Browsing by Subject "F-Box Proteins"
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Item FBXL5 Is Required for the Maintenance of Cellular Andsystemic Iron Homeostasis(2013-01-16) Ruiz, Julio Cesar Francisco; Bruick, Richard Keith, Ph.D.Iron is an essential element for most living organisms. Due to its chemical properties, iron plays an important role in many vital biochemical processes. Both iron excess and deficiency have detrimental effects in human health. Therefore, iron metabolism must be tight regulated. Maintenance of cellular iron homeostasis requires coordinate posttranscriptional regulation of iron metabolism genes by Iron Regulatory Proteins 1 and 2 (IRP1 and IRP2). IRP2 is targeted for proteasomal degradation in iron replete cells by the E3 ubiquitin ligase complex containing F-box and Leucine-rich Repeat Protein 5 (FBXL5). Depletion of FBXL5 leads to aberrant accumulation of IRP2 and misregulation of IRP2 under high iron conditions, underscoring FBXL5 importance in regulation of iron metabolism. Interestingly, FBXL5 is regulated in an inverse fashion to IRP2 as it is stabilized under iron-replete conditions and preferentially degraded when iron or oxygen becomes limiting. However, FBXL5Õs iron- and oxygen-dependent regulation and its role in the maintenance of systemic iron homeostasis are poorly understood. Biochemical and molecular biology assays revealed that FBXL5 features a hemerythrin-like domain that serves as a direct sensor of cellular iron as well as oxygen availability and subsequently governs FBXL5Õs own stability. Importantly, in vivo deletion of the ubiquitously-expressed murine Fbxl5 gene results in a failure to sense increased cellular iron availability, accompanied by constitutive IRP2 accumulation and misexpression of IRP2 target genes. FBXL5-null mice die during embryogenesis, though viability is restored by simultaneous deletion of the IRP2, but not IRP1, gene. Fbxl5 heterozygous mice behave like their wild type littermates when fed an iron-sufficient diet. However, unlike wild type mice that manifest decreased hematocrit and hemoglobin levels when fed a low-iron diet, Fbxl5 heterozygotes maintain normal hematologic values due to increased iron absorption. IRP2Õs responsiveness to low iron is specifically enhanced in the duodena of the heterozygotes and is accompanied by increased expression of the Divalent Metal Transporter-1. These results confirm FBXL5Õs role in the in vivo maintenance of cellular and systemic iron homeostasis and reveal a privileged role for the intestine in their regulation by virtue of its unique FBXL5 iron sensitivity.Item FBXL5: Sensor and Regulator of Mammalian Iron Homeostasis(2011-08-26T17:34:52Z) Salahudeen, Ameen Abdulla; Bruick, Richard K.While iron is an important cofactor for many proteins, the chemical properties of iron that favor its biological roles can lead to toxic side reactions that damage macromolecules. Cellular iron homeostasis is maintained by the coordinate posttranscriptional regulation of gene products responsible for iron uptake, release, utilization, and storage. This process is mediated by Iron Regulatory Proteins (IRPs) that bind to Iron Responsive Elements (IREs) in the mRNAs of these genes. When iron bioavailability is low IRPs bind IREs within these mRNAs, affecting their subsequent translation or stability. When cellular free iron availability is high, IRPs are preferentially degraded by the proteasome. An SCF E3 ubiquitin ligase complex containing the FBXL5 protein regulates this process as a function of cellular iron and oxygen concentrations. This process occurs through the stability of FBXL5, which accumulates under iron and oxygen replete conditions and is targeted for degradation upon iron depletion. FBXL5 contains an iron- and oxygen -sensing hemerythrin domain that acts as a ligand-binding regulatory switch mediating its stability. As a result, FBXL5 directly senses iron and oxygen levels to serve as a regulator of cellular iron homeostasis.Item An Iron Sensing E3 Ubiquitin Ligase Regulates Iron Homeostasis(2011-08-26T17:35:16Z) Thompson, Joel William; Bruick, Richard K.Human iron homeostasis must be tightly regulated to provide sufficient iron for vital cellular processes while preventing the toxic accumulation of free iron. IRP2 plays a critical role in cellular iron homeostasis by coordinating the posttranscriptional regulation of a variety of genes involved in iron metabolism. Posttranslational regulation of IRP2 is essential for its ability to maintain cellular iron homeostasis. The protein is stabilized under iron deficient conditions but polyubiquitinated and degraded by the proteasome when iron is plentiful. However, the E3 ubiquitin ligase that targets IRP2 for degradation is unknown. Moreover, the mechanisms cells use to sense iron levels and correlate changes of this metabolite to differences in IRP2 stability remain poorly understood. To identify the E3 ubiquitin ligase responsible for IRP2 degradation, a high throughput RNAi screen was conducted. The top hit from the screen, FBXL5, interacts with and polyubiquitinates IRP2. Interestingly, FBXL5 is inversely regulated to IRP2. The protein is stabilized under conditions of excess iron and destabilized when iron is limiting. Deletion experiments identified the N terminus of FBXL5 as the region of the protein required for its iron dependent regulation. Bioinformatics predicted the N terminus encodes an iron binding hemerythrin domain. Consistent with this prediction X-ray crystallography demonstrated that the FBXL5 N-terminal domain adopts a hemerythrin fold with a diiron center. Mutation of iron ligating residues in the hemerythrin domain to abolish iron binding leads to constitutive destabilization of FBXL5. Collectively, these findings indicate that the hemerythrin domain acts as a ligand dependent regulatory switch controlling FBXL5Õs expression. Moreover, these data suggest that iron dependent regulation of FBXL5 exerts reciprocal effects on IRP2 stability. Thus, FBXL5 possesses an iron binding hemerythrin domain enabling cells to gauge bioavailable iron levels and control IRP2 expression accordingly, resulting in a tightly regulated circuit in the maintenance of iron homeostasis.