Apolipoprotein (apo) B is the major apoprotein in the triglyceride-rich lipoproteins, chylomicrons, very low-density lipoproteins (VLDL), and intermediate-density lipoproteins (IDL), as well as low-density lipoproteins (LDL) and lipoprotein (a). ApoB exists in two forms, apoB-100, produced in the liver and present in all these lipoprotein classes except chylomicrons, and apoB-48, the chylomicron-specific apoprotein. Both apoBs are central to lipid transport and homeostasis and apoB-48 is essential for intestinal fat absorption. ApoB-48 production is unique in that its mRNA is the edited product of apoB-100 mRNA. ApoB mRNA editing is a process whereby the cytidine (C) in a CAA codon in apoB-100 mRNA, encoding Gln-2153, is deaminated to a uridine (U), producing an in-frame UAA stop codon in apoB-48 mRNA. APOBEC-1 has been identified as the C6U RNA editing protein that mediates this reaction. Auxiliary proteins are required for the efficient editing of apoB mRNA by APOBEC-1. During the last funding cycle, the investigators have cloned three APOBEC-1 binding proteins, called ABBP-1, -2, and -3, and have documented their auxiliary activity in cultured cells. Furthermore, the investigators have cloned an APOBEC-1 homolog that the investigators have called APOBEC-2. APOBEC-2, like APOBEC-1, has cytidine deaminase activity. It is expressed exclusively in heart and skeletal muscle and displays much higher sequence similarity to APOBEC-1 than to cytidine deaminase. There are four Specific Aims: (a) To characterize the function of auxiliary proteins involved in apoB mRNA editing cloned in the investigator's laboratory by studying their functional role by conditional targeted inactivation in mice. (2) To define the substrate mRNA for APOBEC-2 in muscle cells. (3) To study the functional role of APOBEC-2 in vivo by genetic inactivation by gene targeting. (4) To study the functional role of APOBEC-2 in vivo by overexpression by gene targeting. To date, apoB mRNA editing is the only example of C6U editing described in mammals. The proposed experiments define the role of (auxiliary) proteins other than APOBEC-1 in the process. APOBEC-2-mediated editing constitutes the first example of non-apoB C6U RNA editing that will be characterized. Thus, the overall project is highly relevant to lipid nutrient absorption and homeostasis. Significantly, it also provides important information on a newly described pathway in posttranscriptional gene regulation.
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