Iron homeostasis is tightly regulated to ensure that cells have sufficient iron for their needs but at the same time limit toxicity due to iron overload. Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are RNA-binding proteins that bind to iron-responsive elements (IREs), which are located in the untranslated regions of mRNAs, to regulate the posttranscriptional expression of proteins required for iron homeostasis. IRP RNA-binding activity is reduced during iron-replete conditions due to the assembly of a [4Fe-4S] cluster in the RNA-binding pocket of IRP1 and the proteasomal degradation of IRP2. Irp1 -/- and Irp2-/- mouse models have revealed that IRP2 functions as the major RNA-binding protein in vivo. In this thesis, we explore the posttranslational mechanisms that regulate IRP2 iron degradation and RNA-binding activity. We show that the putative E3-ubiquitin ligase HOIL-1, which has been suggested to mediate IRP2 degradation, is not required for the iron-dependent degradation of IRP2. Our data also confirm previous studies showing that a unique 73-amno acid region of IRP2 is not required for IRP2 iron degradation. Instead, we find that the overall tertiary structure and lack of [4Fe-4S] cluster binding, and not a specific region of IRP2, are important requirements for targeting IRP2 for proteasomal degradation. We also focus on the role of cysteine residues in regulating IRP2 RNA-binding activity. We show that IRP2-C512 and IRP2-C516 are located in the RNA-binding pocket of IRP2, and propose that a reversible disulfide bond is formed between these cysteines during oxidative stress in vivo. We show that the formation of this putative disulfide bond inhibits IRP2 RNA binding in the absence of protein degradation, and results in altered regulation of an IRE-containing mRNA. In addition, our studies suggest that the presence of two cysteines residues in the RNA-binding pocket of IRP2 may result in a constricted RNA-binding pocket and contribute to the selectivity of IRP2-IRE interactions. Collectively, we have identified a novel mechanism for regulating IRP2 RNA-binding activity, and iron homeostasis in response to oxidative stress.(A) Schematic diagram comparing cysteine residues in human IRP1 and IRP2. Conserved cysteines are connected by vertical lines. IRP1 cysteines required for [4Fe- 4S] cluster binding are italicized and bolded (C437, C503, and C506).
|Title||:||Posttranslational Regulation of Iron Regulatory Protein 2|
|Publisher||:||ProQuest - 2009|