The very-low-density-lipoprotein receptor (VLDLR) is a recently described lipoprotein receptor that shows considerable similarity to the low-density-lipoprotein receptor (LDLR). This receptor has been suggested to be important for the metabolism of apoprotein-E-containing triacylglycerolrich lipoproteins, such as very-low-density-lipoprotein (VLDL), P-migrating VLDL and intermediate-density lipoprotein. cDNA clones that code for the VLDLR were isolated from a mouse heart cDNA library. The deduced amino acid sequence predicts a mature protein of 846 amino acids preceded by a 27-residue signal peptide. Three mRNA species for the VLDLR with sizes of 3.9, 4.5 and 7.9 kilobases were present in high concentration in heart and muscle, which utilize triacylglycerols as an energy source. VLDLR mRNA is also detected in decreasing amounts in kidney, brain, ovary, testis, lung and adipose tissue. It is essentially absent in liver and small intestine. The amino acid sequence of the VLDLR is highly conserved among rabbit, human and mouse. VLDLR contains five structural domains very similar to those in LDLR, except that the ligand-binding domain in VLDLR has an eightfold repeat instead of a sevenfold repeat in LDLR. Sequence conservation among animal species is much higher for the VLDLR than the LDLR. Sequences of the VLDLR from three vertebrate species and the LDLR from five vertebrate species were aligned and a phylogenetic tree was reconstructed. Although both receptors contain five domains and share amino acid sequence similarity, our computations showed that they diverged before the divergence between mammals and amphibians. In addition, sequence comparison of both receptor sequences suggests that the rabbit is evolutionarily closer to man than to the mouse. These results are consistent with the hypothesis that the VLDLR and the LDLR have evolved from a common ancestral gene to play distinct roles in lipoprotein metabolism and that the metabolic handling of triacylglycerol by the body via the VLDLR is a highly conserved mechanism.Lipoprotein receptors play pivotal roles in the metabolism of triacylglycerols and cholesterol . The best characterized of the lipoprotein receptors is the low-density-lipoprotein receptor (LDLR). The LDLR binds to the apolipoprotein (apo) B-100-containing low-density-lipoproteins (LDL), as well as apoprotein-Econtaining lipoproteins such as intermediate-density lipoproteins (IDL), P-migrating very-low-density lipoproteins @-VLDL) and a cholesterol-induced high-density lipoprotein, which contains apoE as its sole apolipoprotein (Esser et al.,
The 3.7-kilobase cDNA clone isolated contains a 1620-base pair open reading frame which encodes a protein of 540 amino acids. The predicted mouse ACAT protein is 87% identical to the protein product of human ACAT K1 and shares many of the same secondary structural features, including two transmembrane domains, a leucine heptad motif consistent with dimer or multimer formation, and five regions homologous to the "signature sequences" found in other enzymes that catalyze acyl adenylation followed by acyl thioester formation and acyl transfer. Using the cDNA as a hybridization probe, we mapped the gene encoding mouse ACAT to chromosome 1 in a region syntenic to human chromosome 1 where the ACAT gene is located. Northern blot analysis and RNase protection assays of mouse tissues revealed that ACAT mRNA is expressed most highly in the adrenal gland, ovary, and preputial gland and is least abundant in skeletal muscle, adipose tissue, heart, and brain. To study the dietary regulation of ACAT mRNA expression in mouse tissues, we fed C57BL/6J mice a high-fat, highcholesterol (HF/HC) atherogenic diet for 3 weeks and measured ACAT mRNA levels in various tissues by RNase protection. The HF/HC diet had little effect on ACAT mRNA levels in the small intestine, aorta, adrenal, or peritoneal macrophages, whereas hepatic ACAT mRNA levels were doubled in mice fed the atherogenic diet. ACAT activity in liver microsomes was similarly increased in cholesterol-fed mice, suggesting that mouse ACAT is regulated at least in part at the level of mRNA abundance. Additionally, a significant positive correlation was observed between ACAT activity and microsomal free cholesterol levels in chow-and cholesterol-fed mice, supporting the concept of cholesterol availability as a regulator of ACAT. To further investigate the regulation of ACAT activity under controlled conditions, ACAT-deficient Chinese hamster ovary cells were stably transfected with the mouse ACAT cDNA clone driven by a cytomegalovirus promoter. Two transfected Chinese hamster ovary cell lines that expressed the mouse ACAT transgene regained the ability to esterify cholesterol. Cholesterol esterification activity in both of these cell lines was further increased by exposure of these cells to low density lipoprotein. Thus we have demonstrated that mouse ACAT expression in vivo and in vitro is regulated by at least two mechanisms: control of mRNA abundance and post-transcriptional regulation of the enzyme activity, probably by cholesterol availability.The process of cholesterol homeostasis in extrahepatic tissues such as the fibroblast involves the uptake of lipoproteins by cell surface receptors, lysosomal hydrolysis of lipoproteinderived cholesteryl esters to yield free cholesterol, and reesterification of free cholesterol in the endoplasmic reticulum for storage in cytoplasmic lipid droplets. The re-esterification step is crucial to prevent excess free cholesterol from disrupting cell membranes and is carried out by the enzyme acyl-CoA: cholesterol acyltransferase (ACAT) 1 (for...
APOBEC-1 is a catalytic subunit of an apolipoprotein B (apoB) mRNA editing enzyme complex. In humans it is expressed only in the intestine, whereas in mice it is expressed in both the liver and intestine. APOBEC-1 exists as a spontaneous homodimer (Lau, P. P., Zhu, H.-J., Baldini, A., Charnsangavej, C., and Chan, L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8522-8526). We tested the editing activity and dimerization potential of three different mouse APOBEC-1 mutants using in vitro editing activity assay and immunoprecipitation in the presence of epitope-tagged APOBEC-1. One catalytically inactive mutant, mu1 (H61K/C93S/C96S), that retains its capacity to dimerize with wild-type APOBEC-1 was found to inhibit the editing activity of the latter and was thus a dominant negative mutant. Two other inactive mutants that dimerized poorly with APOBEC-1 failed to inhibit its activity. Intravenous injection of a mu1 adenovirus, Admu1, in C57BL/6J mice in vivo resulted in liver-specific expression of mu1 mRNA. On days 4 and 9 after virus injection, endogenous hepatic apoB mRNA editing was 23.3 ؎ 5.0 and 36.8 ؎ 5.7%, respectively, compared with 65.3 ؎ 11 and 71.3 ؎ 5.2%, respectively, for luciferase adenovirus-treated animals. Plasma apoB-100 accounted for 95 and 93% of total plasma apoB in Admu1 animals on days 4 and 9, respectively, compared with 78 and 72% in luciferase adenovirus animals. Plasma cholesterol on day 9 was 98 ؎ 17 mg/dl in the mu1-treated animals, substantially higher than phosphate-buffered saline-treated (57 ؎ 9 mg/dl) or luciferase-treated (71 ؎ 12 mg/dl) controls. Fast protein liquid chromatography analysis of mouse plasma showed that the intermediate density/low density lipoprotein fractions in the animals treated with the dominant negative mutant adenovirus were much higher than those in controls. We conclude that active APOBEC-1 functions as a dimer and its activity is inhibited by a dominant negative mutant. Furthermore, apoB mRNA editing determines the availability of apoB-100, which in turn limits the amount of intermediate density/low density lipoprotein that can be formed in mice. Liver-specific inhibition of apoB mRNA editing is an important component of any strategy to enhance the value of mice as a model for human lipoprotein metabolism.The mouse is a useful animal model for lipoprotein metabolism and atherosclerosis (1, 2). Its value as a model for human disease, however, is limited by the fact that there is a substantial difference in lipoprotein metabolism between the two species. One major difference is the presence of high levels of apolipoprotein B (apoB) 1 mRNA editing in the liver in mice but not in humans.ApoB mRNA editing is a process by which apoB-100 mRNA is converted to apoB-48 mRNA (3, 4) (reviewed in Ref. 5). It involves the conversion of the first base of the codon CAA, encoding glutamine 2153 in apoB-100, to UAA, a stop codon, in apoB-48 mRNA. In humans, editing occurs exclusively in the small intestine but not in the liver. Therefore, the human liver produces apoB-100 and ...
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