A series of natural peptides and mutants, derived from the Alzheimer -amyloid peptide, was synthesized, and the potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. These peptide domains were identified by computer modeling and correspond to respectively the C-terminal (e.g. residues 29 -40 and 29 -42) and a central domain (13-28) of the -amyloid peptide. The C-terminal peptides are predicted to insert in an oblique way into a lipid membrane through their N-terminal end, while the mutants are either parallel or perpendicular to the lipid bilayer. Peptide-induced vesicle fusion was demonstrated by several techniques, including lipid-mixing and core-mixing assays using pyrene-labeled vesicles. The effect of peptide elongation toward the N-terminal end of the entire -amyloid peptide was also investigated. Peptides corresponding to residues 22-42 and 12-42 were tested using the same techniques. Both the 29 -40 and 29 -42 -amyloid peptides were able to induce fusion of unilamellar lipid vesicles and calcein leakage, and the amyloid 29 -42 peptide was the most potent fusogenic peptide. Neither the two mutants or the 13-28 -amyloid peptide had any fusogenic activity. Circular dichroism measurements showed an increase of the ␣-helical content of the two C-terminal peptides at increasing concentrations of trifluoroethanol, which was accompanied by an increase of the fusogenic potential of the peptides. Our data suggest that the ␣-helical content and the angle of insertion of the peptide into a lipid bilayer are critical for the fusogenic activity of the C-terminal domain of the amyloid peptide. The differences observed between the fusogenic capacity of the amyloid 29 -40 and 29 -42 peptides might result from differences in the degree of penetration of the peptides into the membrane and the resulting membrane destabilization. The longer peptides, residues 22-42 and 12-42, had decreased, but significant, fusogenic properties associated with perturbation of the membrane permeability. These data suggest that the fusogenic properties of the C-terminal domain of the -amyloid peptide might contribute to the cytotoxicity of the peptide by destabilizing the cell membrane.The amyloid peptide (A), 1 a 39 -43-residue peptide, is a normal 4-kDa derivative of a large transmembrane glycoprotein, the amyloid  precursor protein. The A peptide is found in an aggregated, poorly soluble form in extracellular amyloid deposition in the brains and leptomeniges of patients with Alzheimer's disease (1). In contrast, it occurs in a soluble form in several biological fluids, including the cerebrospinal fluid, where it is produced by glial cells and neurons and where it circulates at nanomolar concentrations (2). The mechanism by which A causes cell death and exerts its cytotoxicity effect remains unclear, and controversies still exist concerning the cytotoxic action of A on neuronal cells. A number of in vitro studies with the synthetic A peptide have shown that this peptide aggregates easily and...
Familial hypercholesterolemia (FH) results from genetic defects in the LDL receptor, and is characterized by a marked elevation in plasma LDL and by qualitative abnormalities in LDL particles. Because LDL particles are major acceptors of cholesteryl esters (CEs) from HDL, significant changes occur in the flux of CE through the reverse cholesterol pathway. To evaluate the effects of an HMG-CoA reductase inhibitor, pravastatin, on CE transfer from HDL to apo B-containing lipoproteins and on plasma lipoprotein subspecies profile in subjects with heterozygous FH, we investigated the transfer of HDL-CE to LDL subfractions and changes in both concentration and chemical composition of the apo B- and the apo AI-containing lipoproteins. After pravastatin treatment (40 mg/d) for a 12-week period, plasma LDL concentrations (mean +/- SD, 745.4 +/- 51.9 mg/dL) were reduced by 36% in patients with FH (n = 6). By contrast, the qualitative features of the density profile of LDL subspecies in patients with FH, in whom the intermediate (d = 1.029 to 1.039 g/mL) and dense (d = 1.039 to 1.063 g/mL) subspecies were significantly increased relative to a control group, were not modified by pravastatin. In addition, no significant effect on the chemical composition of individual LDL subfractions was observed. Furthermore, plasma HDL concentrations were not modified, although the density distribution of HDL was normalized. Indeed, the HDL density peak was shifted towards the HDL2 subfraction (ratios of HDL2 to HDL3 were 0.7 and 1.1 before and after treatment, respectively). Evaluation of plasma CE transfer protein (CETP) mass was performed with an exogenous CE transfer assay. Under these conditions, no modification of plasma CETP protein mass was induced by pravastatin administration. However, the rate of CE transfer from HDL to LDL was reduced by 24% by pravastatin (61 +/- 17 micrograms CE.h-1.mL-1 plasma; P < .0005), although intermediate and dense LDL subfractions again accounted for the majority (71%) of the total CE transferred to LDL. Thus, pravastatin induced reduction of plasma CETP activity without change in the preferential targeting of the transfer of HDL-CE towards the denser LDL subfractions. In conclusion, pravastatin reduces the elevated flux of CE from HDL to apo B-containing lipoproteins in subjects with heterozygous FH as a result of a reduction in the LDL particle acceptor concentration.
Human apolipoprotein A-I1 (apo A-11) consists of three potential amphipathic helices of 17 residues each, which contribute to the lipid-binding properties of this apolipoprotein. The conformation and lipidbinding properties of these peptides, either as single-helix or as two-helix peptides, were investigated by turbidity, fluorescence, electron-microscopy and circular-dichroism measurements, and are compared in this article. The lipid affinity of shorter C-terminal segments of apo A-I1 was compared with those of the single-helix or two-helix peptides, to define the minimal peptide length required for stable complex formation. The properties of the apo-A-II-(13-48)-peptide were further compared with those of the same segment after deletion of the Ser31 and Pro32 residues, because the deleted apo-A-I1-(13 -30)-(33 -48)-peptide, is predicted to form a long uninterrupted helix.The single helices of apo A-I1 could not form stable complexes with phospholipids, and the helixturn-helix segment spanning residues 13 -48 was not active either. The apo-A-II-(37-77)-peptide and the apo-A-II-(40-73)-peptide could form complexes with lipids, which appear as discoidal particles by negative-staining electron microscopy. The shortest C-terminal domain of apo A-11, able to associate with lipids to form stable complexes was the apo-A-II-(40-73)-peptide, which consisted of the C-terminal helix, a &turn and part of the preceding helix. The shorter apo-A-II-(49-77)-peptide, and the helical apo-A-11-( 13 -30)-(33 -48 j-peptide, could also associate with phospholipids. The complexes formed were, however, less stable, as they dissociated outside the transition temperature range of the phospholipid. These data suggest that the C-terminal pair of helices of apo A-11, which is the most hydrophobic pair, is responsible for the lipid-binding properties of the entire protein. The N-terminal pair of helices of apo A-I1 at residues 13-48 does not associate tightly with lipids. The degree of internal similarity and the cooperativity between the helical segments of apo A-I1 is thus less pronounced than in apo A-I or apo A-IV. The N-terminal and C-terminal domains of apo A-I1 appear to behave as two distinct entities with regard to lipid-protein association.
Twenty-eight male Sprague Dawley rats were divided into two groups: a control group (C) of 15 animals and a streptozotocin-induced diabetes group mildly balanced by insulin (D) of 13 animals. After 15 weeks, plasma and high-density lipoprotein (HDL) lipids were determined in each group. Apoprotein A-I concentration was evaluated in HDL fractions. The capacity of the HDL fraction to inhibit thrombin and ADP-induced aggregation of normal platelets was determined for each rat, and in an additional experiment the relation dose-effect of HDL was established. The effect of HDL of the two groups on the stabilization of prostacyclin was compared by aggregation bioassay. After 15 weeks, HDL cholesterol (free + esterified). After 15 weeks, HDL cholesterol (free + esterified) tended to increase in group D compared with group C (P < 0.08). By contrast, apoprotein A-I was very significantly decreased in HDL-D compared with HDL-C (P < 0.001). These alterations were accompanied by a significantly decreased capacity of HDL (60 micrograms/ml platelet suspension) to inhibit ADP-induced aggregation (P< 0.0001) in group D compared with group C. Furthermore, HDL-D incubated 45 or 90 min with prostacyclin showed a significantly decreased capacity to stabilize prostacyclin compared with HDL-C (P<0.04; P <0.03, respectively). These alterations in HDL could be involved in thrombosis and atheromatous complications associated with this disease.
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