METABOLIC REGULATION OF APOLIPOPROTEIN B MRNA EDITING

载脂蛋白B（apoB）mRNA编辑涉及载脂蛋白的位点特异性脱氨基作用。 核苷酸6666处的胞苷，将CAA谷氨酰胺密码子转化为UAA 翻译终止密码子 脂蛋白大颗粒组装体（apoB 100） 或来自未编辑和编辑mRNA的小（apoB 48）翻译产物 （分别），具有不同的结构和功能特性， 影响他们的新陈代谢。 具有特别生物医学意义的是 发现含有B48的脂蛋白颗粒从 不代谢成低密度脂蛋白（LDL）， 动脉粥样硬化性疾病危险因素。 拟议研究的广泛目标 是评估apoB mRNA编辑的机制如何作为一种 组织特异性和代谢调节的控制点 脂蛋白生成。 检验的具体假设是， 导致编辑体组装的蛋白质因子的相互作用是 动态的，并且可以被细胞修饰以调节apoB的量。 mRNA编辑。 已发表的和初步的数据表明， 五种或更多的蛋白质。 胞苷脱氨酶（27 kDa） 和三个RNA结合蛋白（66，44和40 kDa）被提出来服务于 酶促C->U转换和apoB mRNA编辑位点的各自作用 识别. 240和49 kDa蛋白的参与和功能是 更多的推测性，主要在于这些蛋白质的能力， 提高体外编辑效率。 初步数据显示， 与p240和p44反应的抗体的产生、克隆和测序 p44和p27的cDNA。 具体目标建议开发抗体 以及参与apoB mRNA编辑的因子的cDNA表征， 转染研究，以评估每个因子的发生及其 在apoB mRNA编辑的要求。 反义寡核苷酸抑制 将使用培养细胞中的翻译和基因敲除来确认 每个因素的要求。 McArdle和HepG 2细胞将用作试验 系统，它们分别代表;（1）编辑系统， 活性很容易检测，但可以通过实验增强， 减少，和（2）一个系统，其中编辑不能被检测到，但可以被 通过单次或多次转染实验诱导。 体外 apoB mRNA编辑系统将补充转染研究， 评估编辑因子函数。 中各因素的要求 将根据体外编辑的结果评价编辑 通过免疫耗竭去除因子和添加重组体的活性 因子回到提取物中。 建议的结构相互作用，如 p27和RNA结合蛋白之间的那些，将被直接解决 在体外使用可逆的化学和光化学交联 结合对角2D凝胶的蛋白质印迹分析的策略 和免疫吸附测定。 通过拟议的研究， 可以确定哪些蛋白质和相互作用是必要的， 足以进行编辑，以及哪些因素和相互作用 参与调节编辑水平。

Apolipoprotein B (apoB) mRNA editing involves site-specific deamination of a cytidine at nucleotide 6666, converting a CAA glutamine codon to a UAA translation stop codon. Lipoprotein particles assembled by large (apoB100) or small (apoB48) translation products from unedited and edited mRNA (respectively), have different structural and functional properties which affect their metabolism. Of particular biomedical significance is the finding that B48-containing lipoprotein particles are rapidly cleared from the serum and are not metabolized to low density lipoprotein (LDL), an atherogenic disease risk factor. The broad goal of the proposed research is to evaluate how the mechanism of apoB mRNA editing might serve as a controlling point for tissue-specific and metabolically regulated lipoprotein production. The specific hypothesis tested is that the interactions of protein factors leading to the assembly of editosomes are dynamic, and can be modified by the cell to regulate the amount of apoB mRNA edited. Published and preliminary data suggest the involvement of five or more proteins in the editing process. Cytidine deaminase (27 kDa) and three RNA-binding proteins (66,44 & 40 kDa) are proposed to serve the respective roles of enzymatic C->U conversion and apoB mRNA editing site recognition. The participation and function of 240 and 49 kDa proteins are more speculative and lie largely in the ability of these proteins to enhance in vitro editing efficiency. Preliminary data demonstrate production of antibodies reactive with p240 and p44, cloning and sequencing of cDNAs for p44 and p27. The Specific Aims propose antibody development and cDNA characterization for factors involved in apoB mRNA editing and transfection studies to evaluate the occurrence of each factor and its requirement in apoB mRNA editing. Antisense oligo nucleotide inhibition of translation and gene knockout in cultured cells will be used to confirm each factor's requirement. McArdle and HepG2 cells will be used as test systems for they represent respectively; (1) a system where editing activity is readily detectable but could be experimentally enhanced or diminished, and (2) a system where editing can not be detected but may be induced experimentally through single or multiple transfections. In vitro systems for apoB mRNA editing will complement transfection studies in assessing editing factor functions. The requirement of each factor in editing will be evaluated in terms of the consequence to in vitro editing activity of removing factors by immunodepletion and of adding recombinant factors back to the extracts. Proposed structural interactions, such as those between p27 and the RNA-binding protein, will be directly addressed in vitro using reversible chemical and photochemical cross-linking strategies in conjunction with western blot analysis of diagonal 2D gels and immunoadsorption assays. Through the proposed research, it should be possible to determine which proteins and interactions are necessary and sufficient for editing to occur, and which factors and interactions are involved in modulating the level of editing.