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Molecular Mechanisms for delta-Aminolevulinate Synthase Gene

δ-氨基乙酰丙酸合酶基因的分子机制

基本信息

批准号：
08457044
负责人：
FUJITA Hiroyoshi
金额：
$ 4.93万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

It is generally accepted that almost 75% of heme produced in mammals are supplied for hemoglobin biosynthesis in the bone marrow. During erythroid differentiation the key enzyme for heme biosynthesis in the bone marrow, erythroid specific delta-aminolevulinate synthase, is regulated by erythroid type transcription factors, such as GATA-1 and NF-E2.When we analyzed bone marrow ALAS-E activity from a patient with X-linked sideroblastic anemia, the level was as low as 10% of that in normal control. Western blot analysis, however, demonstrated a remarkable decrease in enzyme protein proportional to the enzyme activity defect. Thus, it is likely the patient has a mutation (s) to reduce the enzyme protein. Further investigations indicated that ALAS-E gene of the patient has Asp190Val mutation, whose protein expressed in QT-6 cells revealed a specific activity as high as the normal control enzyme with a relatively similar transcription-translation activity. We, therefore, examined effects of the mutation for mitchondrial transport of the enzyme. Although the mutated amino acid located approx. 100 amino acids downstream of the processing site of ALAS-E protein, the mutation resulted in 1.0 and 1.5 kDa larger size mature enzyme when compared with the normal protein (Furuyama, 1987).We have also revealed that NF-E2, globin, and ALAS-E are controlled by Ras-Raf-MAP kinase signal transduction. Our analyzes clarified activate domain and suppress domain of NF-E2 were located within p45 and p18, respectively (Nagai, 1988).As far as we examined nonspecific delta-aminolevulinate synthase (ALAS-N), the gene was negatively regulated via NF-KB binding sequence (Fujita, 1998).

人们普遍认为，哺乳动物产生的血红素几乎 75% 用于骨髓中的血红蛋白生物合成。在红细胞分化过程中，骨髓中血红素生物合成的关键酶，红细胞特异性 δ-氨基乙酰丙酸合酶，受到红细胞类型转录因子（例如 GATA-1 和 NF-E2）的调节。当我们分析 X 连锁铁粒幼细胞贫血患者的骨髓 ALAS-E 活性时，其水平低至 10%。在正常控制下。然而，蛋白质印迹分析表明，与酶活性缺陷成比例的酶蛋白显着下降。因此，患者很可能具有减少酶蛋白的突变。进一步研究发现，该患者的ALAS-E基因存在Asp190Val突变，其在QT-6细胞中表达的蛋白显示其比活性与正常对照酶一样高，且转录翻译活性相对相似。因此，我们检查了突变对酶线粒体运输的影响。虽然突变的氨基酸位于大约。 ALAS-E 蛋白加工位点下游 100 个氨基酸处，与正常蛋白相比，突变导致成熟酶尺寸增大 1.0 和 1.5 kDa (Furuyama, 1987)。我们还揭示了 NF-E2、球蛋白和 ALAS-E 受 Ras-Raf-MAP 激酶信号转导控制。我们的分析阐明了 NF-E2 的激活结构域和抑制结构域分别位于 p45 和 p18 内（Nagai，1988）。就我们检查非特异性 δ-氨基乙酰丙酸合酶（ALAS-N）而言，该基因通过 NF-KB 结合序列受到负调控（Fujita，1998）。