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

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。我们研究了食品源性功能剂对花生四烯酸级联反应的调控机制。作为食品制剂的一个相互作用点，我们重点研究了前列腺素生物合成途径中限速酶环氧化酶（考克斯）的两种异构体的基因表达调控。为了以更高的灵敏度特异性测定它们的水平，开发了使用逆转录酶-聚合酶链反应的方法，以cRNA作为内标。该方法使我们能够确定Madin-Darby犬肾细胞和小鼠Swiss 3 T3细胞中2型亚型水平的变化。促进肿瘤的佛波醇二酯引起考克斯-2水平的增加。用n-6和n-3必需脂肪酸修饰细胞膜磷脂，使考克斯异构体的基因表达受不饱和脂肪酸类型的不同而不同，利用重组DNA技术和蛋白质工程研究了佛波醇二酯诱导的新型脂氧合酶（LOX）的结构和功能。将8 S-LOX的C-末端三分之一交换为相应的人类同源物15 S-LOX-2产生了具有排他性15 S-脂氧合酶活性的嵌合酶。立体专一性为S构型。这些结果表明，C-末端区域的脂氧合酶的位置特异性的决定因素。通过在大肠杆菌中进行原位嵌合，然后进行定点突变，确定了一对氨基酸，酪氨酸-603和组氨酸-604为位置决定簇。8 S-LOX中的那些突变为相应的15 S-LOX-2残基转化为表达几乎15 S-脂氧合酶活性的酶。

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.