Multicopper oxidases (MCOs) couple the 4e- reduction of dioxygen to 2H2O with the oxidation of 4 equivalents of a 1-electron donor. A sub-family of these ubiquitous enzymes possesses specificity towards FeII that makes them essential to iron homeostasis in their respective organisms. Our hypothesis is that these ferroxidases function by channeling their FeIII product to a down-stream partner in their iron metabolic pathways. This program has delineated the structure and function in the Fet3p, Ftr1p high-affinity Fe-uptake complex in the Saccharomyces cerevisiae (Sc) plasma membrane (PM). The two fundamental questions addressed in this work are: 1) what structural motifs confer on an MCO this specificity for FeII as substrate;and 2) how is the Fet3p ferroxidase reaction coupled kinetically and physically to the membrane permeation of FeIII by Ftr1p. In this application we propose three specific aims.
In Aim I we will test our hypothesis of what structural motifs define a ferroxidase, specifically that a cohort of carboxylate side chains maximize three factors that determine e- transfer from FeII: FeII binding, FeII redox potential and electronic matrix coupling of the FeII and the type 1 CuII (T1Cu) in the ferroxidase. We will do this by interconverting laccase and ferroxidase enzymes based on our design principles. In addition, we will test our hypothesis that another cohort of carboxylate side chains stabilizes the increasing negative charge on dioxygen as it is reduced in 2, 2e- steps at the trinuclear cluster (TNC). Last we will test our hypothesis that the coordination changes associated with O2 reduction at the TNC trigger e- transfer from the T1 Cu via the canonical MCO His-Cys-His motif that connects the two.
In Aim II we propose to test our hypothesis that Fox1 in Chlamydomonas reinhardtii (Cr) is a human ceruloplasmin-like ferroxidase. We will characterize the Fox1 protein, demonstrating that it has the ferroxidase-specificity motifs that support both FeII oxidation and FeIII trafficking in a Fox1, Ftr1 complex in the Cr PM. We will construct mutants of both Fox1 and Ftr1 that we predict will be sensitive to a FeIII-chelator acting as a metabolite trap in Fe-trafficking between the two proteins in Fe-uptake;we have used this classic test of channeling in the Sc Fet3, Ftr1p complex. We propose that the Fe-trafficking between Fox1 and Ftr1 in Cr provides a realistic model of the putative FeIII-trafficking between hCp and transferrin.
In Aim III, we will test our hypothesis that the two human fungal pathogens, Candida albicans and Cryptococcus neoformans express an equivalent PM Fet, Ftr high-affinity Fe-uptake complex. We will quantify the 59Fe-uptake kinetics via these complexes both in situ and in recombinant form in Sc. Targeting specific ferroxidase and Fe-trafficking residues in the Ca and Cn proteins, we will test our hypothesis that these mutants exhibit the channeling defect exhibited by the Sc homologues. We propose that strains expressing these channeling mutants will exhibit a reduced virulence in vitro and in vivo. Last, using these chelator-sensitive clones, the NCI diversity collection will be mined for compounds with the potential as inhibitors of Fet, Ftr Fe-uptake in these fungal pathogens.

Public Health Relevance

All oxygen-utilizing organisms from fungi to humans require the activity of a copper oxidase enzyme - a multicopper oxidase - to manage their metabolism of the essential nutrient, iron. Fungi from baker's yeast to the human pathogens C. albicans and C. neoformans use these enzymes to acquire the iron they need to thrive and survive. The goal of this research is to take a snap-shot of how these enzymes work and then to use this knowledge to block their trafficking of iron as way of suppressing the virulence of pathogenic fungi in both otherwise healthy and immunocompromised patients.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK053820-11
Application #
7825303
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Sechi, Salvatore
Project Start
1999-05-01
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2011-03-31
Support Year
11
Fiscal Year
2010
Total Cost
$311,678
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; Kosman, Daniel J (2015) Molecular mechanisms of non-transferrin-bound and transferring-bound iron uptake in primary hippocampal neurons. J Neurochem 133:668-83
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) Mechanisms and regulation of iron trafficking across the capillary endothelial cells of the blood-brain barrier. Front Mol Neurosci 8:31
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) Glial cell ceruloplasmin and hepcidin differentially regulate iron efflux from brain microvascular endothelial cells. PLoS One 9:e89003
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
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
Kosman, Daniel J (2013) Iron metabolism in aerobes: managing ferric iron hydrolysis and ferrous iron autoxidation. Coord Chem Rev 257:210-217
McCarthy, Ryan C; Kosman, Daniel J (2013) Ferroportin and exocytoplasmic ferroxidase activity are required for brain microvascular endothelial cell iron efflux. J Biol Chem 288:17932-40

Showing the most recent 10 out of 41 publications