METABOLIC REGULATION OF APOLIPOPROTEIN B MRNA EDITING

载脂蛋白B(ApoB)的mRNA编辑涉及位点特异性脱氨基 第666位核苷酸上的胞苷，将CAA谷氨酰胺密码子转换为UAA 翻译终止密码子大分子(ApoB100)组装的脂蛋白颗粒 或来自未经编辑和编辑的mRNA的小(ApoB48)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。具有特殊生物医学意义的是 发现含有B48的脂蛋白颗粒被迅速清除 血清和不代谢为低密度脂蛋白(低密度脂蛋白)和 动脉粥样硬化性疾病危险因素。拟议研究的总体目标是 是评估apoB mRNA编辑机制如何作为一种 组织特异性和代谢调节的控制点 脂蛋白的产生。经过检验的具体假设是 导致编辑小体组装的蛋白质因素的相互作用是 动态的，并且可以被细胞修饰来调节载脂蛋白B的量 信使核糖核酸编辑。已公布的和初步的数据表明， 在编辑过程中有五个或更多蛋白质。胞苷脱氨酶(27 KDa) 并提出了三种RNA结合蛋白(66、44和40 kDa)来服务于 酶C-gt；U转换和apoB mRNA编辑位点的各自作用 承认。240和49 kDa蛋白的参与和功能是 更具投机性，很大程度上取决于这些蛋白质的能力 提高体外编辑效率。初步数据显示 P240和p44抗体的制备、克隆和测序 P44和p27的cDNA。具体目的是促进抗体的开发 和参与apoB mRNA编辑和转录的因子的c DNA特征 转染法研究以评估每种因子的发生及其 ApoB信使核糖核酸编辑要求。反义寡核苷酸抑制血管紧张素转换酶 在培养细胞中的翻译和基因敲除将被用于证实 每个因素的要求。将使用McArdle和HepG2细胞作为测试 它们分别代表的系统；(1)编辑的系统 活性很容易被检测到，但可以通过实验增强或 减少，以及(2)其中编辑不能被检测到但可以 通过单次或多次转染法实验诱导的。离体 ApoB mRNA编辑系统将补充 评估编辑因子功能。每项要素的要求 编辑将根据对体外编辑的影响进行评估 免疫耗竭和添加重组蛋白去除因子的活性 这些因素又回到了提取物中。建议的结构相互作用，例如 P27和RNA结合蛋白之间的那些，将直接解决 体外可逆化学和光化学交联法 结合对角2D凝胶的蛋白质印迹分析的策略 和免疫吸附试验。通过建议的研究，应该是 有可能确定哪些蛋白质和相互作用是必要的 足以进行编辑，以及哪些因素和交互作用 参与调整编辑级别的。

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.