MOLECULAR REMEDY OF MITOCHONDRIAL DEFECTS

据报道，许多人类疾病与线粒体质子转运NADH-泛醌(UQ)氧化还原酶(又称复合体I)的缺陷有关，复合体I的缺陷导致以下三个问题：(1)呼吸链将NADH氧化回NAD的能力受损；(2)该酶泵出质子的能力受损；(3)产生活性氧(ROS)。这项赠款申请的总体目标是确定由功能失调的复合体I引起的疾病的潜在治疗和/或补救领域。在上述三个问题中，与线粒体无法氧化NADH和/或ROS造成的损害相比，三个质子转运点中任何一个的质子泵功能受损似乎不会对健康造成严重危害。酵母(Saccharmyces Cerevisiae)线粒体缺乏复合体I，但含有由单亚基(Ndi1)组成的NADH-UQ氧化还原酶。在解决与功能障碍复合体I相关的问题的初步尝试中，我们试图利用酵母Ndil在缺乏功能复合体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可以在复合体I缺陷的中国仓鼠突变细胞(CCL16-B2)、复合体I缺陷的人类细胞(C4T)和人胚胎肾293细胞(HEK 293)中功能表达。在所有情况下，表达的Ndil都正确地定位在线粒体中。这些结果表明，ND11基因为复合碘缺乏引起的线粒体疾病的基因治疗提供了一种潜在的有用工具。在这一赠款期间计划进行的研究如下。(1)ND11基因在生长受阻的哺乳动物细胞中的功能表达。(2)ND11基因对哺乳动物线粒体ROS的抑制作用。(3)构建含ND11基因的转基因小鼠。(4)修复编码复合体I的MWFE亚单位的NDUFA1基因点突变。

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.