Iron is an abundant metal in the environment providing challenges to human health by contributing to envi- ronmental toxicity. 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. Chelatable iron promotes oxidative stress by catalyzing the Fen- ton reaction, which produces highly reactive hydroxyl radicals that damage DNA, proteins and membranes. Sub- stantial evidence implicates mitochondrial iron as an important contributor to toxicity, but the molecular pathways of mitochondrial iron uptake are controversial. The prevailing view is that mitoferrins 1 and 2 (Mfrn1/2), proteins localized in the mitochondrial inner membrane, are responsible for mitochondrial iron transport. However, previ- ous studies from ~40 years ago show that the classical electrogenic mitochondrial calcium uniporter also cata- lyzes uptake of Fe2+ but not Fe3+ driven by the mitochondrial membrane potential, a conclusion supported by our own recent studies in intact hepatocytes. Our preliminary pull-down and Duolink studies indicate a physical as- sociation between Mfrn2, the predominant isoform in non-erythropoietic cells, and MCU, the core protein of the uniporter complex, which brings us to the fundamental questions to be addressed by this proposal: 1) Does electrogenic mitochondrial Fe2+ and Ca2+ uptake occur via two independent pathways: one mediated by Mfrn1/2 and another by MCU; or does electrogenic uptake of both cations occur exclusively by one or the other carrier? 2) Alternatively, do Mfrn and the calcium uniporter act cooperatively in mediating up- take of both iron and calcium? Therefore, the studies proposed here are intended to address these questions utilizing Mfrn1/2 single knockout (KO) and double KO (DKO) hepatocytes and their wild type (WT) counterparts.
In Aim 1, mitochondrial Fe2+ uptake will be measured in permeabilized mouse hepatocytes by confocal micros- copy using a newly developed mitochondria-specific Fe2+-indicating fluorophor, mitoferrofluor.
In Aim 2, mito- chondrial Ca2+ uptake will be measured using Ca2+-indicating Fluo5N. If there are two independent pathways (#1 above), then Mfrn2 deficiency will prevent Fe2+ but not Ca2+ uptake, and MCU deficiency will prevent Ca2+ but not Fe2+ uptake, whereas if Mfrn2 and MCU reside in a single complex (#2), then the kinetics of both Fe2+ and Ca2 uptake should be altered by Mfrn2 deficiency, and both Fe2+ and Ca2 uptake will be blocked by MCU deficiency. The concept that MCU and Mfrn are essential for both Fe2+ and Ca2 electrogenic uptake is novel, innovative and paradigm-changing. 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 this process will eventually lead to new more specific interventions against toxicities promoted by iron overload.

Public Health Relevance

Mitochondrial iron uptake and consequent stimulation of reactive oxygen species formation contributes to number of diseases and pathological states. The proposed study will elucidate the molecular pathways of mito- chondrial iron uptake, specifically the role of mitoferrins and the mitochondrial calcium uniporter complex. Infor- mation gained from this study will impact our understanding of a number of pathological conditions where in- creased mitochondrial iron uptake is involved.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21ES031335-02
Application #
10077480
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Shaughnessy, Daniel
Project Start
2020-01-01
Project End
2021-12-31
Budget Start
2021-01-01
Budget End
2021-12-31
Support Year
2
Fiscal Year
2021
Total Cost
Indirect Cost
Name
Medical University of South Carolina
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29407