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

基本信息

批准号：
10349589
负责人：
John J Lemasters
金额：
$ 45.58万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
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/10349589
关键词：
Acetaminophen Address Affect Affinity Area Binding Binding Sites Bioenergetics Biological Assay Biology Cations Cell Cycle Kinetics Cell membrane Cells Chelating Agents Citrates Complex Confocal Microscopy Core Protein DNA Damage Data Divalent Cations Endosomes Ferritin Generations Genetic Models Heme Iron Hepatocyte Hepatotoxicity Homeostasis Hydroxyl Radical Image In Vitro Inner Limiting Membrane Inner mitochondrial membrane Intervention Iron Iron Chelating Agents Iron Overload Knock-out Knowledge Lead Liver diseases Lysosomes Measurement Mediating Membrane Membrane Potentials Membrane Proteins Microscopy Mitochondria Mitochondrial Proteins Modeling Molecular Mutation Oxidation-Reduction Oxidative Stress Pathway interactions Physiological Play Predisposition Process Protein Isoforms Proteins Reaction Reactive Oxygen Species Resolution Role SLC11A2 gene Sulfur Time Toxic effect Transition Elements calcium uniporter clinically relevant experimental study hepatocellular injury imaging genetics in vivo innovation insight iron metabolism mitochondrial dysfunction mitochondrial membrane new therapeutic target novel novel therapeutics prevent tool uptake

项目摘要

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。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，而非螯合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，产生高活性羟基自由基，损害 DNA、蛋白质和细胞膜。很多有证据表明线粒体铁是毒性的重要贡献者，但其分子途径线粒体铁的吸收尚不完全清楚。目前的教条是线粒体铁蛋白（Mfrn1 和 2），线粒体内膜蛋白，负责线粒体铁转运。然而，来自 45 项研究多年前表明，经典的产电线粒体钙单向转运体 (MCU) 复合物也催化由线粒体膜电位驱动的 Fe2+ 而非 Fe3+ 的吸收，这一结论得到了我们自己的支持对完整细胞和透化细胞的研究。我们的初步下拉、Duolink 和超分辨率显微镜研究表明，Mfrn2（非红系细胞中的主要亚型）与 MCU（核心）存在物理关联 MCU 复合体的蛋白质。这给我们带来了该提案要解决的基本问题：1) 线粒体是否通过两条独立的途径积累铁：由 Mfrn 介导的非产电途径以及由 MCU 催化的生电途径？ 2）替代Mfrn和MCU的分子组件存在于介导 Fe2+ 和 Ca2+ 吸收的单一复合物中吗？ 3) Mfrn 是交换者吗，例如 Fe2+/Na+(H+) 交换剂类似于 Ca2+/Na+、Mg2+/Na+ 和 Na+/H+ 交换剂？ 4）MCU和Mfrn如何做，以及二价金属转运蛋白 1 (DMT1) 会导致肝毒性吗？在目标 1 中，我们将描述质膜透化野生型 (WT)、MCU 敲除 (KO) 中线粒体 Fe2+ 的摄取和交换和 Mfrn1/2 双 KO (DKO) 肝细胞。目标 2 将使用以下方法识别 Mfrn2 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 Mfrn2 和 MCU 是否是真实的结合伙伴，并确定 Mfrn2 的关联与其他新颖的合作伙伴。目标 3 将评估靶向突变如何影响对乙酰氨基酚的敏感性 (APAP) 毒性，由铁介导。具体来说，我们将确定 MCU、Mfrn2 和 DMT1 在体外和体内影响 APAP 诱导的线粒体功能障碍和肝细胞杀伤。我们期望这些研究定义了 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 对于线粒体 Fe2+ 摄取和稳态稳态都是必需的，这一概念是新颖的、创新的和范式转变的。该项目将提供对未探索领域的见解生物学并填补了我们对线粒体铁吸收和代谢途径的理解的一个重要空白铁依赖性毒性。更好地理解该过程最终将导致更具体的干预措施对抗铁超负荷引起的肝脏疾病。