal (CT) domain. The objective of the current study is to understand the functional and structural role of the domains of these proteins by “domain swapping” using apoE3 and apoAI, both of which bear a 4-helix bundle in their NT domain, and apolipophorin III (apoLp-III), a monomeric one-domain model apolipoprotein. A series of chimeric apolipoproteins were generate: apoE3-NT/apoAI-CT, apoAI-NT/apoE-CT and apoLp-III/apoA-I-CT. The α-helical content of the chimeras was comparable to that of the parent proteins. Unfolding studies indicated that addition of CT-apoE3 to NT-apoAI conferred more stability to apoAI. While addition of CT-apoAI to NT-apoE3 had no significant effect on the stability of apoE3, addition of NT-apoAI to apoLp-III increased the stability of apoLp-III. Moreover, apoLp-III switched from a monomeric to an oligomeric protein upon addition of CT-apoAI, showing that CT-apoAI has a strong tendency to self-associate. Addition of CT-apoAI to NT-apoE or apoLp-III increased the lipid binding activity by ~ 10-fold, while addition of apoE-CT to apoA-I-NT had no measurable effect on the lipid binding activity. All three chimeras promote ABCA1-mediated cholesterol efflux from J774 macrophages, with the efflux capability being highest for apoAI-NT/apoE-CT. Whereas apoE3-NT/apoAI-CT elicits LDLr binding ability, apoAI-NT/apoE-CT did not. These results suggest that CT of apoAI is a strong promoter of lipid binding, while CT of apoE3 can improve cholesterol efflux ability of apoAI.

Pyrene is a spatially sensitive probe that displays an ensemble of monomeric fluorescence emission peaks (375 – 405 nm), and an additional band (called excimer) at ~460 nm when two fluorophores are spatially proximal. We examined if there is a correlation between distance between two pyrenes on an α-helical structure and excimer/monomer (e/m) ratio. Using structure-guided design, pyrene maleimide was attached to pairs of Cys located in increments of ~ 5 Å apart on helix 2 of the N-terminal domain of apolipoprotein E3 (apoE3). Fluorescence spectral analysis revealed an intense excimer band when the probes were ~ 5 Å from each other with an e/m ratio of ~3.0, which decreased to ~1.0 at 20 Å. An inverse correlation between e/m ratio and the distance between pyrenes was observed, with the probe and helix flexibility also contributing to the extent of excimer formation. We verified this approach by estimating the distance between T57C and C112 (located on helices 2 and 3, respectively) to be 5.2 Å (4.9 Å from NMR and 5.7 Å from X-ray structure). Excimer formation was also noted to a significant extent with probes located in the linker segment suggesting spatial proximity (10 Å to 15 Å) from corresponding sites on neighboring molecules in the tetrameric configuration of apoE. We infer that oligomerization via the C-terminal domain juxtaposes the linker segments from neighboring apoE molecules. This study offers new insights into the conformation of tetrameric apoE and presents the use of pyrene as a powerful probe to study protein spatial organization.

The review focuses on the unique spectral features of pyrene that can be utilized to investigate protein structure and conformation. Pyrene is a fluorescent probe that can be attached covalently to protein side chains, such as sulfhydryl groups. The spectral features of pyrene are exquisitely sensitive to the microenvironment of the probe: it exhibits an ensemble of monomer fluorescence emission peaks that report on the polarity of the probe microenvironment, and an additional band at longer wavelengths, the appearance of which reflects the presence of another pyrene molecule in spatial proximity (~10 A). Its high extinction coefficient allows us to study labeled proteins in solution at physiologically relevant concentrations. The environmentally- and spatially-sensitive features of pyrene allow monitoring protein conformation, conformational changes, protein folding and unfolding, protein-protein, protein-lipid and protein-membrane interactions.

ATI-5261 is a 26-mer peptide that stimulates cellular cholesterol efflux with high potency. This peptide displays high aqueous solubility, despite having amphipathic α-helix structure and a broad non-polar surface. These features suggested to us that ATI-5261 may adopt a specific form in solution, having favorable structural characteristics and dynamics. To test this, we subjected ATI-5261 to a series of biophysical studies and correlated self-association with secondary structure and activity. Gel-filtration chromatography and native gel electrophoresis indicated ATI-5261 adopted a discrete self-associated form of low molecular weight at concentrations > 1 mg/ml. Formation of a discrete molecular species was verified by small angle X-ray scattering (SAXS), which further revealed the peptide formed a tetrameric assembly having an elongated shape and hollow central core. This assembly dissociated to individual peptide strands upon dilution to concentrations required for promoting high-affinity cholesterol efflux from cells. Moreover, the α-helical content of ATI-5261 was exceptionally high (74.1±6.8%) regardless of physical form and concentration. Collectively, these results indicate ATI-5261 displays oligomeric behavior generally similar to native apolipoproteins and dissociates to monomers of high α-helical content upon dilution. Optimizing self-association behavior and secondary structure may prove useful for improving the translatability and efficacy of apolipoprotein mimetic peptides.

Curcumin 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.

Apolipoproteins are the protein components of lipoproteins that have the innate ability to inter convert between a lipid-free and a lipid-bound form in a facile manner, a remarkable property conferred by the helix bundle motif. Composed of a series of four or five amphipathic alpha-helices that fold to form a helix bundle, this motif allows the en face orientation of the hydrophobic faces of the alpha-helices in the protein interior in the lipid-free state. A conformational switch then permits helix-helix interactions to be substituted by helix-lipid interactions upon lipid binding interaction. This review compares the apolipoprotein high-resolution structures and the factors that trigger this switch in insect apolipophorin III and the mammalian apolipoproteins, apolipoprotein E and apolipoprotein A-I, pointing out the commonalities and key differences in the mode of lipid interaction. Further insights into the lipid-bound conformation of apolipoproteins are required to fully understand their functional role under physiological conditions.

Apolipoprotein E was found to protect against the neurotoxic effects of a dimeric peptide derived from the receptor-binding region of this protein (residues 141–149). Both apoE3 and apoE4 conferred protection but the major N-terminal fragment of each isoform did not. Nor was significant protection provided by bovine serum albumin or apoA-I. Full-length apoE3 and apoE4 also inhibited the uptake of a fluorescent-labeled derivative of the peptide, suggesting that the mechanism of inhibition might involve competition for cell surface receptors/proteoglycans that mediate endocytosis and/or signaling pathways. These results might bear on the question of the role of apoE in neuronal degeneration, such as occurs in Alzheimer’s disease where apoE4 confers a significantly greater risk of pathology.

Apolipoprotein (apo) E is an exchangeable apolipoprotein that plays an integral role in cholesterol transport in the plasma and the brain. It is also associated with protein misfolding or amyloid proteopathy of the beta amyloid peptide (Abeta) in Alzheimer's disease (AD) and cerebral amyloid angiopathy. The C-terminal domain (CT) of apoE encompasses two types of amphipathic alpha helices: a class A helix (residues 216-266) and a class G* helix (residues 273-299). This domain also harbors high-affinity lipoprotein binding and apoE self-association sites that possibly overlap. The objective of this study is to examine if the neurotoxic oligomeric Abeta interacts with apoE CT and if this association affects the lipoprotein binding function of recombinant human apoE CT. Site-specific fluorescence labeling of single cysteine-containing apoE CT variants with donor probes were employed to identify the binding of Abeta bearing an acceptor probe by intermolecular fluorescence resonance energy-transfer analysis. A higher efficiency of energy transfer was noted with probes located in the class A helix than with those located in the class G* helix of apoE CT. In addition, incubation of apoE CT with Abeta severely impaired the lipid binding ability and the overall amount of lipid-associated apoE CT. However, when apoE CT is present in a lipid-bound state, Abeta appears to be localized within the lipid milieu of the lipoprotein particle and not associated with any specific segments of the protein. When our data are taken together, they suggest that Abeta association compromises the fundamental lipoprotein binding function of apoE, which may have implications not only in terms of amyloid buildup but also in terms of the accumulation of cholesterol at extracellular sites.

Cholesterol can be detrimental or vital, and must be present in the right place at the right time and in the right amount. This is well known in the heart and the vascular system. However, in the CNS cholesterol is still an enigma, although several of its fundamental functions in the brain have been identified. Brain cholesterol has attracted additional attention owing to its close connection to ApoE, a key polymorphic transporter of extracellular cholesterol in humans. Indeed, both cholesterol and ApoE are so critical to fundamental activities of the brain, that the brain regulates their synthesis autonomously. Yet, similar control mechanisms of ApoE and cholesterol homeostasis may exist on either sides of the blood-brain barrier. One indication is that the APOE ε4 allele is associated with hypercholesterolemia and a proatherogenic profile on the vascular side and with increased risk of Alzheimer's disease on the CNS side. In this review, we draw attention to the association between cholesterol and ApoE in the aging and diseased brain, and to the behavior of the ApoE4 protein at the molecular level. The attempt to correlate in vivo and in vitro observations is challenging but crucial for developing future strategies to address ApoE-related aberrations in cholesterol metabolism selectively in the brain.

This study was undertaken to identify the alpha-helical domains of human apoE that mediate cellular cholesterol efflux and HDL assembly via ATP-binding cassette transporter A1 (ABCA1). The C-terminal (CT) domain (residues 222-299) of apoE was found to stimulate ABCA1-dependent cholesterol efflux in a manner similar to that of intact apoE2, -E3, and -E4 in studies using J774 macrophages and HeLa cells. The N-terminal (NT) four-helix bundle domain (residues 1-191) was a relatively poor mediator of cholesterol efflux. On a per molecule basis, the CT domain stimulated cholesterol efflux with the same efficiency (Km approximately 0.2 microM) as intact apoA-I and apoE. Gel filtration chromatography of conditioned medium from ABCA1-expressing J774 cells revealed that, like the intact apoE isoforms, the CT domain promoted the assembly of HDL particles with diameters of 8 and 13 nm. Removal of the CT domain abolished the formation of HDL-sized particles, and only larger particles eluting in the void volume were formed. Studies with CT truncation mutants of apoE3 and peptides indicated that hydrophobic helical segments governed the efficiency of cellular cholesterol efflux and that conjoined class A and G amphipathic alpha-helices were required for optimal efflux activity. Collectively, the data suggest that the CT lipid-binding domain of apoE encompassing amino acids 222-299 is necessary and sufficient for mediating ABCA1 lipid efflux and HDL particle assembly.

The LDL family of receptors and its member low-density lipoprotein receptor-related protein 1 (LRP1) have classically been associated with a modulation of lipoprotein metabolism. Current studies, however, indicate diverse functions for this receptor in various aspects of cellular activities, including cell proliferation, migration, differentiation, and survival. LRP1 is essential for normal neuronal function in the adult CNS, whereas the role of LRP1 in development remained unclear. Previously, we have observed an upregulation of LewisX (LeX) glycosylated LRP1 in the stem cells of the developing cortex and demonstrated its importance for oligodendrocyte differentiation. In the current study, we show that LeX-glycosylated LRP1 is also expressed in the stem cell compartment of the developing spinal cord and has broader functions in the developing CNS. We have investigated the basic properties of LRP1 conditional knockout on the neural stem/progenitor cells (NSPCs) from the cortex and the spinal cord, created by means of Cre-loxp-mediated recombination in vitro. The functional status of LRP1-deficient cells has been studied using proliferation, differentiation, and apoptosis assays. LRP1 deficient NSPCs from both CNS regions demonstrated altered differentiation profiles. Their differentiation capacity toward oligodendrocyte progenitor cells (OPCs), mature oligodendrocytes and neurons was reduced. In contrast, astrocyte differentiation was promoted. Moreover, LRP1 deletion had a negative effect on NSPCs proliferation and survival. Our observations suggest that LRP1 facilitates NSPCs differentiation via interaction with apolipoprotein E (ApoE). Upon ApoE4 stimulation wild type NSPCs generated more oligodendrocytes, but LRP1 knockout cells showed no response. The effect of ApoE seems to be independent of cholesterol uptake, but is rather mediated by downstream MAPK and Akt activation. GLIA 2016 GLIA 2016;64:1363-1380.

Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma cholesterol levels and are considered anti-atherogenic. ApoE3 (299 residues, ~34 kDa) serves as a ligand for the low density lipoprotein receptor (LDLr) family of proteins that mediate cellular uptake and clearance of plasma lipoproteins. The ability of apoE3 to function as a ligand for LDLr resides in the N-terminal domain (NT) of apoE3. ApoAI (243 residues, ~28 kDa) lowers cholesterol levels through its ability to promote cholesterol efflux from peripheral tissues such as macrophages, forming HDL, which transports cholesterol back to the liver. Residues located in the C-terminal domain (CT) of apoAI mediate this function of promoting cholesterol efflux. The objective of this study is to generate chimeric apolipoproteins composed of apoE3 and apoAI to better understand the role of the C-terminal tail of the parent proteins. Two chimeras were generated by “domain swapping”: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. They were overexpressed in E. coli, isolated and purified. Western blot analysis revealed the presence of epitopes from both parent proteins. Circular dichroism spectroscopy revealed that the α-helical content of apoE3-NT/apoAI-CT and apoAI-NT/E-CT was 43 and 54 %, respectively, which is comparable to that of the parent proteins. Chemical denaturation was used to probe for changes in protein stability. Addition of CT of apoE3 to NT of apoAI conferred more stability to apoAI, while addition of CT of apoAI to NT of apoE3 had no significant effect on apoE3. The rate of apolipoprotein-induced conversion of DMPC vesicles to discoidal structures was followed to assess their lipid binding capability. ApoE3-NT/AI-CT displayed a rate constant that was ~10x higher compared to apoE3, while apoAI-NT/apoE-CT showed a rate constant similar to that of apoAI. Whereas apoE3-NT/apoAI-CT elicits the ability to bind to the LDLr, apoAI-NT/apoE-CT did not. Lastly, both chimeras promote ABCA1-mediated cholesterol efflux from J774 macrophages, with the efflux capability being 2-fold greater for apoAI-NT/apoE-CT. These results show that CT of apoAI can promote lipid binding of apoE3, while CT of apoE3 can improve cholesterol efflux ability of apoAI.

HDL mimetic peptides hold promise for the treatment of cardio-metabolic diseases. Transient increases in plasma TG and/or safety concerns may limit their therapeutic applications however, particularly in high risk patients with hypertriglyceridemia and diabetes. Presently, we sought to identify peptide structural features responsible for these adverse effects. The single helix peptide ATI-5261 was used as a model, since it forms a class A α-helix like many HDL mimetic peptides and has exceptional secondary structure and solubility. High dose ip injection (300 mg/kg) of ATI-5261 induced muscle toxicity in C57Bl/6 mice vs. vehicle (serum CPK@4 h = 32,314±2359 vs 143±44 IU/L), increased ALT and AST activities (233±10 & 4595±933 vs 26±6 & 84±21 IU/L) and elevated plasma TG (1951±77 vs 37±7 mg/dl). A majority (~90%) of the cytotoxicity and TG increase was eliminated by removing phenylalanine residues from the apolar face of ATI-5261. Similar results were obtained by removing cationic arginine residues from the lipid-water interface. Thus both features were necessary, but each not sufficient, to induce cytotoxicity and TG elevations. Based on this, a peptide (CS6253) was created that was non-toxic (no adverse effect level >500 mg/kg; t1/2 =7.0±0.6 h in rats), retained secondary structure (75±5% α-helicity), high solubility and ability to stimulate ABCA1-dependent cholesterol efflux efficiently (Km=0.80±0.40 vs. 0.86±0.25 μg/ml ATI-5261). In vivo CS6253 was TG neutral, promoted macrophage RCT, and reduced (32%) substantial atherosclerosis in apoE KO mice fed western diet 10 wks (lesion area = 22±4 vs. 15±2% with 30 mg CS6253/kg, ip injection every 48 h for 6 wks, p

Apolipoprotein E (apoE) serves as a ligand for the low density lipoprotein receptor (LDLR) only when bound to lipid. The N-terminal domain of lipid-free apoE exists as globular 4-helix bundle that is conferred with LDLR recognition ability after undergoing a lipid binding-induced conformational change. To investigate the structural basis for this phenomenon, site-directed spin label electron paramagnetic resonance spectroscopy experiments were conducted, focusing on the region near the C-terminal end of helix 4 (Ala-164). Using C112S apoE-N-terminal as template, a series of single cysteine substitution variants (at sequence positions 161, 165, 169, 173, 176, and 181) were produced, isolated, and labeled with the nitroxide probe, methane thiosulfonate. Electron paramagnetic resonance analysis revealed that lipid association induced fixed secondary structure in a region of the molecule known to exist as random coil in the lipid-free state. In a complementary approach, site-directed fluorescence analysis using an environmentally sensitive probe indicated that the lipid-induced transition of this region of the protein to α helix was accompanied by relocation to a more hydrophobic environment. In studies with full-length apoE single Cys variants, a similar random coil to stable backbone transition was observed, consistent with the concept that lipid interaction induced an extension of helix 4 beyond the boundary defining its lipid-free conformation. This structural transition likely represents a key conformational change necessary for manifestation of the LDLR recognition properties of apoE.

The potential neurotoxicity of soluble forms of amyloid beta peptide (Abeta) as a key factor in early pathogenesis of Alzheimer's disease is being recognized. In addition, there is growing evidence of the essential role of apolipoprotein E (apoE) in amyloid formation, although molecular details of apoE/Abeta interaction are poorly understood. We employed apoE C-terminal (CT) domain comprising residues 201-299 to identify binding location of Abeta(1-42) by fluorescence resonance energy transfer (FRET) and quenching analyses. Native tryptophan (Trp) residues in the apoE CT domain served as FRET donor, whereas N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) covalently attached to a unique cysteine residue substituted at position 4 of Abeta(1-42) (AEDANS-F4C-Abeta(1-42)) served as FRET acceptor. Fluorescence analysis verified that the oligomerization behavior of AEDANS-F4C-Abeta(1-42) was not abrogated by covalent attachment of AEDANS and that apoE CT domain/AEDANS-F4C-Abeta(1-42) association results in formation of a soluble complex. A large decrease in Trp fluorescence emission was noted in mixtures containing apoE CT domain and AEDANS-F4C-Abeta(1-42), accompanied by appearance of sensitized fluorescence emission of AEDANS as a result of intermolecular FRET. An average distance of separation of 22.6 Angstroms between donors and acceptor was calculated. Fluorescence quenching by potassium iodide (KI) did not reveal significant differences in apoE CT domain Trp microenvironment in the absence or the presence of Abeta(1-42). A twofold increase in quenching constant was noted for KI quenching of AEDANS fluorescence emission in the presence of apoE CT domain, indicative of alterations in Abeta conformation upon interaction with apoE CT domain. We propose intermolecular FRET analysis as a discriminating approach to examine apoE/Abeta interaction, a potentially critical factor in early events involved in amyloid formation.