METABOLIC REGULATION OF APOLIPOPROTEIN B MRNA EDITING

性状（改编自申请人的描述）：载脂蛋白B（apo B） 是乳糜微粒的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apoB的两种变体（B100和B48）在不同的细胞中表达。 由于apoB mRNA的物种和组织特异性编辑，APOBEC-1是 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UAA的翻译截短 apoB 100至B48。肝脏编辑活性导致B48 VLDL分泌， 降低血清LDL水平。人类缺乏apoB mRNA编辑 肝脏和以血清升高为代表的阳性致动脉粥样硬化危险因素 LDL的研究主要集中在apoB mRNA编辑的蛋白质因子上 和他们的规则。除了APOBEC-1，辅助蛋白介导 作为称为编辑体的全酶复合物的一部分进行编辑。这个的目标 研究是分离和测序编码这些辅助蛋白的cDNA， 证明它们在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。具体目标重点 分离和鉴定辅助蛋白并克隆同源蛋白 使用酵母三杂交和遗传选择的cDNA。每一个的功能 候选蛋白质的机制和调控编辑将进行评估 在细胞系中和在限定的体外条件下使用消耗和 添加回研究。辅助因子的组织特异性表达将是 通过北方和西方印迹法检测。肝脏代谢调节 通过改变辅助蛋白表达的编辑活性， 相互作用或亚细胞定位将在McArdle细胞中进行检查 细胞和用乙醇处理的大鼠原代肝细胞。这些研究将 扩展参与apoB mRNA编辑机制的蛋白质的知识， 监管，这将导致对这些因素的重大新的理解 在人类肝脏中进行高保真编辑所需的，并可能揭示分子 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.