Motyka Shawn A, Drew Mark E, Yildirir Gokben, Englund Paul T
Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
J Biol Chem. 2006 Jul 7;281(27):18499-506. doi: 10.1074/jbc.M602880200. Epub 2006 May 10.
The mitochondrial genome of trypanosomes, termed kinetoplast DNA (kDNA), contains thousands of minicircles and dozens of maxicircles topologically interlocked in a network. To identify proteins involved in network replication, we screened an inducible RNA interference-based genomic library for cells that lose kinetoplast DNA. In one cloned cell line with inducible kinetoplast DNA loss, we found that the RNA interference vector had aberrantly integrated into the genome resulting in overexpression of genes down-stream of the integration site (Motyka, S. A., Zhao, Z., Gull, K., and Englund, P. T. (2004) Mol. Biochem. Parasitol. 134, 163-167). We now report that the relevant overexpressed gene encodes a mitochondrial cytochrome b(5) reductase-like protein. This overexpression caused kDNA loss by oxidation/inactivation of the universal minicircle sequence-binding protein, which normally binds the minicircle replication origin and triggers replication. The rapid loss of maxicircles suggests that the universal minicircle sequence-binding protein might also control maxicircle replication. Several lines of evidence indicate that the cytochrome b(5) reductase-like protein controls the oxidization status of the universal minicircle sequence-binding protein via tryparedoxin, a mitochondrial redox protein. For example, overexpression of mitochondrial tryparedoxin peroxidase, which utilizes tryparedoxin, also caused oxidation of the universal minicircle sequence-binding protein and kDNA loss. Furthermore, the growth defect caused by overexpression of cytochrome b(5) reductase-like protein could be partially rescued by simultaneously overexpressing tryparedoxin.
锥虫的线粒体基因组，称为动质体DNA（kDNA），包含数千个微小环和几十个大环，它们在一个网络中拓扑互锁。为了鉴定参与网络复制的蛋白质，我们针对失去动质体DNA的细胞筛选了一个基于诱导性RNA干扰的基因组文库。在一个具有诱导性动质体DNA缺失的克隆细胞系中，我们发现RNA干扰载体异常整合到基因组中，导致整合位点下游基因的过表达（莫蒂卡，S.A.，赵，Z.，古尔，K.，和英格伦德，P.T.（2004年）《分子生物化学寄生虫学》134，163 - 167）。我们现在报告，相关的过表达基因编码一种线粒体细胞色素b(5)还原酶样蛋白。这种过表达通过普遍微小环序列结合蛋白的氧化/失活导致kDNA丢失，该蛋白通常结合微小环复制起点并触发复制。大环的快速丢失表明普遍微小环序列结合蛋白可能也控制大环复制。几条证据表明，细胞色素b(5)还原酶样蛋白通过锥虫硫氧还蛋白（一种线粒体氧化还原蛋白）控制普遍微小环序列结合蛋白的氧化状态。例如，利用锥虫硫氧还蛋白的线粒体锥虫硫氧还蛋白过氧化物酶的过表达也导致普遍微小环序列结合蛋白的氧化和kDNA丢失。此外，同时过表达锥虫硫氧还蛋白可以部分挽救由细胞色素b(5)还原酶样蛋白过表达引起的生长缺陷。
