METABOLIC REGULATION OF APOLIPOPROTEIN B MRNA EDITING

描述(改编自申请人的描述)：载脂蛋白B(ApoB) 是乳胶粒的一种不可交换的结构成分， 密度(VLDL)、中密度(IDL)和低密度(LDL)脂蛋白 粒子。载脂蛋白B的两个变异体(B100和B48)在不同的 由于特定物种和组织对apoB mRNA的编辑而导致的水平。APOBEC-1 IS 负责编辑CAA谷氨酰胺密码子的胞苷脱氨酶 UAA翻译终止密码子，因此翻译截断 ApoB100至B48。肝脏编辑活动导致B48极低密度脂蛋白和a 降低血清低密度脂蛋白的含量。人类apoB基因编辑缺失的研究 肝脏和以血清升高为代表的阳性动脉粥样硬化危险因素 低密度脂蛋白将重点放在参与apoB mRNA编辑的蛋白质因素上 以及他们的规定。除APOBEC-1外，辅助蛋白还参与 编辑是称为编辑小体的全酶复合体的一部分。这样做的目的是 研究是分离和测序编码这些辅助蛋白的cDNA， 展示它们在apoB RNA编辑中的功能或调节作用，以及 确定它们在编辑体中的结构相互作用。具体目标和重点 辅助蛋白的分离鉴定及同源基因的克隆 利用酵母三杂交和遗传选择的cDNA。每一项的功能 对候选蛋白质在编辑机制和调控中的作用进行评价 在细胞系中，并在确定的体外条件下使用耗竭和 附加研究。辅助因子的组织特异性表达将是 经Northern和Western blotting检测。肝脏的代谢调节 通过改变辅助蛋白表达的编辑活动， 将在McArdle细胞中检查相互作用或亚细胞定位 乙醇处理大鼠原代肝细胞。这些研究将 扩展apoB mRNA编辑机制和相关蛋白质的知识 监管，这将导致对这些因素的重大新理解 在人类肝脏中进行高保真编辑所需的，可能会揭示分子 动脉粥样硬化性疾病治疗干预的靶点。

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.