Mechanisms of Fungal Iron Regulation and Thiol Redox Metabolism

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PROJECT SUMMARY Iron and thiol redox homeostasis have interdependent roles in cellular metabolism. Iron serves as a cofactor for a wide variety of proteins and enzymes in essential biochemical pathways, but excess iron can be damaging to cells by catalyzing formation of reactive oxygen species 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 proteins. The tripeptide glutathione (GSH) and glutaredoxin (Grx) proteins function together in both thiol redox control and iron homeostasis by facilitating redox reactions and participating in iron- sulfur (Fe-S) cluster biogenesis pathways. Our previous work in the non-pathogenic yeast S. cerevisiae and S. pombe have revealed the molecular mechanisms by which a subclass of Grxs, known as monothiol Grxs, bind and deliver GSH-ligated Fe-S clusters to communicate iron bioavailability to the transcription factors Aft1/Aft2 in S. cerevisiae and Php4 in S. pombe that regulate iron acquisition and utilization pathways. Furthermore, we have used molecular genetics and cell biology approaches coupled with in vivo redox measurements via genetically- encoded fluorescent redox sensors to characterize GSH subcellular trafficking pathways that impact both iron homeostasis and redox regulation in S. cerevisiae. Here we will extend these findings by studying the impact of GSH and Grxs on the Fe-S binding properties and DNA binding affinity of the S. pombe transcription factor Fep1 that represses Fe uptake pathways during iron repletion. Furthermore, we will define the molecular details of iron regulation pathways in pathogenic yeast (Candida glabrata, Candida albicans) that express homologs of monothiol Grxs, Aft1/2, Fep1, and Php4, but for which little mechanistic information is available. In parallel, we will characterize GSH:GSSG flux between subcellular compartments in yeast cells and measure the impact of GSH deficiency, excess, or impaired trafficking on essential metal metabolism. Our innovative approach to accomplish these goals is to combine yeast molecular genetics and cell biology techniques with biochemical, structural, and biophysical methods (UV-visible absorption and CD spectroscopy, EXAFS, X-ray crystallography, Mössbauer, EPR, and single cell ICP-TOF-MS). The in vitro biochemical, structural, and biophysical studies will be used to probe protein-protein, metal-protein, and protein-DNA interactions in iron sensing pathways to uncover the molecular details of iron signaling and to monitor single cell metallomic changes in yeast populations in response to alterations in iron or GSH metabolism. The genetics and cell biology studies test how these molecular interactions and metallome changes influence the in vivo functions and dynamic localization of iron signaling and GSH metabolism factors. Overall, this multidisciplinary research program is designed to tease out the mechanistic details of iron regulation and subcellular thiol redox control at the cellular and molecular level. By studying both pathogenic and non-pathogenic fungi we will compare and contrast different strategies for adapting to redox perturbations and high/low iron environments.