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Biosynthetic mechanism of informational lipid molecules in the signal transduction of mammalian cells and its molecular regulation by food functional agents

哺乳动物细胞信号转导中信息脂质分子的生物合成机制及其食品功能剂的分子调控

基本信息

批准号：
09660139
负责人：
YOKOTA Kazushige
金额：
$ 2.11万
依托单位：
Shimane University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

Arachidonate metabolites function as informational lipid molecules involved in the signal transduction of mammalian cells. We studied the regulatory mechanism of the arachidonate cascade by food-derived functional agents. As an interacting point of the food agents, we focused the regulation of gene expression of two isoforms of cyclooxygenase (COX), an rate-limiting enzyme in the biosynthetic pathway of prostanoids. To determine their levels specifically with higher sensitivity, the method using reverse transcriptase-polymerase chain reaction was developed with cRNA as internal standards. This method enabled us to determine the change in the levels of 2-type isoform in Madin-Darby canine kidney cells and mouse Swiss 3T3 cells. Tumor-promoting phorbol diesters caused an increase in the level of COX-2. The modification of the membrane phospholipids with n-6 and n-3 essential fatty acids resulted in the distinct regulation of the gene expression of COX isoforms depending on the types of unsaturated fatty acids.The structures and function of phorbol diester-inducible novel lipoxygenase (LOX)s were studied by the recombinant DNA technology and protein engineering. The exchange of the C-terminal one-third of the 8S-LOX for the corresponding human homologue, 15S-LOX-2 produced a chimeric enzyme with exclusive 15S-lipoxygenase activity. The stereospecificity was found to be S configuration. These findings suggested that the C-terminal regions were determinant for the positional specificity of the lipoxygenases. By using the in situ chimeragenesis in Escherichia coli followed by the site-directed mutagenesis, a pair of amino acids, Tyrosine-603 and Histidine-604 was identified as the positional determinant. The mutation of those in 8S-LOX to the corresponding 15S-LOX-2 residues converted to the enzyme expressing almost 15S-lipoxygenase activity.

花生四烯酸代谢物作为信息脂质分子参与哺乳动物细胞的信号转导。我们研究了食品衍生功能剂对花生四烯酸级联的调节机制。作为食品制剂的相互作用点，我们重点关注环氧合酶（COX）两种亚型的基因表达调控，环氧合酶是前列腺素类生物合成途径中的限速酶。为了以更高的灵敏度特异性地测定它们的水平，开发了以cRNA作为内标的逆转录酶-聚合酶链式反应的方法。该方法使我们能够确定 Madin-Darby 犬肾细胞和小鼠 Swiss 3T3 细胞中 2 型异构体水平的变化。促进肿瘤的佛波醇二酯导致 COX-2 水平升高。用n-6和n-3必需脂肪酸对膜磷脂进行修饰，导致COX异构体的基因表达根据不饱和脂肪酸的类型进行不同的调节。通过重组DNA技术和蛋白质工程研究了佛波二酯诱导型新型脂氧合酶（LOX）的结构和功能。将 8S-LOX 的 C 端三分之一替换为相应的人类同源物 15S-LOX-2，产生了具有独有的 15S-脂氧合酶活性的嵌合酶。发现立体特异性为S构型。这些发现表明，C 末端区域是脂氧合酶位置特异性的决定因素。通过在大肠杆菌中使用原位嵌合，然后进行定点诱变，一对氨基酸，酪氨酸-603 和组氨酸-604 被鉴定为位置决定簇。 8S-LOX 中的那些突变为相应的 15S-LOX-2 残基，转化为几乎表达 15S-脂氧合酶活性的酶。