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STUDIES ON THE DEVELOPMENT OF METHODS TO REMOVE THE MUTANT MITOCHONDRIAL GENOME FOR THERAPY OF MITOCHONDRIAL DESEASE

开发去除突变线粒体基因组用于治疗线粒体疾病的方法的研究

基本信息

批准号：
08457196
负责人：
OHTA Shigeo
金额：
$ 5.06万
依托单位：
NIPPON MEDICAL SCHOOL INSTITUTE OF GERONTOLOGY
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 1998
项目状态：
已结题

项目摘要

Several mutations in the mitochondrial genome are responsible for mitochondrial encephalomyopathy. It is in principle impossible to cure the disease by introducing normal genes into affected cells, because of the multicopic mitochondrial genome, and because of greater propagation of the mutant genome than of the normal one. Thus, if the cells accumulated with mutant mtDNA can be eliminated by apoptosis, the mutant mtDNA was removed with the cells themselves, and expectedly compensate by growing the normal cells. For this purpose, we found that Fas gene responds against mitochondrial dysfunction. In addition, three genes that express more by the mitochondrial dysfunction are isolated by the differential display method. One is a kind DNA ligase and another belongs to the DNA modifying enzyme, and the other is novel gene.In studies on apoptosis, structure of a regulator of apoptosis, Bcl-x was determined by x-ray crystallography and the structure of Bax, an inducer of apoptosis was modeled by the analogy modeling method. The structure of Bax was speculated to have a pore forming domain, and turned out to be responsible for apoptosis. Without the dimer forming domains with the other BcI-2 family proteins, only the pore forming domain induced apoptosis. This finding indicates that the pore forming domain should be a good protein for inducing apoptosis in the cells accumulated with the mutant mitochondrial DNA.Furthermore, in order to find the molecular mechanism of the pathogenesis of mitochondrial encephalomyopathy, the mutant mitochondrial tRNAs were purified and determined the nucleotide sequences, including modified bases. We found that the first letters of the anticodon are commonly deficient in the modification. This result suggest that this defect on the modification causes misincorporahion of amino acid into the proteins encoded by the mitochondria genome, indicating the novel method to cure the disease by forced modification of anticodon.

线粒体基因组中的几个突变是线粒体脑肌病的原因。由于线粒体基因组是多位点的，而且突变基因组比正常基因组繁殖得更快，因此原则上不可能通过将正常基因导入患病细胞来治愈疾病。因此，如果积累有突变mtDNA的细胞可以通过凋亡消除，那么突变mtDNA与细胞本身一起被去除，并通过生长正常细胞来补偿。为此，我们发现Fas基因对抗线粒体功能障碍。此外，通过差异显示方法，分离了三个因线粒体功能障碍而表达更多的基因。在细胞凋亡的研究中，用X射线晶体学方法测定了细胞凋亡调控因子Bcl-x的结构，用类比模拟法模拟了细胞凋亡诱导因子Bax的结构。推测Bax的结构具有孔形成结构域，并且被证明负责细胞凋亡。在没有与其他BcI-2家族蛋白的二聚体形成结构域的情况下，仅孔形成结构域诱导细胞凋亡。这一发现表明，孔形成结构域是一个很好的蛋白质，诱导细胞凋亡的积累与突变的线粒体DNA。此外，为了寻找线粒体脑肌病发病的分子机制，突变的线粒体tRNA的纯化和确定的核苷酸序列，包括修改的碱基。我们发现反密码子的第一个字母通常在修饰中有缺陷。这一结果表明，这种修饰上的缺陷导致氨基酸错配到线粒体基因组编码的蛋白质中，提示通过强制性反密码子修饰来治疗疾病的新方法。