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Results: 7

1.
FIGURE 5.

FIGURE 5. From: Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins.

Overlay of DUOX1 and DUOX2 peroxidase-like domain model structures. The predicted structures of hDUOX1 (cyan) and hDUOX2 (green) N-terminal regions were overlaid to demonstrate the difference in the relative positions of the conserved cysteine residues Cys364 (red) of hDUOX1 and Cys370 (purple) of hDUOX2.

Jennifer L. Meitzler, et al. J Biol Chem. 2013 March 8;288(10):7147-7157.
2.
FIGURE 2.

FIGURE 2. From: Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins.

Partial alignment of the DUOX maturation factors. The sequence alignment shown, generated by the ClustalW program, encompasses the soluble domain of DUOX maturation factors from both human (h) and mouse (m). Soluble domains were determined by the TMHMM 2.0 Server; SolhDUOXA1: 75–183 AA, SolhDUOXA2: 75–175, SolmDUOXA1: 75–181, SolmDUOXA2: 74–175. The conserved cysteine residues (Cys167) found in both DUOXA1 and DUOXA2 are highlighted in red.

Jennifer L. Meitzler, et al. J Biol Chem. 2013 March 8;288(10):7147-7157.
3.
FIGURE 4.

FIGURE 4. From: Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins.

Gel filtration analysis of DUOX truncated proteins. A, comparison of hDUOX11–593 wild-type (WT), reduced (+βME), and C364G proteins (left) and hDUOX21–599 WT, C351G, and C582G (right). Arrows highlight the varying amounts of the dimeric species. B, direct comparison of hDUOX11–593 and hDUOX21–599 by size exclusion (left) and SDS-PAGE analysis (right) of hDUOX11–593 unreduced WT (lane 1) and reduced WT protein with βME (lane 2). After reductant removal, incubation at room temperature without (lane 3) or with diamide addition (lane 4) demonstrated hDUOX11–593 dimer reformation.

Jennifer L. Meitzler, et al. J Biol Chem. 2013 March 8;288(10):7147-7157.
4.
FIGURE 7.

FIGURE 7. From: Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins.

Model of potential hDUOX1 protein and maturation factor association in vivo. Interactions of the peroxidase-like domain of hDUOX1 (green) with both a soluble extracellular region of the hDUOXA1 maturation factor (orange) and itself are demonstrated. Cys345, Cys364, Cys565, and Cys579 are highlighted, demonstrating their relative positions within the peroxidase-like domain of DUOX1. A potential disulfide bonding interaction may exist between a cysteine within the hDUOXA1 domain and a hDUOX1 cysteine (possibly Cys579). The loss of in vitro dimerization upon mutation of Cys364 within the N-terminal region of hDUOX1 suggests that this residue was responsible for self-association of the hDUOX1 peroxidase-like domain through a disulfide interaction, illustrated here in the context of the full-length protein.

Jennifer L. Meitzler, et al. J Biol Chem. 2013 March 8;288(10):7147-7157.
5.
FIGURE 1.

FIGURE 1. From: Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins.

Comparison of sequence and structural localization of critical cysteine residues in hDUOX isoforms. A, sequence alignment of classic peroxidase domains with the hDUOX proteins. The highlighted segments shown focus on the regions encompassing the cysteine residues under study: Cys345/Cys351, Cys364/Cys370, Cys565/Cys568, Cys579/Cys582 (hDUOX1/hDUOX2). B, structural model of hDUOX21–599 produced by the SWISS-MODEL program server and PyMOL. Left, view of model structure highlighting residues investigated by point mutation in orange. Right, 45-degree y axis rotation of the model structure, illustrating residue solvent exposure and localization along one plane of the DUOX protein structure.

Jennifer L. Meitzler, et al. J Biol Chem. 2013 March 8;288(10):7147-7157.
6.
FIGURE 6.

FIGURE 6. From: Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins.

Biosynthesis and activity of hDUOX1 mutants. A, wild-type nontransfected HEK (NT) and transfected HEK cells expressing normal (hDUOX1/A1) or hDUOX1 mutants were pulse-labeled for 2 h, harvested (0 h chase), or chased (18 h) with cold methionine, and immunoprecipitated with anti-hDUOX1 antibody. Immunoprecipitates were separated by SDS-PAGE and subjected to autoradiography. DUOX1-related proteins were quantitated by a PhosphorImager. The gel is a representative of two independent experiments. B, amounts of radiolabeled mutant hDUOX1 were compared with the amount of normal hDUOX1 synthesized under the same conditions (0 h chase). Values are normalized to that for normal hDUOX1, and the data are the mean ± S.D. (error bars) for two independent pulse-chase experiments.

Jennifer L. Meitzler, et al. J Biol Chem. 2013 March 8;288(10):7147-7157.
7.
FIGURE 3.

FIGURE 3. From: Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins.

Structural characterization of DUOX mutants. A, tryptophan fluorescence emission spectra of DUOX11–593 cysteine mutants (C345G, diamond; C364G, circle; C565G, square; C579G, triangle), collected at 341 nm (pH 4.0, solid line) and 348 nm (pH 7.0, dashed line). B, CD spectra of hDUOX11–593 and hDUOX21–599 at 20 °C with their respective cysteine mutants compared at pH 7.0, with the spectra overlaid for comparison. C, CD spectral comparison of hDUOX11–593 versus C579G (left) and hDUOX21–599 versus C582G (right) at 35 °C, collected during temperature gradient studies. All CD experiments were conducted in 10 mm phosphate buffer. D, top, hDUOX11–593 cysteine mutants under reducing (left) and nonreducing (right) conditions. An asterisk highlights the upper proposed dimer band, seen to vary in intensity, most notably for C364G (arrow). D, bottom, hDUOX21–599 cysteine mutant set studied under reduced (left) and nonreduced (right) conditions. Obvious dimerization appears absent from wild-type and mutant proteins except for C351G (arrow).

Jennifer L. Meitzler, et al. J Biol Chem. 2013 March 8;288(10):7147-7157.

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