Molecular biological studies on congenital lactic acidemia due to pyruvate dehydrogenase deficiency

丙酮酸脱氢酶缺乏所致先天性乳酸血症的分子生物学研究

• 批准号: 02670443
• 负责人: KURODA Yasuhiro
• 金额: $ 1.47万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02670443/
• 关键词: metabolic inborn errors; lactic acidemia; pyruvate dehydrogenase; pyruvate dehydrogenase phosphatase; pyruvate; lactate; ビルビン酸; 活性化障害; DNA; 塩基配列

Pyruvate dehydrogenase(PDH)complex is a mitochondrial multiple enzyme complex and catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. PDH complex consists of six components, PDH, lipoate acetyltransferase, lipoamide dehydrogenase, PDH phosphatase, PDH kinase and protein-X. The activity of PDH complex is regulated by dephosphorylation(activation)and phosphorylation(inactivation), catalyzed by PDH phosphatase and PDH kinase, respectively. The defect of PDH complex and the defect in the activation of PDH complex are common causes of the disorders leading to congenital actic acidemia.The PDH phosphatase activities in cultured skin fibroblasts from three patients with lactic acidemia due to the defect of the activation of PDH complex were determined by our newly developed assay method. The enzyme activities were significantly reduced in the three patients. The markedly reduced amounts of alpha and beta subunits of PDH which was a substrate for PDH phosphatase, were revealed i … More n the fibroblasts from two of the three patients by the immunoblot technique. In contrast, the fibroblasts from one of the three patients had approximately similar amounts of the alpha and beta subunits to control. These results suggest that the defect of the activation of PDH complex might be primarily due to the abnormalities in PDH and PDH phosphatase in the two patients and the other patient, respectively.A mutation, 4-bp insertion, in the gene for alpha subunit of PDH was found in a female patient with PDH deficiency due to the rapid degradation of alpha and beta subunit proteins of PDH. This 4-bp insertion caused frameshift that altered amino acid sequence and created the premature stop codon. This female patient was a heterozygote of the normal and this mutant alleles. However, most of cultured skin fibroblasts from this patient expressed the mutant allele. These results suggested that in this female patient, X chromosome containing the normal allele was predominantly inactivated so that she showed lactic acidemia and neurological abnormalities in spite of a heterozygote of the normal and the mutant alleles and that the mutant alpha subunit protein failed to form a stable structure of PDH and both alpaha and beta subunit proteins were degraded rapidly. Less

丙酮酸脱氢酶复合体是一种线粒体多酶复合体，催化丙酮酸氧化脱羧生成乙酰辅酶A。PDH复合物由6种组分组成，分别为PDH、硫辛酰胺脱氢酶、硫辛酰胺乙酰转移酶、PDH磷酸酶、PDH激酶和蛋白X。PDH复合物的活性受去磷酸化（活化）和磷酸化（失活）的调节，分别由PDH磷酸酶和PDH激酶催化。PDH复合物的缺陷和PDH复合物的活化缺陷是导致先天性乳酸血症的常见原因，本文用我们建立的检测方法，测定了3例因PDH复合物活化缺陷而导致先天性乳酸血症的皮肤成纤维细胞中PDH磷酸酶活性。三名患者的酶活性明显降低。PDH磷酸酶的底物PDH的α和β亚基的量显著减少， ...更多信息 用免疫印迹技术对其中2例患者的成纤维细胞进行免疫组化染色。相比之下，来自三名患者之一的成纤维细胞具有与对照组大致相似的α和β亚基量。结果提示，PDH复合物活化缺陷可能主要是由于PDH和PDH磷酸酶的异常所致;在1例女性PDH缺陷症患者中，由于PDH α和β亚基蛋白快速降解，导致PDH α亚基基因发生4-bp插入突变。这个4-bp的插入引起移码，改变了氨基酸序列，并产生了提前终止密码子。这名女性患者是正常和突变等位基因的杂合子。然而，来自该患者的大多数培养的皮肤成纤维细胞表达突变等位基因。这些结果表明，在这名女性患者中，含有正常等位基因的X染色体主要失活，因此，尽管存在正常和突变等位基因的杂合子，但她仍表现出乳酸血症和神经系统异常，并且突变的α亚基蛋白未能形成PDH的稳定结构，α和β亚基蛋白均迅速降解。少

项目成果

Ito M,Huq AHMM,Naito E,Saijo T,Takeda E,Kuroda Y: "Detection of the mutation of Ela gene in a female patient with pyruvate dehydrogenase deficiency due to the rapid degradation of El protein" J Inher Metab Dis.

Ito M，Huq AHMM，Naito E，Saijo T，Takeda E，Kuroda Y：“由于 El 蛋白快速降解导致丙酮酸脱氢酶缺乏症女性患者 Ela 基因突变的检测”J Inher Metab Dis。

小橋 秀彰,伊藤 道徳,マ-ブブル ホク,西條 隆彦,横田 一郎,内藤 悦雄,武田 英二,黒田 泰弘: "ピルビン酸脱水素酵素複合体活性化障害の病因に関する研究ーピルビン酸脱水素酵素ホスファタ-ゼ活性低下ー" 日本小児科学会雑誌. 95. 1525-1531 (1991)

Hideaki Kobashi、Noriyoshi Ito、Hoku Mable、Takahiko Saijo、Ichiro Yokota、Etsuo Naito、Eiji Takeda、Yasuhiro Kuroda：“丙酮酸脱氢酶复合体激活障碍的发病机制研究 - 丙酮酸脱氢酶磷酸酶活性” 95. 1525-1531 (1991)

Ito,M.: "Molecular basis of defectire actiration of Pyruvate dehydrogenase in cultured skin fibroflasts from patiente with congenital lactic acidemia" Pediatric Research.

Ito,M.：“先天性乳酸血症患者培养的皮肤成纤维细胞中丙酮酸脱氢酶激活缺陷的分子基础”儿科研究。

Kobashi H, Ito M, Huq AHMM, Saijo T, Yokota I, Naito E, Takeda E, Kuroda Y: "Studies on the causes of defect of the activation of pyruvate dehydrogenase complex in congenital lactic acidemia : Decrease of pyruvate dehydrogenase phosphatase activity in cul

Kobashi H、Ito M、Huq AHMM、Saijo T、Yokota I、Naito E、Takeda E、Kuroda Y：“先天性乳酸血症中丙酮酸脱氢酶复合物活化缺陷的原因研究：丙酮酸脱氢酶磷酸酶活性降低

小橋 秀彰,伊藤 道徳,マ-ブブル・ホク,西條 隆彦,横田 一郎,内藤 悦雄,武田 英二,黒田 泰弘: "ピルビン酸脱水素酵素複合体活性化障害の病因に関する研究ーピルビン酸脱水素酵素ホスファタ-ゼ活性低下ー" 日本小児科学会雑誌. 95. 1525-1531 (1991)

Hideaki Kobashi、Noriyoshi Ito、Hoku Mable、Takahiko Saijo、Ichiro Yokota、Etsuo Naito、Eiji Takeda、Yasuhiro Kuroda：“丙酮酸脱氢酶复合物激活障碍 - 丙酮酸脱氢酶磷酸酶的发病机制研究”“活性下降”日本儿科杂志社会。95。1525-1531（1991）