# Mechanisms of Mitochondrial Iron Uptake: New Therapeutic Targets in Hepatotoxicity

> **NIH NIH R01** · MEDICAL UNIVERSITY OF SOUTH CAROLINA · 2022 · $455,807

## Abstract

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 o...

## Key facts

- **NIH application ID:** 10349589
- **Project number:** 5R01DK119523-02
- **Recipient organization:** MEDICAL UNIVERSITY OF SOUTH CAROLINA
- **Principal Investigator:** John J Lemasters
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $455,807
- **Award type:** 5
- **Project period:** 2021-04-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10349589

## Citation

> US National Institutes of Health, RePORTER application 10349589, Mechanisms of Mitochondrial Iron Uptake: New Therapeutic Targets in Hepatotoxicity (5R01DK119523-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10349589. Licensed CC0.

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