segment selection  (Bio-Rad)

Journal: Frontiers in Chemistry

Article Title: The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 phox

doi: 10.3389/fchem.2015.00003

Figure Lengend Snippet: The enhanced binding of p67 phox to Nox2 peptides with an intramolecular disulfide bond is maximal when the cysteines are separated by a single glycine . The binding of p67 phox (1–212), in solution, to surface-attached native and modified Nox2 peptide 24 (357–371), with an N-terminal biotin tag, was measured. The native peptide comprises the 369 CysGlyCys 371 triad at the C-terminus, in the dithiol form (labeled 24N CGC), and a disulfide form was synthesized, with an intramolecular disulfide bond between cysteines C369 and C371 (labeled 24N CGC S-S). Two more peptides were synthesized in which the CGC triad was replaced with CysGlyHisCys (labeled 24N CGHC) or CysGlyProCys (labeled 24N CGPC), to mimic the catalytic sites of PDI and thioredoxin, respectively. These two peptides were also synthesized in the dithiol form and with an intramolecular disulfide bond between cysteines 369 and 372. The methodology is described in the Materials and Methods Section. Results represent means ± SEM of three experiments.

Article Snippet: Synthetic peptides corresponding to selected sequence segments of Nox2 were made by two companies: Mimotopes (Clayton, Victoria, Australia), and Bachem (Bubendorf, Switzerland).

Techniques: Binding Assay, Modification, Labeling, Synthesized

Journal: Frontiers in Chemistry

Article Title: The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 phox

doi: 10.3389/fchem.2015.00003

Figure Lengend Snippet: Treatment of p67 phox with N -ethylmaleimide (NEM), diamide, or H 2 O 2 reverses the enhanced binding to Nox2 peptides containing an intramolecular disulfide bond . p67 phox (1–526) (1.5 μM) was incubated with NEM (0.1 mM) (A) or diamide (5 mM) (B) , for 1 h at room temperature. p67 phox (1–212) (1.5 μM) was exposed to H 2 O 2 (0.1, 1, or 10 mM) for 2 h at 4°C (C) . Control preparations were supplemented with equal volumes of PBS for the same time interval. The binding of treated and untreated p67 phox , in solution, to surface-attached Nox2 peptide 24 (357–371), with an N-terminal biotin tag, was measured. Binding to peptide 24 in the dithiol form (labeled 24N) and/or only (in the case of exposure to H 2 O 2 ) to the peptide with an intramolecular disulfide bond between cysteines C369 and C371 (labeled 24N S-S) was assessed. Results shown in all panels represent means ± SEM of three experiments.

Article Snippet: Synthetic peptides corresponding to selected sequence segments of Nox2 were made by two companies: Mimotopes (Clayton, Victoria, Australia), and Bachem (Bubendorf, Switzerland).

Techniques: Binding Assay, Incubation, Control, Labeling

Journal: Frontiers in Chemistry

Article Title: The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 phox

doi: 10.3389/fchem.2015.00003

Figure Lengend Snippet: Recombinant NusA-Nox2 proteins used in the present work . A number of NusA-Nox2 fusion proteins, consisting of NusA and segments of the dehydrogenase region of Nox2, were constructed (see Materials and Methods and Supplementary Material). The figure shows a schematic representation of the serial N-terminal truncations of the Nox2 part of the fusion proteins. It also illustrates the location of peptides 24 (357–371) and 28 (369–383); the position of the FAD- and NADPH-binding sites, and the epitopes of the two monoclonal anti-Nox2 antibodies used in the present work. The truncation most used (357–570) is marked by a red-colored ellipse.

Article Snippet: Synthetic peptides corresponding to selected sequence segments of Nox2 were made by two companies: Mimotopes (Clayton, Victoria, Australia), and Bachem (Bubendorf, Switzerland).

Techniques: Recombinant, Construct, Binding Assay

Journal: Frontiers in Chemistry

Article Title: The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 phox

doi: 10.3389/fchem.2015.00003

Figure Lengend Snippet: Immunoblot of NusA-Nox2 fusion protein truncations using anti Nox2 monoclonal antibodies 54.1 and NL7. (A) The figure illustrates the fact that antibody 54.1 (its epitope represented by Nox2 residues 381–390) recognizes Nox2 truncations 357–570 and 372–570, and reacts weakly with truncation 387–570. (B) Antibody NL7 (its epitope represented by Nox2 residues 498–506) recognizes all truncations, from 357–570 to 462–570.

Article Snippet: Synthetic peptides corresponding to selected sequence segments of Nox2 were made by two companies: Mimotopes (Clayton, Victoria, Australia), and Bachem (Bubendorf, Switzerland).

Techniques: Western Blot, Bioprocessing

Journal: Frontiers in Chemistry

Article Title: The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 phox

doi: 10.3389/fchem.2015.00003

Figure Lengend Snippet: p67 phox (1–526) and p67 phox (1–212) bind to NusA-Nox2(328–570) and NusA-Nox2(357–570) but not to shorter Nox2 truncations . Binding of NusA-Nox2 fusion proteins and NusA, as a control, in the fluid phase, to surface-bound p67 phox (1–526) (A) and p67 phox (1–212) (B) was measured as described in the Materials and Methods Section. All proteins were purified by gel filtration on Superdex 200 and the homodimer-containing fractions were used in the binding studies. The results represent one characteristic experiment. The binding of p67 phox to NusA-Nox2(328–570) and NusA-Nox2(357–570) and the lack of binding to the five other truncations, which did not exceed the binding to NusA, was a constant feature of all experiments.

Article Snippet: Synthetic peptides corresponding to selected sequence segments of Nox2 were made by two companies: Mimotopes (Clayton, Victoria, Australia), and Bachem (Bubendorf, Switzerland).

Techniques: Binding Assay, Control, Purification, Filtration

Journal: Frontiers in Chemistry

Article Title: The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 phox

doi: 10.3389/fchem.2015.00003

Figure Lengend Snippet: PDI reductase activity of NusA-Nox2(357–570). (A) Recombinant NusA-Nox2(357–570) (2 μM) was assayed for disulfide reductase activity on dieosin glutathione disulfide (DE-GSSG), in the presence of DTT, as described by Raturi and Mutus and detailed in the Materials and Methods Section. Briefly, the reaction mixtures contained 800 nM DE-GSSG and 12.5 μM DTT and the kinetics of the increase in fluorescence were followed for 30 min, using an excitation wavelength of 519 nm and an emission wavelength of 545 nm. Results are expressed as V max (milli relative fluorescence units per min). (B) The dose dependence of the PDI reductase activity of NusA-Nox2(357–570) was assayed on DE-GSSG in the presence of DTT, as described in (A) . The concentration of NusA-Nox2(357–570) was varied from 1 to 4 μM. The results of one characteristic experiment are illustrated. (C) The absence of the 369 CysGlyCys 371 triad in NusA-Nox2(372–570) or mutating Cys 369 and Cys 371 to Arg in NusA-Nox2(357–570) eliminates PDI reductase activity. NusA-Nox2(357–570), NusA-Nox2(372–570), and NusA-Nox2(357–570) C369R, C371R (all, at a concentration of 2 μM) were assayed for disulfide reductase activity on DE-GSSG in the presence of DTT, as described in (A) . The results of one characteristic experiment are illustrated. (D) The PDI inhibitor phenylarsine oxide (PAO) interferes with the PDI activity of NusA-Nox2(357–570). Recombinant NusA-Nox2(357–570) (2 μM) was assayed for disulfide reductase activity on DE-GSSG in the presence of DTT, in the absence and presence of 50 μM PAO, as described in (A) . The results of one characteristic experiment are illustrated.

Article Snippet: Synthetic peptides corresponding to selected sequence segments of Nox2 were made by two companies: Mimotopes (Clayton, Victoria, Australia), and Bachem (Bubendorf, Switzerland).

Techniques: Activity Assay, Recombinant, Fluorescence, Concentration Assay

Journal: Frontiers in Chemistry

Article Title: The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 phox

doi: 10.3389/fchem.2015.00003

Figure Lengend Snippet: PDI reductase activities of NusA-Nox2(357–570) compared to those of the NusA-Nox2(357-570) C369R, C371R mutant and to PDIA1 and PDIA3 . (A) PDI reductase activity of recombinant NusA-Nox2(357–570) was assayed in a concentration range of 1–4 μM and plotted as non-linear regression (one site binding equation). (B) PDI reductase activity of recombinant NusA-Nox2(357–570) C369R, C371R mutant was assayed in a concentration range of 1–4 μM and plotted as a cubic spline curve. (C) PDI reductase activity of recombinant PDIA1 was assayed in a concentration range of 10–80 nM and plotted as linear regression. (D) PDI reductase activity of recombinant PDIA3 was assayed in a concentration range of 1–10 nM and plotted as linear regression. The assays were performed as described by Raturi and Mutus and detailed in the Materials and Methods Section. Results represent means ± SEM of 9 (NusA-Nox2(357–570), 4 (NusA-Npx2(357–570) C369R, C371R), 4 (PDIA1), and 3 (PDIA3) experiments.

Article Snippet: Synthetic peptides corresponding to selected sequence segments of Nox2 were made by two companies: Mimotopes (Clayton, Victoria, Australia), and Bachem (Bubendorf, Switzerland).

Techniques: Mutagenesis, Activity Assay, Recombinant, Concentration Assay, Binding Assay

Journal: Frontiers in Chemistry

Article Title: The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 phox

doi: 10.3389/fchem.2015.00003

Figure Lengend Snippet: Amino acid sequence similarities between the dehydrogenase region of Nox2 and PDIA3 . The figure illustrates three small regions (3–6 residues) exhibiting partial sequence identities between Nox2 and PDIA3. The representation of the motifs in non-phagocytic Noxes (Noxes 1, 3, 4, and 5) is also shown. Monoclonal anti-Nox2 antibody 54.1 recognizes Nox2 residues 381 KLPKIAVDGP 390 and might recognize PDIA3 residues 114 AYDGP 118 (see Figure ). Monoclonal anti-Nox2 antibody NL7 recognizes Nox2 residues 498 EKDVITGLK 506 and might recognize PDIA3 residues 252 KDLIQG 257 (see Figure ). Polyclonal anti-PDIA3 antibody H-220 was raised against PDIA3 residues 108–207 and could potentially recognize PDIA3 114 AYDGP 118 and 156 IVG 158 and Nox2 residues 386 AVDGP 390 and 357 IVG 359 , respectively (see Figure ). The regions of Nox2/PDIA3 homology do not exist in the sequence of PDIA1, in good agreement with the fact that the two anti-Nox2 antibodies do not recognize PDIA1 (see Figures ) and a polyclonal anti-PDIA1 antibody does not recognize Nox2 (see Figure ). Identical residues in different proteins are in red font; the CGC triad in Nox2 is in orange font.

Article Snippet: Synthetic peptides corresponding to selected sequence segments of Nox2 were made by two companies: Mimotopes (Clayton, Victoria, Australia), and Bachem (Bubendorf, Switzerland).

Techniques: Sequencing

Journal: Frontiers in Chemistry

Article Title: The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 phox

doi: 10.3389/fchem.2015.00003

Figure Lengend Snippet: Two monoclonal anti-Nox2 antibodies react with PDIA3 but not with PDIA1 by immunoblotting. (A) Monoclonal anti-Nox2 antibody 54.1 (epitope, residues 381–390) reacts with recombinant NusA-Nox2(357–570) and NusA-Nox2(372–570) but not with NusA. It reacts strongly with recombinant PDIA3 but not with recombinant PDIA1. It recognizes a protein of about 54–58 kDa (diffuse band) in the guinea pig macrophage membrane, corresponding to Nox2 (Knoller et al., ). (B) Monoclonal anti-Nox2 antibody NL7 (epitope, residues 498–506) reacts with recombinant NusA-Nox2(357–570) and NusA-Nox2(372–570) but not with NusA. It reacts moderately with recombinant PDIA3 but not with recombinant PDIA1. It recognizes a protein of about 54–58 kDa (diffuse band) in the guinea pig macrophage membrane, corresponding to Nox2. Nox2(357–570) numbered 1, 2, and 3 represent three batches of NusA-Nox2(357–570).

Article Snippet: Synthetic peptides corresponding to selected sequence segments of Nox2 were made by two companies: Mimotopes (Clayton, Victoria, Australia), and Bachem (Bubendorf, Switzerland).

Techniques: Western Blot, Recombinant, Membrane

Journal: Frontiers in Chemistry

Article Title: The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 phox

doi: 10.3389/fchem.2015.00003

Figure Lengend Snippet: Cross-reactivity of some anti-Nox2 antibodies with PDIA3 and of some anti-PDIA3 antibodies with Nox2 .

Article Snippet: Synthetic peptides corresponding to selected sequence segments of Nox2 were made by two companies: Mimotopes (Clayton, Victoria, Australia), and Bachem (Bubendorf, Switzerland).

Techniques: Recombinant, Membrane

Journal: Frontiers in Chemistry

Article Title: The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 phox

doi: 10.3389/fchem.2015.00003

Figure Lengend Snippet: A polyclonal anti-PDIA3 antibody but not a polyclonal anti-PDIA1 antibody reacts with Nox2 by immunoblotting . (A) Polyclonal anti-PDIA3 antibody H-220 reacts with recombinant NusA-Nox2(357–570) but not with NusA-Nox2(372–570) and NusA. It reacts strongly with recombinant PDIA3 but not with recombinant PDIA1. It also recognizes a protein in the guinea pig macrophage membrane (sharp single band). (B) Polyclonal anti-PDIA1 antibody H-160 does not react with recombinant NusA-Nox2(357–570) and NusA-Nox2(372–570). It reacts with recombinant PDIA1 and weakly, with recombinant PDIA3. It also recognizes a protein in the guinea pig macrophage membrane (sharp double band). Nox2(357–570) numbered 1, 2, and 3 represent three batches of NusA-Nox2(357–570).

Article Snippet: Synthetic peptides corresponding to selected sequence segments of Nox2 were made by two companies: Mimotopes (Clayton, Victoria, Australia), and Bachem (Bubendorf, Switzerland).

Techniques: Western Blot, Recombinant, Membrane

Journal: Frontiers in Chemistry

Article Title: The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 phox

doi: 10.3389/fchem.2015.00003

Figure Lengend Snippet: Detection of PDIA3 in macrophage membranes by both anti-Nox2 and anti-PDIA3 antibodies . Monoclonal anti-Nox2 antibody 54.1, which recognizes recombinant PDIA3 (see Figure ), is shown to detect in macrophage membranes an antigen most likely to be PDIA3. This appears in four distinct membrane batches as a sharply defined band on the background of the more diffuse Nox2; the overlap is caused by the almost identical size of guinea pig Nox2 and PDIA3. A polyclonal anti-PDIA3 antibody (HPA003230), found not to cross-react with Nox2 (see Table ), detects PDIA3 in macrophage membranes. The position and character of the bands detected by anti-PDIA3 strengthens the proposal that the sharp band detected by anti-Nox2 represents PDIA3.

Article Snippet: Synthetic peptides corresponding to selected sequence segments of Nox2 were made by two companies: Mimotopes (Clayton, Victoria, Australia), and Bachem (Bubendorf, Switzerland).

Techniques: Recombinant, Membrane

Journal: Frontiers in Chemistry

Article Title: The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 phox

doi: 10.3389/fchem.2015.00003

Figure Lengend Snippet: Mutating all four cysteines in p67 phox (1–212) eliminates its ability to bind to Nox2 peptide 24 . Binding of wild type p67 phox (1–212) and p67 phox in which 40 Cys, 45 Cys, 121 Cys, and 165 Cys were mutated to Ser, in solution, to surface-attached Nox2 peptide 24 (357–371), with an N-terminal biotin tag, in the dithiol form (labeled 24N CGC), and with an intramolecular disulfide bond between cysteines C369 and C371 (labeled 24N CGC S-S), was assessed. The methodology is described in the Materials and Methods Section. (A) Binding of the mutant protein to both forms of the peptide is markedly reduced. Results represent means ± SEM of 3 experiments. (B) End point view of the wells of a 10 min kinetic experiment (A) . The depth of the blue-green color, representing oxidized TMB, is proportional with the binding of wild type and mutant p67 phox to the peptides. Results are those of a single representative experiment.

Article Snippet: Synthetic peptides corresponding to selected sequence segments of Nox2 were made by two companies: Mimotopes (Clayton, Victoria, Australia), and Bachem (Bubendorf, Switzerland).

Techniques: Binding Assay, Labeling, Mutagenesis