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Results: 5

1.
FIGURE 1.

FIGURE 1. From: Structural and Molecular Characterization of Iron-sensing Hemerythrin-like Domain within F-box and Leucine-rich Repeat Protein 5 (FBXL5).

EPR spectra of FBXL5 Hr domain. Spectra 1 and 2, the 20 K EPR spectrum of the Hr domain following purification under ambient oxygen conditions (spectrum 1) and upon the addition of excess reducing agent (spectrum 2). Comparison with a canonical Hr from P. gouldii is shown in the inset.

Joel W. Thompson, et al. J Biol Chem. 2012 March 2;287(10):7357-7365.
2.
FIGURE 3.

FIGURE 3. From: Structural and Molecular Characterization of Iron-sensing Hemerythrin-like Domain within F-box and Leucine-rich Repeat Protein 5 (FBXL5).

Regulation of FBXL5 is dependent on assembly of diiron center within hemerythrin domain. Eight hours after transfection, HEK 293T cells were treated with either 100 μm DFO or 100 μm FAC for 16 h, and FBXL5 accumulation was assessed by immunoblot analysis. A, analysis of mutations to residues comprising the primary iron coordination shell. B, analysis of FBXL5 Glu-58 mutations.

Joel W. Thompson, et al. J Biol Chem. 2012 March 2;287(10):7357-7365.
3.
FIGURE 5.

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

FBXL5 Hr domain does not appear to bind oxygen ligand. A, comparison of the FBXL5 and DcrH diiron centers. Shown in stereo is a superposition of the bound iron atoms (dark blue balls), bridging oxygen (dark red ball), and first coordination sphere iron ligands (green) of FBXL5 Hr (PDB code 3V5Y) with corresponding atoms (pale balls) and ligands (yellow) of deoxy-DcrH Hr (PDB code 2AWC). B, stopped-flow kinetics of reduced anaerobic FBXL5 Hr rapidly mixed with O2 saturated buffer. Fitting of the reoxidation of the Hr domain at 340 nm required three summed exponentials to adequately account for the observed data (inset), indicating a complex oxidation process. Abs, absorbance.

Joel W. Thompson, et al. J Biol Chem. 2012 March 2;287(10):7357-7365.
4.
FIGURE 2.

FIGURE 2. From: Structural and Molecular Characterization of Iron-sensing Hemerythrin-like Domain within F-box and Leucine-rich Repeat Protein 5 (FBXL5).

Crystal structure of FBXL5 hemerythrin domain. A, ribbon representation of the hemerythrin domain from H. sapiens FBXL5 (residues 5–159; PDB code 3V5X). Helices are shown in green, and loops are in shown in red. The additional fifth C-terminal helix found is shown in yellow. The dotted lines represent the disordered residues 75–76 and 81–83. B, the first coordination sphere iron ligands of the FBXL5 Hr diiron center are shown as green sticks, and the conserved second coordination sphere iron ligands are shown as pink sticks. Iron ligands and hydrogen bonds are shown as dotted black lines. C, diiron center of FBXL5 Hr Native 1, monomer A with superimposed FoFc simulated annealing omit electron density map (gray, contoured at 3.0 σ) calculated for the bridging oxygen (red) and the side chains (green) of the first coordination sphere (atoms beyond the C-β for the amino acids were omitted). An anomalous difference Fourier electron density map (red, contoured at 15.0 σ) is shown for the iron atoms (cyan).

Joel W. Thompson, et al. J Biol Chem. 2012 March 2;287(10):7357-7365.
5.
FIGURE 4.

FIGURE 4. From: Structural and Molecular Characterization of Iron-sensing Hemerythrin-like Domain within F-box and Leucine-rich Repeat Protein 5 (FBXL5).

FBXL5 degradation under iron-deplete conditions requires functional ubiquitin-proteasome system and regulatory sequence. A, immunoblot analysis of immunoprecipitated (IP) FLAG-FBXL5 from stably transfected HEK 293T cells treated with FAC or DFO in the absence or presence of the proteasome inhibitor MG132. B, ubiquitination of FBXL5 Hr is not dependent on FBXL5. HEK 293 cells stably expressing the FBXL5 Hr domain were transfected with a negative control siRNA (NT) or siRNA targeting endogenous FBXL5 prior to treatment with FAC or DFO in the absence or presence of MG132. C, immunoblot analysis of FLAG-Hr-HA accumulation in ts-20 BALB/c 3T3 cells incubated with FAC or DFO for 6 h followed by a switch to either E1-permissive (35 °C) or E1-restrictive (39 °C) temperatures for 24 h in medium containing FAC or DFO as indicated. D, immunoblot analysis of FLAG-Hr-HA accumulation in ts-20 BALB/c 3T3 cells. Cells were incubated with FAC for 6 h and maintained in 1 or 21% O2 at 35 °C or 39 °C for 24 h. E and F, immunoblot analysis of FBXL5 accumulation from expression constructs having increasingly longer N-terminal deletions (E) or short internal deletions (F). FL, full length.

Joel W. Thompson, et al. J Biol Chem. 2012 March 2;287(10):7357-7365.

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