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) 可去除线粒体 DNA (mtDNA) 中的负超螺旋，已被确定为人类线粒体疾病候选基因。然而，尽管负线粒体DNA超螺旋显着增加，但TOP1MT-/-小鼠并未表现出明显的线粒体功能障碍。因此，TOP1MT 被认为是负 mtDNA 超螺旋的调节因子，但该功能在线粒体稳态中的作用仍不清楚。我们已经证明 TOP1MT 作为 mtDNA 转录的直接负调节因子。在分离的线粒体中，可以通过刺激内源性 ROS 产生来抑制该功能。一旦表达抗氧化 TOP1MT 突变体，该效应就被消除。我们的观察结果支持双负环模型，其中 TOP1MT 负向​​调节 mtDNA 转录，进而受到氧化的负向调节。我们的目标是提供推测的调节环路的因果证据（目标 1），描述所涉及的成分和分子相互作用（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 转录的氧化还原调节在 TOP1MT 耗尽或删除时会丢失，并且不会被无活性或抗氧化的 TOP1MT 突变体补充，并且（ii）线粒体 ROS 急剧增加减少 TOP1MT 向重链和轻链启动子的募集，减少 TOP1MT 的核结合并增加负 mtDNA 超螺旋，并且这些反应在TOP1MT0/0 细胞由野生型 TOP1MT 组成，但不是失活或抗氧化的 TOP1MT 突变体。然后，我们将通过将上述互补模型置于急性和/或慢性间歇性 UVA 照射下，继续确定线粒体应激反应中 TOP1MT 依赖性机制，并监测 UVA 对 ROS 水平、mtDNA 转录、mtDNA 拷贝数、核线粒体的影响-生物发生、核和线粒体DNA编码的呼吸复合物的平衡以及呼吸功能。我们最终将通过研究从老年和年轻人类供体的暴露于阳光和防晒的皮肤中分离出的原代成纤维细胞的状态来验证 TOP1MT 依赖性适应过程的生理相关性。最终，我们的目标是通过在小鼠中诱导性 TOP1MT 敲除来验证我们的结果，但这些实验超出了本应用的范围。我们期望揭示一种提供 mtDNA 转录局部氧化还原调节的机制。我们期望阐明这种机制是否将呼吸能力调整到线粒体基质内的氧化还原状态，并用于微调线粒体生物遗传应激反应中的线粒体核通讯。我们可能会证实，推测的调节机制的破坏与 UVA 诱导的和与年龄相关的人类真皮成纤维细胞线粒体功能障碍有关。