Regulatory mechanism of Enzymes of Cholesterol Biosynthesis

胆固醇生物合成酶的调控机制

• 批准号: 07044234
• 负责人: ONO Teruo
• 金额: $ 4.86万
• 依托单位: Niigata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044234/
• 关键词: squalene epoxidase(SE); benzophenone derivatives; HMG-CoA reductase; LDL-receptor; gene expression; chromosome 8 q24.13; D8S508; SRE; NF-Y; コレステロール; スクアレン; スクアレン・エポキシダーゼ; 阻害剤; 動脈硬化; 高脂血症; スクリーニング; バイオセンサー

We have studied on structure and regulation aspects of mammalian squalene epoxidase (SE), the late stage of cholesterol biosynthetic enzyme.1. Determination of Functional Domains of SE : Using benzophenone derivatives of FAD,trinorsqualene alcohol and NB-598, we have determined putative binding domains for substrate and NB-598. We also found that terbinafine resistant gene in yeast replaced the only one amino acid compared to wild type yeast (L*F).2. Regulation of SE : Regulation of SE gene expression was studied in comparison with those of HMG-CoA reductase and LDL-receptor using HeLa cells. The results suggest that sterol produced endogenously can regulate SE expression at the level of transcription.3. Structure of human SE genomic DNA : Total length of human SE gene is about 17 kilobase including 11 exons. The introns are ranging from 87 to 3.2 kb. Exon I and II contained putative memmbrane binding domain and FAD-binding followed substrate binding domains, respectively. The identica … More l undecapeptide in common with yeast and mammlaina SE is localized in the exon VIII.4. Chromosomal localization of human SE gene : We have dteremined the regional localization of human SE by using Stanford G3 radiation hybrid panel. Computer analysis of the PCR products for each panel suggests that human SE sequence tagged site is linked with micro-satellite marker D8S508. The marker is localiz-ed q24.13-q telomere of chromosome 8. It is possble that Langer-Giedion syndromeis one of the candidate for SE defect.5. Analyzes of promoter regions of human and rat SE genes : We have determined 5'-sequenec of rat and human SE genes from each genomic clones and aligned 5'-flanking sequences of initiation codons. In both human and rat, the well-conserved region is moticed at the location of 700 or 800 bp upstream from initiation codon. Rat transcriptional initiation site that determined by primer extension method was in 3'-region of the conserved region. To determine the regulatory element of human SE promoter, we preparaed several chimeric lucife-rase plasmids and fusion constructs with deleted human SE promoter and transfected in HeLa cells. The two limited conserved region, a classical SRE-like region and NY-F regions of rat and human may be cruscial for regulation of SE gene by sterols. Less

本论文对哺乳动物胆固醇生物合成的后期酶鲨烯环氧酶（SE）的结构和调控进行了研究. SE的功能结构域的确定：使用FAD的二苯甲酮衍生物、三去甲角鲨烯醇和NB-598，我们已经确定了底物和NB-598的推定结合结构域。我们还发现，特比萘芬抗性基因在酵母菌中仅替换了一个氨基酸（L*F）. SE的调节：使用HeLa细胞研究了SE基因表达的调节，并与HMG-CoA还原酶和LDL受体的调节进行了比较。结果表明，内源产生的甾醇可以在转录水平上调控SE的表达.人SE基因组DNA的结构：人SE基因全长约17个外显子，包括11个外显子。内含子的长度为87 ~ 3.2kb。外显子I和II分别含有膜结合结构域和FAD结合后底物结合结构域。身份证 ...更多信息 l十一肽与酵母和酿酒酵母共同定位于外显子VIII。4。人SE基因的染色体定位：我们用斯坦福大学G3辐射杂交板对人SE基因进行了区域定位。对每一组PCR产物的计算机分析表明，人SE序列标记位点与微卫星标记D8 S508连锁。该标记定位于8号染色体q24.13-q端粒。Langer-Giedion综合征可能是SE缺陷的候选者之一.人和大鼠SE基因启动子区的分析：我们确定了大鼠和人SE基因的5 '端序列，并对起始密码子的5'端侧翼序列进行了比对。在人和大鼠中，高度保守的区域位于起始密码子上游700或800 bp处。用引物延伸法确定大鼠转录起始位点位于保守区的3 ′端。为了确定人SE启动子的调控元件，我们构建了几种嵌合荧光素酶质粒和缺失人SE启动子的融合质粒，并转染HeLa细胞。这两个有限的保守区，一个经典的SRE样区和NY-F区的大鼠和人类可能是关键的调控SE基因的甾醇。少

项目成果

Nagai,M.: "Localization of the Squalene epoxidase gene to human chromosome 8q 24.13." Genomics. (in press). (1997)

Nagai,M.：“角鲨烯环氧化酶基因定位于人类染色体 8q 24.13。”

Kosuga,K.: "Nucleotide sequence of a cDNA for mouse squalene epoxidase." Biochim.Biophys.Acta. 1260. 345-348 (1995)

Kosuga,K.：“小鼠角鲨烯环氧化酶 cDNA 的核苷酸序列。”

Kanda, T.: "Effect of bile on the intestinal bile-acid binding protein(I-BABP)expression. In vivo and in vitro studies." FEBS Lett.384. 131-134 (1996)

Kanda, T.：“胆汁对肠道胆汁酸结合蛋白 (I-BABP) 表达的影响。体内和体外研究。”

Kanda, T.: "Effect of bile on the intestinal bile-acid binding protein (I-BABP) expression. In vivo and in vitro studies" FEBA Lett.384. 131-134 (1996)

Kanda, T.：“胆汁对肠道胆汁酸结合蛋白 (I-BABP) 表达的影响。体内和体外研究”FEBA Lett.384。

小野輝夫: "メタボリックマップ" 化学同人(印刷中), (1997)

小野照夫：《代谢图》化学同人（正在出版），（1997）