Biolron, the molecular study of iron in biology and medicine, has experienced an explosion of interest and new information in the last few years with the identification of new genes for iron uptake, transport and storage and mutations related to human diseases. Many proteins and strategies are used for iron uptake and transport but only one, the ferritin protein, is used to concentrate iron the approximately 10/14 fold required by cells; ferritin gene deletion in mice is embryonic lethal. Mutations in the ferritin regulatory and coding sequences are associated with human disease. Iron is concentrated as a mineral (""""""""rust""""""""), in the central cavity (12 nm diameter) of the apoferritin protein. Ferritin controls both Fe2+ entry (oxidation and mineralization) and Fe2+ exit (chelation) via protein pores (8/molecule). Ferroxidase sites (FOXS) use a transient (msec), diferric peroxo (DFP) intermediate. We characterized Fe entry, DFP formation and DFP decay to H202 and Fe3+ mineral precursors by UV-vis, Mossbauer, RR, EXAFS and site-directed mutagenesis (SDM). Designed (SDM) showed that exit pore, Fe++ and Fe+++ chelation are controlled by 3 types of conserved structural motifs. Low concentrations of chaotropes open ferritin pores to chelators, mimicking SDM. Proposed studies include: 1: Analyzing Fe/O2 reactions in variant ferritin proteins (SDM, cytoplasmic H+L, mitochondrial-all H) or varied [Fe] by measuring effects on Fe++ oxidation, Fe3+ -O/OH products and H202 release; 2: Analyzing ferritin pore structure/function with pore """"""""blockers"""""""" by XRD and variant ferritins in vitro and in cells. A natural, combinatorial array of RNA/protein (IRE/IRP) interactions links regulation of ferritin synthesis to proteins for Fe uptake, the TCA cycle and heme synthesis. We showed that IRP2/IRE selectivity required specific IRE stem structure that was H+ sensitive and bound Mg++ in ferritin mRNA, and was context-induced in the multi-IRE + AURE structure of TfR mRNA. Cu-phen, a 3D-sensitive structure probe recognized the IRP2-specific structure in HeLa cells, demonstrating the RNA fold in vivo and establishing the possibilities of targeting 3D RNA in vivo. Experiments proposed include: 1: Analyzing IRE context and elF effects for different IRE-mRNAs; 2: Developing selective, IRE-targeted compounds to manipulate IRE-mRNA in vivo and to model viral and oncogene RNA targets; 3: Exploring, with 3D-sensitive probes, SECIS/protein interactions that control Se-protein synthesis. Outcomes: 1 - Increased understanding of Fe2+ entry, exit and removal from cytoplasmic and mitochondrial ferritin leading to novel Fe chelators for iron overload diseases (sickle cell, beta-Thalassemia); 2- Determination of the role of context-dependent regulation of IRE-mRNA function; 3- Identification of compounds to manipulate 3D mRNA features in vivo, understanding higher order SECIS structure related to protein binding and differential Se control of SECIS mRNA for redox and H202 removal, for Fe/O2 control of IRE-mRNA for Fe homeostasis and responses to oxidative stress.
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