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（TOP1MT）从线粒体DNA（mtDNA）中去除负超胶，并已被鉴定为人类中的线粒体疾病候选基因。但是，尽管MTDNA超螺旋负明显增加，但TOP1MT - / - 小鼠并未表现出明显的线粒体功能障碍。因此，TOP1MT被指示为负mtDNA超螺旋的调节剂，但是该功能在线粒体稳态中的作用尚不清楚。我们已经证明，TOP1MT充当mtDNA转录的直接负调节剂。通过刺激内源性ROS产生，可以在分离的线粒体中抑制此功能。在表达抗氧化的TOP1MT突变体后，效果被废除。我们的观察结果支持双负环的模型，其中TOP1MT对MTDNA转录进行负调控，而又受氧化负面调节。我们旨在提供因果循环的因果证明（目标1），表征了所涉及的成分和分子相互作用（目标2），并解决了该机制在氧化应激适应和与年龄相关的线粒体功能障碍中的作用（目标3）。对于这种成脓液，我们将在孤立的线粒体（i）中证明mtDNA转录的氧化还原调节在消耗或删除TOP1MT时会丢失，并且不补充抗性或氧化的TOP1MT突变体或抗氧化的抗抑郁剂，并且（II）在线粒体上的急剧繁殖量的急性下降量增加了，而又有剂量的繁殖量。 TOP1MT和增加负MTDNA超串联，并且这些响应通过野生型TOP1MT在TOP1MT0/0细胞中补充，但不是抗氧化或耐氧化的TOP1MT突变体。然后，我们将继续前进，通过将上述互补模型对急性和/或慢性间歇性UVA暴露来确定线粒体应力反应中的TOP1MT依赖性机制，并在MTDNA转录，mtDNA拷贝数，核核分解次数和MTDNA的官能化和MTDNNA-ewsirir and nectir and MetDnna-anddnna-anddnna-Anddnne and Mentdnna-anddnna-anddnne-anddnne-anddnna-Anddnna-Anddnne-anddnna-Anddecodecodecodecods and ros和MTDNA转录上监测UVA影响。我们最终将通过在原发性成纤维细胞中解决其状态的隔离形式暴露于阳光暴露和日晒的老年人和年轻人供体的皮肤，从而验证TOP1MT依赖性适应过程的生理相关性。最终，我们旨在通过诱导的TOP1MT在小鼠中验证我们的结果，但是这些实验不在此应用程序的范围之内。我们希望发现一种提供MTDNA转录局部氧化还原调节的机制。我们期望阐明该机制是否可以调整线粒体基质内的氧化还原状态的呼吸能力，并在线粒体生物遗传胁迫反应中微调线粒体 - 核核电通信。我们可能会确认，在人真皮成纤维细胞中，超越调节机制的破坏与UVA诱导的线粒体功能障碍有关。