Lecithin:cholesterol acyltransferase. Functional regions and a structural model of the enzyme

The amino acid sequence of human lecithin:cholesterol acyltransferase has been determined by degradation and alignment of peptides obtained from tryptic and staphylococcal digestions and the cleavage with cyanogen bromide and consisted of 416 amino acid residues. All of the tryptic peptides of lecithin:cholesterol acyltransferase were isolated and sequenced. Peptides resulting from digestion by staphylococcal protease, cyanogen bromide cleavage, or the combination of the two methods were employed to find overlapping segments. The N terminus of human lecithin:cholesterol acyltransferase was determined to be phenylalanine by sequencing the whole protein up to 40 residues while the C terminus was identified as glutamic acid through carboxypeptidase Y cleavage. Cys50 and Cys74 and Cys313 and Cys356 were identified as the two disulfide bridges while the free sulfhydryl groups were located at positions 31 and 184. The N-glycosylated sites of the protein were assigned to asparagines at positions 20, 84, 272, and 384. The active site of lecithin:cholesterol acyltransferase was identified as serine on position 181 according to its homology with other serine-type esterases which have a common structure of glycine-variable amino acid-active serine-variable amino acid-glycine (Gly-X-Ser-X-Gly) with the variable amino acids disrupting the homology. No long internal repeats or homologies with apolipoproteins were found. The secondary structure is consistent with the results of predictive algorithms. A simple model of the enzyme is proposed on the basis of available chemical data and predictive methods.     

Evidence that apoB-100 of low-density lipoproteins is a novelsrc-related protein kinase

Protein-tyrosine kinases of signal transduction pathways occur and function intracellularly. In contrast, the low-density lipoprotein (LDL) particle circulates in plasma, where its function is to solubilize and transport lipid. Recently, several reports showed that LDL may have a role in signal transduction. We have identified a region in the apoB-100 primary structure which shows similarity to Src-homology-1 (SH1) domains, the kinase region of protein-tyrosine kinases. Results obtained in protein kinase assays of highly purified LDL showed that only the apoB-100 was phosphorylated, suggesting that apoB-100 has the capacity to undergo autophosphorylation like known protein-tyrosine kinases. Phosphorylation was not observed for any other apolipoprotein in LDL or for any component of high-density lipoprotein and lipoprotein [a]. Our results suggest that apoB-100 may be a novel and functional member of thesrc protein kinase family.     

Molecular evolution of the MAGUK family in metazoan genomes

Background  

Development, differentiation and physiology of metazoans all depend on cell to cell communication and subsequent intracellular signal transduction. Often, these processes are orchestrated via sites of specialized cell-cell contact and involve receptors, adhesion molecules and scaffolding proteins. Several of these scaffolding proteins important for synaptic and cellular junctions belong to the large family of membrane-associated guanylate kinases (MAGUK). In order to elucidate the origin and the evolutionary history of the MAGUKs we investigated full-length cDNA, EST and genomic sequences of species in major phyla.     

Regulation of peroxisome proliferator-activated receptor-gamma-mediated gene expression. A new mechanism of action for high density lipoprotein

Cellular cholesterol content reflects a balance of lipid influx by lipoprotein receptors and endogenous synthesis and efflux to cholesterol acceptor particles. The beneficial effect of high density lipoprotein (HDL) in protecting against the development of cardiovascular disease is thought to be mediated predominately through its induction of cellular cholesterol efflux and "reverse cholesterol transport" from peripheral tissues to the liver. We tested the hypothesis that HDL could inhibit cellular lipid accumulation by modulating expression of peroxisome proliferator-activated receptor-gamma (PPARgamma)-responsive genes. To this end, we evaluated expression of two PPARgamma-responsive genes, CD36, a receptor for oxidized low density lipoprotein, and aP2, a fatty acid-binding protein. HDL decreased expression of macrophage CD36 and aP2 in a dose-dependent manner. HDL also decreased aP2 expression in fibroblasts, reduced accumulation of lipid, and slowed differentiation of fibroblasts into adipocytes. HDL stimulated mitogen-activated protein (MAP) kinase activity, and inhibition of CD36 expression was blocked by co-incubation with a MAP kinase inhibitor. HDL increased expression of PPARgamma mRNA and protein, induced translocation of PPARgamma from the cytoplasm to the nucleus, and increased PPARgamma phosphorylation. Our data demonstrate that despite induction and translocation of PPARgamma in response to HDL, MAP kinase-mediated phosphorylation of PPARgamma inhibited expression of PPARgamma-responsive genes and suggest mechanisms by which HDL may inhibit cellular lipid accumulation.