Browsing by Subject "Iron"
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Item Downregulation of the Cytosolic Iron-Sulfur Assembly Pathway in Cancer by an E3 Ubiquitin Ligase(2017-06-19) Weon, Jenny Linda; Tu, Benjamin; Potts, Patrick Ryan; Minna, John D.; Liu, YiIron-sulfur (Fe-S) clusters are considered to be one of the oldest cofactors utilized by proteins and are essential for life from bacteria to mammals. Multiple processes in the cell require Fe-S cofactors, such as electron transfer in mitochondrial respiration, enzymatic reactions, and as structural components in DNA repair enzymes. We describe here the first post-translational mechanism to regulate Fe-S assembly and delivery through the ubiquitination and degradation of a key cytosolic iron-sulfur cluster assembly (CIA) pathway component by a MAGE-RING ligase (MRL). The MAGE protein family consists of ~40 members in humans that function in complex with E3 ubiquitin ligases to enhance ubiquitination activity, alter E3 subcellular localization, and/or specify E3 targets. Using biochemical and cellular approaches we have discovered that the MAGE-F1-NSE1 ligase disrupts Fe-S cluster delivery through ubiquitination and degradation of the CIA pathway protein MMS19. MMS19 is a substrate specifying, late-acting component of the CIA pathway that facilitates Fe-S transfer from the multi-component cascade of assembly proteins to specific recipient apoproteins. Notably, many MMS19 targets are enzymes involved in DNA repair. We found that MAGE-F1 directs the E3 ligase NSE1 to target MMS19 for ubiquitination and degradation. Knockdown of MAGE-F1 stabilized MMS19 and overexpression of MAGE-F1 decreased MMS19 levels without affecting MMS19 mRNA levels. We further confirmed MAGE-F1 inhibits Fe-S incorporation into known MMS19-dependent Fe-S proteins, such as FANCJ, POLD1, RTEL1, XPD, and DPYD, but not MMS19-independent Fe-S proteins, such as PPAT. Loss of Fe-S incorporation leads to decreased DNA repair capacity of cells, exemplified by decreased homologous recombination rates and altered sensitivity to DNA damaging agents. Importantly, numerous cancer types harbor copy-number amplification of MAGE-F1, including lung squamous carcinoma and head and neck squamous carcinoma. Consistent with MAGE-F1 inhibitory activity on Fe-S incorporation into key DNA repair enzymes, MAGE-F1-amplified tumors bear a significantly greater mutational burden than non-MAGE-F1-amplified cancers and the expression of MAGE-F1-NSE1 correlates with poor patient prognosis. In summary, we provide the first evidence for post-translational regulatory control of Fe-S cluster assembly and a novel mechanism by which a broad spectrum of DNA repair enzymes can be regulated and lead to genomic instability in cancer.Item FBXL5 Is Required for the Maintenance of Cellular Andsystemic Iron Homeostasis(2013-01-16) Ruiz, Julio Cesar Francisco; Bruick, Richard K.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(2011-08-10) 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 FGF23 at the intersection of phosphate and iron homeostasis(2023-08-25) Wolf, MylesItem Iron deficiency(1975-09-18) Sheehan, Richard G.Item An Iron Sensing E3 Ubiquitin Ligase Regulates Iron Homeostasis(2011-08-10) 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.Item Iron storage disorders(1980-08-14) Ware, Athol J.Item [Southwestern News](2002-03-05) Carter, Wayne