REDOX-regulation of mitochondrial transcription by mitochondrial DNA topoisomerase I

线粒体 DNA 拓扑异构酶 I 对线粒体转录的氧化还原调节

• 批准号: 273662364
• 负责人: Professor Dr. Friedrich Boege
• 金额: --
• 依托单位: Institut für Klinische Chemie und Laboratoriumsdiagnostik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/273662364?language=en
• 关键词: REDOX; regulation; mitochondrial; transcription; DNA

Mitochondrial topoisomerase I (TOP1MT) removes negative supercoils from mitochondrial DNA (mtDNA) and has been identified as mitochondrial disease candidate gene in humans. However, TOP1MT-/- mice do not exhibit overt mitochondrial dysfunction despite a marked increase in negative mtDNA supercoiling. TOP1MT is thus indicated a regulator of negative mtDNA supercoiling, but the role of that function in mitochondrial homeostasis remains unclear.We have demonstrated that TOP1MT acts as direct negative regulator of mtDNA-transcription. This function can be inhibited in isolated mitochondria by stimulating endogenous ROS production. Upon expression of oxidation-resistant TOP1MT mutants the effect is abolished. Our observations support the model of a double negative loop, in which TOP1MT negatively regulates mtDNA-transcription and is in turn negatively regulated by oxidation. We aim to provide causal proof of the surmised regulatory loop (objective 1), characterise the components and molecular interactions involved (objective 2), and address the role of this mechanism in oxidative stress adaptation and age-associated mitochondrial dysfunction (objective 3). For that pupose, we will demonstrate in isolated mitochondria (i) that REDOX-modulation of mtDNA transcription is lost upon depletion or deletion of TOP1MT and not complemented by inactive or oxidation-resistant TOP1MT mutants, and (ii) that acute increases in mitochondrial ROS diminish the recruitment of TOP1MT to heavy and light strand promoters, decrease nucleoid association of TOP1MT and increase negative mtDNA supercoiling, and that these responses are complemented in TOP1MT0/0 cells by wild type TOP1MT but not inactive or oxidation-resistant TOP1MT mutants. We will then move on to identify TOP1MT-dependent mechanisms in mitochondrial stress responses by subjecting the above complementation models to acute and/or chronic intermittent UVA exposure and monitor the UVA-impact on ROS levels, mtDNA-transcription, mtDNA copy number, nuclear mito-biogenesis, the balance of nuclear and mtDNA-encoded respiratory complexes, and respiratory function. We will finally validate the physiological relevance of TOP1MT-dependent adaptation processes by addressing their state in primary fibroblasts isolated form sun-exposed and sun-protected skin of old and young human donors. Ultimately, we aim at validating our results by inducible TOP1MT knock out in mice, but these experiments are outside the purview of this application.We expect to uncover a mechanism that provides local REDOX-regulation of mtDNA transcription. We expect to elucidate whether this mechanism adjusts respiratory capacity to the REDOX state inside the mitochondrial matrix and serves to fine tune mito-nuclear communication in mito-biogenetic stress responses. We will possibly confirm that disruption of the surmised regulatory mechanism is involved in UVA-induced and age-related mitochondrial dysfunction in human dermal fibroblasts.

线粒体拓扑异构酶I（mitochondrialtopoisomerase I，TOP1 MT）是去除线粒体DNA（mitochondrialDNA，mtDNA）中负超螺旋的重要基因，已被确定为人类线粒体疾病的候选基因。然而，TOP1 MT-/-小鼠没有表现出明显的线粒体功能障碍，尽管负mtDNA超螺旋显着增加。因此，TOP1 MT是负性mtDNA超螺旋的调节子，但该功能在线粒体内稳态中的作用尚不清楚。这种功能可以通过刺激内源性ROS产生来抑制分离的线粒体。在抗氧化TOP1 MT突变体的表达后，该效应被消除。我们的观察结果支持双负环模型，其中TOP1 MT负调控mtDNA转录，反过来又受氧化负调控。我们的目标是提供推测的调节环的因果证据（目标1），阐明所涉及的组分和分子相互作用（目标2），并解决该机制在氧化应激适应和年龄相关的线粒体功能障碍中的作用（目标3）。为此，我们将在分离的线粒体中证明：（i）线粒体DNA转录的REDOX调节在TOP1 MT耗尽或缺失时丧失，并且不能被失活或抗氧化的TOP1 MT突变体补充，和（ii）线粒体ROS的急性增加减少了TOP1 MT向重链和轻链启动子的募集，减少了TOP1 MT的类核缔合，并增加了负性线粒体DNA超螺旋，并且这些应答在TOP1 MT 0/0细胞中由野生型TOP1 MT而不是无活性或抗氧化的TOP1 MT突变体补充。然后，我们将继续通过将上述互补模型进行急性和/或慢性间歇性UVA暴露来确定线粒体应激反应中的TOP1 MT依赖机制，并监测UVA对ROS水平、mtDNA转录、mtDNA拷贝数、核有丝分裂生物发生、核和mtDNA编码的呼吸复合物的平衡以及呼吸功能的影响。我们将最终验证TOP1 MT依赖性适应过程的生理相关性，通过解决它们在从老年和年轻人供体的阳光暴露和防晒皮肤分离的原代成纤维细胞中的状态。最终，我们的目标是通过诱导小鼠TOP1 MT基因敲除来验证我们的结果，但这些实验超出了本申请的范围，我们希望揭示一种机制，该机制提供了mtDNA转录的局部REDOX调控。我们希望阐明这种机制是否调整呼吸能力的线粒体基质内的氧化还原状态，并微调线粒体生物应激反应中的线粒体-核通讯。我们将可能证实，在UVA诱导的和年龄相关的人皮肤成纤维细胞的线粒体功能障碍的推测的调节机制的中断。