Structural and Molecular Characterization of Iron-sensing Hemerythrin-like Domain within F-box and Leucine-rich Repeat Protein 5 (FBXL5)

Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2012; 287(10):7357-65. DOI: 10.1074/jbc.M111.308684
Source: PubMed


Mammalian cells maintain iron homeostasis by sensing changes in bioavailable iron levels and promoting adaptive responses.
FBXL5 is a subunit of an E3 ubiquitin ligase complex that mediates the stability of iron regulatory protein 2, an important
posttranscriptional regulator of several genes involved in iron metabolism. The stability of FBXL5 is regulated in an iron-
and oxygen-responsive manner, contingent upon the presence of its N-terminal domain. Here we present the atomic structure
of the FBXL5 N terminus, a hemerythrin-like α-helical bundle fold not previously observed in mammalian proteins. The core
of this domain employs an unusual assortment of amino acids necessary for the assembly and sensing properties of its diiron
center. These regulatory features govern the accessibility of a mapped sequence required for proteasomal degradation of FBXL5.
Detailed molecular and structural characterization of the ligand-responsive hemerythrin domain provides insights into the
mechanisms by which FBXL5 serves as a unique mammalian metabolic sensor.

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Available from: Diana R. Tomchick, Aug 26, 2014
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    • "In vitro analyses suggest that BTS interacts with (Fig. 3) and restricts the accumulation of iron-responsive bHLH transcription factors ILR3 and bHLH115 through its E3 ligase activity and can mediate proteasomal degradation of these targets even in the absence of HHE domains (Fig. 5, A–C). Notably, the mammalian protein FBXL5, unlike BTS, does not contain a RING domain and thus lacks the ability to directly ubiquitinate its targets for degradation via the 26S proteasome pathway (Salahudeen et al., 2009; Vashisht et al., 2009; Thompson et al., 2012). FBXL5 does, however, provide the specificity to the SCF FBXL5 E3 ligase complex to regulate iron homeostasis. "
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    ABSTRACT: Iron uptake and metabolism are tightly regulated in both plants and animals. In Arabidopsis thaliana, BRUTUS (BTS), which contains three hemerythrin (HHE) domains and a Really Interesting New Gene (RING) domain, interacts with bHLH transcription factors that are capable of forming heterodimers with POPEYE, a positive regulator of the iron deficiency response. BTS has been shown to have E3 ligase capacity and to play a role in root growth, rhizosphere acidification, and iron reductase activity in response to iron deprivation. To further characterize the function of this protein we examined the expression pattern of recombinant ProBTS::GUS and found that it is expressed in developing embryos and other reproductive tissues, corresponding with its apparent role in reproductive growth and development. Our findings also indicate that the interactions between BTS and PYE-like (PYEL) bHLH transcription factors occur within the nucleus and are dependent on the presence of the RING domain. We provide evidence that BTS facilitates 26S proteasome-mediated degradation of PYEL proteins in the absence of iron. We also determined that upon binding iron at the HHE domains, BTS is destabilized, and that this destabilization relies on specific residues within the HHE domains. This study reveals an important and novel mechanism for plant iron homeostasis whereby an E3 ubiquitin ligase may post-translationally control components of the transcriptional regulatory network involved in the iron deficiency response. Copyright © 2014, American Society of Plant Biologists.
    Plant physiology 12/2014; 167(1). DOI:10.1104/pp.114.250837 · 6.84 Impact Factor
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    • "FBXL5 exhibits genuine iron-sensing properties , as it is stabilized in the presence of iron and oxygen by forming a Fe-O-Fe center within its N-terminal hemerythrinlike domain. Loss of this center in iron-starved and/or hypoxic cells exposes a degron via a conformational change (Chollangi et al., 2012; Thompson et al., 2012). This allows proteasomal degradation of FBXL5, which leads to concomitant accumulation of IRP2. "
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    ABSTRACT: Iron regulatory proteins 1 and 2 (IRP1 and IRP2) post-transcriptionally control the expression of several mRNAs encoding proteins of iron, oxygen and energy metabolism. The mechanism involves their binding to iron responsive elements (IREs) in the untranslated regions of target mRNAs, thereby controlling mRNA translation or stability. Whereas IRP2 functions solely as an RNA-binding protein, IRP1 operates as either an RNA-binding protein or a cytosolic aconitase. Early experiments in cultured cells established a crucial role of IRPs in regulation of cellular iron metabolism. More recently, studies in mouse models with global or localized Irp1 and/or Irp2 deficiencies uncovered new physiological functions of IRPs in the context of systemic iron homeostasis. Thus, IRP1 emerged as a key regulator of erythropoiesis and iron absorption by controlling hypoxia inducible factor 2α (HIF2α) mRNA translation, while IRP2 appears to dominate the control of iron uptake and heme biosynthesis in erythroid progenitor cells by regulating the expression of transferrin receptor 1 (TfR1) and 5-aminolevulinic acid synthase 2 (ALAS2) mRNAs, respectively. Targeted disruption of either Irp1 or Irp2 in mice is associated with distinct phenotypic abnormalities. Thus, Irp1(-/-) mice develop polycythemia and pulmonary hypertension, while Irp2(-/-) mice present with microcytic anemia, iron overload in the intestine and the liver, and neurologic defects. Combined disruption of both Irp1 and Irp2 is incombatible with life and leads to early embryonic lethality. Mice with intestinal- or liver-specific disruption of both Irps are viable at birth but die later on due to malabsorption or liver failure, respectively. Adult mice lacking both Irps in the intestine exhibit a profound defect in dietary iron absorption due to a "mucosal block" that is caused by the de-repression of ferritin mRNA translation. Herein, we discuss the physiological function of the IRE/IRP regulatory system.
    Frontiers in Pharmacology 07/2014; 5:176. DOI:10.3389/fphar.2014.00176 · 3.80 Impact Factor
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    • "To eliminate possible bacterial contamination, we checked the gene structure and confirmed that the Acanthamoeba gene has introns within the hemerythrin domain. The two major clades (A, nonrespiratory, and B, respiratory) are separated with high nodal support, which is in agreement with observed structural differences (Histidine 74 being only present in clade B) (Thompson et al. 2012). Interestingly, clade B hemerythrins seem to be more common and highly diversified in prokaryotes (French et al. 2008) than in eukaryotes. "
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    ABSTRACT: Hemerythrins and hemocyanins are respiratory proteins present in some of the most ecologically diverse animal lineages; however the precise evolutionary history of their enzymatic domains (hemerythrin, hemocyanin M and tyrosinase) is still not well understood. We survey a wide dataset of prokaryote and eukaryote genomes and RNAseq data to reconstruct the phylogenetic origins of these protiens. We identify new species with hemerythrin, hemocyanin M and tyrosinase domains in their genomes, particularly within animals, and demonstrate that the current distribution of respiratory proteins is due to several events of lateral gene transfer and/or massive gene loss. We conclude that the last common metazoan ancestor had at least two hemerythrin domains, one hemocyanin M domain and six tyrosinase domains. The patchy distribution of these proteins among animal lineages can be partially explained by physiological adaptations, making these genes good targets for investigations into the interplay between genomic evolution and physiological constraints.
    Genome Biology and Evolution 07/2013; 5(7):1435-144. DOI:10.1093/gbe/evt102 · 4.23 Impact Factor
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