HEPATIC REGULATION OF THE HUMAN APOB GENE

人类 APOB 基因的肝脏调节

基本信息

批准号：
6530662
负责人：
HRIDAY K DAS
金额：
$ 11.92万
依托单位：
UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-09-01 至 2003-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6530662
关键词：
Baculoviridae DNA footprinting affinity chromatography apolipoproteins blood lipoprotein biosynthesis cardiovascular disorder cholesterol disease /disorder proneness /risk gel mobility shift assay genetic mapping genetic transcription intermolecular interaction laboratory rabbit laboratory rat liver metabolism polymerase chain reaction transcription factor yeast two hybrid system

项目摘要

ApoB, the only protein of low density lipoprotein (LDL) is synthesized primarily in human liver. Plasma levels of LDL cholesterol and apoB correlate directly with atherosclerosis susceptibility and premature heart disease. Moderately decreased levels of LDL cholesterol and hypobetalipoproteinemia are generally associated with decreased risk of coronary artery disease. Regulation of apoB gene transcription is critically important for assembly and secretion of very low density lipoprotein (VLDL), and therefore, may ultimately determine plasma LDL cholesterol. However, we do not yet understand the precise regulatory mechanisms controlling the transcription of the human apoB gene, and in the absence of that information it has been difficult to develop effective therapy directed toward transcriptional regulation of the apoB gene. The experiments proposed in this application will elucidate the regulatory mechanisms of apoB gene transcription. A secondary aim of this proposal tests the hypothesis that trans-acting factors that regulate apoB gene transcription may also control plasma LDL cholesterol level. Since trans-acting factors mediate apoB gene transcription by interacting with cis-acting DNA elements as well as with specific co-activators, it is necessary to clone genes encoding these trans-acting factors and their co-activators to elucidate the mechanism of apoB gene transcription in the liver. The proposed specific aims are to (1) clone trans-acting factor BRF-4; (2) map its DNA-binding and trans-activation domains; (3) elucidate the mechanism of interaction between HNF-4 and BRF-4 as well as HNF-4 and BRF-2, and their roles in apoB secretion; (4) identify co-activators of BRF-4 and elucidate their roles in apoB transcription and secretion. BRF-4 will be purified from rat liver by DNA-specific affinity column and characterized by SDS/PAGE, gel mobility shift, DNase I footprinting, and in vitro transcription assay. RACE PCR will be used to clone the gene encoding BRF-4. DNA-binding and trans-activation domains of BRF- 4 will be determined by expressing mutated or truncated BRF-4 in a recombinant baculovirus expression system. Co-activators will be identified by a yeast two-hybrid system. Understanding the mechanism of apoB transcription may someday lead to the control of apoB expression and consequently to the control of LDL cholesterol levels.

APOB，是低密度脂蛋白（LDL）的唯一蛋白质主要合成在人肝脏中。 LDL胆固醇和APOB的血浆水平直接与动脉粥样硬化的敏感性和过早心脏病相关。 LDL胆固醇水平中度降低，低钙蛋白血症通常与冠状动脉疾病的风险降低有关。 APOB基因转录的调节对于非常低密度脂蛋白（VLDL）的组装和分泌至关重要，因此最终可能确定血浆LDL胆固醇。但是，我们尚不了解控制人APOB基因转录的确切调节机制，在没有该信息的情况下，很难开发针对APOB基因转录调控的有效治疗。本应用中提出的实验将阐明APOB基因转录的调节机制。该提案的次要目的检验了以下假设：调节APOB基因转录的跨作用因子也可能控制血浆LDL胆固醇水平。由于反式作用因子通过与顺式作用DNA元件以及特定的共激活剂相互作用来介导APOB基因转录，因此有必要克隆编码这些反式作用因子及其共激活因子以阐明肝脏中APOB基因转录的机制。提出的特定目的是（1）克隆跨作用因子BRF-4；（2）绘制其DNA结合和反式激活域；（3）阐明HNF-4和BRF-4以及HNF-4和BRF-2之间相互作用的机制及其在APOB分泌中的作用；（4）识别BRF-4的共激活因子，并阐明其在APOB转录和分泌中的作用。 BRF-4将通过DNA特异性亲和力柱从大鼠肝脏中纯化，并以SDS/PAGE，凝胶迁移率，DNase I足迹和体外转录测定为特征。种族PCR将用于克隆编码BRF-4的基因。 BRF-4的DNA结合和反式激活结构域将通过在重组杆状病毒表达系统中表达突变或截断的BRF-4来确定。共激活因子将通过酵母两杂交系统识别。了解APOB转录的机制可能有一天会导致APOB表达的控制，从而控制LDL胆固醇水平。