Iron and thiol redox homeostasis are intimately connected in cellular metabolism. Iron is an essential cofactor for proteins and enzymes in numerous biochemical pathways, but when left unchecked, excess iron catalyzes formation of reactive oxygen species (ROS) that disrupt thiol redox homeostasis. Intracellular thiol-disulfide balance is critical, in turn, for the activity of proteins with functionally important cysteine residues, which includes many Fe-binding enzymes. Thus, iron homeostasis and maintenance of thiol-disulfide balance are mutually dependent processes that are critical for cell survival. Th tripeptide glutathione (GSH) and glutaredoxin (Grx) proteins function together in both thiol redox control and iron homeostasis by catalyzing thiol-disulfide exchange reactions and participating in Fe-S cluster biogenesis pathways. Maintenance of GSH and iron homeostasis in the mitochondrion is especially important since this organelle is the primary site for Fe- S cluster and heme biogenesis, as well as the main source and target of ROS production. However, there are significant gaps in understanding both iron regulation mechanisms and mitochondrial thiol redox control pathways at the cellular and molecular level that require further study. The long term goals of this research program are: (1) to identify the mechanisms for maintaining adequate intracellular levels of the essential metal iron, and (2) to characterize intracellular factors that control mitochondrial thiol redox balance and GSH flux between subcellular compartments. Providing mechanistic insight into these critical cellular functions is essential for preventing and treating diseases of iron overload, oxidative stress, and mitochondrial redox imbalance. For the iron regulation project, the innovative approach to accomplish these goals is to use a combination of protein biochemistry, mutagenesis, yeast genetics and cell biology, and biophysical methods (UV-visible absorption, CD, resonance Raman, EXAFS, Mssbauer, EPR, NMR spectroscopy, SAXS, and X-ray crystallography). The in vitro biochemical and biophysical studies will probe protein-protein, metal-protein, and protein-DNA interactions in iron sensing pathways to uncover the molecular details of iron signaling, while the genetics and cell biology studies test how these molecular interactions influence the in vivo functions and dynamic localization of iron signaling factors. For the mitochondrial redox project, a molecular genetics approach will be used to manipulate gene expression and protein localization, coupled with in vivo thiol redox measurements using targeted GFP-based redox sensors, to identify factors that influence thiol-disulfide balance and control GSH flux between subcellular compartments. Both projects exploit yeast model systems since these simple eukaryotes are easy to maintain and genetically manipulate in the lab, yet expresses many of the same redox and metal homeostasis systems as human cells. Overall, this multidisciplinary research program is designed to tease out the mechanistic details of both iron regulation and subcellular thiol redox control at the cellular and molecular level.

Public Health Relevance

Disruptions in redox regulation and iron metabolism have been implicated in numerous human diseases including cancer, neurodegenerative diseases, mitochondrial dysfunction disorders, and iron overload disorders. Understanding the fundamental mechanisms for maintaining redox and iron homeostasis at the cellular and molecular level is essential for designing prevention and treatment strategies for these disorders. The goals of this proposal are: (1) to identify the mechanisms for maintaining adequate intracellular levels of the essential metal iron, and (2) to characterize intracellular factors that control subcellular redox balance using yeast as a model system.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Unknown (R35)
Project #
5R35GM118164-02
Application #
9271203
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Anderson, Vernon
Project Start
2016-05-10
Project End
2021-04-30
Budget Start
2017-05-01
Budget End
2018-04-30
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of South Carolina at Columbia
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041387846
City
Columbia
State
SC
Country
United States
Zip Code
29208
Albetel, Angela-Nadia; Outten, Caryn E (2018) Characterization of Glutaredoxin Fe-S Cluster-Binding Interactions Using Circular Dichroism Spectroscopy. Methods Enzymol 599:327-353
Outten, Caryn E (2017) Checks and balances for the iron bank. J Biol Chem 292:15990-15991
Ponsero, Alise J; Igbaria, Aeid; Darch, Maxwell A et al. (2017) Endoplasmic Reticulum Transport of Glutathione by Sec61 Is Regulated by Ero1 and Bip. Mol Cell 67:962-973.e5
Dlouhy, Adrienne C; Beaudoin, Jude; Labbé, Simon et al. (2017) Schizosaccharomyces pombe Grx4 regulates the transcriptional repressor Php4 via [2Fe-2S] cluster binding. Metallomics 9:1096-1105
Dlouhy, Adrienne C; Li, Haoran; Albetel, Angela-Nadia et al. (2016) The Escherichia coli BolA Protein IbaG Forms a Histidine-Ligated [2Fe-2S]-Bridged Complex with Grx4. Biochemistry 55:6869-6879
Scian, Michele; Guttman, Miklos; Bouldin, Samantha D et al. (2016) The Myeloablative Drug Busulfan Converts Cysteine to Dehydroalanine and Lanthionine in Redoxins. Biochemistry 55:4720-30