Mechanisms of Mitochondrial Iron Uptake: New Therapeutic Targets in Hepatotoxicity

线粒体铁摄取机制：肝毒性的新治疗靶点

• 批准号: 10597049
• 负责人: John J Lemasters
• 金额: $ 45.62万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10597049
• 关键词: Acetaminophen; Address; Affect; Affinity; Area; Binding; Binding Sites; Bioenergetics; Biological Assay; Biology; Cations; Cell Membrane Permeability; Cell membrane; Cells; Citrates; Complex; Confocal Microscopy; Core Protein; DNA Damage; Data; Divalent Cations; Endosomes; Ferritin; Generations; Genetic Models; Heme; Hepatocyte; Hepatotoxicity; Homeostasis; Hydroxyl Radical; Image; In Vitro; Inner Limiting Membrane; Inner mitochondrial membrane; Intervention; Iron; Iron Overload; Kinetics; Knock-out; Knowledge; Liver diseases; Lysosomes; Measurement; Mediating; Membrane; Membrane Potentials; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Mutation; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Permeability; Physiological; Play; Predisposition; Process; Protein Isoforms; Proteins; Reaction; Reactive Oxygen Species; Role; SLC11A2 gene; Sulfur; Time; Toxic effect; Transition Elements; acetaminophen-induced liver injury; calcium uniporter; clinically relevant; cofactor; experimental study; hepatocellular injury; in vivo; innovation; insight; iron metabolism; mitochondrial dysfunction; mitochondrial membrane; new therapeutic target; novel; novel therapeutics; prevent; superresolution microscopy; tool; uptake

Iron is a transition metal that exists in two pools within cells. Chelatable iron comprises free iron and iron loosely bound to anionic metabolites like ATP and citrate, whereas non-chelatable iron is tightly bound to ferritin, heme and iron-sulfur clusters. Redox active chelatable iron promotes oxidative stress by catalyzing the Fenton reaction, which produces highly reactive hydroxyl radicals that damage DNA, proteins and membranes. Much evidence implicates mitochondrial iron as an important contributor to toxicity, but the molecular pathways of mitochondrial iron uptake are incompletely understood. Current dogma is that mitoferrin (Mfrn1 and 2), a mitochondrial inner membrane protein, is responsible for mitochondrial iron transport. However, studies from 45 years ago show that the classical electrogenic mitochondrial calcium uniporter (MCU) complex also catalyzes uptake of Fe2+ but not Fe3+ driven by the mitochondrial membrane potential, a conclusion supported by our own studies in intact and permeabilized cells. Our preliminary pull-down, Duolink and super-resolution microscopy studies show a physical association of Mfrn2, the predominant isoform in non-erythroid cells, with MCU, the core protein of the MCU complex. This brings us to the fundamental questions to be addressed by this proposal: 1) Do mitochondria accumulate iron via two independent pathways: a non-electrogenic pathway mediated by Mfrn and an electrogenic pathway catalyzed by MCU? 2) Alternatively do Mfrn and the molecular components of MCU exist within a single complex mediating both Fe2+ and Ca2+ uptake? 3) Is Mfrn an exchanger, such as an Fe2+/Na+(H+) exchanger in analogy to Ca2+/Na+, Mg2+/Na+, and Na+/H+ exchangers? 4) How do MCU and Mfrn, as well as divalent metal transporter 1 (DMT1), contribute to hepatotoxicity? In Aim 1, we will characterize mitochondrial Fe2+ uptake and exchange in plasma membrane-permeabilized wildtype (WT), MCU knockout (KO) and Mfrn1/2 double KO (DKO) hepatocytes. Aim 2 will identify interactions of Mfrn2 with other proteins using an unbiased enrichment-mass spectrometric (AE-MS) approach, DuoLink and super-resolution microscopy to establish whether or not Mfrn2 and MCU are authentic binding partners and also to identify associations of Mfrn2 with other novel partners. Aim 3 will assess how targeted mutations affect susceptibility to acetaminophen (APAP) toxicity, which is mediated by iron. Specifically, we will determine how deficiencies of MCU, Mfrn2 and DMT1 affect APAP-induced mitochondrial dysfunction and hepatocellular killing in vitro and in vivo. We expect these studies to define the specific roles of MCU, Mfrn2 and DMT1 in this clinically relevant model of hepatocellular injury. The concept that Mfrn and MCU are both essential for both mitochondrial Fe2+ uptake and homeo-stasis is novel, innovative and paradigm-shifting. The project will provide insights into an unexplored area of biology and fill an important gap in our understanding of the pathways involved in mitochondrial iron uptake and iron-dependent toxicities. Better understanding of the process will eventually lead to more specific interventions against hepatic diseases promoted by iron overload.

铁是一种过渡金属，存在于细胞内的两个池中。可螯合铁包括游离铁和铁 与阴离子代谢物如ATP和柠檬酸盐松散结合，而不可螯合的铁与铁蛋白紧密结合， 血红素和铁硫簇。氧化还原活性螯合铁通过催化芬顿促进氧化应激 反应，产生高活性的羟基自由基，破坏DNA，蛋白质和膜。多 有证据表明线粒体铁是毒性的重要贡献者，但线粒体铁的分子途径 线粒体铁摄入还不完全清楚。目前的教条是，线粒体铁蛋白（Mfrn 1和2）， 线粒体内膜蛋白，负责线粒体铁转运。研究显示，45 多年前的研究表明，经典的产电线粒体钙单向转运体（MCU）复合物也催化 线粒体膜电位驱动Fe 2+而不是Fe 3+的摄取，这一结论得到了我们自己的支持。 对完整和透化细胞的研究。我们初步的下拉式、Duolink和超分辨率显微镜 研究表明，Mfrn 2（非红系细胞中的主要同种型）与MCU（核心细胞）之间存在物理联系。 MCU复合物的蛋白质。这就引出了本提案所要解决的基本问题：1） 线粒体是否通过两条独立的途径积累铁：一条由Mfrn介导的非产电途径 和MCU催化的产电途径2)或者做Mfrn和MCU的分子组分 存在于一个单一的复杂介导的Fe 2+和Ca 2+的吸收？3)Mfrn是一种交换器，例如 Fe 2 +/Na+（H+）交换剂类似于Ca 2 +/Na+、Mg 2 +/Na+和Na+/H+交换剂？4)MCU和Mfrn如何， 以及二价金属转运蛋白1（DMT 1），有助于肝毒性？在目标1中，我们将描述 质膜透性野生型（WT）、MCU敲除（KO）中的线粒体Fe 2+摄取和交换 和Mfrn 1/2双KO（DKO）肝细胞。目的2将使用一种新的方法来鉴定Mfrn 2与其他蛋白质的相互作用。 无偏富集质谱（AE-MS）方法，DuoLink和超分辨率显微镜， 确定Mfrn 2和MCU是否是真实的结合配偶体，并且还鉴定Mfrn 2的关联 与其他新的合作伙伴。目标3将评估靶向突变如何影响对乙酰氨基酚的易感性 （APAP）毒性，其由铁介导。具体来说，我们将确定MCU、Mfrn 2和 DMT 1影响APAP诱导的线粒体功能障碍和肝细胞杀伤在体外和体内。我们预计 这些研究旨在定义MCU、Mfrn 2和DMT 1在这种临床相关模型中的具体作用， 肝细胞损伤Mfrn和MCU对于线粒体Fe 2+摄取和稳态都是必不可少的概念是新颖的、创新的和范式转变的。该项目将提供一个未探索的领域的见解， 填补了我们对线粒体铁摄取途径的理解中的一个重要空白， 铁依赖性毒性更好地理解这一过程将最终导致更具体的干预措施 对抗由铁超载引起的肝脏疾病。