Results: 5

1.
Fig. 5.

Fig. 5. From: Posttranslational hydroxylation of ankyrin repeats in I?B proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH).

MS demonstrating hydroxylation of endogenous IκBα. MS/MS spectra are depicted showing the N-terminally modified *CGADVNR peptide, derived by 6-exo-trig cyclization from the peptide S-alkylated with iodoacetamide. (Left) Fragments of the unhydroxylated IκBα peptide, [M+H]+ = m/z 774.3. (Right) Fragments of the hydroxylated IκBα peptide, [M+H]+ = m/z 790.3; a +16-Da shift is observed in the y-ion series, appearing at the y2 ion, which corresponds to fragments containing Asn-244.

Matthew E. Cockman, et al. Proc Natl Acad Sci U S A. 2006 October 3;103(40):14767-14772.
2.
Fig. 2.

Fig. 2. From: Posttranslational hydroxylation of ankyrin repeats in I?B proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH).

Recombinant p105 and IκBα promote decarboxylation of 2-OG in assays of FIH activity. 2-OG decarboxylation is measured by 14CO2 formation. (A) Reactions containing the indicated GST-tagged proteins: HIF-1α 775–826 (HIF-1α C-terminal activation domain); p105 ARD 537–809 (p105 ARD); p105 ARD Asn-678 mutant (p105 ARD(N678A)). Wild type (p105 ARD) but not the N678A mutant promoted 2-OG decarboxylation. Higher background was observed with p105 ARD(N678A) because of less pure preparation of protein. (B) Experiments comparing p105 ARD with full-length IκBα and IκBβ proteins. IκBα, but not IκBβ, promotes 2-OG decarboxylation.

Matthew E. Cockman, et al. Proc Natl Acad Sci U S A. 2006 October 3;103(40):14767-14772.
3.
Fig. 1.

Fig. 1. From: Posttranslational hydroxylation of ankyrin repeats in I?B proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH).

Interactions between p105 and FIH. (A) Interaction of [35S]methionine-labeled p105 polypeptides with extract from control cells (−) or cells induced with doxycycline (DOX) to express FLAG-FIH (+). Anti-FLAG immunoprecipitations; coprecipitating products are visualized by autoradiography. Residues 650–684 are essential for FIH interaction. (B) Alignment of residues 667–690 (fourth ankyrin repeat) of p105 with the ARD of FEM1β and UACA as well as the HIF-1α hydroxylation site (∗). (C) Interaction between FIH and transfected full-length PK-tagged p105, Asn-678 mutant p105 (N678A), in 293T cells exposed to DMOG (+) or vehicle control (−). EV, empty vector control. Anti-PK immunoprecipitates; coprecipitating FIH is detected by anti-FIH mAb. (D) Coimmunoprecipitation of endogenous FIH with p105 from HeLa cell extract. p105 IP, antibody to the C terminus of p105; CON IP, preimmune serum.

Matthew E. Cockman, et al. Proc Natl Acad Sci U S A. 2006 October 3;103(40):14767-14772.
4.
Fig. 3.

Fig. 3. From: Posttranslational hydroxylation of ankyrin repeats in I?B proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH).

Mass spectrometric analysis of FIH-mediated hydroxylation of p105 and IκBα in vivo. (A) LC/MS spectrum from a tryptic digest of PK-tagged p105 immunopurified from transfected 293T cells. Peaks at m/z = 763.87 and 771.87 correspond to [M + 2H]2+ of unhydroxylated and hydroxylated LLVAAGADVNAQEQK peptides, respectively. Mutations (L668K/R870A) were introduced into p105 to enable LC/MS and MS/MS analysis of tryptic peptides. (B) MS/MS spectra of the m/z 763.87 (Left) and m/z 771.87 (Right) parent ions assign Asn-678 as the site of hydroxylation in p105. A +16-Da shift appears in the y-ion series appearing at y6, corresponding to fragments containing Asn-678. (C) Extracted ion chromatograms illustrating the effect of FIH intervention on IκBα hydroxylation at Asn-244. (Left) Standards for the hydroxylated and unhydroxylated modified tryptic peptides (see Fig. 9 for MS/MS assignments). (Right) Tryptic digest of HA-tagged IκBα immunopurified from transfected 293T cells; FIH overexpression increases, whereas FIH siRNA decreases, the ratio of hydroxylated to unhydroxylated peptide. (∗C indicates derivatization on cysteine; see Experimental Procedures.)

Matthew E. Cockman, et al. Proc Natl Acad Sci U S A. 2006 October 3;103(40):14767-14772.
5.
Fig. 4.

Fig. 4. From: Posttranslational hydroxylation of ankyrin repeats in I?B proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH).

Effects of FIH on IκB/NF-κB complex formation and activity. (A) EMSA of NF-κB DNA-binding activity in nuclear extracts of TNF-α-stimulated HeLa cells. Effect of recombinant IκBα, GST-IκBα, and GST-IκBα incubated in vitro with FIH, FIH plus inhibitor (N-oxalylglycine, NOG), or mutant D201A FIH (mut FIH). Slightly more potent inhibitory effects of hydroxylated IκBα (lane 4) were consistently observed. Arrow indicates the NF-κB DNA-binding activity. IκBα inputs were normalized but not shown. (B) Interaction of recombinant GST-IκBα, pretreated with either wild type or mutant D201A FIH (mut FIH), with 35S-labeled p50 and/or p65 produced by IVTT in wheat germ lysate. Interaction was performed in the presence of BSA, assayed by GST pull-down. Coprecipitating p50 and p65 are detected by autoradiography. GST-IκBα input is normalized but not shown. (C) Association of IκBα with p65 and p50 in cells. U2OS cells were transfected with control or FIH siRNA and stimulated with TNF-α. Anti-IκBα immunoprecipitates were immunoblotted with anti-IκBα, anti-p50, or anti-p65 antibodies. Cells were harvested either at time 0 (untreated), after a 30-min exposure to TNF-α (TNF-α), or at the indicated times (in minutes) after removal of TNF-α. (D) NF-κB DNA-binding activity. EMSA of nuclear extracts from serum-deprived 293T cells transfected with control (−) or FIH-specific (+) siRNA duplexes and treated with TNF-α for 30 min. Arrow indicates the NF-κB DNA-binding activity. (E) NF-κB reporter activity. 293T cells were treated with FIH or control siRNA then transfected with NF-κB (κB LUC) or control (CON LUC) reporter plasmids. Cells were stimulated with TNF-α for 6 h to induce NF-κB activity, and the ability of cotransfected IκBα to suppress reporter gene activity was assessed. Relative luciferase activity is shown as the mean ± 1 SD of triplicate samples from a representative experiment.

Matthew E. Cockman, et al. Proc Natl Acad Sci U S A. 2006 October 3;103(40):14767-14772.

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