Iron in Mitochondrial Physiology and Disease

铁在线粒体生理学和疾病中的作用

• 批准号: 8632557
• 负责人: PAUL A. LINDAHL
• 金额: $ 29.9万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8632557
• 关键词: Address; Adenine; Binding; Biological Assay; Cell Extracts; Cell physiology; Cells; Characteristics; Chelating Agents; Communities; Complex; Coupled; Cysteine; Cytosol; Development; Disease; Electron Microscopy; Funding; Genes; Health; Heme; Homeostasis; Human; Ions; Iron; Jurkat Cells; Liquid Chromatography; Mass Spectrum Analysis; Metabolism; Methods; Mitochondria; Mitochondrial Diseases; Monitor; Mossbauer Spectroscopy; Nutrient; Organelles; Oxygen; Pathway interactions; Phenotype; Physiology; Plasma; Play; Process; Proteins; Public Health; RNA Interference; Reactive Oxygen Species; Regulation; Research; Roentgen Rays; Role; Sampling; Services; Spectrum Analysis; Sulfur; System; Systems Biology; Time; Transferrin; Transition Elements; Translational Research; United States National Institutes of Health; Yeasts; absorption; biophysical techniques; cell injury; frataxin; genetic strain; genetically modified cells; heme biosynthesis; insight; interest; knock-down; mass spectrometer; molecular mass; nanoparticle; novel; overexpression; particle; programs; public health relevance; time use; trafficking

Iron in Mitochondrial Physiology and Disease (PI: Paul Alan Lindahl) Project Summary Iron plays critical roles in cellular metabolism, human health and disease. Fe/S clusters and hemes are synthesized in mitochondria and thus much cellular Fe is imported into this organelle. One aim of this translational research program is to elucidate the Fe-associated players involved in these processes, as this will provide new mechanistic insights into cellular Fe metabolism and help develop new strategies to treat Fe- related diseases. A great challenge in Fe trafficking is to determine whether low-molecular-mass (LMM) Fe complexes participate. Using a liquid chromatography system with an on-line ICP-MS, we have detected numerous LMM Fe complexes in yeast and human Jurkat cells. We are particularly interested in cytosolic Fe species that are imported into mitochondria, and in mitochondrial Fe species that are used for Fe/S cluster and/or heme biosynthesis. We will characterize and identify these LMM Fe complexes using M¿ssbauer (MB), EPR, mass spectrometry, NMR and X-ray absorption spectroscopy. We will probe the cellular functions of these species and identify associated Fe pathways leading into and operating within mitochondria. We will use WT cells grown under different conditions, and various genetic strains in which Fe metabolism has been altered. Fe trafficking will also be examined on the systems biology level using an ensemble of interrelated spectroscopic probes centered on MB spectroscopy. The distribution of Fe in WT and genetically modified cells and their organelles will be used to address the mechanism of Fe trafficking. We will examine strains that either accumulate or deplete Fe from the cytosol or mitos. We will characterize the mechanism of Fe/S cluster and heme biosynthesis by developing an assay for these processes using intact isolated mitos. We will determine whether a MB-detectable pool of mitochondrial FeII is used for Fe/S cluster and/or heme biosynthesis, and determine the function of each component of the pool. We will evaluate whether Fe is exported from mitochondria. We will investigate Fe trafficking in human cells that have been genetically modified to allow RNAi knockdowns and overexpressions of genes involved in Fe metabolism. We will knock- down frataxin and examine the phenotype that develops over time and with the extent of knock-down at long times. We will determine the order in which various Fe-accumulation characteristics develop and establish a mechanism for the genesis of the resulting diseased state. Other genes involved in Fe metabolism and disease will be knocked-down and/or overexpressed. Finally, we will dedicate a portion of our MB time to collecting and interpreting spectra of samples from other NIH-supported labs who study cellular Fe metabolism. Relevance of this research to public health. Numerous iron-associated diseases are related to problems in transporting iron into different parts of the cell. Iron often accumulates in the mitochondria, generating very small iron particles and highly reactive oxygen species that damage cells. Analogous diseased states will be recreated in yeast and human cells, and studied using sophisticated biophysical and bioanalytical methods. 1

铁在线粒体生理学和疾病（PI：保罗艾伦林达尔） 项目摘要 铁在细胞代谢、人体健康和疾病中起着关键作用。Fe/S簇合物和血红素是 在线粒体中合成，因此大量的细胞铁被输入到这个细胞器中。这样做的目的之一 翻译研究计划是阐明铁相关的球员参与这些过程，因为这 将为细胞铁代谢提供新的机制见解，并有助于开发治疗铁代谢的新策略。 相关疾病。铁运输的一个巨大挑战是确定低分子量（LMM）铁是否 综合体参与。使用液相色谱系统与在线ICP-MS，我们已经检测到 在酵母和人Jurkat细胞中存在大量LMM Fe复合物。我们对胞质Fe特别感兴趣 线粒体中的Fe种主要用于Fe/S簇 和/或血红素生物合成。我们将使用穆斯堡尔谱（MB）表征和鉴定这些LMM Fe络合物， EPR、质谱、NMR和X射线吸收光谱。我们将探索 这些物种，并确定相关的铁途径，导致和线粒体内运作。我们将使用 WT细胞生长在不同的条件下，和各种遗传菌株，其中铁代谢已被破坏。 改变了还将在系统生物学水平上使用相互关联的 以MB光谱为中心的光谱探针。铁在WT和转基因细胞中的分布 其细胞器将被用来解决铁的运输机制。我们将检测 从细胞质或线粒体中积累或消耗铁。我们将对Fe/S团簇的形成机理进行研究 和血红素生物合成的方法。我们将 确定线粒体Fe II的MB可检测池是否用于Fe/S簇和/或血红素 生物合成，并确定池的每个组分的功能。我们将评估Fe是否 由线粒体输出。我们将研究铁在人类细胞中的运输， 修饰以允许RNAi敲除和过量表达参与Fe代谢的基因。我们会敲门- 下调共济失调蛋白，并检查随着时间的推移和长期敲低的程度而发展的表型。 次我们将确定各种铁积累特征发展的顺序，并建立一个 导致疾病状态发生的机制。其他参与铁代谢和疾病的基因 将被敲低和/或过表达。最后，我们将把一部分MB时间用于收集和 解释来自其他NIH支持的研究细胞铁代谢的实验室的样品光谱。 这项研究与公共卫生的相关性。许多与铁相关的疾病与以下问题有关： 将铁转运到细胞的不同部位。铁通常积聚在线粒体中， 小的铁颗粒和高活性氧物质，破坏细胞。类似的疾病状态 在酵母和人类细胞中重建，并使用复杂的生物物理和生物分析方法进行研究。 1