Redox status of serum apolipoprotein E and its impact on HDL cholesterol levels.

BACKGROUND: Apolipoprotein E (apoE) is closely involved in the pathogenesis of apoE-related diseases, such as Alzheimer's disease and cardiovascular disease. The redox modulation of cysteine-thiols in a protein is involved in various pathophysiological regulations; however, that of apoE has not been studied in detail. Herein, we devised an analytical method to determine the redox status of serum apoE and assessed its relation to serum cholesterol levels and apoE phenotype. METHODS: The present method was based on a band shift assay, using a photocleavable maleimide-conjugated polyethylene glycol. RESULTS: The basic characteristics of the present method were found to be satisfactory to determine the redox status of serum apoE quantitatively. Serum apoE was separated into its reduced-form (r-), non-reduced-form (nr-), apoE-AII complex, and homodimer using this method. R-apoE could be detected as a 40-kDa band, whereas nr-apoE remained as monomeric apoE. R-apoE displayed a preference for VLDL; however, the levels showed the correlation with HDL-cholesterol levels (p<0.005). Redox status of serum apoE was significantly different among apoE phenotypes. The quantitative ratios of nr-apoE to total apoE in serum from subjects with apoE4/E3 were higher than in serum from subjects with apoE3/E3 (p<0.0001) and apoE3/E2 (p<0.001). CONCLUSION: The redox status of serum apoE might be related to the synthesis of HDL. The information concerning the redox status of serum apoE provided by the present method may be a potent indicator to evaluate various apoE-related diseases.

Effects of different isoforms of apoE on aggregation of the α-synuclein protein implicated in Parkinson's disease.

Parkinson's disease is a progressive brain disorder due to the degeneration of dopaminergic neurons in the substantia nigra. The accumulation of aggregated forms of α-synuclein protein into Lewy bodies is one of the characteristic features of this disease although the pathological role of any such protein deposits in causing neurodegeneration remains elusive. Here, the effects of different apolipoprotein E isoforms (apoE2, apoE3, apoE4) on the aggregation of α-synuclein in vitro were examined using thioflavin T assays and also an immunoassay to detect the formation of multimeric forms. Our results revealed that the aggregation of α-synuclein is influenced by apoE concentration. At low concentrations of apoE (<15nM), all of the isoforms were able to increase the aggregation of α-synuclein (50µM), with apoE4 showing the greatest stimulatory effect. This is in contrast to a higher concentration (>15nM) of these isoforms, where a decrease in the aggregation of α-synuclein was noted. The data show that exceptionally low levels of apoE may seed α-syn aggregation, which could potentially lead to the pathogenesis of α-synuclein-induced neurodegeneration. On the other hand, higher levels of apoE could potentially lower the degree of α-synuclein aggregation and confer protection. The differential effects noted with apoE4 could explain why this particular isoform results in an earlier age of onset for Parkinson's disease.

Preparation, characterization and in vitro-targeted delivery of novel Apolipoprotein E-based nanoparticles to C6 glioma with controlled size and loading efficiency.

Apolipoprotein E (APOE) with its extraordinary features is readily assembled with hydrophobic compounds via its compact hydrophobic units (CHUs). These assemblies can then be converted to stable particles by protein-protein interactions via coiled coil regions (CCRs) which exist in APOE structure. Applying these features of APOE, we prepared novel nanoparticles called NAPOE, using no cross-linker. Vitamin D3 - a hydrophobic antitumor model - was loaded within the nanoparticles (NPs). The NPs were mostly spherical with the mean diameter and zeta potential of 94.39 ± 5.71 nm and -20 ± 0.3 mV, respectively. The molar ratio of VD3/APOE in NPs was 37.2 ± 0.61. The NPs targeted C6 glioma cells in vitro via over-expressed LDLRs. The efficiency of the NPs uptake to malignant C6 glioma cells was remarkable compared to non-tumor glial cells (p < 0.05). The releasing rate of hydrophobic cargo from the particles was high (p < 0.05) and reached to maximum, 12 h after targeting C6 cells. The size and drug loading of NPs were found to be controlled by the definite numbers of CCRs and CHUs in APOE. In conclusion, it is suggested that NAPOE NPs can facilitate the controlled delivery of hydrophobic drugs to the malignant C6 glioma cells according to the degree of invasiveness.

Chemical cross-linking/mass spectrometry maps the amyloid ß peptide binding region on both apolipoprotein E domains.

Apolipoprotein E (apoE) binds the amyloid ß peptide (Aß), one of the major culprits in Alzheimer's disease development. The formation of apoE:Aß complexes is implicated in both Aß clearance and fibrillization. However, the binding interface between apoE and Aß is poorly defined despite substantial previous research efforts, and the exact role of apoE in the pathology of Alzheimer's disease remains largely elusive. Here, we compared the three main isoforms of apoE (E2, E3, and E4) for their interaction with Aß1-42 in an early stage of aggregation and at near physiological conditions. Using electron microscopy and Western blots, we showed that all three isoforms are able to prevent Aß fibrillization and form a noncovalent complex, with one molecule of Aß bound per apoE. Using chemical cross-linking coupled to mass spectrometry, we further examined the interface of interaction between apoE2/3/4 and Aß. Multiple high-confidence intermolecular apoE2/3/4:Aß cross-links confirmed that Lys16 is located in the region of Aß binding to apoE2/3/4. Further, we demonstrated that both N- and C-terminal domains of apoE2/3/4 are interacting with Aß. The cross-linked sites were mapped onto and evaluated in light of a recent structure of apoE. Our results support binding of the hydrophobic Aß at the apoE domain-domain interaction interface, which would explain how apoE is able to stabilize Aß and thereby prevent its subsequent aggregation.

Investigation of APOE isoforms and the association between APOE E3 and E4 with migraine in the Australian Caucasian population.

Migraine is a debilitating neurovascular disease that is associated with pulsating head pain accompanied by nausea, vomiting, photophobia, phonophobia and sometimes visual sensory disturbances. Because of its role in nitric oxide regulation and interleukin release, apolipoprotein E (APOE) has been suggested to play a role in the migraine pathogenesis pathway. This study evaluated the potential role of three APOE variants in an Australian population and the role that they may play in susceptibility to migraine. The study found no significant association between the tested variants and migraine for any of the APOE variants investigated.

Mass spectrometry-based protein footprinting characterizes the structures of oligomeric apolipoprotein E2, E3, and E4.

The three common isoforms of apolipoprotein E (ApoE) differ at two sites in their 299 amino acid sequence; these differences modulate the structure of ApoE to affect profoundly the isoform associations with disease. The ε4 allele in particular is strongly associated with Alzheimer's disease. The study of the structural effects of these mutation sites in aqueous media is hampered by the aggregation proclivity of each ApoE isoform. Hence, understanding the differences between isoforms has thus far relied on lower resolution biophysical measurements, mutagenesis, homology studies, and the use of truncated ApoE variants. In this study, we report two comparative studies of the ApoE family by using the mass spectrometry-based protein footprinting methods of FPOP and glycine ethyl ester (GEE) labeling. The first experiment examines the three full-length WT isoforms in their tetrameric state and finds that the overall structures are similar, with the exception of M108 in ApoE4 which is more solvent-accessible in this isoform than in ApoE2 and ApoE3. The second experiment provides clear evidence, from a comparison of the footprinting results of the wild-type proteins and a monomeric mutant, that several residues in regions 183-205 and 232-251 are involved in self-association.

S-nitrosylation of ApoE in Alzheimer's disease.

The mechanism by which apolipoprotein E (ApoE) isoforms functionally influence the risk and progression of late-onset Alzheimer's disease (LOAD) remains hitherto unknown. Herein, we present evidence that all ApoE isoforms bind to nitric oxide synthase 1 (NOS1) and that such protein-protein interaction results in S-nitrosylation of ApoE2 and ApoE3 but not ApoE4. Our structural analysis at the atomic level reveals that S-nitrosylation of ApoE2 and ApoE3 proteins may lead to conformational changes resulting in the loss of binding to low-density lipoprotein (LDL) receptors. Collectively, our data suggest that S-nitrosylation of ApoE proteins may play an important role in regulating lipid metabolism and in the pathogenesis of LOAD.

The association−dissociation behavior of the ApoE proteins: kinetic and equilibrium studies.

The apolipoprotein E family consists of three major protein isoforms: apolipoprotein E4 (ApoE4), ApoE3, and ApoE2. The isoforms, which contain 299 residues, differ only by single-amino acid changes, but of the three, only ApoE4 is a risk factor for Alzheimer's disease. At micromolar concentrations, lipid-free ApoE exists predominantly as tetramers. In more dilute solutions, lower-molecular mass species predominate. Using fluorescence correlation spectroscopy (FCS), intermolecular fluorescence resonance energy transfer (FRET), and sedimentation methods, we found that the association−dissociation reaction of ApoE can be modeled with a monomer−dimer−tetramer process. Equilibrium constants have been determined from the sedimentation data, while the individual rate constants for association and dissociation were determined by measurement of the kinetics of dissociation of ApoE and are in agreement with the equilibrium constants. Dissociation kinetics as measured by intermolecular FRET show two phases reflecting the dissociation of tetramer to dimer and of dimer to monomer, with dissociation from tetramer to dimer being more rapid than the dissociation from dimer to monomer. The rate constants differ for the different ApoE isoforms, showing that the association−dissociation process is isoform specific. Strikingly, the association rate constants are almost 2 orders of magnitude slower than expected for a diffusion-controlled process. Dissociation kinetics were also monitored by tryptophan fluorescence in the presence of acrylamide and the data found to be consistent with the monomer−dimer−tetramer model. The approach combining multiple methods establishes the reaction scheme of ApoE self-association.

The apolipoprotein E2 isoform is associated with accelerated onset of coronary artery disease in systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a highly prevalent autoimmune disease and coronary artery disease (CAD) is a complication of SLE which is often crucial for the patient's prognosis. It is hypothesized that apolipoprotein E (Apo E), which is involved in cholesterol metabolism, might play a role in this process. MATERIAL/METHODS: Patients with SLE registered at the University of Toronto Lupus Clinic who had DNA available for study had their Apo E genotype determined. Each case was assessed for the presence of CAD, and Apo E allele frequencies in patients with SLE were compared with data from the general population. Age at onset and disease duration of CAD were also recorded and compared between groups. RESULTS: DNA was stored from 152 patients, of whom 38 (25%) had CAD. There was no difference in the frequencies of the Apo E isoforms between SLE patients and the general population. Patients with the E2 allele developed CAD after a mean +/-SD of 6.0+/-1.9 yrs compared with 14.5+/-5.4 yrs in those with E3/3 (p<0.01). CONCLUSIONS: The distribution of Apo E genotypes in SLE is not significantly different from that of the North American population. In SLE, Apo E2 was associated with a more rapid development of CAD. Therefore, Apo E2 might interact with other disease-related factors to accelerate the onset of CAD in some patients with SLE and as such might be an additional marker of risk in this population.

Residues Leu261, Trp264, and Phe265 account for apolipoprotein E-induced dyslipidemia and affect the formation of apolipoprotein E-containing high-density lipoprotein.

Overexpression of apolipoprotein E (apoE) induces hypertriglyceridemia in apoE-deficient mice, which is abrogated by deletion of the carboxy-terminal segment of residues 260-299. We have used adenovirus-mediated gene transfer in apoE-/- and apoA-I-/- mice to test the effect of three sets of apoE mutations within the region of residues 261-265 on the induction of hypertriglyceridemia, the esterification of cholesterol of very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL), and the formation of spherical or discoidal apoE-containing HDL. A single-amino acid substitution (apoE4[Phe265Ala]) induced hypertriglyceridemia in apoE-/- or apoA-I-/- mice, promoted the accumulation of free cholesterol in the very low-density lipoprotein (VLDL) and HDL region, and decreased HDL cholesterol levels. A double substitution (apoE4[Leu261Ala/Trp264Ala]) induced milder hypertriglyceridemia and increased HDL cholesterol levels. A triple substitution (apoE4[Leu261Ala/Trp264Ala/Phe265Ala] or apoE2[Leu261Ala/Trp264Ala/Phe265Ala]) did not induce hypertriglyceridemia and increased greatly the HDL cholesterol levels. Electron microscopy (EM) analysis of the HDL fractions showed that apoE4[Leu261Ala/Trp264Ala/Phe265Ala] and apoE2[Leu261Ala/Trp264Ala/Phe265Ala] contained spherical HDL, apoE4[Leu261Ala/Trp264Ala] contained mostly spherical and few discoidal HDL particles, and apoE4[Phe265Ala] contained discoidal HDL. We conclude that residues Leu261, Trp264, and Phe265 play an important role in apoE-induced hypertriglyceridemia, the accumulation of free cholesterol in VLDL and HDL, and the formation of discoidal HDL. Substitution of these residues with Ala improves the apoE functions by preventing hypertriglyceridemia and promoting formation of spherical apoE-containing HDL.