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4种同工酶（DD1-DD4）具有83-97%的氨基酸序列同源性，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，这表明它们各自的同工酶存在遗传多态性。本研究旨在阐明四种同工酶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。所得结果总结如下：1。重组DD3和AKRlC3的动力学分析表明，它们在功能上是相同的，并且3个残基都不参与活性位点。底物特异性研究表明，该酶在生理上具有前列腺素D2 11 -酮还原酶的作用，而不是羟基类固醇脱氢酶。同工酶的定点突变和嵌合酶的形成表明，有几种氨基酸残基与底物和调节剂相互作用，并负责辅酶和激活剂的结合和/或定向。抗高血脂的氯贝特衍生物和抗炎的2-芳基丙酸衍生物被确定为DD4特异性的激活剂。此外，甲状腺素，一种甲状腺激素，被发现是这种同工酶的生理激活剂。这些化合物的激活机制被动力学地阐明，并在同工酶的激活剂结合位点的残基被位点定向诱变提出。我们建立了RT-PCR和RFLP方法来鉴别报道的13个二氢二醇脱氢酶同工酶的cdna。在50份人体组织样本中，分别检测到dd1、DD2和DD3的1种mRNA。结果表明，DDI、DD2和DD3的cdna是这三个基因的主要等位基因，而其他报道的cdna来源于各自同工酶基因的罕见变异或可能是测序错误。另一方面，约20%的样本显示出DD4的mrna杂合表达，以及与DD4不同2个氨基酸的变体。137份血液样本的基因组DNA分析证实了DD4基因的变异，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的出现频率约为9%。该变异基因的生理作用及其与肝脏疾病的关系目前尚不清楚。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。