METABOLIC REGULATION OF APOLIPOPROTEIN B MRNA EDITING

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DESCRIPTION (adapted from the applicant's description): Apolipoprotein B (apoB) is a non-exchangeable structural component of chylomicrons and of very low density (VLDL), intermediate density (IDL), and low density (LDL) lipoprotein particles. Two variants of apoB (B100 and B48) are expressed at different levels due to species- and tissue-specific editing of apoB mRNA. APOBEC-1 is the cytidine deaminase responsible for the editing of a CAA glutamine codon to a UAA translation stop codon and consequently the translational truncation of apoB100 to B48. Hepatic editing activity results in secretion of B48 VLDL and a reduction in the amount of serum LDL. The lack of apoB mRNA editing in human liver and the positive atherogenic risk factor represented by elevated serum LDL has focused research on the protein factors involved on apoB mRNA editing and their regulation. In addition to APOBEC-1, auxiliary proteins mediate editing as part of a holoenzyme complex termed the editosome. The goals of this research are to isolate and sequence cDNAs encoding these auxiliary proteins, demonstrate their functional or regulatory role in apoB RNA editing, and determine their structural interactions in the editosome. Specific aims focus on isolating and characterizing auxiliary proteins and cloning the cognate cDNAs using yeast three-hybrid and genetic selections. The function of each candidate protein in the mechanism and regulation of editing will be evaluated in cell lines and under defined in vitro conditions using depletion and add-back studies. Tissue-specific expression of auxiliary factors will be examined by Northern and Western blotting. Metabolic regulation of hepatic editing activity through alteration in auxiliary protein expression, interactions, or subcellular localization will be examined in McArdle cell lines and rat primary hepatocytes treated with ethanol. These studies will extend knowledge of proteins involved in apoB mRNA editing mechanisms and regulation, which will lead to significant new understanding of the factors required for high fidelity editing in human liver and may reveal molecular targets for therapeutic intervention in atherogenic diseases.