References of "Vanloo, B"
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See detailStructural investigation of reconstituted high density lipoproteins by scanning tunnelling microscopy
Culot, C.; Durant, F.; Lazarescu, S. et al

in Applied Surface Science (2004), 203

Being able to participate in the reverse cholesterol transport (RCT), high density lipoproteins (HDL) are known to be anti-atherogenic. In order to understand such a process, it is thus essential to have ... [more ▼]

Being able to participate in the reverse cholesterol transport (RCT), high density lipoproteins (HDL) are known to be anti-atherogenic. In order to understand such a process, it is thus essential to have a detailed knowledge of the structure and molecular organisation of HDL. Reconstituted nascent high density lipoproteins (r-HDL), consisting of synthetic phospholipids together with different apolipoproteins (apo A-I, A-IV and E), were thus analysed by scanning tunnelling microscopy (STM). Both shape and dimensions of the discoidal HDL particles measured by this technique were found in good agreement with the data available from the literature. The accuracy of the STM pictures presented in this paper enables for the first time the visualisation of the molecular organisation of such macromolecules. The arrangement of the protein as antiparallel helical segments, is consistent with the general mode of organisation of apolipoprotein/phospholipid discoidal particles previously reported. [less ▲]

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See detailContribution Of The Hydrophobicity Gradient To The Secondary Structure And Activity Of Fusogenic Peptides
Decout, A.; Labeur, C.; Vanloo, B. et al

in Molecular Membrane Biology (1999), 16(3), 237-46

Fusogenic peptides belong to a class of helical amphipathic peptides characterized by a hydrophobicity gradient along the long helical axis. According to the prevailing theory regarding the mechanism of ... [more ▼]

Fusogenic peptides belong to a class of helical amphipathic peptides characterized by a hydrophobicity gradient along the long helical axis. According to the prevailing theory regarding the mechanism of action of fusogenic peptides, this hydrophobicity gradient causes the tilted insertion of the peptides in membranes, thus destabilizing the lipid core and, thereby, enhancing membrane fusion. To assess the role of the hydrophobicity gradient upon the fusogenic activity, two of these fusogenic peptides and several variants were synthesized. The LCAT-(57-70) peptide, which is part of the sequence of the lipolytic enzyme lecithin cholesterol acyltransferase, forms stable beta-sheets in lipids, while the apolipoprotein A-II (53-70) peptide remains predominantly helical in membranes. The variant peptides were designed through amino acid permutations, to be either parallel, perpendicular, or to retain an oblique orientation relative to the lipid-water interface. Peptide-induced vesicle fusion was monitored by lipid-mixing experiments, using fluorescent probes, the extent of peptide-lipid association, the conformation of lipid-associated peptides and their orientation in lipids, were studied by Fourier Transformed Infrared Spectroscopy. A comparison of the properties of the wild-type and variant peptides shows that the hydrophobicity gradient, which determines the orientation of helical peptides in lipids and their fusogenic activity, further influences the secondary structure and lipid binding capacity of these peptides. [less ▲]

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See detailCharacterization Of Functional Residues In The Interfacial Recognition Domain Of Lecithin Cholesterol Acyltransferase (Lcat)
Peelman, F.; Vanloo, B.; Perez-Mendez, O. et al

in Protein Engineering (1999), 12(1), 71-8

Lecithin cholesterol acyltransferase (LCAT) is an interfacial enzyme active on both high-density (HDL) and low-density lipoproteins (LDL). Threading alignments of LCAT with lipases suggest that residues ... [more ▼]

Lecithin cholesterol acyltransferase (LCAT) is an interfacial enzyme active on both high-density (HDL) and low-density lipoproteins (LDL). Threading alignments of LCAT with lipases suggest that residues 50-74 form an interfacial recognition site and this hypothesis was tested by site-directed mutagenesis. The (delta56-68) deletion mutant had no activity on any substrate. Substitution of W61 with F, Y, L or G suggested that an aromatic residue is required for full enzymatic activity. The activity of the W61F and W61Y mutants was retained on HDL but decreased on LDL, possibly owing to impaired accessibility to the LDL lipid substrate. The decreased activity of the single R52A and K53A mutants on HDL and LDL and the severer effect of the double mutation suggested that these conserved residues contribute to the folding of the LCAT lid. The membrane-destabilizing properties of the LCAT 56-68 helical segment were demonstrated using the corresponding synthetic peptide. An M65N-N66M substitution decreased both the fusogenic properties of the peptide and the activity of the mutant enzyme on all substrates. These results suggest that the putative interfacial recognition domain of LCAT plays an important role in regulating the interaction of the enzyme with its organized lipoprotein substrates. [less ▲]

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See detailThe C-terminal helix of human apolipoprotein AII promotes the fusion of unilamellar liposomes and displaces apolipoprotein AI from high-density lipoproteins.
Lambert, Géraldine ULg; Decout, A.; Vanloo, B. et al

in European Journal of Biochemistry (1998), 253(1), 328-38

To assess the functional properties of apolipoprotein (apo) AII and to investigate the mechanism leading to the displacement of apo AI from native and reconstituted high-density lipoproteins (HDL and r ... [more ▼]

To assess the functional properties of apolipoprotein (apo) AII and to investigate the mechanism leading to the displacement of apo AI from native and reconstituted high-density lipoproteins (HDL and r-HDL) by apo AII, wild-type and variant apo AII peptides were synthesized. The wild-type peptides, residues 53-70 and 58-70, correspond to the C-terminal helix of apo AII and are predicted to insert at a tilted angle into a lipid bilayer. We demonstrate that both the apo AII-(53-70) peptide, and to a lesser extent the apo AII-(58-70) peptide are able to induce fusion of unilamellar lipid vesicles together with membrane leakage, and to displace apo AI from HDL and r-HDL. Two variants of the apo AII-(53-70)-wild-type (WT) peptide, designed either to be parallel to the water/lipid interface [apo AII-(53-70)-0 degrees] or to retain an oblique orientation [apo AII-(53-70)-30 degrees], were synthesized in order to test the influence of the obliquity on their fusogenic properties and ability to displace apo AI from HDL. The parallel variant did not bind lipids, due to its self-association properties. However, the apo AII-(53-70)-30 degrees variant was fusogenic and promoted the displacement of apo AI from HDL. Moreover, the extent of fusion of the apo AII-(53-70)-WT, apo AII-(58-70)-WT and apo AII-(53-70)-30 degrees peptides was related to the alpha-helical content of the lipid-bound peptides measured by infrared spectroscopy. Infrared measurements using polarized light also confirmed the oblique orientation of the helical component of the three peptides. In native and r-HDL, the tilted insertion of the C-terminal helix of apo AII resulting in a partial destabilization of the HDL external lipid layer might contribute to the displacement of apo AI by apo AII. [less ▲]

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See detailContribution Of The Hydrophobicity Gradient Of An Amphipathic Peptide To Its Mode Of Association With Lipids
Perez-Mendez, O.; Vanloo, B.; Decout, A. et al

in European Journal of Biochemistry (1998), 256(3), 570-9

A class of peptides that associate with lipids, known as oblique-orientated peptides, was recently described [Brasseur R., Pillot, T., Lins, L., Vandekerckhove, J. & Rosseneu, M. (1997) Trends Biochem ... [more ▼]

A class of peptides that associate with lipids, known as oblique-orientated peptides, was recently described [Brasseur R., Pillot, T., Lins, L., Vandekerckhove, J. & Rosseneu, M. (1997) Trends Biochem. Sci. 22, 167-171]. Due to an asymmetric distribution of hydrophobic residues along the axis of the alpha-helix, such peptides adopt an oblique orientation which can destabilise membranes or lipid cores. Variants of these oblique peptides, designed to have an homogeneous distribution of hydrophobic and hydrophilic residues along the helical axis, are classified as regular amphipathic peptides. These peptides are expected to lie parallel to the polar/apolar interface with their hydrophobic residues directed towards the apolar and their hydrophilic residues towards the polar phase. An hydrophobic, oblique-orientated peptide was identified at residues 56-68 in the sequence of the lecithin-cholesterol acyltransferase (LCAT), enzyme. This peptide is predicted to penetrate a lipid bilayer at an angle of 40 degrees through its more hydrophobic C-terminal end and thereby induce the destabilisation of a membrane or a lipid core. The LCAT-(56-68) wild-type peptide was synthesised together with the LCAT-(56-68, 0 degrees) variant, in which the hydrophobicity gradient was abolished through residue permutations. In two other variants, designed to keep their oblique orientation, the W61 residue was shifted either towards the more hydrophilic N-terminal at residue 57, or to position 68 at the hydrophobic C-terminal end of the peptide. Peptide-induced vesicle fusion was demonstrated by fluorescence measurements using pyrene-labeled vesicles and by monitoring of vesicle size by gel filtration. The interaction between peptides and lipids was monitored by measurement of the intrinsic tryptophan fluorescence emission of the peptides. Fluorescence polarisation measurements, using diphenyl hexatriene, were carried out to follow changes in the lipid fluidity. The LCAT-(56-68) wild-type peptide and the two oblique variants, induced fusion of unilamellar dimyristoylglycerophosphocholine vesicles. Tryptophan fluorescence emission measurements showed a 12-14 nm blue shift upon addition of the wild-type peptide and of the W61-->68 variant to lipids, whereas the fluorescence of the W61-->57 variant did not change significantly. This observation supports the insertion of the more hydrophobic C-terminal residues into the lipid phase, as predicted by the theoretical calculations. In contrast, the 0 degrees variant peptide had no fusogenic activity, and it associated with lipids to form small discoidal lipid/peptide complexes. The phospholipid transition temperature was decreased after addition of the wild-type, the W61-->68 and W61-->57 fusogenic peptides, whereas the opposite effect was observed with the 0 degrees variant. The behaviour of the wild-type and variant LCAT-(56-68) peptides stresses the contribution of the hydrophobicity gradient along the axis of an amphipathic peptide to the mode of association of this peptide with lipids. This parameter consequently influences the structural modifications occurring to lipids upon association with amphipathic peptides. [less ▲]

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See detailA Novel Bacteriocin With A Ygngv Motif From Vegetable-Associated Enterococcus Mundtii: Full Characterization And Interaction With Target Organisms
Bennik, Mhj.; Vanloo, B.; Brasseur, Robert ULg et al

in Biochimica et Biophysica Acta-Biomembranes (1998), 1373(1), 47-58

A novel broad-spectrum antimicrobial peptide produced by vegetable-associated Enterococcus mundtii was purified and characterized, and designated mundticin. To our knowledge, this is the first report on ... [more ▼]

A novel broad-spectrum antimicrobial peptide produced by vegetable-associated Enterococcus mundtii was purified and characterized, and designated mundticin. To our knowledge, this is the first report on bacteriocin production by this organism. The elucidation of the full primary amino acid sequence of mundticin (KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATGGAAGWSK) revealed that this antimicrobial peptide belongs to the class IIa bacteriocins of lactic acid bacteria which share a highly conserved N-terminal 'YGNGV' motif. Data obtained by computer modelling indicated an oblique orientation of the alpha-helical regions of mundticin and homologous class IIa bacteriocins at a hydrophobic-hydrophilic interface, which may play a role in the destabilization of phospholipid bilayers. The average mass of mundticin, as determined by electron spray mass spectrometry, was found to be 4287.21+/-0.59 Da. With respect to its biological activity, mundticin was shown to inhibit the growth of Listeria monocytogenes, Clostridium botulinum and a variety of lactic acid bacteria. Moreover, it was demonstrated to have a bactericidal effect on L. monocytogenes as a result of the dissipation of the membrane potential, and a loss of intracellular ATP in absence of ATP leakage. Its good solubility in water, and its stability over a wide pH and temperature range indicate the potential of this broad spectrum bacteriocin as a natural preservation agent for foods. [less ▲]

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See detailSpecific Modulation Of The Fusogenic Properties Of The Alzheimer Beta-Amyloid Peptide By Apolipoprotein E Isoforms
Pillot, T.; Goethals, M.; Vanloo, B. et al

in European Journal of Biochemistry (1997), 243(3), 650-9

C-terminal fragments of the Alzheimer amyloid peptide (amino acids 29-40 and 29-42) have physico-chemical properties related to those of the fusion peptides of viral proteins and they are able to induce ... [more ▼]

C-terminal fragments of the Alzheimer amyloid peptide (amino acids 29-40 and 29-42) have physico-chemical properties related to those of the fusion peptides of viral proteins and they are able to induce the fusion of liposomes in vitro. We proposed that these properties could mediate a direct interaction of the amyloid peptide with cell membranes and account for part of the cytotoxicity of the amyloid peptide. In view of the epidemiologic and biochemical linkages between the pathology of Alzheimer's disease and apolipoprotein E (apoE) polymorphism, we examined the potential interaction between the three common apoE isoforms and the C-terminal fragments of the amyloid peptide. We show that, at low concentration, only apoE2 and apoE3 are potent inhibitors of the amyloid peptide fusogenic and aggregational properties, whereas the apoE4 isoform has no effect. We further show that the protective effect of apoE is mediated by the formation of stable apoE/amyloid peptide complexes, as determined by tryptophan emission fluorescence measurements and by gel electrophoresis. The interaction specificity between apoE2 and apoE3 and the amyloid fragments is demonstrated here, since other apolipoproteins (e.g. apolipoprotein A-I and A-II), with similar amphipathic structures, do not interact with the amyloid C-terminal fragments. Finally, we show that, reciprocally, the amyloid peptide can interact directly with the apoE2 and apoE3 isoforms to decrease or perturb their normal association with lipids. These data suggest that the 29-40 and 29-42 domains of the amyloid peptide could be critical for the amyloid-apoE interaction, and that apoE2 and apoE3 isoforms, but not apoE4, could play a protective role against the formation of amyloid aggregates and/or against their interaction with cellular membranes. [less ▲]

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See detailDesign Of A New Class Of Amphipathic Helical Peptides For The Plasma Apolipoproteins That Promote Cellular Cholesterol Efflux But Do Not Activate Lcat
Labeur, C.; Lins, Laurence ULg; Vanloo, B. et al

in Arteriosclerosis, Thrombosis, and Vascular Biology (1997), 17(3), 580-8

Amphipathic helical peptides represent the lipid-binding units of the soluble plasma apolipoproteins. Several synthetic peptide analogues have been designed to mimic such structures and have been used to ... [more ▼]

Amphipathic helical peptides represent the lipid-binding units of the soluble plasma apolipoproteins. Several synthetic peptide analogues have been designed to mimic such structures and have been used to unravel some of the mechanisms involved in the physiological function of the apolipoproteins, including lipid binding, LCAT activation, and enhancement of cholesterol efflux from lipid-laden cells. A series of novel synthetic peptides, named ID peptides, was modeled on the basis of the structural properties common to the amphipathic helices of apolipoprotein (apo) A-I. In these new peptides, however, the segregation between hydrophobic and hydrophilic faces of the helices is more pronounced than in apoA-I, so that the surface of the hydrophobic and hydrophilic faces of the amphipathic helices is equal. Moreover, there are fewer negatively charged residues in the center of the hydrophilic face of the helical peptides. Most charged amino acids are located along the edge of the helix and are susceptible to forming salt bridges with residues of an antiparallel helix, such as around a discoidal phospholipid/peptide complex. The physicochemical characteristics of these peptides and their complexes with phospholipids were compared with those of the 18A peptide and its lipid/peptide complex. All ID peptides bind dimyristoylphosphatidylcholine vesicles more rapidly than the 18A peptide to yield discoidal peptide/phospholipid complexes of comparable size. The alpha-helical content of the lipid-free ID peptides is close to that of the 18A peptide and increases slightly on lipid binding. The stability of the ID and 18A peptides and of the phospholipid/peptide complexes against guanidinium hydrochloride denaturation is higher than that of lipid-free and lipid-bound apoA-I. LCAT activation by the 18A/phospholipid/cholesterol complexes equals that of apoA-I/ phospholipid/cholesterol complexes, whereas none of the ID peptides tested is able to activate LCAT to a significant extent. Incubation of the peptide/phospholipid complexes with lipid-laden macrophages induces cellular cholesterol efflux and incorporation of cholesterol into the complexes. The cholesterol efflux capacity of the peptide/phospholipid complexes is comparable among the peptides and higher than that of apoprotein/phospholipid complexes. In conclusion, although the amphipathicity of the new peptides is higher than that of the 18A model peptide, the lack of LCAT activation by the ID peptides suggests that an enhanced segregation of the hydrophobic and hydrophilic residues, equal magnitude of hydrophobic and hydrophilic faces of the helix, and the absence of negatively charged residues in the central part of the hydrophilic face might account for the lack of LCAT activity of these peptides. These parameters do not affect the capacity of the peptide/phospholipid complexes to promote cellular cholesterol efflux. [less ▲]

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See detailThe 118-135 Peptide Lot The Human Prion Protein Forms Amyloid Fibrils And Induces Liposome Fusion
Pillot, T.; Lins, Laurence ULg; Goethals, M. et al

in Journal of Molecular Biology (1997), 274(3), 381-93

The prion protein (PrPC) is a glycoprotein of unknown function normally found at the surface of neurons and of glial cells. It is involved in diseases such as bovine spongiform encephalopathy, and ... [more ▼]

The prion protein (PrPC) is a glycoprotein of unknown function normally found at the surface of neurons and of glial cells. It is involved in diseases such as bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease in the human, where PrPC is converted into an altered form (termed PrPSc). PrPSc is highly resistant towards proteolytic degradation and accumulates in the central nervous system of affected individuals. By analogy with the pathological events occuring during the development of Alzheimer's disease, controverses still exist regarding the relationship between amyloidogenesis, prion aggregation and neuronal loss. To unravel the mechanism of PrP neurotoxicity and understand the interaction of PrP with cellular membranes, a series of natural and variant peptides spanning residues 118 to 135 of PrP was synthesized. The potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. According to computer modeling calculations, the 120 to 133 domain of PrP is predicted to be a tilted lipid-associating peptide, and to insert in a oblique way into a lipid bilayer through its N-terminal end. In addition to amyloidogenic properties exhibited in vitro by these peptides, peptide-induced vesicle fusion was demonstrated by several techniques, including lipid- and core-mixing assays. Elongation of the 120 to 133 peptide towards the N- and C-terminal ends of the PrP sequence showed that the 118 to 135 PrP peptide has maximal fusogenic properties, while the variant peptides had no effect. Due to their high hydrophobicity, all peptides tested were able to interact with liposomes to induce leakage of encapsulated calcein. We demonstrate also that the propensity of the peptides to fold as an alpha-helix increases their fusogenic activity, thus accounting for the maximal fusogenic activity of the most stable helix at residues 118 to 135. These data suggest that, by analogy with the C-terminal domain of the beta-amyloid peptide, the fusogenic properties exhibited by the prion peptides might contribute to the neurotoxicity of these peptides by destabilizing cellular membranes. [less ▲]

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See detailStructural And Functional Properties Of The 154-171 Wild-Type And Variant Peptides Of Human Lecithin-Cholesterol Acyltransferase
Peelman, F.; Goethals, M.; Vanloo, B. et al

in European Journal of Biochemistry (1997), 249(3), 708-15

The 154-171 segment of the human lecithin-cholesterol acyltransferase (LCAT) enzyme was identified as the most stable amphipathic helix in the LCAT sequence. Its mean hydrophobicity, hydrophobic moment ... [more ▼]

The 154-171 segment of the human lecithin-cholesterol acyltransferase (LCAT) enzyme was identified as the most stable amphipathic helix in the LCAT sequence. Its mean hydrophobicity, hydrophobic moment and its orientation at a lipid/water interface are similar to those of some of the helical repeats of apolipoprotein A-IV and E. This domain was therefore proposed as a candidate peptide accounting for the association between LCAT and its lipid substrate. To investigate this hypothesis we synthesized the LCAT-(154-171)-peptide, two variants containing the natural Y156N and R158C mutations and a variant with increased hydrophobicity through Y156I, L160I, L163I and Y171W substitutions. The structural and lipid-binding properties of these synthetic peptides were investigated by turbidity, fluorescence, electron microscopy and circular dichroism. The wild-type peptide, the R158C variant in its dimeric form, as well as the more hydrophobic peptide, associated with phospholipids, whereas the Y156N and the R158C variant in its monomeric form did not. However, only the complexes generated with the hydrophobic variant were stable enough to resist dissociation during gel filtration. The wild-type peptide and hydrophobic variant formed discoidal complexes with dimyristoylglycerophosphocholine (Myr2GroPCho) as shown by negative staining electron microscopy. Comparison of the properties of the wild-type and hydrophobic variant LCAT-(154-171)-peptide stresses the contribution of the hydrophobic face of the amphipathic helix to the formation and stabilization of the peptide/lipid complexes. This is further confirmed by the decreased affinity of the Y156N variant peptide for lipids, as this mutation decreased the mean hydrophobicity of the hydrophobic face of the amphipathic helix. These results support the hypothesis that the 154-171 segment of LCAT might be involved in the interaction of the enzyme with its lipid substrate and suggest that the decreased activity of the Y156N natural LCAT mutant might result from a decreased affinity of this mutant for lipids. [less ▲]

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See detailFusogenic Properties Of The C-Terminal Domain Of The Alzheimer Beta-Amyloid Peptide
Pillot, T.; Goethals, M.; Vanloo, B. et al

in Journal of Biological Chemistry (1996), 271(46), 28757-65

A series of natural peptides and mutants, derived from the Alzheimer beta-amyloid peptide, was synthesized, and the potential of these peptides to induce fusion of unilamellar lipid vesicles was ... [more ▼]

A series of natural peptides and mutants, derived from the Alzheimer beta-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 beta-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 beta-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 beta-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 beta-amyloid peptide had any fusogenic activity. Circular dichroism measurements showed an increase of the alpha-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 alpha-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 beta-amyloid peptide might contribute to the cytotoxicity of the peptide by destabilizing the cell membrane. [less ▲]

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See detailAssociation Of Synthetic Peptide-Fragments Of Human Apolipoprotein-A-I With Phospholipids
Vanloo, B.; Demoor, L.; Boutillon, C. et al

in Journal of Lipid Research (1995), 36(8), 1686-96

The sequences of the plasma apolipoproteins have a high degree of internal homology as they contain several 22-mer internal repeats. These amphipathic helical repeats are considered as the structural and ... [more ▼]

The sequences of the plasma apolipoproteins have a high degree of internal homology as they contain several 22-mer internal repeats. These amphipathic helical repeats are considered as the structural and functional units of this class of proteins. We proposed that the 22-mer repeats of the plasma apolipoproteins consist of 17-mer helical segments separated by extended beta-strands comprising five amino acid residues with a proline in the center of this segment. These beta-strand segments help reverse the orientation of the consecutive helices of apoA-I, A-IV, and E in a discoidal apolipoprotein-phospholipid complex. In order to support this hypothesis, we synthesized apoA-I fragments consisting of, respectively, one putative helix (residues 166-183), one helix plus a beta-strand (residues 161-183), and a pair of helices separated by a beta-strand (residues 145-183). The structural and lipid-binding properties of these peptides were investigated by turbidity, fluorescence, binding studies with unilamellar phospholipid vesicles, electron microscopy, and circular dichroism measurements. Our data show that one single putative helical segment or one helical segment plus one extended beta-strand do not form stable complexes with phospholipids. The addition of a second adjacent helix has no influence on the lipid affinity of the apoA-I 145-183 peptide compared to the shorter segments but substantially improves the stability of the complexes. The helical content of the peptide increases upon lipid association as observed with apoA-I. The complexes generated with the apoA-I 145-183 peptide appear as discoidal particles by negative staining electron microscopy, with heterogeneous sizes ranging between 250 and 450 A. The relative orientation of the peptide and the phospholipid is the same as in a DMPC/apoA-I complex as the helices are oriented parallel to the acyl chains of the phospholipid. However, the stability of these complexes is significantly lower than that of the corresponding DMPC/apoA-I complexes. The transition temperature, fluidity, and cooperativity of the phospholipid bilayer are only weakly affected by the association with the apoA-I 145-183 peptide. These data suggest that a pair of helical peptides linked through a beta-strand associates more tightly with lipids and can form discoidal lipid-peptide complexes, than a single helix. A comparison with the properties of native apoA-I suggests, however, that the cooperativity between pairs of helices in native apoA-I further contributes to strengthen the lipid-protein association. [less ▲]

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See detailHelix Helix Interactions In Reconstituted High-Density-Lipoproteins
Lins, Laurence ULg; Brasseur, Robert ULg; De Pauw, Mc. et al

in Biochimica et Biophysica Acta-Lipids and Lipid Metabolism (1995), 1258(1), 10-8

In this work we calculated the ionic interactions between adjacent amphipathic helices of apo A-I and apo A-IV. The calculation of the electrostatic potential around the helices helps identify the charged ... [more ▼]

In this work we calculated the ionic interactions between adjacent amphipathic helices of apo A-I and apo A-IV. The calculation of the electrostatic potential around the helices helps identify the charged residues susceptible to form salt bridges between adjacent helices. An estimation of the stability of the different pairs of helices is derived from the calculation of the energy of interaction between contiguous helices at a water/lipid interface after energy minimization. The most stable energetic conformation corresponds to the 17-residue helices oriented anti-parallel and separated by a stretch of 5 residues in an extended beta-strand conformation, as calculated through the 'stereo alphabet' calculation procedure. In a pair of helices, the hydrophobic faces are directed towards the lipid core of the discoidal phospholipid-apolipoprotein complex and the hydrophobic lipid-protein interactions are major determinants for the stability of the complex. Interactions between polar residues located on the opposite face of the helix and water molecules can also contribute to the overall energy of the system. Finally, salt bridge formation between residues of opposite charge along the edge of the helical segments contribute to the cooperativity of the phospholipid-apolipoprotein complex formation. The mode of assembly of the amphipathic helical repeats of the apolipoproteins around the edge of a discoidal complex is therefore determined both by the hydrophobic character of the residues and by the charge complementarity along the edge of the helices which increases the structural stability and determines the relative orientation of the helices. [less ▲]

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See detailPhospholipid-Binding And Lecithin-Cholesterol Acyltransferase Activation Properties Of Apolipoprotein-A-I Mutants
Holvoet, P.; Zhao, Za.; Vanloo, B. et al

in Biochemistry (1995), 34(41), 13334-42

Recombinant human apolipoprotein A-I (apo A-I) and three deletion mutants: apo A-I(delta Leu44-Leu126), apo A-I(delta Glu139-Leu170), and apo A-I(delta Ala190-Gln243), purified from the periplasmic space ... [more ▼]

Recombinant human apolipoprotein A-I (apo A-I) and three deletion mutants: apo A-I(delta Leu44-Leu126), apo A-I(delta Glu139-Leu170), and apo A-I(delta Ala190-Gln243), purified from the periplasmic space of Escherichia coli, were studied. The rate of turbidity decrease following mixing of apo A-I(delta Ala190-Gln243) with dimyristoylphosphatidylcholine (DMPC) vesicles at 23 degrees C was 10-fold lower than that of the other apo A-I proteins, confirming that the carboxy-terminal region of apo A-I plays a role in rapid lipid binding. The Stokes radii of reconstituted high-density lipoproteins (rHDL), containing dipalmitoylphosphatidylcholine and cholesterol, were larger for the three apo A-I mutants [6.3 nm for apo A-I(delta Leu44-Leu126), 6.1 nm for apo A-I(delta Glu139-Leu170), and 6.5 nm for apo A-I(delta Ala190-Gln243)] than for intact apo A-I (5.0 nm). The mutant rHDL all contained 4 apo A-I molecules per particle as compared to 2 for intact apo A-I. Circular dichroism measurements revealed 8 alpha-helices per apo A-I molecule, 5 per apo A-I(delta Leu44-Leu126), 6 per apo A-I(delta Glu139-Leu170), and 4 per apo A-I(delta Ala190-Gln243) molecule as compared to predicted values of 8, 5, 6, and 6 alpha-helices, respectively. [less ▲]

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See detailEnzymatic hydrolysis of reconstituted dimyristoylphosphatidylcholine-apo A-I complexes.
Lins, Laurence ULg; Piron, S.; Conrath, K. et al

in Biochimica et Biophysica Acta (1993), 1151(2), 137-42

Apolipoproteins share a common structural feature, their interaction with phospholipids. It is believed that amphipathic helical sequences enable apolipoproteins to bind to lipid bilayer and to form ... [more ▼]

Apolipoproteins share a common structural feature, their interaction with phospholipids. It is believed that amphipathic helical sequences enable apolipoproteins to bind to lipid bilayer and to form discoidal particles of defined dimensions. While the knowledge of the apo A-I sequence and secondary structure has been used to make predictions about its mode of association with lipids, the available experimental data necessary to propose a precise model of these discoidal structures are still limited. An important step in our understanding of these structures would be to identify the apolipoprotein lipid-associated domains. Proteolysis of apo A-I-DMPC reconstituted HDL (rHDL) and free apo A-I is used here to identify lipid-protected domains of apo A-I. Free cleaved peptides were separated from rHDL associated peptides by density gradient centrifugation. The lipid-associated peptides were further analyzed by SDS-PAGE and transferred by Western blot to a ProBlott membrane for sequencing. Cleavage occurred at residue 43 with proteinase K, 46 with trypsin and residue 47 or 48 with pronase. A large domain from about residue 45 to the C-terminal remains highly protected against hydrolysis eventhough it contains several bonds susceptible to proteolytic cleavage. No protected fragments were detected by SDS-PAGE after enzymatic cleavage of free apo A-I in identical experimental conditions. [less ▲]

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See detailSynthetic model peptides for apolipoproteins. I. Design and properties of synthetic model peptides for the amphipathic helices of the plasma apolipoproteins.
Brasseur, Robert ULg; Vanloo, B.; Deleys, R. et al

in Biochimica et Biophysica Acta (1993), 1170(1), 1-7

Amphipathic helical peptides are the lipid-binding motives of the plasma apolipoproteins, and synthetic peptide analogs have been used to unravel the mechanism of lipid association within this class of ... [more ▼]

Amphipathic helical peptides are the lipid-binding motives of the plasma apolipoproteins, and synthetic peptide analogs have been used to unravel the mechanism of lipid association within this class of proteins. Hydrophobic interactions between the apolar amino acid residues belonging to the hydrophobic face of the amphipathic helices and the lipids are the major driving forces in the peptide-lipid association to form discoidal complexes. Ionic interactions and salt bridge formation between contiguous peptide chains in the complex can, however, contribute to the overall stability of the lipid-protein particle. This was studied by designing peptide analogs to the helical repeats of the apolipoproteins with variable degrees of salt bridge formation between adjacent peptide chains. The most stable conformation for pairs of synthetic peptides was calculated by energy minimisation together with the energy of interaction between peptides. The sequence of the peptides was derived from that of the 18A peptide synthesized by Segrest et al., and the theoretical calculations confirmed that ionic interactions between residues close to each other, along the edge of two adjacent anti-parallel peptides, can significantly contribute towards the stability of a peptide-phospholipid complex. [less ▲]

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See detailSynthetic model peptides for apolipoproteins. II. Characterization of the discoidal complexes generated between phospholipids and synthetic model peptides for apolipoproteins.
Corijn, J.; Deleys, R.; Labeur, C. et al

in Biochimica et Biophysica Acta (1993), 1170(1), 8-16

The structure, composition and physico-chemical properties of complexes generated between phospholipids and synthetic model peptides for the amphipathic helices of the plasma apolipoproteins were studied ... [more ▼]

The structure, composition and physico-chemical properties of complexes generated between phospholipids and synthetic model peptides for the amphipathic helices of the plasma apolipoproteins were studied. The sequences of the peptides were derived from that of the 18A peptide and designed to either enhance or decrease ionic interactions between pairs of peptides, as described in the accompanying paper. Complexes were prepared with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or with DPPC and cholesterol, and isolated on a Superose 6HR column. Association kinetics for the DMPC-peptides complexes were followed by measuring the turbidity as a function of the temperature. The diameters of the DPPC-peptide complexes, measured by gradient gel electrophoresis (GGE), were about 120 A. Fluorescence polarization measurements after labeling with diphenyl hexatriene (DPH) yielded transition temperatures of, respectively, 40.6, 41.5 and 41.8 degrees C for the DPPC/18AM1-, DPPC/18AM4- and DPPC/18A-peptide complexes. These values were confirmed by differential scanning calorimetry. Circular dichroism and infrared spectroscopy revealed that the peptides adopt an alpha-helical structure in solution and this percentage increased from 30-40% in the free peptides up to 50-60% in the complexes. Attenuated total reflection (ATR) infrared measurements of the complexes indicated that the peptides are oriented parallel to the acyl chains of the phospholipid bilayer. Denaturation of the peptides and of the peptide-lipid complexes was monitored by Trp fluorescence under addition of increasing amounts of GdmCl. The mid-points of the denaturation curves lie at, respectively, 0.05, 0.25 and 0.35 M GdmCl for the 18AM4, 18A and 18AM1 peptide and are shifted towards higher GdmCl concentrations after peptide-lipid binding. GdmCl denaturation decreased the alpha-helical content of the peptides and of the complexes, as monitored by circular dichroism measurement. The helix to random coil structure transition occurred at, respectively, 2.1, 2.2, and 2.0 M GdmCl for 18A, 18AM1 and 18AM4, compared to 5.1, 5.0, and 5.3 M in the corresponding complexes. These data suggest altogether that the structural properties, the mode of lipid-protein association and the stability of the phospholipid-peptide complexes are similar to those of native plasma apolipoproteins. The 18A and 18AM4 peptides which contain charged residues along the edge of the helix, leading to salt bridge formation between peptides were shown to mimic the amphipathic helices of the plasma apolipoproteins. [less ▲]

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See detailStructure of the apolipoprotein A-IV/lipid discoidal complexes: an attenuated total reflection polarized Fourier transform infrared spectroscopy study.
Lins, Laurence ULg; Brasseur, Robert ULg; Rosseneu, M. et al

in Biochimica et Biophysica Acta (1993), 1149(2), 267-77

Discoidal lipid particles were prepared from a reaction mixture containing apo A-IV and dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) in the molar ratio of 185:1 (lipid ... [more ▼]

Discoidal lipid particles were prepared from a reaction mixture containing apo A-IV and dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) in the molar ratio of 185:1 (lipid/protein). The complexes were isolated by gel filtration and characterized in terms of composition and size. Infrared attenuated total reflection spectroscopy was used to estimate the secondary structure of apolipoprotein A-IV and the orientation of its amphipathic alpha-helices with respect to the lipid hydrocarbon chains. In addition, infrared spectra were analyzed in terms of the conformation and organization of different regions of the lipid molecules in the particles. This approach has been applied successfully to reconstituted HDL particles prepared from a reaction mixture containing DPPC and apo A-I in the molar ratio of 150:1 (Wald, J.H., Goormaghtigh, E., De Meutter, J., Ruysschaert, J.M. and Jonas, A. (1990) J. Biol. Chem. 265, 20044-20050). Apo A-IV helicity increased for the protein bound to DMPC or DPPC but the increase was more pronounced for the apo A-IV/DMPC particles. In both complexes, the alpha helical amphipathic segments of the protein were parallel to the lipid acyl chains and no significant modification of the overall organization of the lipid molecules in the lipid bilayer was observed. The presence of apo A-IV seems only to affect the conformation of the lipid hydrocarbon chains in close contact with the protein in the discoidal particles. [less ▲]

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See detailStructural and functional properties of apolipoprotein B in chemically modified low density lipoproteins.
Vanderyse, L.; Devreese, A. M.; Baert, J. et al

in Atherosclerosis (1992), 97(2-3), 187-99

The structural and compositional changes occurring during in vitro chemical modification of apolipoprotein B-100 (apo B), the apolipoprotein component of low density lipoproteins (LDL), were investigated ... [more ▼]

The structural and compositional changes occurring during in vitro chemical modification of apolipoprotein B-100 (apo B), the apolipoprotein component of low density lipoproteins (LDL), were investigated in this study. The functional properties of chemically modified apo B and especially its potential to induce accumulation of cholesterol esters in macrophages were related to the structural changes of apo B. Acetylation, maleylation or malondialdehyde conjugation did not significantly affect the lipid composition of LDL. However, the unsaturated cholesteryl esters content, especially that of cholesteryl arachidonate was significantly decreased through Cu-oxidation. The number of reactive lysine residues in apo B was decreased by Cu-catalyzed LDL oxidation, acetylation, maleylation and by malondialdehyde conjugation. The number of free cysteines decreased from six in native apo B-100 to three in Cu-oxidized LDL. The tryptophan fluorescence intensity decreased most in malondialdehyde-conjugated LDL and in Cu-oxidized LDL, compared with acetylated and maleylated LDL. The secondary structure of native and chemically modified LDL was measured by attenuated total reflection infrared spectroscopy and by circular dichroism. No significant changes were observed in the secondary structure of any of the modified LDL. These data suggest that neither acetylation, malondialdehyde treatment or even Cu-oxidation substantially altered the secondary structure of apo B, in spite of significant modifications in the primary structure. Incubation of chemically modified LDL with J774 macrophages induced an accumulation of cellular cholesteryl esters and foam cell formation. The highest cholesterol accumulation was induced after malondialdehyde treatment of LDL. These data suggest that the cellular uptake and accumulation of modified LDL is not modulated by changes in the apo B structure. Rather it seems dependent upon the net charge of the apo B protein and probably involves the modification of critical lysine residues. [less ▲]

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See detailMolecular modeling of the amphipathic helices of the plasma apolipoproteins.
Brasseur, Robert ULg; Lins, Laurence ULg; Vanloo, B. et al

in Proteins (1992), 13(3), 246-57

In this paper we propose a classification of the amphipathic helical repeats occurring in the plasma apolipoprotein sequences. It is based upon the calculation of the molecular hydrophobicity potential ... [more ▼]

In this paper we propose a classification of the amphipathic helical repeats occurring in the plasma apolipoprotein sequences. It is based upon the calculation of the molecular hydrophobicity potential around the helical segments. The repeats were identified using a new autocorrelation matrix, based upon similarities of hydrophobic and hydrophilic properties of the amino acid residues within the apolipoprotein sequences. The helices were constructed by molecular modeling, the molecular hydrophobicity potential was calculated, and isopotential contour lines drawn around the helices yielded a three-dimensional visualization of the hydrophobicity potential. Two classes of apolipoproteins could be differentiated by comparing the hydrophobic angles obtained by projection of the isopotential contour lines on a plane perpendicular to the long axis of the helix. The isopotential contour lines around apo AI, AIV, and E are more hydrophilic than hydrophobic, whereas they are of similar intensity for apo AII, CI, and CIII. In both cases discoidal lipid-protein complexes are generated, with the amphipathic helices around the edge of the lipid core. The long axis of the helices is oriented parallel to the phospholipid acyl chains and the hydrophilic side of the helix toward the aqueous phase. As a result of the differences in hydrophobicity potential, the contact between the hydrophobic side of the helices and the phospholipid acyl chains is larger for apo AII, CI, and CIII than for the other apolipoproteins. This might account for the greater stability of the discoidal complexes generated between phospholipids and these apoproteins. [less ▲]

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