Red cell maturation depends on increased expression of proteins for iron uptake, transport, concentration, and heme synthesis to produce hemoglobin. Ferritin plays a key role in red cells and iron homeostasis: concentrating iron as a mineral and protecting the mineral from cell reductants. Coregulation of ferritin mRNAs, FTH and FTL, with iron trafficking proteins depends on a group of mRNA structures, the iron regulatory element (IRE) that binds regulatory proteins IRP1 and 2. The crystal structure of a ferritin-IRE-RNA/IRP1 complex facilitates designs for kinetic/equilibrium studies of IRE binding to IRP peptide and translation factors. Recent observations that FTH and FTL genes are coregulated during transcription with MARE/ARE antioxidant response genes led to identification of Bach1 as the FTH and FTL repressor. Ferritin minimizes oxidative stress and stores iron for proteins. Ferritin protein structure and function are very complex. The protein nanocages self-assemble with a cavity (8 nm) for Fe/O minerals. Fe2+ ions enter and leave the ferritin protein cage through pores. Ferritin catalysis relates to other di-iron cofactor protein catalysts. Intermediate steps and mechanisms between Fe2+ entry, Fe2+/O2 catalysis, mineral formation/dissolution, and Fe2+ exit are only partly characterized. Rates of Fe2+ exit from the ferritin protein cage, as chelated/bound Fe2+ after reduction of the mineral, depend on localized protein folding/unfolding of the cage pores. Two regulating peptides were identified in vitro that may model regulatory proteins. The chelator-peptide conjugate that increased iron chelation in solution has potential uses in removing excess iron in human disease. The Aims for this grant period, are to: 1. understand Fe pathways for Fe2+ entry, O2 reactions, transit to the cavity, pore gating (folding/unfolding) and Fe2+ release to carriers/chelators protein;2. understand the differential expression of IRE-containing mRNAs;and 3. determine the effect of MafK and 5-methylcytosine (5mC) on ferritin DNA expression/Bach1 binding. Design: 1A. Identify Fe-protein intermediates by mutating covarying residues;1B. Characterize binding peptides in solution and putative regulators in vivo. 2A,B. Analyze different IRE-RNAs translation and kinetics of binding to initiation factors and/or IRP1 and designed peptides from IRP1, 2. 3A. Analyze Bach1 1 MafK binding to MARE/ARE-DNAs 1 5mC;3B. Analyze iron effects on FTL/FTH DNA 5mC. Techniques: Biochemistry (binding kinetics/equilibria, UV-vis fluorescence MCD/CD, NMR spectroscopies, and X-ray crystallography), Molecular Biology (in vitro translation, mutagenesis, protein expression, EMSA, DNA-5mC analysis), and Cell Biology (RT-PCR, immunoblotting). The results on iron homeostasis, ferritin function, protein catalysis, protein pores, mRNA function, and protein synthesis can also translate to iron in diseases, e.g., HH, SCD, Thalassemia and malaria. PUBLIC HEALTH RELEVANCE: Basic studies of iron homeostasis, gene and mRNA regulation, and host/pathogen diiron catalysis in ferritin can be translated to drug targeting, and for the linked peptide-chelator, to iron chelation in Sickle Cell Disease, Thalassemia, Hereditary Hemochromatosis, and to the emerging awareness of iron in malaria and diabetes.

R01DK020251

1977-08-01

2012-08-31