Characterizing the biochemical regulation of mitochondrial one-carbon metabolism

线粒体一碳代谢的生化调节特征

• 批准号: 10410227
• 负责人: Owen Samuel Skinner
• 金额: $ 2.35万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10410227
• 关键词: Aging; Automobile Driving; Biochemical; Biochemistry; Biological; Carbon; Cell Proliferation; Cell physiology; Cells; Cellular Stress; Collection; Complex; Consumption; Coupled; Couples; Cytoplasm; Cytosol; Data Set; Defect; Dependence; Disease; Drug Targeting; Drug usage; Electron Transport; Enzymes; Folic Acid; Functional disorder; Genetic; Genetic Models; Glycine; Health; Health Benefit; Hela Cells; Homeostasis; Human; Human Development; Hypoxia; Inborn Errors of Metabolism; Incubated; Knock-out; Laboratories; Lead; Lesion; Link; Logic; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Metabolic; Metabolic Pathway; Metabolism; Methotrexate; Mitochondria; Mitochondrial Diseases; Mitochondrial complex I deficiency; Modeling; NADH; NADP; Neural Tube Defects; Organelles; Oxidases; Oxidation-Reduction; Oxides; Oxidoreductase; Oxygen; Pathway interactions; Pharmaceutical Preparations; Play; Production; Reaction; Reactive Oxygen Species; Regulation; Reporting; Resolution; Role; SLC19A1 gene; Serine; Stable Isotope Labeling; Testing; Therapeutic Intervention; Time; Twin Multiple Birth; Water; Work; base; cancer cell; cell type; cofactor; cytotoxic; developmental disease; established cell line; folic acid metabolism; human disease; metabolome; metabolomics; mitochondrial dysfunction; oxidation; rare genetic disorder; tool; virtual

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.

项目总结/摘要 众所周知，一碳（1C）或叶酸代谢的功能障碍对人类的有害影响 发育，导致神经管缺陷。然而，这一途径也涉及到两个线粒体 功能障碍（见于衰老以及罕见的遗传性疾病）和许多癌症。由于中心的 1C代谢在这些不同的人类疾病，它已经是药物的目标，如甲氨蝶呤。 然而，该途径的潜在生化逻辑仍然非常复杂，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。 导致1C代谢复杂性的是其亚细胞区室化和氧化还原依赖性。 1C代谢有两个平行的分支，一个在胞质溶胶中，一个在细胞质中，这一点还没有得到很好的认识。 线粒体，其由亚细胞氧化还原状态的NADH和NADPH辅因子控制。因此，我们认为， 为了充分描述驱动1C代谢的生化逻辑，有必要使用工具来精确地干扰 这些辅因子的亚细胞氧化还原状态。穆萨实验室最近的工作提供了工具， 确切地说：四种水形成氧化酶（NOX）的集合，可以选择性地氧化NADH或 细胞质或线粒体中的NADPH池。 这项提议旨在利用这些强大的遗传工具来首次破译生物化学的逻辑 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 扰动是探测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。缺氧也是 显示显著重塑线粒体1C分支并另外产生细胞毒性活性氧 物种（ROS）。为了更好地表征1C代谢，亚细胞氧化还原状态， 和活性氧，该提案将利用高分辨率质谱来测量全代谢组 扰动最后，该提议将代谢组学数据集与细胞毒性的测量相结合。 活性氧，并使用一个已经建立的细胞系缺乏一个关键的线粒体1C酶， 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。但其 潜在的生化调节仍然知之甚少。利用最近开发的遗传工具， 调节亚细胞氧化还原稳态与高分辨率代谢组学，这项建议的目的是破译 1C代谢途径的生化逻辑与人类当前和紧迫问题的启示 健康和疾病。