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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