Eukaryotic iron metabolism involves two processes: redox cycling and trafficking. The transport of 'free' iron across eukaryotic plasma and some intracellular membranes is a paradigm of this metabolism. Thus, uptake of environmental Fe3+ involves first its reduction by a plasma membrane ferrireductase. The Fe2+ produced can be substrate for a multicopper oxidase - a ferroxidase - that couples the reduction of O2 to the production of 4Fe3+. This ferric iron is then ligand for an iron permease that transports the iron across the plasma membrane. High affinity iron uptake in the yeast, Saccharomyces cerevisiae, exhibits all of these features. The metalloreductase, Fre1p, produces the Fe2+ that is substrate for ferroxidation by Fet3p, a ceruloplasmin ortholog, with permeation facilitated by Ftr1p. In yeast, as in the intestinal epithelium, the ferroxidation and permeation steps are coupled in the strict metabolic sense: permeation requires ferroxidation. This coupling suggests a primary hypothesis of this research: in the Fet3p, Ftr1p system the ferric iron product of the Fet3p ferroxidase reaction is channeled to Ftr1p for subsequent transmembrane trafficking. A template for this model is the movement of iron into and out of the ferritin (Ft) core. This hypothesis requires that both Fet3p and Ftr1p possess amino acid residues that participate in this channeling process, in addition to those structural motifs required for ferroxidation and permeation per se. There also may be motifs associated with the coupling of these two processes. The objective of this research is a full and detailed structure-function analysis of the Fet3p, Ftr1 system using biochemical, biophysical, genetic and cell biology approaches. These include: kinetic, spectral and crystallographic studies of wild type and mutant Fet3 proteins; iron uptake kinetic analysis of Ftr1p iron trafficking mutants; biochemical, genetic and fluorescence analysis of the physical and functional interaction between Fet3p and Ftr1p; and kinetic and electrophysiologic analysis of the coupling of ferroxidation and uptake. This structure-function characterization of the Fet3p, Ftr1p system will provide significant new understanding of eukaryotic iron trafficking.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK053820-08
Application #
7162119
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Sechi, Salvatore
Project Start
1999-05-01
Project End
2008-03-31
Budget Start
2006-12-01
Budget End
2008-03-31
Support Year
8
Fiscal Year
2007
Total Cost
$241,769
Indirect Cost
Name
State University of New York at Buffalo
Department
Biochemistry
Type
Schools of Medicine
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
Ji, Changyi; Steimle, Brittany L; Bailey, Danielle K et al. (2018) The Ferroxidase Hephaestin But Not Amyloid Precursor Protein is Required for Ferroportin-Supported Iron Efflux in Primary Hippocampal Neurons. Cell Mol Neurobiol 38:941-954
Kosman, Daniel J (2018) The teleos of metallo-reduction and metallo-oxidation in eukaryotic iron and copper trafficking. Metallomics 10:370-377
Ji, Changyi; Kosman, Daniel J (2015) Molecular mechanisms of non-transferrin-bound and transferring-bound iron uptake in primary hippocampal neurons. J Neurochem 133:668-83
McCarthy, Ryan C; Kosman, Daniel J (2015) Mechanisms and regulation of iron trafficking across the capillary endothelial cells of the blood-brain barrier. Front Mol Neurosci 8:31
Bailão, Elisa Flávia L C; Lima, Patrícia de Sousa; Silva-Bailão, Mirelle G et al. (2015) Paracoccidioides spp. ferrous and ferric iron assimilation pathways. Front Microbiol 6:821
McCarthy, Ryan C; Kosman, Daniel J (2015) Iron transport across the blood-brain barrier: development, neurovascular regulation and cerebral amyloid angiopathy. Cell Mol Life Sci 72:709-27
McCarthy, Ryan C; Park, Yun-Hee; Kosman, Daniel J (2014) sAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin. EMBO Rep 15:809-15
McCarthy, Ryan C; Kosman, Daniel J (2014) Activation of C6 glioblastoma cell ceruloplasmin expression by neighboring human brain endothelia-derived interleukins in an in vitro blood-brain barrier model system. Cell Commun Signal 12:65
McCarthy, Ryan C; Kosman, Daniel J (2014) Glial cell ceruloplasmin and hepcidin differentially regulate iron efflux from brain microvascular endothelial cells. PLoS One 9:e89003
Kjaergaard, Christian H; Qayyum, Munzarin F; Augustine, Anthony J et al. (2013) Modified reactivity toward O2 in first shell variants of Fet3p: geometric and electronic structure requirements for a functioning trinuclear copper cluster. Biochemistry 52:3702-11

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