Characterizing the biochemical regulation of mitochondrial one-carbon metabolism

线粒体一碳代谢的生化调节特征

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要一碳 (1C) 或叶酸代谢功能障碍因其对人体的有害影响而闻名发育，导致神经管缺陷。然而，该途径也涉及线粒体功能障碍（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目标。然而，该途径的潜在生化逻辑仍然极其复杂，使其其基本功能难以理解，因此限制了其进一步药物靶向的能力。 1C 代谢的复杂性归因于其亚细胞区室化和氧化还原依赖性。人们还没有充分认识到 1C 代谢有两个平行的分支，一个在细胞质中，一个在细胞质中。线粒体，由 NADH 和 NADPH 辅因子的亚细胞氧化还原状态控制。所以，为了充分表征驱动1C代谢的生化逻辑，需要有工具来精确扰动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正是这样：四种水形成氧化酶 (NOX) 的集合，可以选择性地氧化 NADH 或 NADPH 库位于细胞质或线粒体中。该提案旨在利用这些强大的遗传工具首次破译生化逻辑两种细胞应激状态下的 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，同时减少线粒体和胞质 NADH 池。也曾缺氧显示可显着重塑线粒体 1C 分支并额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 ROS，该提案将利用高分辨率质谱来测量整个代谢组扰动。最后，该提案将代谢组学数据集与细胞毒性测量结合起来活性氧并使用缺乏关键线粒体 1C 酶的已建立细胞系分离 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发挥着至关重要的作用。然而，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通过高分辨率代谢组学调节亚细胞氧化还原稳态，该提案旨在破译 1C代谢途径的生化逻辑对人类当前和紧迫问题的影响健康和疾病。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

An engineered enzyme that targets circulating lactate to alleviate intracellular NADH:NAD+ imbalance.

一种针对循环乳酸以缓解细胞内 NADH:NAD 失衡的工程酶。

DOI：
发表时间：
2020
期刊：
影响因子：
46.9
作者：
Patgiri, Anupam;Skinner, Owen S;Miyazaki, Yusuke;Schleifer, Grigorij;Marutani, Eizo;Shah, Hardik;Sharma, Rohit;Goodman, Russell P;To, Tsz;Robert Bao, Xiaoyan;Ichinose, Fumito;Zapol, Warren M;Mootha, Vamsi K
通讯作者：
Mootha, Vamsi K

Genetic Screen for Cell Fitness in High or Low Oxygen Highlights Mitochondrial and Lipid Metabolism.

高氧或低氧条件下细胞健康度的遗传筛选突出了线粒体和脂质代谢。