Ferritin links Fe homeostasis and antioxidant protection though Fe and O2 sensitive mRNA regulation and Fe and O2 catalytic substrates in the protein nanocage. Complex gene regulation and conserved protein structure emphasize the biological importance of ferritin. Outcomes in the grant period under review and results are: 1) Increased understanding of Fe2+ entry, exit and removal from cytoplasmic and mitochondrial ferritin leading to novel Fe chelators for iron overload diseases (SCD, 2-THL): Fe2+ entry: We identified the active site ligands for Fe2+ required for catalysis (DFP formation). Fe2+ exit: We identified small molecules and peptides that control the gated protein pores. When coupled to desferal, the peptides increase Fe chelation 8-fold. 2) Determination of the role of context-dependent regulation of IRE-mRNA function: We identified downstream, 5'UTR sequences selectively controlling H and L ferritin (FTH and FTL) mRNA, and a ferritin regulatory synergy mediated by heme /Bach1)/DNA-MARE/ARE that complements heme/IRP/RNA-IRE. Ferritin DNA is linked to 2-globin, heme oxygenase, quinone reductase, and thioredoxin reductase through Bach1/DNA repression. 3) Identification of compounds to manipulate 3D mRNA features in vivo. A small natural product, yohimbine, bound the IRE-RNA and increased mRNA translation. X-ray crystallography of the ferritin-IRE/IRP1 complex revealed induced fitting of both RNA and protein, the protein-RNA contact surface, a possible role for of apo-IRP in regulation, and possible sites of eIF-interactions. We now propose experiments to: 1) Connect the steps in the Fe2+ cycle through ferritin by analysis of the multiple Fe/O2 reactions controlled in the protein; 2) Characterize pore-altering-peptide interactions with ferritin; 3) Analyze novel iron chelators in mouse models of iron overload; and 4) Identify ferritin interactions in cells. We also propose to learn mechanisms of ferritin mRNA and DNA (gene) regulation by: 1) Measuring kinetics of """"""""weak"""""""" or """"""""strong"""""""" IRE-IRP1-eIF(s) binding/release 1 PolyA Binding Protein (PABP); 2) Determining effects of PABP and eIFs on translation rates of human IRE-mRNAs (FTH, FTL, mt-acon); 3) Searching for IRE-RNA binding peptides; and 4) Examining DNA methylation. ? ?
The results will provide mechanistic understanding of IRE-mRNA translation important in iron homeostasis and in disease, such as eIF4F mediated apoptosis and selective mRNA translation, and determine the role of DNA methylation in tissue-specific ferritin expression. In addition, studies of ferritin protein structure/function will determine Fe pathways (entry, catalysis, and exit) within the ferritin protein nanocages and identify novel, ferritin-targeted chelators in mouse models for iron overload in 2-Thalassemia and Sickle cell disease. ?
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