Characterizing the biochemical regulation of mitochondrial one-carbon metabolism

项目摘要/摘要 众所周知，一碳(1C)或叶酸代谢障碍对人类有害 发育，导致神经管缺陷。然而，该途径也牵涉到线粒体和 功能障碍(发现于衰老和罕见的遗传性疾病)，以及许多癌症。因为它的中心地位 1C代谢跨越这些不同的人类疾病，它已经是甲氨蝶呤等药物的靶点。 然而，该途径的潜在生化逻辑仍然令人难以置信地复杂，使其 基本功能难以理解，因此限制了其被进一步药物靶向的能力。 1C代谢的复杂性在于它的亚细胞区隔和氧化还原依赖性。 人们还没有很好地认识到，1C代谢有两个平行的分支，一个在胞浆中，另一个在胞浆中 线粒体，由NADH和NADPH辅助因子的亚细胞氧化还原状态控制。因此， 为了充分描述驱动1C新陈代谢的生化逻辑，有必要有工具来精确地干扰 这些辅因子的亚细胞氧化还原状态。Mootha实验室最近的工作提供了工具来完成 的确如此：四种水形成氧化酶(NOXE)的集合，它们可以选择性地氧化NADH或 NADPH池位于细胞质或线粒体中。 这项提议旨在首次使用这些强大的遗传工具来破译生化逻辑 在两种细胞应激状态下的潜在1C代谢：线粒体功能障碍和缺氧。这些 微扰是探索1C代谢活性的良好模型。线粒体功能障碍上调 途径，并同时减少线粒体和胞质NADH池。低氧也一直是 研究表明，显著重塑线粒体1C分支，并额外产生细胞毒性活性氧 种(ROS)。为了更好地描述1C代谢、亚细胞氧化还原状态、 和ROS，这项提议将利用高分辨率质谱学来测量全代谢组 微扰。最后，这项提议将代谢组学数据集与细胞毒性的测量相结合 活性氧物种，并使用缺乏关键线粒体1C酶的已建立的细胞系来 分离1C对ROS的贡献。 1C代谢在人类发育、癌症和线粒体功能障碍中起着关键作用。然而，它的 潜在的生化调控仍然知之甚少。利用最近开发的遗传工具来 用高分辨率代谢组学调节亚细胞氧化还原动态平衡，这一建议旨在破译 1C代谢途径的生化逻辑及其对人类当前和紧迫问题的启示 健康和疾病。

Project Summary/Abstract Dysfunctions in one carbon (1C), or folate, metabolism are well-known for their deleterious effects on human development, causing neural tube defects. However, the pathway is also implicated in both mitochondrial dysfunction (found in aging as well as rare, genetic disorders), and many cancers. Because of the centrality of 1C metabolism across these diverse human diseases, it is already the target of drugs such as methotrexate. Nevertheless, the underlying biochemical logic of the pathway remains incredibly complex, rendering its fundamental functions difficult to understand, and therefore limiting its ability to be targeted by further drugs. Contributing to the complexity of 1C metabolism are its subcellular compartmentalization and redox dependency. It is not well appreciated that there are two parallel branches of 1C metabolism, one in the cytosol and one in the mitochondria, which are controlled by the subcellular redox state of NADH and NADPH cofactors. Therefore, to fully characterize the biochemical logic driving 1C metabolism, it is necessary to have tools to precisely perturb the subcellular redox state of these cofactors. Recent work by the Mootha laboratory has provided tools to do exactly that: a collection of four water-forming oxidases (NOXes) that can selectively oxidize the NADH or NADPH pool in the cytoplasm or the mitochondria. This proposal aims to use these powerful genetic tools to decipher, for the first time, the biochemical logic underlying 1C metabolism in two states of cellular stress: mitochondrial dysfunction and hypoxia. These perturbations are good models to probe the activity of 1C metabolism. Mitochondrial dysfunction upregulates the pathway, and simultaneously reduces both the mitochondrial and cytosolic NADH pools. Hypoxia has also been shown to significantly remodel the mitochondrial 1C branch and additionally produces cytotoxic reactive oxygen species (ROS). To better characterize the complex interactions between 1C metabolism, subcellular redox state, and ROS, this proposal will leverage high-resolution mass spectrometry to measure whole-metabolome perturbations. Finally, this proposal will couple the metabolomics dataset with measurements of cytotoxic reactive oxygen species and use an already-established cell line lacking a critical mitochondrial 1C enzyme to isolate the contributions of 1C on ROS. 1C metabolism plays a critical role in human development, cancer, and mitochondrial dysfunction. However, its underlying biochemical regulation remains poorly understood. Leveraging recently developed genetic tools to modulate subcellular redox homeostasis with high-resolution metabolomics, this proposal aims to decipher the biochemical logic of the 1C metabolic pathway with implications for current and pressing problems in human health and disease.