METABOLIC HEPATIC FLUX OF ISOPRENOIDS

描述（改编自申请人的摘要）：本申请的主要目的 建议是研究胆固醇中的第一种特定酶 生物合成途径，角鲨烯合成酶，以了解 其调控机制。 这一目标现在可以实现，因为在 在项目的前三年，研究人员获得了cDNA， 大鼠肝酶和人肝酶的克隆以及 人基因的5'侧翼序列的基因组克隆。 基于 在当前资助期内完成的工作，我们现在申请5年续期 研究人员提议调查其潜在机制 参与固醇介导的该基因的转录调节。 研究人员已经鉴定了一个69 bp的启动子序列， 负责这项规定，并建议调查和澄清 参与这种调节的顺式作用元件的结构。 进一步提出研究细胞反式作用蛋白 参与该基因调控的因素，并比较 他们，和监管机制，以监管其他高度 调节胆固醇生物合成途径中的酶。 这将是 通过分离和克隆这些蛋白质因子和 详细研究了它们与编码角鲨烯的基因的相互作用 合成酶 根据本补助期内取得的成果， 研究人员建议调查存在的意义， 角鲨烯合成酶mRNA的不同大小类别异构体在调节 在不同的胆固醇生成条件下，这种肝酶的活性。 最后，研究人员建议验证人类的调节 角鲨烯合酶基因在转基因小鼠中的表达。 我们也 建议研究这种酶过度生产的含义， 转基因小鼠，对甾醇和非甾醇的总体代谢 类异戊二烯 成功完成上述研究， 资助申请应加强对分子基础的理解， 肝胆固醇生成。

DESCRIPTION (Adapted from applicant's Abstract): The main objective of this proposal is to study the first specific enzyme in the cholesterol biosynthetic pathway, squalene synthase, in order to understand the mechanisms of its regulation. This objective is now obtainable since in the first three years of the project the investigators have obtained the cDNA clones of both the rat hepatic and the human hepatic enzymes as well as genomic clones of the 5' flanking sequences of the human gene. Based on work completed in the current grant period we now apply for a 5-year renewal in which the investigators propose to investigate the underlying mechanisms involved in the sterol-mediated transcriptional regulation of this gene. The investigators have identified a 69 bp promoter sequence which is responsible for this regulation and propose to investigate and elucidate the structure of the cis-acting elements which are involved in this regulation. It is further proposed to investigate the cellular trans-acting protein factors that are involved in the regulation of this gene and to compare them, and the regulatory mechanism to the regulation of other highly regulated enzymes in the cholesterol biosynthetic pathway. This will be achieved by the isolation and cloning of these protein factors and a detailed study of their interaction with the gene encoding squalene synthase. Based on results obtained in the current grant period the investigators propose to investigate the significance of the existence of different size-classes isoforms of squalene synthase mRNA in the regulation of this hepatic enzyme activity under varying cholesterogenic conditions. Finally, the investigators propose to verify the regulation of the human squalene synthase gene by its expression in transgenic mice. We also propose to study the implication of over production of this enzyme, in transgenic mice, on the overall metabolism of sterol and non sterol isoprenoids. The successful completion of these studies outlined in the grant application should enhance the understanding of the molecular basis of hepatic cholesterol production.