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。 松散地与阴离子代谢物如三磷酸腺苷和柠檬酸盐结合，而非螯合铁与铁蛋白紧密结合， 血红素和铁硫团簇。氧化还原活性络合铁通过催化Fenton促进氧化应激 反应，产生高活性的羟基自由基，破坏DNA、蛋白质和细胞膜。大有可为 有证据表明线粒体铁是毒性的重要贡献者，但 线粒体的铁摄取尚不完全清楚。目前的教条是丝裂铁蛋白(Mfrn1和2)，a 线粒体内膜蛋白，负责线粒体铁的运输。然而，来自45个国家的研究 几年前的研究表明，经典的线粒体钙单一转运体(Mcu)复合体也催化 线粒体膜电位驱动对Fe2+的摄取而不是Fe3+的摄取，这一结论得到了我们自己的支持 在完整和通透性细胞中的研究。我们的初步下拉、双链接和超分辨率显微镜 研究表明，非红系细胞中的主要亚型Mfrn2与核心MCU之间存在物理联系 MCU复合体的蛋白质。这将我们带到该提案要解决的基本问题上：1) 线粒体是否通过两条独立的途径积累铁：Mfrn介导的非生电途径 和MCU催化的一种电生成途径？2)交替DO Mfrn和MCU的分子组成 存在于同时调节Fe2+和Ca~(2+)吸收的单一复合体中？3)Mfrn是交换器，如 Fe2+/Na+(H+)交换器类似于Ca2+/Na+、Mg2+/Na+和Na+/H+交换器？4)MCU和Mfrn、 和二价金属转运蛋白1(DMT1)一样，也参与了肝毒性？在目标1中，我们将描述 线粒体Fe2+在质膜通透性野生型(WT)、MCU基因敲除(KO)中的摄取和交换 和Mfrn1/2双KO(DKO)肝细胞。AIM 2将使用一种 无偏富集-质谱仪(AE-MS)、DuoLink和超分辨显微镜 确定Mfrn2和MCU是否是可信的绑定伙伴，并识别Mfrn2的关联 和其他新奇的伙伴。AIM 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，由铁介导。具体地说，我们将确定MCU、Mfrn2和 DMT1在体内外影响APAP诱导的线粒体功能障碍和肝细胞杀伤。我们预计 这些研究旨在确定MCU、Mfrn2和DMT1在这一临床相关模型中的具体作用 肝细胞损伤。Mfrn和MCU对线粒体Fe2+摄取和内稳态都是必不可少的这一概念是新颖的、创新的和范式转换的。该项目将提供对未勘探区域的洞察 生物学，填补了我们对线粒体铁摄取途径的了解的一个重要空白 铁依赖的毒性。更好地理解这一过程最终将导致更具体的干预措施 对抗铁超载所促进的肝病。