Domain Of apoE Research Articles

Structure–function properties of the apoE-dependent COX-2 pathway in vascular smooth muscle cells

Apolipoprotein (apoE) E is a multifunctional protein that plays a critical role in atherogenesis, in part by regulating the intimal proliferation of vascular smooth muscle cells. Recently, a novel cyclooxygenase (COX)-2 pathway was shown to contribute to the anti-proliferative action of human apoE3 in vascular smooth muscle cells (VSMC). Here, we provide insight into the structure–function properties by which apoE mediates these effects. ApoE3 is most effective in promoting COX-2 expression as a lipid-free protein and is less active after lipidation. Alterations in the stability of the helix bundle N-terminal domain of apoE that contains the binding site for the low density lipoprotein (LDL) receptor and heparin do not affect the up-regulation of the COX-2 pathway. In addition, the apoE2, 3, and 4 isoforms are all capable of up-regulating the COX-2 pathway. Finally, the effect of apoE on COX-2 was found to be independent of expression on the VSMC surface of the LDL receptor and heparan sulfate proteoglycans (HSPG). In summary, our data indicates that apoE, particularly in the lipid-free state, can up-regulate COX-2 in murine vascular smooth muscle cells apparently independently of binding to the LDLR, LRP or HSPG.

The C-Terminal Lipid-Binding Domain of Apolipoprotein E Is a Highly Efficient Mediator of ABCA1-Dependent Cholesterol Efflux that Promotes the Assembly of High-Density Lipoproteins

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.

Lipid-induced Extension of Apolipoprotein E Helix 4 Correlates with Low Density Lipoprotein Receptor Binding Ability

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 alpha 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.

Amino-terminal Domain Stability Mediates Apolipoprotein E Aggregation into Neurotoxic Fibrils

The three isoforms of apolipoprotein (apo) E are strongly associated with different risks for Alzheimer's disease: apoE4 > apoE3 > apoE2. Here, we show at physiological salt concentrations and pH that native tetramers of apoE form soluble aggregates in vitro that bind the amyloid dyes thioflavin T and Congo red. However, unlike classic amyloid fibrils, the aggregates adopt an irregular protofilament-like morphology and are seemingly highly α-helical. The aggregates formed at substantially different rates (apoE4 > apoE3 > apoE2) and were significantly more toxic to cultured neuronal cells than the tetramer. Since the three isoforms have large differences in conformational stability that can influence aggregation and amyloid pathways, we tested the effects of mutations that increased or decreased stability. Decreasing the conformational stability of the amino-terminal domain of apoE increased aggregation rates and vice versa. Our findings provide a new perspective for an isoform-specific pathogenic role for apoE aggregation in which differences in the conformational stability of the amino-terminal domain mediate neurodegeneration.

Atherosclerosis and Vascular Disease: Effects of Peptide Mimetics of Apolipoproteins

Levels of high density lipoprotein (HDL) and its major protein component, apolipoprotein (apo) A-I, are strongly inversely correlated to risk of atherosclerosis and other vascular diseases. A number of properties of apo A-I may contribute to this protection, including removal of cholesterol from peripheral tissues to the liver (reverse cholesterol transport), anti-inflammatory and anti-oxidative activities, and modulation of vascular function. Apo A-I has lipid-associating domains that form class A amphipathic helices. Peptide analogs that have no sequence homology to the domains in apo A-I but possess the class A motif have been shown to not only associate with phospholipid but also mimic several of the functional properties of apo A-I. Peptide 4F, with four phenylalanines on the non-polar face, was found to be maximally effective in mimicking the positive qualities of apo A-I; this peptide inhibited atherosclerosis, reduced inflammation and oxidation, and improved vascular function in a number of animal models, and when synthesized with D-amino acids is orally bioavailable. Several other classes of peptide mimetics are now being studied, and may contribute to our understanding of the functions of apo E and apo J. The use of peptide mimetics to study apolipoprotein function has proved to be a powerful tool, and may lead to novel therapeutic agents in the prevention of atherosclerosis and other vascular diseases.

Characterization of low density lipoprotein receptor ligand interactions by fluorescence resonance energy transfer

The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. A soluble low density lipoprotein receptor (sLDLR) and a tryptophan (Trp)-deficient variant human apolipoprotein E3 (apoE3) N-terminal domain (NT) were used in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine (AEDANS), and the protein was complexed with lipid. Incubation of sLDLR with AEDANS-Trp-null apoE3-NT dimyristoylphosphatidylcholine (DMPC) disks, but not lipid-free AEDANS-apoE, induced an enhancement in AEDANS fluorescence emission intensity (excitation, 280 nm) consistent with intermolecular energy transfer from excited Trp in sLDLR to receptor-bound apoE. Ligand binding to sLDLR required calcium and was saturable. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low density lipoprotein. Fluorescence changes in this system reflected pH-dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Intermolecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for the characterization of the myriad molecular interactions ascribed to this family of receptor.

A fluorescence based assay of apolipoprotein E ‐ low density lipoprotein receptor interactions

Members of the low density lipoprotein receptor (LDLR) family function to maintain plasma cholesterol homeostasis. One of the best characterized LDLR ligands is apolipoprotein E (apoE). Whereas lipid-free apoE does not serve as a ligand, lipid associated apoE binds to LDLR. The NT domain of apoE is responsible for recognition by LDLR. A soluble fragment of LDLR (N-terminal residues 1-699) was expressed in HEK 293 cells and isolated from the culture medium by affinity chromatography. A tryptophan deficient apoE3 N terminal (NT) domain (Trp null apoE3-NT) mutant was expressed in E. coli, isolated and covalently labeled on cysteine 112 with the extrinsic fluorescent probe, N-iodoacety-N’-(5-sulfo-1-naphthyl) ethyeylenediamine (AEDANS). AEDANS Trp null apoE3-NT was complexed with dimyristoylphosphatidylcholine (DMPC) and incubated with LDLR. Enhancement in AEDANS fluorescence emission via energy transfer from LDLR Trp residues (excitation 280 nm) was observed. The enhancement in AEDANS fluorescence was saturable and was abolished by incubation with EDTA or pre-incubation with a non-fluorescent competitor ligand (Trp null apoE3-NT DMPC). The present assay provides a sensitive, convenient and quantitative means to evaluate ligand binding to LDLR.

Modulation of Apolipoprotein E Structure by Domain Interaction: DIFFERENCES IN LIPID-BOUND AND LIPID-FREE FORMS

Interaction of the amino- and carboxyl-terminal domains in apolipoprotein (apo) E, referred to as domain interaction, is predicted to be more pronounced in apoE4 than in apoE3 and to underlie the association of apoE4 with Alzheimer and cardiovascular diseases. However, direct physical proof for the domain interaction concept is lacking. To address this issue, fluorescence resonance energy transfer and electron paramagnetic resonance spectroscopy were used to probe the spatial proximity of the two domains of apoE. Both methods demonstrated that the two domains are closer in both lipid-free and phospholipid-bound apoE4 than in apoE3 as a result of domain interaction. In addition, as shown by electron paramagnetic resonance, the domains of apoE4 move apart to resemble more closely the distance in apoE3 when the isoforms are bound to triglyceride-rich emulsion particles. These results demonstrate that domain interaction is a structural property of apoE4 and that apoE adopts different conformations when complexed to different lipids.

Structural Analysis of Lipoprotein E Particles

Apolipoprotein E (apoE) is a key regulator of cholesterol homeostasis. Human apoE has three common isoforms, each with different risk implications for cardiovascular and neurodegenerative disease. Neither the structure of lipoprotein E particles nor the structural consequences of the isoform differences are known. In this investigation, synthetic lipoprotein particles were prepared by complexing phospholipids with full-length apoE isoforms, or with truncated N-terminal and C-terminal domains of apoE. These particles were examined with calorimetry, electron microscopy, circular dichroism spectroscopy, and internal reflection infrared spectroscopy. Results indicate that particles made with the three full-length apoE isoforms are discoidal in shape, and structurally indistinguishable. Thus, differences in their pathological consequences are not due to gross differences in particle structure. Although apoE is predominantly helical, and the axes of the helices are parallel to the flat surfaces of the particles, the orientational order of lipid acyl chains is low and inconsistent with the belt model of lipoprotein A-I structure. Instead, the data suggest that there are at least two different types of apoE-lipid interactions within lipoprotein E particles. One type occurs between apoE helices and the edge of the lipid bilayer as in the belt model, while a second type involves apoE helices that situate in the plane of the membrane and disturb acyl chain order. These interactions allow LpE particles to form with different protein/lipid ratios, and they account for the structure of LpE particles made with only the truncated domains.

Issue Highlights

HomeCirculationVol. 111, No. 23Issue Highlights Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIssue Highlights Originally published14 Jun 2005https://doi.org/10.1161/circ.111.23.3015Circulation. 2005;111:3015ALDOSTERONE SYNTHASE INHIBITOR AMELIORATES ANGIOTENSIN II–INDUCED ORGAN DAMAGE, by Fiebeler et al.Aldosterone has been shown to contribute to angiotensin II–induced end-organ damage, and clinical studies have confirmed the benefit of adding aldosterone antagonists to angiotensin-converting enzyme inhibitor agents to prevent adverse remodeling. Angiotensin II increases circulating levels of aldosterone levels as well as de novo tissue synthesis of aldosterone by stimulating aldosterone synthase (CYP11B2); however, it is unknown which source of aldosterone plays a greater role in tissue remodeling and damage. In these studies, Fiebeler et al examine this question by utilizing both FAD286, a novel CYP11B2 inhibitor, and adrenalectomy in transgenic rats overexpressing human renin and angiotensinogen genes. Using these models, they are able to isolate the source of aldosterone that modulates some of the adverse cardiac and renal effects associated with hyperaldosteronism. See p 3087.LONG-TERM RESPONSE TO CALCIUM CHANNEL BLOCKERS IN IDIOPATHIC PULMONARY ARTERIAL HYPERTENSION, by Sitbon et al.Characteristics of patients with idiopathic pulmonary arterial hypertension (IPAH) who benefit from long-term calcium channel blockers (CCBs) are unknown. Sitbon and colleagues performed acute pulmonary vasodilator testing with epoprostenol or nitric oxide during initial right heart catheterization in 557 IPAH patients. Acute responders were treated initially with oral CCB. Long-term CCB responders were defined as those being in functional class I or II after at least 1 year on CCB monotherapy. Among the 70 patients (12.6%) who displayed acute pulmonary vasoreactivity and received CCB therapy, 38 (6.7%) improved long term. Long-term CCB responders had less severe disease at baseline and displayed a more pronounced fall in mean pulmonary artery pressure during acute vasodilator testing. Of note, long-term CCB responders represented less than 10% of IPAH patients evaluated in a pulmonary vascular referral center. These data suggest that routine administration of CCB as first-line therapy in IPAH patients may be ill advised. See p 3105.APOLIPOPROTEIN E MIMETIC PEPTIDE DRAMATICALLY LOWERS PLASMA CHOLESTEROL AND RESTORES ENDOTHELIAL FUNCTION IN WATANABE HERITABLE HYPERLIPIDEMIC RABBITS, by Gupta et al.Elevated high-density lipoprotein (HDL) levels are associated with a reduction in the frequency of cardiovascular events. To develop new drugs for the treatment of atherosclerosis, investigators have been evaluating the therapeutic properties of HDL components in both animal models of vascular disease and patients with coronary atherosclerosis. However, some of these agents are structurally complex and difficult to produce. In this issue of Circulation, Gupta et al analyze the atheroprotective properties of a synthetic peptide containing the arginine-rich receptor-binding domain of ApoE fused to an amphipathic helical peptide that possesses ApoA-I–like properties. A single administration of the dual-domain peptide dramatically reduces plasma cholesterol levels and improves vascular reactivity in a dyslipidemic rabbit model. These improvements coincide with a reduction in markers of oxidative stress that are associated with vascular inflammation. These data suggest that this HDL-mimetic peptide could make ‘good‘ cholesterol better and have utility for the treatment of atherosclerosis. See p 3112.Visit http://www.circ.ahajournals.org:Cardiology Patient PageHow to Respond to an Implantable Cardioverter-Defibrillator Shock. See p e380.Images in Cardiovascular MedicineReversible Right Ventricular Hypertrophy Due to Cardiac Sarcoidosis. See p e383. Download figureDownload PowerPointThree-Dimensional Imaging in Rupture of Papillary Muscle After Acute Myocardial Infarction. See p e385.Angiosarcoma of the Pericardium: A Fatal Disease. See p e388.CorrespondenceSee p e390. Previous Back to top Next FiguresReferencesRelatedDetails June 14, 2005Vol 111, Issue 23 Advertisement Article InformationMetrics https://doi.org/10.1161/circ.111.23.3015 Originally publishedJune 14, 2005 PDF download Advertisement

Orientation and mode of lipid-binding interaction of human apolipoprotein E C-terminal domain.

ApoE (apolipoprotein E) is an anti-atherogenic lipid transport protein that plays an integral role in lipoprotein metabolism and cholesterol homoeostasis. Lipid association educes critical functional features of apoE, mediating reduction in plasma and cellular cholesterol levels. The 10-kDa CT (C-terminal) domain of apoE facilitates helix-helix interactions in lipid-free state to promote apoE self-association and helix-lipid interactions during binding with lipoproteins, although the mode of lipid-binding interaction is not well understood. We investigated the mode of lipid-binding interaction and orientation of apoE CT domain on reconstituted lipoproteins. Isolated recombinant human apoE CT domain (residues 201-299) possesses a strong ability to interact with phospholipid vesicles, yielding lipoprotein particles with an apparent molecular mass of approximately 600 kDa, while retaining the overall alpha-helical content. Electron microscopy and non-denaturing PAGE analysis of DMPC (dimyristoylphosphatidylcholine)--apoE CT domain lipoprotein complexes revealed discoidal complexes with a diameter of approx. 17 nm. Cross-linking apoE CT domain on discoidal particles yielded dimeric species as the major product. Attenuated total reflectance Fourier transform IR spectroscopy of phospholipid-apoE CT domain complexes reveals that the helical axis is oriented perpendicular to fatty acyl chains of the phospholipid. Fluorescence quenching analysis of DMPC-apoE CT domain discoidal complexes by spin-labelled stearic acid indicated a relatively superficial location of the native tryptophan residues with respect to the plane of the phospholipid bilayer. Taken together, we propose that apoE CT domain interacts with phospholipid vesicles, forming a long extended helix that circumscribes the discoidal bilayer lipoprotein complex.

The Receptor Binding Domain of Apolipoprotein E Is Responsible for Its Antioxidant Activity

Apolipoprotein E (apoE) is a 34-kDa lipid-associated protein present in plasma and in the central nervous system. Previous studies have demonstrated that apoE has multiple functions, including the ability to transport lipids, regulate cell homeostasis, and inhibit lipid oxidation. The lipid binding domain of apoE has been localized to the carboxyl-terminal domain, whereas a cluster of basic amino acid residues within the N-terminal domain is responsible for its receptor binding activity. This study was undertaken to identify the domain in apoE responsible for its antioxidant activity. Results showed that apoE inhibits Cu(2+)-induced LDL oxidation by delaying conjugated diene formation in a concentration-dependent manner. Reductive methylation of lysine residues or cyclohexanedione modification of arginine residues in apoE abolished its ability to inhibit LDL oxidation. Additional studies showed that a 22-kDa peptide containing the N-terminal domain of apoE3 was more effective than a similar peptide with the apoE4 sequence in inhibiting Cu(2+)-induced LDL oxidation. In contrast, the 10-kDa peptide that contains the C-terminal domain of apoE was ineffective. Inhibition of Cu(2+)-induced LDL oxidation can also be accomplished with a peptide containing either a single sequence or a tandem repeat sequence of the receptor binding domain (residues 141-155) of apoE. Taken together, these results localized the antioxidant domain of apoE to its receptor binding domain and the basic amino acids in this domain are important for its antioxidant activity.

Stimulation of Vascular Smooth Muscle Cell Proliferation and Migration by Apolipoprotein(a) Is Dependent on Inhibition of Transforming Growth Factor-β Activation and on the Presence of Kringle IV Type 9

Elevated plasma concentrations of lipoprotein(a) are a risk factor for the development of a variety of atherosclerotic disorders. Despite intensive study, the mechanisms by which lipoprotein(a) promotes these disorders remain to be unequivocally defined. It has been demonstrated that lipoprotein(a), through its unique constituent apolipoprotein(a) (apo(a)), stimulates vascular smooth muscle cell (SMC) migration and proliferation. These effects arise from the ability of apo(a) to inhibit the formation of active transforming growth factor beta (TGF-beta) from its latent precursor, which in turn is caused by the ability of apo(a) to decrease the formation of plasmin from its precursor plasminogen. We utilized a battery of recombinant apo(a) variants that represent systematic deletions of the various domains in the molecule to further probe the mechanism underlying the effect of apo(a) on SMC responses. All recombinant apo(a) variants that contained kringle IV type 9 were able to stimulate SMC proliferation and migration and to decrease the formation of active TGF-beta; conversely all recombinant apo(a) variants lacking kringle IV type 9 had no effect on these parameters. The kringle IV type 9-dependent effects of apo(a) on SMC proliferation required the presence of plasminogen, suggesting for the first time that this kringle mediates the ability of apo(a) to inhibit pericellular plasmin formation.

Two apolipoprotein E mimetic peptides, ApoE(130-149) and ApoE(141-155)2, bind to LRP1.

LRP1 is a cell surface receptor responsible for clearing some 30 known ligands. We have previously shown that each of the three complete LDL receptor-homology domains of the LRP1 extracellular domain (sLRPs) binds apoE-enriched beta-VLDL particles. Here we show that two peptides from the N-terminal receptor binding domain of apoE, which are known to elicit a number of different cellular responses, bind to LRP1. Solution binding assays show that the two peptides, apoE(130-149) and apoE(141-155)(2), interact with each of the sLRPs (2, 3, and 4). Each peptide was found to exhibit the same solution binding characteristics as apoE-enriched beta-VLDL particles. Surface plasmon resonance analyses of the sLRP-apoE peptide interaction show that both peptides bind the sLRPs with K(D) values in the 100 nM range, a value similar to the effective concentration required for observation of the cellular responses. Consistent with results from mutagenesis studies of binding of apoE to LDLR, apoE(130-149,Arg142Glu) bound with a K(D) similar to that of the wild-type sequence, while apoE(130-149,Lys143Glu) showed a 10-fold decrease in K(D). Each of the peptides bound heparin, and heparin competed for sLRP binding.

Probing the pathways of chylomicron and HDL metabolism using adenovirus-mediated gene transfer.

This review clarifies the functions of key proteins of the chylomicron and the HDL pathways. Adenovirus-mediated gene transfer of several apolipoprotein (apo)E forms in mice showed that the amino-terminal 1-185 domain of apoE can direct receptor-mediated lipoprotein clearance in vivo. Clearance is mediated mainly by the LDL receptor. The carboxyl-terminal 261-299 domain of apoE induces hypertriglyceridemia, because of increased VLDL secretion, diminished lipolysis and inefficient VLDL clearance. Truncated apoE forms, including apoE2-202, have a dominant effect in remnant clearance and may have future therapeutic applications for the correction of remnant removal disorders. Permanent expression of apoE and apoA-I following adenoviral gene transfer protected mice from atherosclerosis. Functional assays, protein cross-linking, and adenovirus-mediated gene transfer of apoA-I mutants in apoA-I deficient mice showed that residues 220-231, as well as the central helices of apoA-I, participate in ATP-binding cassette transporter A1-mediated lipid efflux and HDL biogenesis. Following apoA-I gene transfer, an amino-terminal deletion mutant formed spherical alpha-HDL, a double amino- and carboxyl-terminal deletion mutant formed discoidal HDL, and a carboxyl-terminal deletion mutant formed only pre-beta-HDL. The findings support a model of cholesterol efflux that requires direct physical interactions between apoA-I and ATP-binding cassette transporter A1, and can explain Tangier disease and other HDL deficiencies. New insights are provided into the role of apoE in cholesterol and triglyceride homeostasis, and of apoA-I in the biogenesis of HDL. Clearance of the lipoprotein remnants and increase in HDL synthesis are obvious targets for therapeutic interventions.

Helix Orientation of the Functional Domains in Apolipoprotein E in Discoidal High Density Lipoprotein Particles

Human apolipoprotein E (apoE) mediates high affinity binding to the low density lipoprotein receptor when present on a lipidated complex. In the absence of lipid, however, apoE does not bind the receptor. Whereas the x-ray structure of lipid-free apoE3 N-terminal (NT) domain is known, the structural organization of its lipid-associated, receptor-active conformation is poorly understood. To study the organization of apoE amphipathic alpha-helices in a lipid-associated state, single tryptophan-containing apoE3 variants were employed in fluorescence quenching studies. The relative positions of the Trp residues with respect to the phospholipid component of apoE/lipid particles were established from the degree of quenching by phospholipids bearing nitroxide groups at various positions along their fatty acyl chains. Four apoE3-NT variants bearing Trp reporter groups at positions 141, 148, 155, or 162 within helix 4 and two apoE3 variants containing single Trp at positions 257 or 264 in the C-terminal (CT) domain, were reconstituted into phospholipid-containing discoidal complexes. Parallax analysis revealed that each engineered Trp residue in helix 4 of apoE3-NT, as well as those in the CT domain of apoE, localized approximately 5 A from the center of the bilayer. Circular dichroism studies revealed that lipid association induces additional helix formation in apoE. Protease protection assays suggest the flexible loop segment between the NT and CT domains may transition from unstructured to helix upon lipid association. Taken together, these data support a model wherein the alpha-helices in the receptor-binding region and the CT domain of apoE align perpendicular to the fatty acyl chains of the phospholipid bilayer. In this alignment, the residues of helix 4 are arrayed in a positively charged, curved helical segment for optimal receptor interaction.

Inhibition of nicotinic acetylcholine receptors by apolipoprotein E-derived peptides in rat hippocampal slices

Apolipoprotein E (ApoE) is a well-known genetic risk factor for Alzheimer's disease (AD). Dysfunctions in cholinergic signaling, and in particular in the function of neuronal nicotinic acetylcholine receptors (nAChRs), have also been linked with AD and cognition. To address whether there is a link between ApoE and nAChR function, we used electrophysiological techniques to test the effects of synthetic ApoE-mimetic peptides derived from the low-density lipoprotein receptor (LDLR) binding domain for the ability to modulate nAChR activity in hippocampal interneurons. ApoE 133–149 completely inhibited ACh-evoked responses in a dose-dependent manner, yielding an IC 50 value of 720±70 nM. A shorter peptide spanning residues 141–148 mimicked this effect while a second peptide spanning residues 133–140 was without effect, indicating that the arginine-rich domain is responsible for nAChR interaction. Inhibition of ACh-evoked responses was voltage-independent, and displayed partial receptor specificity as no effect on glycine- or GABA-evoked responses occurred. These results demonstrate that peptides derived from the LDLR binding domain of ApoE block the function of nAChRs in hippocampal slices, an interaction that may have implications for AD.

Domains of apoE required for binding to apoE receptor 2 and to phospholipids: implications for the functions of apoE in the brain.

We have studied the contribution of the carboxy terminal domains of lipid-free apoE isolated from apoE-expressing cell cultures in binding to phospholipids and have determined the affinities of reconstituted POPC-apoE particles for the apoER2. It was found that the initial rate of association of apoE2, apoE3, apoE4, and a mutant form apoE4R158M to multilamellar DMPC vesicles was similar and was reduced and eventually diminished by gradual deletion of the carboxy terminal segments. The truncated apoE forms retained their ability to associate with plasma lipoproteins. Receptor binding studies were performed using the ldlA-7 cells expressing apoER2 and transiently transfected COS-M6 and the appropriate control untransfected cells. Specific binding to apoER2 was obtained by subtracting from the total binding to the receptor-expressing cells the nonspecific binding values of the untransfected cells. POPC-apoE particles generated using apoE3, apoE4, the truncated apoE4-259, apoE4-229, apoE4-202, and apoE-165, and the mutant apoE4R158M all bound tightly to the apoER2 (K(d) range of 12 +/- 3 to 19 +/- 4 microg/mL). POPC-apoE2 bound with reduced affinity (K(d) = 31 +/- 5.3 microg/mL). The findings establish that the apoER2 binding domain of apoE is in the 1-165 amino terminal region, whereas the carboxy terminal 230-299 region of apoE is required for efficient initial association with phospholipids.

Molecular mechanisms of type III hyperlipoproteinemia: The contribution of the carboxy-terminal domain of ApoE can account for the dyslipidemia that is associated with the E2/E2 phenotype.

Apolipoprotein E2, which has an R158 for C substitution, has reduced affinity for the LDL receptor and is associated with type III hyperlipoproteinemia in humans. Consistent with these observations, we have found that following adenovirus-mediated gene transfer, full-length apoE2 aggravates the hypercholesterolemia and induces hypertriglyceridemia in E-deficient mice and induces combined hyperlipidemia in C57BL/6 mice. Unexpectedly, the truncated apoE2-202 form that has an R158 for C substitution when expressed at levels similar to those of the full-length apoE2 normalized the cholesterol levels of E-deficient mice without induction of hypertriglyceridemia. The apoE2 truncation increased the affinity of POPC-apoE particles for the LDL receptor, and the full-length apoE2 had a dominant effect in VLDL triglyceride secretion. Hyperlipidemia in normal C57BL/6 mice was prevented by coinfection with equal doses of each, the apoE2 and the apoE2-202-expressing adenoviruses, indicating that truncated apoE forms have a dominant effect in remnant clearance. Hypertriglyceridemia was completely corrected by coinfection of mice with an adenovirus-expressing wild-type lipoprotein lipase, whereas an inactive lipoprotein lipase had a smaller effect. The findings suggest that the apoE2-induced dyslipidemia is not merely the result of substitution of R158 for C but results from increased secretion of a triglyceride-enriched VLDL that cannot undergo lipolysis, inhibition of LpL activity, and impaired clearance of chylomicron remnants. Infection of E(-)(/)(-)xLDLr(-)(/)(-) double-deficient mice with apoE2-202 did not affect the plasma cholesterol levels, and also did not induce hypertriglyceridemia. In contrast, apoE2 exacerbated the hypercholesterolemia and induced hypertriglyceridemia, suggesting that the LDL receptor is the predominant receptor in remnant clearance.

Inhibition of Angiogenesis and Angiogenesis-dependent Tumor Growth by the Cryptic Kringle Fragments of Human Apolipoprotein(a)

Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain. Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo. However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated. To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli. The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic fibroblast growth factor-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo. The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis. Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-15) tumor growth in nude mice. Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor growth. Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent anti-angiogenic and anti-tumor molecule.