Structures and clinical significance of polymorphism of human dihydrodiol dehydrogenase

人二氢二醇脱氢酶多态性的结构及临床意义

• 批准号: 09672240
• 负责人: HARA Akira
• 金额: $ 2.05万
• 依托单位: Gifu Pharmaceutical 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 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672240/
• 关键词: dihydrodiol dehydrogenase; aldo-keto reductase family; genetic polymorphism; structure-function relationship; prostaglandin D_2; anti-inflammatory drug; anti-hyperlipidemic drug; steroid metabolism; ヒドロキシステロイド脱水素酵素; 肝疾患; 家系調査; 高脂血症薬

Four isozymes (DD1-DD4) of human dihydrodiol dehydrogenase share 83-97% amino acid sequence identity and belong to the aldo-keto reductase fhaiily, but differ in specificity for endogenous substrates and effects by modulators. In addition, nine cDNAs similar to that for the four isozymes have recently been reported, which suggests the existence of genetic polymorphism of the respective isozymes. This study was performed to elucidate structural determinants for the functional difference among the four isozymes, genetic polymorphism of the respective isozymes, and clinical significance of the variant gene. The results obtained are summarized as follows :1. Kinetic analyses of recombinant DD3 and AKRlC3, which differs by 3 amino acids from DD3, indicated that they are functionally identical and the 3 residues are not involved in the active site. Substrate specificity study suggested that the enzymes physiologically act as prostaglandin D2 1 1-ketoreductase rather than hydroxysteroid dehyd … More rogenase.2. Site-directed mutagenesis of the isozymes and formation of chimeric enzymes suggested several amino acid residues, which interact with substrates and modulators, and are responsible for the binding and/or orientation of the coenzyme and activators.3. Anti-hyperlipidemic clofibrate derivatives and anti-inflammatory 2-arylpropionic acid derivatives were identified as activators specific for DD4. In addition, thyroxin, a thyroid hormone, was found to be the physiological activator of this isozyme. The mechanism of activation by these compounds was kinetically elucidated and residues in the activator-binding site of the isozyme were suggested by site-directed mutagenesis.4. We established the RT-PCR and RFLP methods to discriminate the 13 cDNAs reported for the dihydrodiol dehydrogenase isozymes. Only one mRNA species for DDl, DD2 and DD3, respectively, were detected in 50 human tissue samples. The result suggested that the cDNAs for DDI, DD2 and DD3 are the principal alleles of the three genes, and the other cDNAs reported are derived from rare variants of the respective isozyme genes or could be sequencing errors. On the other hand, about 20% of the samples showed heterozygous expression of mRNAs for DD4 and a variant that differs by 2 amino acids from DD4. Analysis of genomic DNA for 137 blood samples confirms this variant DD4 gene, and one of the samples showed homozygous expression of this variant gene. The frequency of this variant allele in Japanese was about 9%. The physiological effect of this variant gene and its relationship to hepatic diseases are not unclear at present. Less

人二氢二醇脱氢酶的四种同工酶（DD 1-DD 4）具有83-97%的氨基酸序列同一性，属于醛酮还原酶家族，但对内源性底物的特异性和调节剂的作用不同。此外，最近还报道了9个与这4种同工酶相似的cDNA，这表明各同工酶存在遗传多态性。本研究旨在阐明四种同工酶之间功能差异的结构决定因素、各同工酶的遗传多态性以及变异基因的临床意义。主要研究结果如下：1.重组DD 3和AKR 1C 3的动力学分析表明，它们在功能上是相同的，并且这3个残基不参与活性位点，AKR 1C 3与DD 3相差3个氨基酸。底物特异性研究表明，该酶在生理上起前列腺素D2 1 1-酮还原酶的作用，而不是羟基类固醇脱氢酶。 ...更多信息 rogenase. 2.同工酶的定点突变和嵌合酶的形成提示了几个氨基酸残基，它们与底物和调节剂相互作用，并负责辅酶和激活剂的结合和/或定位.抗高血压的氯贝特衍生物和抗炎的2-芳基丙酸衍生物被鉴定为对DD 4特异的活化剂。此外，甲状腺素，甲状腺激素，被发现是该同工酶的生理激活剂。动力学上阐明了这些化合物的激活机制，并通过定点突变提出了同工酶激活剂结合位点的残基.我们建立了RT-PCR和RFLP的方法来区分已报道的13个二氢二醇脱氢酶同工酶的cDNA。在50个人组织样品中分别仅检测到DD 1、DD 2和DD 3的一种mRNA种类。结果表明，DD 1、DD 2和DD 3基因的cDNA是这3个基因的主要等位基因，其它已报道的cDNA可能是各自基因的罕见变异体或测序错误。另一方面，约20%的样品显示DD 4和与DD 4相差2个氨基酸的变体的mRNA的杂合表达。对137份血液样本的基因组DNA分析证实了该变异DD 4基因，其中一份样本显示该变异基因的纯合表达。该变异等位基因在日本人中的频率约为9%。目前，该变异基因的生理作用及其与肝脏疾病的关系尚不清楚。少

项目成果

Shiraishi Hiroaki: "Sequence of the cDNA of a human liver dihydrodiol dehydrogenase isoform (AKR1C2) and tissue distribution of its mRNA" Biochem.J.334. 399-405 (1998)

Shiraishi Hiroaki：“人肝二氢二醇脱氢酶异构体 (AKR1C2) 的 cDNA 序列及其 mRNA 的组织分布”Biochem.J.334。

Matsuura Kazuya: "Activation of human 3α-hydroxysteroid dehydrogenase by clofibrate derivatives" J.Pharmacol.Exp.Ther.発表予定(印刷中). (1998)

Matsuura Kazuya：“氯贝特衍生物激活人 3α-羟基类固醇脱氢酶”J.Pharmacol.Exp.Ther. 预定演示（1998 年）。

Matsuura Kazuya: "Identification of a principal mRNA species for human 3α-hydroxysteroid dehydrogenase isoform (AKR1C3) that exhibits high prostaglandin D_2 11-ketoreductase activity" J.Biochem.124. 940-946 (1998)

Matsuura Kazuya：“具有高前列腺素 D_2 11-酮还原酶活性的人 3α-羟基类固醇脱氢酶亚型 (AKR1C3) 的主要 mRNA 种类的鉴定”J.Biochem.124 (1998)。

Matsuura Kazuya: "Identification of a principal mRNA species for human 3α-hydroxysteroid dehydrogenase isoform(AKRIC3)that exhibits high prostaglandin D_2 II-ketoreductase activity" J.Biochem.124・5. 940-946 (1998)

松浦和也：“具有高前列腺素 D_2 II-酮还原酶活性的人 3α-羟基类固醇脱氢酶亚型 (AKRIC3) 的主要 mRNA 种类的鉴定”J.Biochem.124·5 (1998)。

Matsuura Kazuya: "Roles of the C-terminal domains of human dihydrodiol dehydrogenase isoforms In the binding of substrates and modulators" Biochem.J.336. 429-436 (1998)

Matsuura Kazuya：“人二氢二醇脱氢酶亚型的 C 末端结构域在底物和调节剂结合中的作用”Biochem.J.336。