Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: Neuroglobin phosphorylation in vitro and after hypoxia. A, primary amino acid sequence of neuroglobin showing putative phosphorylation sites and binding sites for 14-3-3. B, in vitro phosphorylation of recombinant neuroglobin (Ngb) using purified ERK1 and ERK2 with and without 10 μm U0126 (inhibitor). C, in vitro phosphorylation of recombinant neuroglobin using purified recombinant PKA in the presence and absence of the PKA synthetic peptide inhibitor. D, quantitative analysis of neuroglobin phosphorylation with and without specific respective inhibitors using NIH Image software. E, immunoblot analysis of wild-type and mutant neuroglobin expression in SH-SY5Y neuronal cells that overexpress neuroglobin. F, increased ERK1/2 activity after 3 h of hypoxia as determined by immunoblot analysis using anti-phospho-ERK (pERK) antibody (upper panel). Total ERK1/2 protein was unchanged during hypoxia (middle panel). G, neuroglobin-GFP-expressing cells were subjected to either normoxia or 1% hypoxia for 3 h, metabolically labeled with inorganic orthophosphate, and immunoprecipitated (IP) with anti-neuroglobin antibody. The immunoprecipitated proteins were resolved by SDS-PAGE, and the phosphorylation signals were detected by autoradiogram. Neuroglobin phosphorylation was significantly increased in neuroglobin-GFP-expressing neuronal cells in vivo after 3 h of hypoxia compared with normoxia. GSD, glucose- and serum-deprived medium. H, quantitative analysis of in vivo neuroglobin phosphorylation using NIH Image software.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Phospho-proteomics, In Vitro, Sequencing, Binding Assay, Recombinant, Purification, Software, Western Blot, Mutagenesis, Expressing, Activity Assay, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, In Vivo

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: ERK inhibitor FR180204 inhibits neuroglobin phosphorylation in neuronal cells after hypoxia. A, neuroglobin-expressing cells were cultured and subjected to normoxic and hypoxic conditions in the presence and absence of 0.5 μm FR180204 for 3 h. After 3 h, the cells were lysed, and immunoblotting was performed with anti-phosphoserine antibody (αPS). Ngb, neuroglobin. B, the same blot was stripped and probed with anti-β-actin antibody.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Phospho-proteomics, Expressing, Cell Culture, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: Neuroglobin association with 14-3-3 under hypoxia. A and B, GFP- and neuroglobin (Ngb)-expressing cells were subjected to either normoxia or 1% hypoxia, lysed, and immunoprecipitated (IP) with anti-14-3-3 antibody, followed by probing with anti-pan-14-3-3 and anti-GFP antibodies. C, quantitative analysis revealed that neuroglobin binding to 14-3-3 was increased in neuronal cells during hypoxia. D and E, time-dependent increase in 14-3-3 binding to neuroglobin after hypoxia. Neuroglobin-GFP-expressing cells were subjected to either normoxia or 1% hypoxia at the indicated times, lysed, immunoprecipitated with anti-neuroglobin antibody, and immunoblotted with anti-GFP and anti-14-3-3 antibodies, respectively. F, quantitation of the time-dependent increase in 14-3-3 binding to neuroglobin. G, neuroglobin association with 14-3-3 in intact live cells as determined by FRET analysis. Cells were exposed to normoxia or hypoxia for 3 h prior to analysis for protein-protein interaction by FRET. Cells were either singly transfected (upper panels) or cotransfected with YFP-14-3-3 and CFP-neuroglobin (Neu; middle and lower panels). The panels show CFP, YFP, merge, or FRET (pseudo-color, far right panels); the latter represents intensity of the FRET signal.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Expressing, Immunoprecipitation, Binding Assay, Quantitation Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: Neuroglobin association with 14-3-3 as studied by FRET. Shown in the upper panels are single cell imaging showing cell fluorescence before and after photobleaching for cells transfected with CFP and YFP (A), cells transfected with WT CFP-neuroglobin and YFP-14-3-3 under normoxic conditions (B), cells transfected with WT CFP-neuroglobin and YFP-14-3-3 under hypoxic conditions (C), cells transfected with CFP-S17A neuroglobin and YFP-14-3-3 under hypoxic conditions (D), and cells transfected with CFP-S50A neuroglobin and YFP-14-3-3 under hypoxic conditions (E). In the lower panels of A–E are shown the fluorescence signals from CFP (green traces) and YFP (red traces) measured along the region marked with the red arrows (x axis indicates the progress of the fluorescence detector along the red arrow). When CFP and YFP are not close (and neuroglobin and 14-3-3 are therefore not close), photobleaching is expected to decrease the YFP signal (red) with very little effect on the CFP signal (green). Alternatively, when CFP and YFP are close enough (and neuroglobin and 14-3-3 are therefore close), photobleaching will decrease the YFP signal (red) and increase the CFP signal (green) because CFP cannot transfer energy to YFP anymore (see “Results” for details). The increases in green fluorescence accompanied by larger decreases in red fluorescence are marked in the C and D, indicating protein interaction between 14-3-3 and neuroglobin after hypoxia occurred in WT and S17A neuroglobin. In A, B, and E, there is a decrease in red fluorescence, but the profile for green fluorescence is not changed after photobleaching, indicating poor FRET between CFP and YFP, suggesting very little interaction between 14-3-3 and neuroglobin. These results suggest that neuroglobin Ser50 plays a predominant role in 14-3-3 binding. F, the bar graph shows quantification of FRET efficiency under experimental conditions. FRET efficiency (percent) was calculated and graphed from n = three experiments and >12 randomly selected cells for each condition that was analyzed. S50A mutant neuroglobin showed a significantly reduced interaction with 14-3-3 compared with S17A mutant and wild-type neuroglobin.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Imaging, Fluorescence, Transfection, Binding Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: Expression of endogenous neuroglobin and its association with 14-3-3 in brain. A, neuroglobin expression in different regions of sheep brain as assessed by immunoblotting with neuroglobin-specific polyclonal antibody. B and C, neuroglobin (Ngb) association with 14-3-3. Co-immunoprecipitations were performed using anti-neuroglobin and anti-14-3-3 antibodies from 100 μg of proteins from sheep fetal and adult brainstem and thalamus as described under “Experimental Procedures.” The immune complex was resolved on 4–12% NuPAGE gradient gel, transferred to nitrocellulose, and immunoblotted with anti-neuroglobin antibody (B) and anti-14-3-3 antibody (C).

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: 14-3-3 binding stabilizes neuroglobin phosphorylation. A, protein expression in 14-3-3ϵ, 14-3-3ζ, and control siRNA-silenced neuroglobin-GFP-expressing neuronal cells. mAb, monoclonal antibody. B, β-actin expression in 14-3-3 and control siRNA-silenced neuroglobin-GFP-expressing neuronal cells under the same conditions. C,14-3-3 protein expression is significantly reduced in 14-3-3 siRNA-treated cells as quantitated by NIH Image software. D, control and 14-3-3 siRNA-silenced neuronal cells were metabolically labeled with inorganic phosphate, immunoprecipitated with anti-neuroglobin antibody, and resolved by SDS-PAGE, and the signals were detected by autoradiogram. Shown is the phosphorylation of neuroglobin in 14-3-3 and control siRNA-treated cells in vivo after 3 h of hypoxia. E, quantitative analysis by NIH Image software reveals that neuroglobin (Ngb) phosphorylation is significantly reduced in 14-3-3 siRNA-treated cells.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Binding Assay, Phospho-proteomics, Expressing, Control, Software, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, In Vivo

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: Neuroglobin phosphorylation increases nitrite reductase activity. A, phosphorylation of neuroglobin (Ngb) by ERK2 in vitro. B, bimolecular rate constants (BRC) calculated for the reaction of untreated or phosphorylated neuroglobin with nitrite. Phosphorylation increased nitrite reductase activity by 3-fold. C and D, spectral changes in non-phosphorylated (C) and phosphorylated (D) neuroglobin during the reaction with nitrite. E, absorbance traces indicate the qualitative change in the rates for the reaction of phosphorylated and non-phosphorylated neuroglobin with nitrite. F, phosphorylation increases five-coordination as indicated by faster rates of CO binding. Shown is the normalized decay of deoxyneuroglobin (due to the concomitant increase in neuroglobin-CO) after mixing phosphorylated/non-phosphorylated neuroglobin (5 μm) with half-saturated CO buffer ([CO] ≈ 500 mm). The inset shows representative reaction traces at 425 nm. Abs, absorbance.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Phospho-proteomics, Activity Assay, In Vitro, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide *

doi: 10.1074/jbc.M111.271973

Figure Lengend Snippet: Effect of 14-3-3 binding on nitrite reductase activity. A, non-phosphorylated and phosphorylated neuroglobin (Ngb) with ERK2. B, Coomassie Blue staining of non-phosphorylated and phosphorylated neuroglobin. C, 14-3-3-dependent nitrite reductase activity of phosphorylated and non-phosphorylated neuroglobin after purified 14-3-3 protein addition. BRC, bimolecular reaction rates.

Article Snippet: Human neuroglobin cDNA containing clone SC122910 (NCBI accession number NM_021257 ) was purchased from OriGene (Rockville, MD) and used as a template for generating mutants.

Techniques: Binding Assay, Activity Assay, Staining, Purification

Journal:

Article Title: Neuroglobin is up-regulated by and protects neurons from hypoxic-ischemic injury

doi: 10.1073/pnas.251466698

Figure Lengend Snippet: Neuronal hypoxia and ischemia induce Ngb protein expression. (a) Representative Western blot showing increased Ngb expression in cultured cortical neurons maintained without oxygen for the indicated number of hours (Left). Panel beneath the Western blot shows Ngb mRNA expression over the same time course. Expression of the 17-kDa band (arrow) was quantified by computer densitometry (mean ± SEM, n = 3; *, P < 0.05 relative to 0 h by t test) (Right). (b) Representative Western blots (n = 3) showing increased Ngb expression in cultures treated for 24 h with 300 μM Co2+ or 100 μM Dfx (Left), but no change with 0.1 μM staurosporine (Stauro) or 500 μM SNP (Right). (c) Fluorescence labeling of cultured cortical neurons showing Ngb immunoreactivity (red) in the cytoplasm of cells that express the neuronal nuclear antigen NeuN (green) (Left). Segregation of Ngb expression (green) and DNA damage (detected by labeling with the Klenow fragment of DNA polymerase I, red) into distinct populations, corresponding to viable cells with large nuclei (DAPI staining, blue) and nonviable cells with shrunken nuclei (Center). Preabsorption of the Ab with authentic Ngb peptide antigen abolished immunolabeling (Right). (d) Representative sections from contralateral, nonischemic rat cerebral cortex (Left) and penumbra (Center) or core (Right) of ischemic cerebral cortex at 24 h. Immunostaining for Ngb shows increased Ngb expression in the penumbra; this increased staining is localized to the cytoplasm of normal-appearing, unshrunken cells with neuronal morphology (Center, Insets). Brown, anti-Ngb; blue, cresyl violet. [Original magnification, ×400 (c and Insets to d) and ×200 (d)].

Article Snippet: To evaluate the in vivo expression of Ngb, immunohistochemistry was done on cerebral cortical sections from rats subjected to 90 min of focal cerebral ischemia followed by 4–24 h of reperfusion ( 12 ), using the same anti-Ngb primary Ab described above (1:200) and a horseradish peroxidase-conjugated goat anti-rabbit secondary Ab (1:1000, Santa Cruz Biotechnology). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 Neuronal hypoxia and ischemia induce Ngb protein expression. ( a ) Representative Western blot showing increased Ngb expression in cultured cortical neurons maintained without oxygen for the indicated number of hours ( Left ).

Techniques: Expressing, Western Blot, Cell Culture, Fluorescence, Labeling, Staining, Immunolabeling, Immunostaining

Journal: PLoS ONE

Article Title: Neuroglobin in Breast Cancer Cells: Effect of Hypoxia and Oxidative Stress on Protein Level, Localization, and Anti-Apoptotic Function

doi: 10.1371/journal.pone.0154959

Figure Lengend Snippet: (A ) Typical cytograms of vehicle–, E2- (10 nM), H 2 O 2 - (400 μM), and Pb(IV)- (200 μM) treated MCF-7 cells for 24 h (left) and relative analyses (right) obtained from Annexin V-FITC with PI assays. Data of viable (PI and Annexin V-FITC double negative) and dead (Annexin V-FITC positive and PI AnnexinV-FITC double positive) cells are means ± SD of three different experiments. P< 0.05 was calculated with Student’s t test vs vehicle (*). (B ) Western blot analysis of NGB protein levels performed in vehicle- and E2 (10 nM)- treated control MCF7 cells (ScNGB) and NGB stable silenced cells (ShNGB) treated with selected compounds for 24 h. Typical Western blot representative of three independent experiments. (C ) Western blot analyses of PARP-1 cleavage in MCF-7 cells infected with scramble RNA (ScNGB MCF-7) treated with above reported compounds for 24h. (D ) Analysis of protein PARP-1 cleavage in MCF-7 cells infected with silencing NGB shRNA (ShNGB MCF-7) and incubated with E2 (10 nM), H 2 O 2 (400 μM), and Pb(IV) (200 μM) for 24 h. Western blot analyses of PARP-1 cleavage in MCF-7 cells infected with scramble RNA (ScNGB MCF-7, (E) ) or with silencing NGB shRNA (ShNGB MCF-7, (F) ) and treated with E2 (10 nM, 24 h) before the treatment with Pb(IV) (200 μM, 24 h). (C-F) , Left panels are typical Western blots of three independent experiments. Right panels represent the result of densitometric analyses. The amount of proteins was normalized by comparison with tubulin levels. Data are means ± SD of three different experiments. P<0.05 was determined with Student t-test vs. vehicle (*) and Pb(IV) (°).

Article Snippet: shRNA lentiviral particles transduction was performed using control shRNA Lentiviral particles (Santa Cruz sc-108080) and Neuroglobin shRNA lentiviral particles (Santa Cruz sc-42081-v) according to manufacturer’s instructions as previously described [ ].

Techniques: Western Blot, Control, Infection, shRNA, Incubation, Comparison