MOLECULAR REMEDY OF MITOCHONDRIAL DEFECTS

线粒体缺陷的分子修复

• 批准号: 6752052
• 负责人: TAKAO YAGI
• 金额: $ 25.56万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6752052
• 关键词: HeLa cells; NAD(P)H dehydrogenase; Saccharomyces cerevisiae; cell line; cellular respiration; electron transport; enzyme activity; enzyme complex; free radical oxygen; gene expression; gene therapy; hydrogen transport; immunocytochemistry; laboratory mouse; laboratory rabbit; mitochondrial disease /disorder; mitochondrial membrane; point mutation; transfection; ubiquinone

It has been reported that many human diseases are associated with defects in the mitochondrial protontranslocating NADH-ubiquinone (UQ) oxidoreductase, also known as complex I. Defects in complex I, which render it dysfunctional, result in the following three problems: (1) impaired ability of the respiratory chain to oxidize NADH back to NAD; (2) impaired ability of this enzyme to pump protons; (3) production of reactive oxygen species (ROS). The overall goal of this grant application is to identify potential areas of treatment and/or remedies for the diseases that result from dysfunctional complex I. Of the three problems described above, impairment of proton pumping at any one of the three proton translocation sites does not appear to present a severe health hazard when compared to the inability of mitochondria to oxidize NADH and/or damage caused by ROS. Yeast (Saccharomyces cerevisiae) mitochondria lack complex I but contain instead a NADH-UQ oxidoreductase composed of a single- subunit (Ndi1). In an initial attempt to tackle the problems associated with dysfunctional complex I, we have attempted to employ the yeast Ndil to transmit electrons from NADH to UQ in mammalian mitochondria lacking a functional complex I. We have demonstrated that the Ndil can be functionally expressed in complex I-deficient Chinese hamster mutant cells (CCL16-B2), complex I deficient human cells (C4T), and human embryonal kidney 293 cells (HEK 293). In all cases the expressed Ndil was correctly localized in the mitochondria. These results indicate that the ND11 gene provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency. The studies planned during this grant period are as follows. (1) Functional expression of the ND11 gene in growth-arrested mammalian cells. (2) Suppression of the ROS in mammalian mitochondria by ND11 transfection. (3) Construction of transgenic mice containing the ND11 gene. (4) Repair of a point mutation in the NDUFA1 gene encoding the MWFE subunit of complex I.

据报道，许多人类疾病与线粒体质子转移的缺陷有关NADH-纤维酮（UQ）氧化还原酶，也称为复杂I中的复杂I。 （2）该酶泵送质子的能力受损； （3）产生活性氧（ROS）。该赠款应用的总体目的是确定由功能失调的复合物I I.在上述三个问题中引起的疾病的潜在领域和/或补救措施，与Mitochondizia nadh和ROS氧化相比，在三个质子易位站点中的任何一个质子泵送的损害似乎都不是严重的健康危害。酵母（酿酒酵母）线粒体缺乏复合物I，但含有由单个亚基（NDI1）组成的NADH-UQ氧化还原酶。 为了解决与功能失调复合物I相关的问题的最初尝试，我们试图使用酵母NDIL将电子从NADH传播到缺乏功能性复合物的哺乳动物线粒体中的NADH到UQ。 293个细胞（HEK 293）。 在所有情况下，表达的NDIL都正确定位在线粒体中。 这些结果表明，ND11基因为由复杂的I缺乏引起的线粒体疾病的基因治疗提供了潜在的有用工具。在此赠款期间计划的研究如下。 （1）ND11基因在生长促进的哺乳动物细胞中的功能表达。 （2）通过ND11转染抑制ROS在哺乳动物线粒体中的抑制。 （3）构建含有ND11基因的转基因小鼠。 （4）修复编码复合物I的MWFE亚基的NDUFA1基因中的点突变。