The applicants note that distinctive features of ferritin mRNA (IRE, iron regulatory element), protein structure and gene regulation combine to create the cell-specific biological system required for concentrating iron one hundred billion times above the free ion for use in proteins of DNA synthesis, respiration, and detoxification. Conditions altering ferritin function or iron metabolism include thalassemia, sickle cell anemia, hemochromatosis, inflammation, and hemodialysis. Studies by the applicants over the past eight years, using amphibian red cell ferritin cDNAs, engineered for mRNA and recombinant H and L subunit protein, have led to a variety of new information. Ferritin mRNA: IRE binding of a trans factor blocks ribosome binding; IREs have a specific context (flanking regions, cap distance), conserved sequence and encode both positive and negative control. Higher order structure of the IRE region, probed with novel transition metal complexes and NMR revealed a folded, heat-stable hairpin loop (HL) structure, with subdomains, long-range interactions (dependent on a G in the HLI as well as a protein (IRP) binding site (""""""""footprint""""""""). Ferritin Protein: Fe(III) linked to a ferritin tyrosine is an early Fe intermediate in H-ferritin and revealed conformational changes during Fe uptake. High resolution X- ray crystallography of L and L(Glu/Ala) ferritins also showed conformational flexibility; EXAFS showed specific protein effects on mineralization. XAS studies with natural ferritins, before availability of recombinant protein, showed an Fe(III)-oxo cluster on carboxylate ligands, heterogeneity of Fe reduction rates, alternate mineral structures, and the ability to store Fe(II) under limiting conditions; common Fe uptake features in H- or L-subunit rich ferritins were also observed. Ferritin Genes: conservation of sequence and regulatory signals was observed in plants and animals but IRE regulation of mRNA was animal-specific. The applicants now propose to continue RNA studies combining IRE mutagenesis (ferritin, transferrin receptor) (site-specific and random) with analysis of RNA/protein interactions (IRP, eIFs) by CD, EPR, binding, mRNA translation, mRNA stability (assay development) and in collaboration, NMR spectroscopy. Protein studies proposed by the applicants combine site-mutagenesis of H and L red cell ferritin subunits with UV-vis spectroscopy and EXAFS to understand how the protein influences rapid and slow iron mineralization, the structure of the iron core, iron release, and the role of coordinated water in Fe transport through ferritin (Cr and Co complexes used as Fe analogs). Muteins accumulating Fe intermediates will be analyzed, in collaboration, by resonance Raman and Mossbauer spectroscopy, and by X- ray crystallography. Gene Regulation: To understand developmental expression of ferritin, the applicants plan to examine trans factors for the genes in embryonic and adult red cells.
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