Selective Translation Initiations Of Trans-spliced Human Acyl-coenzyme A: Cholesterol Acyltransferase-1 MRNA

Acyl-CoA: cholesterol acyltransferase (ACAT) is an intracellular enzymepresent in various eukaryotic cells and catalyzes the formation of cholesterolesters (CE) from long-chain fatty acyl coenzyme A and cholesterol. Up to now,two ACAT genes have been identified in mammals. In adult human tissues,ACAT1 protein is found in almost all of the cells and tissues examined, andbelieved to be crucial for maintaining cholesterol homeostasis. On the otherhand, ACAT2 is selectively expressed in liver and small intestine, mainlyinvolving in the dietary cholesterol absorption and assembly of apoB-containinglipoprotein. ACATs slao play important roles in some serious human diseasesincluding atherosclerosis, Alzheimer's disease (AD) and gallstone. For thesereasons, ACAT has been considered as one of major pharmaceutical targets fordeveloping CE-lowering, anti-atherosclerosis and/or anti-AD drugs. In collaboration with Professor Chang TY in Dartmouth Medical School, wehave first reported the organization of human ACAT1 genomic DNAs. Thehuman ACAT1 gene is located in two different chromosomes (7 and 1), with eachchromosome containing a disparate ACAT1 promoter (P7 and P1). Our furtherexperiments show that the human ACAT1 mRNA corresponding to cDNA K1 isproduced by a novel RNA trans-splicing. Using ACAT1 N-terminal 131 aminoacids as antigen, coded from 5'-part of cDNA K1 open reading frame (ORF,residues 1397-3049), specific anti ACAT1 antibody DM10 was prepared. With - 6 -Abstractthis antibody in Western blot analysis, a major 50 kDa ACAT1 protein has beenobserved in human cells and transfected AC29 cells with cDNA K1 ORFsequence. Based on the previous study, this work is mainly focused ontranslation of the trans-spliced human ACAT1 mRNA and the relative molecularmechanism. Western blot analysis with DM10 illustrates that a larger 56 kDa humanACAT1 protein together with the 50 kDa one can be expressed in AC29 cellstransfected with full-length cDNA K1 located in two chromosomes. Furtherevidences from serial 5'-deletions in cDNA K1 reveal that both chromosome 7and 1 sequences of cDNA K1 contribute to translation of the larger 56 kDaACAT1 protein, while only chromosome 1 sequence of cDNA K1 is needed forproduction of the 50 kDa ACAT1 protein. Moreover, two ACAT1 N-terminalproducts (ACAT1-NTPs) are detected in several cell lines transfected with3'-truncated cDNA K1, and show the same expression patterns, suggesting that thedifferences of these two ACAT1 proteins may exist in their N-terminus. Forfurther study, another anti-ACAT1 antibody DM58 was raised from 40 aminoacids encoded by 120 nucleotides upstream to and in frame with cDNA K1 ORF.Using DM58 in Western blot analysis, only the larger ACAT1 protein (56/25 kDa)could be detected in transfected AC29 cells, indicating that the larger ACAT1protein contains an additional amino acid sequence upstream to N-terminal of 50kDa one. Different N-terminal amino acid sequences of 50 and 56 kDa ACAT1proteins cause their different enzymatic activities, but have no effect on theirsubcellular locations tested with immunoelectron microscopy. In addition,endogenous 56 kDa ACAT1 protein together with 50 kDa one is also detected inhuman monocyte (THP-1)-derived macrophages, and the amount of the 56 kDaACAT1 protein increases with the PMA-treated time prolonged. Endogenousexpression of 56 kDa ACAT1 protein is examined in human heart tissues andprimary cultured human smooth muscle cells. To our knowledge, our current - 7 -Abstractresults provide the first mammalian example that a functional protein, the 56 kDaACAT1, can be produced from trans-spliced mRNA. Based on the elucidation of two human ACAT1 proteins with differentenzymatic activities translated from trans-spliced ACAT1 mRNA, their translationinitiations and molecular mechanisms are then further investigated. The predicated human ACA...