Biomedical subjects

M Rosseneu

Publications and source records attributed to M Rosseneu.

At least 19 recordsLinked to original sources

PubMed2004

Penetratin-membrane association: W48/R52/W56 shield the peptide from the aqueous phase.

Using molecular dynamics simulations, we studied the mode of association of the cell-penetrating peptide penetratin with both a neutral and a charged bilayer. The results show that the initial peptide-lipid association is a fast process driven by electrostatic interactions. The homogeneous distribution of positively charged residues along the axis of the helical peptide, and especially residues K46, R53, and K57, contribute to the association of the peptide with lipids. The bilayer enhances the stability of the penetratin helix. Oriented parallel to the lipid-water interface, the subsequent insertion of the peptide through the bilayer headgroups is significantly slower. The presence of negatively charged lipids considerably enhances peptide binding. Lateral side-chain motion creates an opening for the helix into the hydrophobic core of the membrane. The peptide aromatic residues form a pi-stacking cluster through W48/R52/W56 and F49/R53, protecting the peptide from the water phase. Interaction with the penetratin peptide has only limited effect on the overall membrane structure, as it affects mainly the conformation of the lipids which interact directly with the peptide. Charge matching locally increases the concentration of negatively charged lipids, lateral lipid diffusion locally decreases. Lipid disorder increases, through decreased order parameters of the lipids interacting with the penetratin side chains. Penetratin molecules at the membrane surface do not seem to aggregate.

Binding Sites↗

PubMed2001

Specific docking of apolipoprotein A-I at the cell surface requires a functional ABCA1 transporter.

The identification of defects in ABCA1 as the molecular basis of Tangier disease has highlighted its crucial role in the loading with phospholipids and cholesterol of nascent apolipoprotein particles. Indeed the expression of ABCA1 affects apolipoprotein A-I (apoA-I)-mediated removal of lipids from cell membranes, and the possible role of ABCA1 as an apoA-I surface receptor has been recently suggested. In the present study, we have investigated the role of the ABCA1 transporter as an apoA-I receptor with the analysis of a panel of transfectants expressing functional or mutant forms of ABCA1. We provide experimental evidence that the forced expression of a functional ABCA1 transporter confers surface competence for apoA-I binding. This, however, appears to be dependent on ABCA1 function. Structurally intact but ATPase-deficient forms of the transporter fail to elicit a specific cell association of the ligand. In addition the diffusion parameters of membrane-associated apoA-I indicate an interaction with membrane lipids rather than proteins. These results do not support a direct molecular interaction between ABCA1 and apoA-I, but rather suggest that the ABCA1-induced modification of the lipid distribution in the membrane, evidenced by the phosphatidylserine exofacial flopping, generates a biophysical microenvironment required for the docking of apoA-I at the cell surface.

ATP Binding Cassette Transporter 1↗

PubMed2001

Comparison of the aggregation properties, secondary structure and apoptotic effects of wild-type, Flemish and Dutch N-terminally truncated amyloid beta peptides.

The Dutch (E22Q) and Flemish (A21G) mutations in the betaAPP region of the amyloid precursor protein (APP) are associated with familial forms of Alzheimer dementia. However, patients with these mutations express substantially different clinical phenotypes. Therefore, secondary structure and cytotoxic effects of the three Abeta(12-42) variants [wild-type (WT), Dutch and Flemish] were tested. At a concentration of 5 microM the aggregation of these peptides followed the order: Abeta(1-42) WT > Abeta(12-42) WT > Abeta(12-42) Flemish > Abeta(12-42) Dutch. The stability of the secondary structure of these peptides upon decreasing the trifluoroethanol (TFE) concentration in the buffer was followed by circular dichroism measurements. WT peptides progressively lost their alpha-helical structure; this change occurred faster for both the Flemish and Dutch peptides, and at higher percentages of TFE in the buffer, and was accompanied by an increase in beta-sheet and random coil content. Apoptosis was induced in neuronal cells by the Abeta(12-42) WT and Flemish peptides at concentrations as low as 1-5 microM, as evidenced by propidium iodide (PI) staining, DNA laddering and caspase-3 activity measurements. Even when longer incubation times and higher peptide concentrations were applied the N-truncated Dutch peptide did not induce apoptosis. Apoptosis induced by the full length Abeta(1-42) peptide was weaker than that induced by its N-truncated variant. These data suggest that N-truncation enhanced the cytotoxic effects of Abeta WT and Flemish peptides, which may play a role in the accelerated progression of dementia.

Alzheimer Disease↗

PubMed2001

ApoE protects cortical neurones against neurotoxicity induced by the non-fibrillar C-terminal domain of the amyloid-beta peptide.

Although the genetic link between the epsilon 4 allele of apolipoprotein E (apoE) and Alzheimer's disease (AD) is well established, the apoE isoform-specific activity underlying this correlation remains unclear. We have recently characterized the interaction of the soluble the amyloid-beta peptide (A beta) with model membrane and demonstrated that non-fibrillar A beta peptide, including N-terminal truncated forms of A beta, induced apoptotic cell death in primary rat cortical neurones in vitro. To further investigate the potential interaction between apoE and A beta in the pathogenesis of AD, we have determined the effect of apoE isoforms on the neurotoxicity of non-fibrillar A beta peptides. We demonstrate here that the apoE2 and E3 isoforms protect cortical neurones against apoptotic cell death induced by a non-fibrillar form of the A beta(1-40), A beta(12-42), A beta(29-40) and A beta(29-42) peptides, whereas apoE4 had no effect. This effect involves the formation of stable complexes between apoE and the C-terminal domain (e.g. amino acids 29-40) of A beta(1-40). Interestingly, apoE had no effect on the toxicity induced by aggregated A beta peptides, suggesting a lack of interaction between apoE and amyloid fibrils. Our results provide evidence that interaction with the C-terminal domain of A beta, apoE2 and E3, but not apoE4, inhibits the interactions of the non-fibrillar A beta peptide with the plasma membrane of neurones, A beta peptide aggregation and subsequent neurotoxicity.

Amino Acid Sequence↗

PubMed2001

Three arginine residues in apolipoprotein A-I are critical for activation of lecithin:cholesterol acyltransferase.

Previous studies have suggested that the helical repeat formed by residues 143;-164 of apolipoprotein A-I (apoA-I) contributes to lecithin:cholesterol acyltransferase (LCAT) activation. To identify specific polar residues involved in this process, we examined residue conservation and topology of apoA-I from all known species. We observed that the hydrophobic/hydrophilic interface of helix 143;-164 contains a cluster of three strictly conserved arginine residues (R149, R153, and R160), and that these residues create the only significant positive electrostatic potential around apoA-I. To test the importance of R149, R153, and R160 in LCAT activation, we generated a series of mutant proteins. These had fluorescence emission, secondary structure, and lipid-binding properties comparable to those of wild-type apoA-I. Mutation of conserved residues R149, R153, and R160 drastically decreased LCAT activity on lipid-protein complexes, whereas control mutations (E146Q, D150N, D157N, R171Q, and A175R) did not decrease LCAT activity by more than 55%. The markedly decreased activities of mutants R149, R153, and R160 resulted from a decrease in the maximal reaction velocity V(max) because the apparent Michaelis-Menten constant K(m) values were similar for the mutant and wild-type apoA-I proteins. These data suggest that R149, R153, and R160 participate in apoA-I-mediated activation of LCAT, and support the "belt" model for discoidal rHDL. In this model, residues R149, R153, and R160 do not form salt bridges with the antiparallel apoA-I monomer, but instead are pointing toward the surface of the disc, enabling interactions with LCAT. - Roosbeek, S., B. Vanloo, N. Duverger, H. Caster, J. Breyne, I. De Beun, H. Patel, J. Vandekerckhove, C. Shoulders, M. Rosseneu, and F. Peelman. Three arginine residues in apolipoportein A-I are critical for activation of lecithin:cholesterol acyltransferase J. Lipid Res. 2001. 42: 31;-40.

Amino Acid Sequence↗

PubMed2001

Effect of mutations of N- and C-terminal charged residues on the activity of LCAT.

On the basis of structural homology calculations, we previously showed that lecithin:cholesterol acyltransferase (LCAT), like lipases, belongs to the alpha/beta hydrolase fold family. As there is higher sequence conservation in the N-terminal region of LCAT, we investigated the contribution of the N- and C-terminal conserved basic residues to the catalytic activity of this enzyme. Most basic, and some acidic residues, conserved among LCAT proteins from different species, were mutated in the N-terminal (residues 1;-210) and C-terminal (residues 211;-416) regions of LCAT. Measurements of LCAT-specific activity on a monomeric substrate, on low density lipoprotein (LDL), and on reconstituted high density lipoprotein (rHDL) showed that mutations of N-terminal conserved basic residues affect LCAT activity more than those in the C-terminal region. This agrees with the highest conservation of the alpha/beta hydrolase fold and structural homology with pancreatic lipase observed for the N-terminal region, and with the location of most of the natural mutants reported for human LCAT. The structural homology between LCAT and pancreatic lipase further suggests that residues R80, R147, and D145 of LCAT might correspond to residues R37, K107, and D105 of pancreatic lipase, which form the salt bridges D105-K107 and D105-R37. Natural and engineered mutations at residues R80, D145, and R147 of LCAT are accompanied by a substantial decrease or loss of activity, suggesting that salt bridges between these residues might contribute to the structural stability of the enzyme.

Amino Acid Sequence↗

PubMed2000

A hydrophobic cluster at the surface of the human plasma phospholipid transfer protein is critical for activity on high density lipoproteins.

The plasma phospholipid transfer protein (PLTP) belongs to the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family, together with the cholesteryl ester transfer protein, the lipopolysaccharide binding protein (LBP) and the bactericidal permeability increasing protein (BPI). In the present study, we used the crystallographic data available for BPI to build a three-dimensional model for PLTP. Multiple sequence alignment suggested that, in PLTP, a cluster of hydrophobic residues substitutes for a cluster of positively charged residues found on the surface of LBP and BPI, which is critical for interaction with lipopolysaccharides. According to the PLTP model, these hydrophobic residues are situated on an exposed hydrophobic patch at the N-terminal tip of the molecule. To assess the role of this hydrophobic cluster for the functional activity of PLTP, single point alanine mutants were engineered. Phospholipid transfer from liposomes to high density lipoprotein (HDL) by the W91A, F92A, and F93A PLTP mutants was drastically reduced, whereas their transfer activity toward very low density lipoprotein and low density lipoprotein did not change. The HDL size conversion activity of the mutants was reduced to the same extent as the PLTP transfer activity toward HDL. Based on these results, we propose that a functional solvent-exposed hydrophobic cluster in the PLTP molecule specifically contributes to the PLTP transfer activity on HDL substrates.

Acute-Phase Proteins↗

PubMed2000

A mechanism of membrane neutral lipid acquisition by the microsomal triglyceride transfer protein.

The microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) belong to the vitellogenin (VTG) family of lipid transfer proteins. MTP is essential for the intracellular assembly and secretion of apoB-containing lipoproteins, the key intravascular lipid transport proteins in vertebrates. We report the predicted three-dimensional structure of the C-terminal lipid binding cavity of MTP, modeled on the crystal structure of the lamprey VTG gene product, lipovitellin. The cavity in MTP resembles those found in the intracellular lipid-binding proteins and bactericidal/permeability-increasing protein. Two conserved helices, designated A and B, at the entrance to the MTP cavity mediate lipid acquisition and binding. Helix A (amino acids 725-736) interacts with membranes in a manner similar to viral fusion peptides. Mutation of helix A blocks the interaction of MTP with phospholipid vesicles containing triglyceride and impairs triglyceride binding. Mutations of helix B (amino acids 781-786) and of N780Y, which causes abetalipoproteinemia, have no impact on the interaction of MTP with phospholipid vesicles but impair triglyceride binding. We propose that insertion of helix A into lipid membranes is necessary for the acquisition of neutral lipids and that helix B is required for their transfer to the lipid binding cavity of MTP.

Amino Acid Sequence↗

PubMed2000

Characterization of recombinant wild type and site-directed mutations of apolipoprotein C-III: lipid binding, displacement of ApoE, and inhibition of lipoprotein lipase.

The physicochemical properties of recombinant wild type and three site-directed mutants of apolipoprotein C-III (apoC-III), designed by molecular modeling to alter specific amino acid residues implicated in lipid binding (L9T/T20L, F64A/W65A) or LPL inhibition (K21A), were compared. Relative lipid binding efficiencies to dimyristoylphosphatidylcholine (DMPC) were L9T/T20L > WT >K21A > F64A/W65A with an inverse correlation with size of the discoidal complexes formed. Physicochemical analysis (Trp fluorescence, circular dichroism, and GdnHCl denaturation) suggests that L9T/T20L forms tighter and more stable lipid complexes with phospholipids, while F64A/W65A associates less tightly. Lipid displacement properties were tested by gel-filtrating apoE:dipalmitoylphosphatidylcholine (DPPC) discoidal complexes mixed with the various apoC-III variants. All apoC-III proteins bound to the apoE:DPPC complexes; the amount of apoE displaced from the complex was dependent on the apoC-III lipid binding affinity. All apoC-III proteins inhibited LPL in the presence or absence of apoC-II, with F64A/W65A displaying the most inhibition, suggesting that apoC-III inhibition of LPL is independent of lipid binding and therefore of apoC-II displacement. Taken together. these data suggest that the hydrophobic residues F64 and W65 are crucial for the lipid binding properties of apoC-III and that redistribution of the N-terminal helix of apoC-III (L9T/T20L) enhances the stability of the lipid-bound protein, while LPL inhibition by apoC-III is likely to be due to protein:protein interactions.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2000

Headgroup specificity of lecithin cholesterol acyltransferase for monomeric and vesicular phospholipids.

In this study, we investigated how the nature of the phospholipid head group and the macromolecular structure of the phospholipid, either as a monomer or incorporated into a lipid matrix, influence the activity of lecithin cholesterol acyltransferase (LCAT). As substrates we used 1,2-bis-(1-pyrenebutanoyl)-phosphatidylcholine, 1, 2-bis-(1-pyrenebutanoyl)-phosphatidylethanolamine and 1, 2-bis-(1-pyrenebutanoyl)-phosphatidyl-alcohols, either as monomers or incorporated into small unilamellar vesicles consisting of dipalmitoylphosphatidylcholine ether. The rate of hydrolysis of the pyrene-labeled phospholipids was determined both by fluorescence and by high performance liquid chromatography. V(max) and K(m) were calculated for the different substrates. The data show that V(max) is 10- to 30-fold higher for the hydrolysis of monomeric phosphatidylcholine (PC) compared to phosphatidylethanolamine (PE) and the phosphatidylalcohols, while K(m) values are comparable. When the fluorescent substrates were incorporated into dipalmitoylphosphatidylcholine ether vesicles, we observed a 4- to 10-fold increase of V(max) for PE and the phosphatidylalcohols, and no significant change for K(m). V(max) for PC remained the same. Natural LCAT mutants causing Fish-Eye Disease (FED) and analogues of these mutants expressed in Cos-1 cells, had similar activity on monomeric PC and PE. These data suggest that the activity of LCAT is determined both by the molecular structure of the phospholipid and by its macromolecular properties. The LCAT activity on monomeric substrates decreases as: phosphatidylcholine&z. Gt;phosphatidylethanolamine congruent withphosphatidylpropanol congruent withphosphatidylethanol congruent withphosphatidylethyleneglycol. The incorporation of PE and the phosphatidylalcohols into a matrix of dipalmitoylphosphatidylcholine decreases the specificity of the phospholipid head group.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2000

A nonfibrillar form of the fusogenic prion protein fragment [118-135] induces apoptotic cell death in rat cortical neurons.

Neuronal loss is a salient feature of prion diseases. However, its cause and mechanism, particularly its relationship with the accumulation and precipitation of the pathogenic, protease-resistant isoform PrP(Sc) of the cellular prion protein PrP(C), are still an enigma. Several studies suggest that neuronal loss could occur through a process of programmed cell death, which is consistent with the lack of inflammation in these conditions. By analogy with the pathological events occurring during the development of Alzheimer's disease, controversies still exist regarding the relationship between amyloidogenesis, prion aggregation, and neuronal loss. We recently demonstrated that a prion protein fragment (118-135) displayed membrane-destabilizing properties and was able to induce, in a nonfibrillar form, the fusion of unilamellar liposomes. To unravel the mechanism of prion protein neurotoxicity, we characterize the effects of the human Pr[118-135] peptide on rat cortical neurons. We demonstrate that low concentrations of the Pr[118-135] peptide, in a nonfibrillar form, induce a time- and dose- dependent apoptotic cell death, including caspase activation, DNA condensation, and fragmentation. This toxicity might involve oxidative stress, because antioxidant molecules, such as probucol and propyl gallate, protect neurons against prion peptide toxicity. By contrast, a nonfusogenic variant Pr[118-135, 0 degrees ] peptide, which displays the same amino acid composition but several amino acid permutations, is not toxic to cortical neurons, which emphasizes the critical role of the fusogenic properties of the prion peptide in its neurotoxicity. Taken together, our results suggest that the interaction between the Pr[118-135] peptide and the plasma membrane of neurons might represent an early event in a cascade leading to neurodegeneration.

Amino Acid Chloromethyl Ketones↗

PubMed2000

Structure and function of lecithin cholesterol acyl transferase: new insights from structural predictions and animal models.

The enzyme lecithin cholesterol acyl transferase is responsible for the synthesis of most of the cholesteryl esters in plasma, and therefore plays a key role in lipoprotein metabolism. The relationship between the structure and function of lecithin cholesterol acyl transferase has been extensively studied in the past years, and new data appeared in 1999 documenting the substrate specificity and physiological role of lecithin cholesterol acyl transferase. The discovery of natural mutants, together with the proposal of a three-dimensional model for the enzyme, has provided new tools to unravel the function of specific residues of lecithin cholesterol acyl transferase. The use of transgenic animals and the production of knock-out lecithin cholesterol acyl transferase mice has further contributed to the understanding of the lecithin cholesterol acyl transferase 'in vivo' function. Evidence for a protective role of lecithin cholesterol acyl transferase against the development of atherosclerosis through the hydrolysis of oxidized lipids was recently proposed. Lecithin cholesterol acyl transferase patterns in several pathologies were further clarified. These newer developments are reviewed here.

Animals↗

PubMed2000

Relationship between structure and biochemical phenotype of lecithin:cholesterol acyltransferase (LCAT) mutants causing fish-eye disease.

In order to test the hypothesis that fish-eye disease (FED) is due to a deficient activation of lecithin:cholesterol acyltransferase (LCAT) by its co-factor apolipoprotein (apo) A-I, we overexpressed the natural mutants T123I, N131D, N391S, and other engineered mutants in Cos-1 cells. Esterase activity was measured on a monomeric phospholipid enelogue, phospholipase A(2) activity was measured on reconstituted high density lipoprotein (HDL), and acyltransferase activity was measured both on rHDL and on low density lipoprotein (LDL). The natural FED mutants have decreased phospholipase A(2) activity on rHDL, which accounts for the decreased acyltransferase activity previously reported. All mutants engineered at positions 131 and 391 had decreased esterase activity on a monomeric substrate and decreased acyltransferase activity on LDL. In contrast, mutations at position 123 preserved these activities and specifically decreased phospholipase A(2) and acyltransferase activites on rHDL. Mutations of hydrophilic residues in amphipathic helices alpha 3;-4 and alpha His to an alanine did not affect the mutants' activity on rHDL. Based upon the 3D model built for human LCAT, we designed a new mutant F382A, which had a biochemical phenotype similar to the natural T123I FED mutant. These data suggest that residues T123 and F382, located N-terminal of helices alpha 3-4 and alpha His, contribute specifically to the interaction of LCAT with HDL and possibly with its co-factor apoA-I. Residues N131 and N391 seem critical for the optimal orientation of the two amphipathic helices necessary for the recognition of a lipoprotein substrate by the enzyme.

Animals↗

PubMed2000

Characterization and functional studies of lipoproteins, lipid transfer proteins, and lecithin:cholesterol acyltransferase in CSF of normal individuals and patients with Alzheimer's disease.

We investigated the lipoprotein distribution and composition in cerebrospinal fluid (CSF) in a group of patients with Alzheimer's disease (AD) or affected by other types of dementia in comparison to non-demented controls. We found slightly decreased apolipoprotein (apo)E and cholesterol concentrations in CSF of AD patients and moderately increased apoA-I concentrations, while in patients suffering from other types of dementia the apoA-I CSF concentration was increased. ApoA-IV concentrations varied widely in human CSF, but were not associated with any clinical condition. HDL(2)-like apoE-containing lipoproteins represent the major lipoprotein fraction. In CSF of normal controls, only a minor HDL(3)-like apoA-I-containing lipoprotein fraction was observed; this fraction was more prevalent in AD patients. ApoA-II was recovered mostly in the HDL(3) density range, while apoA-IV was not associated with lipoproteins but appeared in a lipid-free form, co-localizing with LCAT immunoreactivity. Bi-dimensional analysis demonstrated pre-beta and alpha apoA-I-containing particles; apoE and apoA-II were detected only in alpha-migrating particles. ApoA-IV distributed both to pre-beta and gamma-migrating particles; the LCAT signal was co-localized in this gamma-migrating fraction. Enzymatically active LCAT was present in human CSF as well as PLTP activity and mass; no CETP mass was detected. In CSF from AD patients, LCAT activity was 50% lower than in CSF from normal controls. CSF lipoproteins induced a significant cholesterol efflux from cultured rat astrocytes, suggesting that they play an active role in maintaining the cholesterol homeostasis in brain cells.

Alzheimer Disease↗

PubMed2000

Characterization of the lipid-binding properties and lipoprotein lipase inhibition of a novel apolipoprotein C-III variant Ala23Thr.

We have identified a G-to-A transition in exon 3 of the APOC3 gene resulting in a novel Ala23Thr apolipoprotein (apo) C-III variant, associated with apoC-III deficiency in three unrelated Yucatan Indians. The Ala23Thr substitution modifies the hydrophobic/hydrophilic repartition of the helical N-terminal peptide and hence could disturb the lipid association. In vitro expression in Escherichia coli of wild-type and mutant apoC-III enabled the characterization of the variant. Compared with wild-type apoC-III-Ala23, the mutant apoC-III-Thr23 showed reduced affinity for dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles with higher amounts of free apoC-III. Displacement of apoE from discoidal apoE:dipalmitoylphosphatidycholine (DPPC) complex by apoC-III-Thr23 was comparable to wild type but the less efficient binding of the apoC-III-Thr23 to the discoidal complex resulted in a higher apoE/apoC-III (mol/mol) ratio (34%) than with wild-type/apoE:DPPC mixtures. The inhibition of lipoprotein lipase (LPL) by apoC-III-Thr23 was comparable to that of wild type, and therefore effects on LPL activity could not explain the lower triglyceride (Tg) levels in Thr-23 carriers. Thus, these in vitro results suggest that in vivo the less efficient lipid binding of apoC-III-Thr23 might lead to a faster catabolism of free apoC-III, reflected in the reduced plasma apoC-III levels identified in Thr-23 carriers, and poorer competition with apoE, which might enhance clearance of Tg-rich lipoproteins and lower plasma Tg levels seen in Thr-23 carriers.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1999

Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein.

The microsomal triglyceride transfer protein (MTP) complexed to protein disulphide isomerase (PDI) is obligatory for the assembly of chylomicrons and very-low-density lipoproteins. The determination of the atomic structure of the MTP-PDI heterodimer has important implications for the treatment of those forms of hyperlipidaemia associated with the overproduction of very-low-density lipoproteins, which predispose to premature coronary heart disease. To perform structural studies of the human MTP-PDI complex it was necessary to produce milligram quantities of pure protein. We chose the baculovirus expression system for this purpose. Insects cells were co-infected with recombinant viruses encoding FLAG-tagged MTP and His-tagged PDI; the resulting heterodimer was purified by affinity chromatography. From 5 litres of insect cells, 4-6 mg of more than 95% pure recombinant protein was obtained. CD and attenuated total reflection Fourier-transform infrared spectroscopy indicate that the purified protein has around 34% alpha-helical and 33% beta-structure content. The recombinant protein had a comparable triglyceride transfer activity to that of bovine MTP-PDI. The production of polyclonal antibodies raised against the MTP and PDI subunits of the purified protein is described. The present study demonstrates the feasibility of expressing two proteins at high levels in insect cells and describes a transferable methodology for the purification of the resulting protein complex.

Amino Acid Sequence↗

PubMed1999

The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins.

The assembly of atherogenic lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein (apo)B, a microsomal triglyceride transfer protein (MTP) and protein disulphide isomerase (PDI). Here we show by molecular modelling and mutagenesis that the globular amino-terminal regions of apoB and MTP are closely related in structure to the ancient egg yolk storage protein, vitellogenin (VTG). In the MTP complex, conserved structural motifs that form the reciprocal homodimerization interfaces in VTG are re-utilized by MTP to form a stable heterodimer with PDI, which anchors MTP at the site of apoB translocation, and to associate with apoB and initiate lipid transfer. The structural and functional evolution of the VTGs provides a unifying scheme for the invertebrate origins of the major vertebrate lipid transport system.

Amino Acid Sequence↗

PubMed1999

Abnormal capacity to induce cholesterol efflux and a new LpA-I pre-beta particle in type 2 diabetic patients.

In this study, we first characterized the lipoprotein components of serum samples obtained from a group of well-controlled diabetic patients and from healthy subjects in fasting and postprandial states. We then explored some aspects of reverse cholesterol transport in the same population. Patients showed high levels of fasting triglycerides, postprandial triglyceride responses and LpC-III levels (3.18+/-0.86 vs 2.17+/-0.54 mg/dl, P < 0.001). There were also positive correlations between LpC-III and fasting triglycerides (r = 0.82, P < 0.001), total triglyceride area (r = 0.75, P < 0.001) and incremental triglyceride area (r = 0.54, P < 0.001). HDL-C and apo A-I were significantly decreased in diabetic patients due to a selective reduction in LpA-I subfraction, whose antiatherogenic role is generally accepted (37.4+/-8.0 vs 49.2+/-12.5 mg/dl, P < 0.001). In addition, HDL from patients proved to be triglyceride enriched and cholesteryl ester depleted, alterations which were further amplified in the postprandial state. The molar ratio HDL-C/apo A-I + apo A-II, already defined as a predictor of apo A-I fractional catabolic rate, was significantly diminished in the patient group (15.1+/-2.2 vs 20.8+/-3.3, P < 0.001), thus suggesting an accelerated catabolism of apo A-I. For the first time, we describe here the presence of a small apo A-I-containing particle, isolated by two-dimensional electrophoresis and characterized by immunoblotting, only in samples from diabetic patients. This particle that we named pre-beta0, has an apparent molecular weight of 40 kDa. As regards the capacity of serum samples to promote cholesterol efflux from [3H]cholesterol-labeled Fu5AH rat hepatoma cells, patient samples were found to induce significantly lower cholesterol efflux than controls only in the postprandial state (21.2+/-3.3 vs 23.8+/-1.8%, P = 0.012). The presence of pre-beta0 in samples from diabetic patients might therefore be associated to an altered capacity of these serum samples to promote cellular cholesterol efflux. Overall, these abnormalities may contribute to a delay in the reverse cholesterol transport pathway in type 2 diabetic patients.

Adult↗