The maintenance of cellular iron homeostasis is essential to cell viability. Iron is required for numerous intracellular metabolic activities, but excess iron is a potent toxin because it cam become involved in inducing free radicals leading to oxidative injury. Iron uptake is regulated in most cells by transferrin receptors and intracellular iron is store and detoxified by ferritin. The synthesis of transferring receptors and ferritin is concordantly regulated by cytoplasmic iron regulatory proteins (IRPs) which bind to specific sequences known as iron regulatory elements (IREs) on the mRNAs of ferritin and transferring receptors. Consequently, IRPs are key components of cellular iron regulation. Cellular iron dysregulation has become a major health issue in a broad range of common diseases including heart disease and stroke. Iron toxicity is the lading cause of poison-related deaths in children. Hemochromatosis, an iron over-load disorder, is associated with numerous diseases including late onset diabetes and cancer. It is thus imperative to a healthy society that cellular iron regulation be understood and interventions designed where possible. Although the interaction between IRPs and IREs has been under investigation for a number of years, we propose the novel hypothesis that the function of IRPs is related to their intracellular distribution and that translocation of the IRPs is a means of post-transcriptional regulation of these proteins. IRPs are thought to exist in 3 pools in the cell: 1) bound to IRE, 2) unbound but available, 3) unbound and unavailable. We will first demonstrate the intracellular distribution of the IRPs using both microscopic and biochemical analyses. The microscopic analyses will include fusion of IRPs with green fluorescent protein enabling examination of our hypothesis in live cells. We will exploit the known response of IRPs to iron exposure and the known response of cellular ferritin to pro-inflammatory agents to test our global hypothesis in Aims 2 and 3 of this application.
In Aim 4, we will determine the biological consequences of cytokine exposure on cellular iron homeostasis in relation to IRP/IRE interaction. The significance of the proposed studies lives in providing novel insight into the pathway of the critical cellular iron regulatory system. In addition, the data generated herein will be of significant interest to general studies of RNA/protein interaction and intracellular translocation of RNA. As gene therapies move toward consideration of regulation t the RNA level, these data could have profound effects as the IRP/IRE system is considered a model for post- transcriptional regulation of RNA.
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