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 lipidprotein 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-Imediated 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.

In a previous characterization of the ABCA subfamily of the ATP-binding cassette (ABC) transporters, we identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters (Peelman, F., Labeur, C., Vanloo, B., Roosbeek, S., Devaud, C., Duverger, N., Denefle, P., Rosier, M., Vandekerckhove, J., and Rosseneu, M. (2003) J. Mol. Biol. 325, 259 -274). These phosphorylation residues are located in the conserved cytoplamic R1 and R2 domains, downstream of the nucleotide binding domains NBD1 and NBD2. To study the possible regulation of the ABCA1 transporter by CK2, we expressed the recombinant cytoplasmic domains of ABCA1, NBD1؉R1 and NBD2؉R2. We demonstrated that in vitro ABCA1 NBD1؉R1, and not NBD2؉R2, is phosphorylated by CK2, and we identified Thr-1242, Thr-1243, and Ser-1255 as the phosphorylated residues in the R1 domain by mass spectrometry. We further investigated the functional significance of the threonine and serine phosphorylation sites in NBD1 by site-directed mutagenesis of the entire ABCA1 followed by transfection into Hek-293 Tet-Off cells. The ABCA1 flippase activity, apolipoprotein AI and AII binding, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine and serine residues. This was confirmed by the effect of specific protein kinase CK2 inhibitors upon the activity of wild type and mutant ABCA1 in transfected Hek-293 Tet-Off cells. The activities of the mutants mimicking threonine phosphorylation were close to that of wild type ABCA1. Our data, therefore, suggest that besides protein kinase A and C, protein kinase CK2 might play an important role in vivo in regulating the function and transport activity of ABCA1 and possibly of other members of the ABCA subfamily.The role of the ABCA1 1 transporter, a member of the subfamily A of the ATP binding cassette transporters, in the efflux of cellular phospholipids and cholesterol has become well established (2). Several studies link ABCA1 mutations to impaired cellular cholesterol and phospholipid efflux characteristic of Tangier disease and high density lipoprotein-deficiency patients (3-5). Expression of WT and mutant ABCA1 in cultured cells demonstrated the correlation between the level of expression and activity of the ABCA1 transporter, the extent of binding to the apolipoprotein AI (apoAI) acceptor and the efflux of cellular lipid (6).Human ABC transporters consist of a cytoplasmic nucleotide binding domain (NBD), which binds and hydrolyzes ATP, and of a membrane-spanning domain through which the substrate is translocated (7-8). Besides these elements, regulatory domains with putative phosphorylation sites were described in several human transporters (7). We carried out an extensive analysis of the subfamily A of the ABC transporters and showed that this subfamily consists of 13 human ABCA and of many eukaryotic and prokaryotic ABCA homologues (1). Multiple alignments of the subfamily A transporters de...

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.

In this study the plasma lipid and apoprotein concentrations have been assayed in 80 full-term newborns, at 0, 7 and 30 days of life, and the data have been analyzed as a function of the composition of the diet. The total cholesterol, HDL cholesterol, the apo A-I, A-II and B protein concentrations were followed in 4 groups of infants receiving respectively breast-feeding, adapted formulae I, II with a P/S ratio close to that of maternal milk and a formula III enriched with polyunsaturated fatty acids. After 7 and 30 days the infants receiving the adapted formulae I and II have plasma lipid and apoprotein values similar to those of the breast-fed infants indicating a parallel evolution of the lipids and apoproteins in the three groups. The lipid and apoprotein patterns were significantly different in the group of infants receiving a diet enriched with polyunsaturated fatty acids. The total and VLDL-LDL cholesterol and the apo B protein concentrations are significantly lower than in the breast-fed infants after 7 days, and these differences become more pronounced after 30 days. These results suggest that the fatty acid composition of the diet influences the lipid and lipoprotein synthesis in newborns, specially by decreasing the lipid and apoprotein concentrations of the VLDL-LDL fraction.

ABSTRACT. In this study the lipid and apoprotein profiles were investigated in newborns at 0, 7, and 30 days of life. The plasma lipoproteins were separated both by ultracentrifugation and gel filtration in order to compare the patterns obtained by the two techniques. At birth, the apo E concentration is comparable to that measured in adults, but its distribution among lipoproteins is significantly different as more than 80% of the plasma apo E belongs to high-density lipoproteins (HDL). At 7 and 30 days the plasma apo E concentrations are close to the values at birth, but a significant redistribution occurs from HDL to very low-density lipoproteins. By analogy with apo B, the plasma apo CIII concentration is low at birth and increases between 0 and 7 days by a factor of about two. Plasma triglycerides increase significantly during the first week of life so that the apo CIII increase is most pronounced in very low-density lipoproteins. These lipoproteins therefore become enriched in apo E, apo CIII and triglycerides between 0 and 7 days. At birth, a distinct HDL fraction, enriched in apo E, apo A11 and cholesterol (HDLE), could be detected. To compensate for the low LDL levels, this HDLE fraction might function as an additional source for cholesterol delivery to peripheral tissues via the apo (B, E) receptor. At later age, low-density lipoprotein synthesis is enhanced, apo E is transferred to very low-density lipoproteins, and cholesterol delivery via the HDL, becomes less important. These data demonstrate that significant differences occur in the plasma concentration and distribution of the apo CIII and E proteins during the initial period of life, and that these apoproteins fulfill an important metabolic role. (Pediatr Res 20: 324-328, 1986) Abbreviations HDL, high-density lipoproteins VLDL, very low-density lipoproteins LDL, low-density lipoproteins During the perinatal period, major shifts in nutritional supply cause changes in substrate utilization by the fetus and newborn. Unimpeded substrate flow from placenta to fetus is required to meet the energy requirements for growth and for fuel storage, primarily of glycogen and fat. During the intra-and postpartum periods, this constant flow is interrupted so that glycogen is required to maintain serum glucose levels. Glycogen stores are then depleted and active glycogenesis starts. In the perinatal period, fatty acid and ketone body oxidation become important energy sources, and during this period an adaptation to a high fat milk diet becomes therefore necessary.These adaptations are controlled by changes in substrate flow and hormonal milieu of the fetus and newborn. Such changes n substrate result in rapid shifts in the synthesis and metabolism of lipid transporting particles, whose composition then changes from that before birth.In newborns, plasma lipid and lipoprotein patterns also are significantly different from those observed in adults, both in their concentration and distribution. Newborn plasma lipoproteins consist mostly of HDL, while VLDL and LDL are...