MOLECULAR REMEDY OF MITOCHONDRIAL DEFECTS

据报道，许多人类疾病与线粒体质子转运辅酶Q-NADH氧化还原酶（也称为复合物I）的缺陷有关。 复合物I的缺陷使其功能失调，导致以下三个问题：（1）呼吸链将NADH氧化回NAD的能力受损;（2）该酶泵送质子的能力受损;（3）活性氧物质（ROS）的产生。这项资助申请的总体目标是确定治疗和/或补救由功能失调的复合体I引起的疾病的潜在领域。在上述三个问题中，当与线粒体不能氧化NADH和/或由ROS引起的损伤相比时，在三个质子易位位点中的任何一个处的质子泵送的损伤似乎不存在严重的健康危害。酵母（酿酒酵母）线粒体缺乏复合物I，但含有由单个亚基（Ndi 1）组成的NADH-UQ氧化还原酶。 在解决与功能失调的复合物I相关的问题的初步尝试中，我们已经尝试使用酵母Ndil在缺乏功能性复合物I的哺乳动物线粒体中将电子从NADH传递到UQ。 我们已经证明，Ndil可以在复合物I缺陷型中国仓鼠突变体细胞（CCL 16-B2）、复合物I缺陷型人细胞（C4 T）和人胚肾293细胞（HEK 293）中功能性表达。 在所有情况下，表达的Ndil正确定位于线粒体中。 这些结果表明，ND 11基因提供了一个潜在的有用的工具，基因治疗线粒体疾病引起的复合物I缺陷。计划在赠款期内进行的研究如下。 (1)ND 11基因在生长停滞的哺乳动物细胞中的功能性表达。 (2)通过ND 11转染抑制哺乳动物线粒体中的ROS。 (3)ND 11转基因小鼠的构建 (4)修复编码复合物I的MWFE亚基的NDUFA 1基因中的点突变。

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.