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: 10210670
• 负责人: John J Lemasters
• 金额: $ 45.32万
• 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/10210670
• 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

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，存在于细胞内两个池中。可螯合的铁包括自由铁和铁 与ATP和柠檬酸盐等阴离子代谢产物松散结合，而不可螯合的铁紧密结合到铁蛋白， 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 反应产生高反应性羟基自由基，损害DNA，蛋白质和膜。很多 证据暗示线粒体铁是毒性的重要因素，但分子途径的分子途径 线粒体铁的摄取尚不完全了解。当前的教条是丝曲霉（MFRN1和2），a 线粒体内膜蛋白负责线粒体铁转运。但是，来自45的研究 几年前表明，经典的电源线粒体钙Uniporter（MCU）复合物也催化 通过线粒体膜电位驱动的Fe2+的吸收，而不是Fe3+的吸收，这是我们自己支持的结论 完整和透化细胞的研究。我们的初步下拉，Duolink和超分辨率显微镜 研究表明，MFRN2的物理相关性是非果蝇细胞中主要的同工型，MCU（MCU）（核心） MCU复合物的蛋白质。这使我们得出了该提议要解决的基本问题：1） 线粒体是否通过两种独立途径积累铁：MFRN介导的非电动途径 MCU催化的电源途径？ 2）或者MCU的MFRN和分子成分 存在于介导Fe2+和Ca2+摄取的单个复合物中？ 3）是mfrn的交换器，例如 Fe2+/Na+（H+）交换器类似于Ca2+/Na+，Mg2+/Na+和Na+/H+交换器？ 4）MCU和MFRN如何 以及二价金属转运蛋白1（DMT1），有助于肝毒性？在AIM 1中，我们将描述 线粒体Fe2+质膜 - 渗透性野生型（WT），MCU敲除（KO）中的摄取和交换 和MFRN1/2双KO（DKO）肝细胞。 AIM 2将使用一个 无偏的富集质量光谱法（AE-MS）方法，Duolink和超分辨率显微镜至 确定MFRN2和MCU是否是真实的约束伙伴，并且还确定MFRN2的关联 与其他小说合作伙伴。 AIM 3将评估靶向突变如何影响对对乙酰氨基酚的易感性 （APAP）毒性，由铁介导。具体而言，我们将确定MCU，MFRN2和 DMT1会影响APAP诱导的线粒体功能障碍和体外和体内肝细胞杀死。我们期望 这些研究以定义MCU，MFRN2和DMT1在此临床相关模型中的特定作用 肝细胞损伤。 MFRN和MCU对于线粒体Fe2+摄取和Homo-Stasis都是新颖，创新和范式转移的概念。该项目将提供有关未开发领域的见解 生物学并填补了我们对线粒体摄取涉及的途径的理解和 铁依赖性毒性。更好地了解该过程将最终导致更具体的干预措施 针对铁超负荷促进的肝病。