ENTEROHEPATIC LIPID FLUX AND APOPROTEIN BIOSYNTHESIS

肠肝脂质通量和脱辅基蛋白生物合成

• 批准号: 2218729
• 负责人: Nicholas O. Davidson
• 金额: $ 38.03万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2218729
• 关键词: apolipoproteins; blood lipoprotein biosynthesis; fusion gene; gene complementation; gene expression; gene mutation; gene targeting; genetic regulation; genetic transcription; genome; immunoaffinity chromatography; laboratory rat; lipid transport; liver metabolism; messenger RNA; molecular cloning; molecular genetics; posttranscriptional RNA processing; posttranslational modifications; protein structure function; site directed mutagenesis; western blottings

Apolipoprotein B (apo B) is an obligate structural component of both intestinal and hepatic triglyceride-rich lipoprotein and plays a central role in their catabolism. Two distinct isomorphs (apo B100 and apo B48) are synthesized, in a tissue-specific manner, from a common gene. Apo B48 is produced in mammalian small intestine and arises as a result of a site-specific, posttranscriptional, cytidine deamination of apo B m RNA, referred to as apo B mRNA editing. In this reaction, a CAA (glutamine) codon in apo B100 is edited to a UAA, stop codon and results in apo B48 synthesis. The truncated protein lacks the domains, present in apo B100, for binding to the LDL receptor and also for interaction with apo (a) which leads to the formation of Lp(a). As a result, apo B mRNA editing assumes central importance in determining the atherogenic potential of apo B. Apo B mRNA editing is mediated by protein factor(s), which together constitute the apo B RNA editing enzyme. One of the components, REPR, (rat apo B mRNA editing protein) has been cloned by the investigator, REPR is most likely the catalytic subunit of the apo B mRNA editing enzyme complex and has an absolute requirement for additional protein components [complementation factors] in order to mediate apo B RNA editing. However, there is little information concerning the nature and identity of these complementation factors. Additionally, although REPR has been shown to function as a component of the apo B mRNA editing enzyme through complementation and transfection studies, its role is not established to be indispensable. Accordingly, the objectives of this proposal are, first, to identify proteins which interact with REPR and complement apo B mRNA editing. In the second aim, we will mutate critical residues within a conserved Zn-binding domain in REPR, present in other cytidine deaminases. Using a functional OST-REPR fusion protein, we will determine the effects of these mutations on apo B RNA editing. Additionally, in studies which represent the major focus of this second aim, we will determine the biological importance of REPR to ap0 B gene expression. This will be achieved by means of targeted gene disruption both in ES cells and in selected cell lines, using genomic clones isolated and characterized by the investigator. This approach will allow a definitive assessment of the importance of REPR and its homologs in apo B mRNA editing and in the regulation of apo B gene expression. Taken together, this approach should allow elucidation of the molecular mechanisms underlying this novel pathway for gene regulation in the mammalian small intestine.

载脂蛋白B（APO B）是两者的义务结构成分 肠道和肝甘油三酸酯富含脂蛋白，并发挥中心 在其分解代谢中的作用。两个不同的异构词（APO B100和APO B48） 以组织特异性的方式从一个共同基因合成。 APO B48 是在哺乳动物小肠中产生的，由于 位点特异性，转录后，胞苷脱氨酸的Apo B M RNA， 称为Apo B mRNA编辑。在此反应中，CAA（谷氨酰胺） Apo B100中的密码子已编辑为UAA，停止密码子并导致Apo B48 合成。截短的蛋白质缺乏Apo B100中存在的结构域， 用于与LDL受体结合以及与Apo的相互作用（a） 导致LP（a）的形成。结果，Apo B mRNA编辑 在确定的动脉粥样硬化潜力方面具有至关重要的重要性 apo B. apo b mRNA编辑是由蛋白质因子介导的，s） 共同构成Apo B RNA编辑酶。其中一个组件， ret，（大鼠apo b mRNA编辑蛋白）已被克隆 研究者，反复是Apo B mRNA的催化亚基 编辑酶复合物，并具有额外的绝对要求 蛋白质成分[互补因子]以介导Apo B RNA编辑。但是，关于性质的信息很少 以及这些补充因素的身份。另外，虽然 reth已显示作为APO B mRNA编辑的组成部分 酶通过互补和转染研究，其作用不是 确定是必不可少的。因此，目标的目标 提案首先是确定与reper相互作用的蛋白质和 补充Apo B mRNA编辑。在第二个目标中，我们将变为关键 保守的Zn结合域中的残留物在其他中存在 胞苷脱氨酶。使用功能性OST-REPR融合蛋白，我们将 确定这些突变对APO B RNA编辑的影响。 此外，在代表第二个重点的研究中 目的，我们将确定reter对AP0 B基因的生物学重要性 表达。这将通过靶向基因破坏来实现 在ES细胞和选定的细胞系中，都使用基因组克隆 孤立并以研究者为特征。这种方法将允许 对RETER及其在APO中的同源物的重要性的确切评估 B mRNA编辑和APO B基因表达的调节。拍摄 总之，这种方法应允许阐明分子 这种新型基因调节途径的机制 哺乳动物小肠。