Journal: The Journal of Biological Chemistry

Article Title: Activity-dependent Regulation of Histone Lysine Demethylase KDM1A by a Putative Thiol/Disulfide Switch *

doi: 10.1074/jbc.M116.734426

Figure Lengend Snippet: FAD-dependent amine oxidases generate H2O2 to deaminate their substrates. The nuclear (nuc) amine oxidases KDM1A/B remove methyl groups from lysine residues on histone H3, as well as other nuclear proteins. The mitochondrial (mito) amine oxidases MAO-A/B catabolize small molecule amines such as neurotransmitters. All four enzymes share considerable structural homology and sequence identity (% seq ID) in their amine oxidase catalytic domains.

Article Snippet: Membranes were probed for KDM1A (1:5000, Cell Signaling, catalogue no. 2184S, lot no. 1), KDM1B (1:5000, Abcam catalogue no. ab52001, lot no. GR81275-1), MAO-A (1:1000, Santa Cruz, catalogue no. sc-20156, lot no. D2710) or MAO-B (1:1000, Abcam catalogue no. ab125010, lot no. GR188691-2).

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Activity-dependent Regulation of Histone Lysine Demethylase KDM1A by a Putative Thiol/Disulfide Switch *

doi: 10.1074/jbc.M116.734426

Figure Lengend Snippet: Chemical structures of FAD-directed and thiol-reactive KDM1A inhibitors. Thiol-reactive probes form disulfide bonds that are readily reversed in reducing environments, or essentially irreversible thioether bonds.

Article Snippet: Membranes were probed for KDM1A (1:5000, Cell Signaling, catalogue no. 2184S, lot no. 1), KDM1B (1:5000, Abcam catalogue no. ab52001, lot no. GR81275-1), MAO-A (1:1000, Santa Cruz, catalogue no. sc-20156, lot no. D2710) or MAO-B (1:1000, Abcam catalogue no. ab125010, lot no. GR188691-2).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Activity-dependent Regulation of Histone Lysine Demethylase KDM1A by a Putative Thiol/Disulfide Switch *

doi: 10.1074/jbc.M116.734426

Figure Lengend Snippet: KDM1A is reversibly inhibited by thiol-reactive small molecules. A, in vitro enzymatic assays with recombinant GST-tagged KDM1A/B or MAO-A/B reveal potent inhibition of KDM1A by reversible and essentially irreversible thiol-reactive small molecules. Apparent IC50 values reported in μm for 20 min (KDM1A) and 60 min (KDM1B, MAO-A/B) reactions. B, inhibition of KDM1A by disulfide forming thiol-reactive small molecules (disulfiram, 2,2′-DPS) is reversible by addition of DTT. Covalent modification by Np-Mal or RN1 is not reversible by DTT. Compounds (1 μm) were pre-incubated with KDM1A for 10 min prior to reduction, and demethylation of H3K4me2 starting material was detected by LC-MS. Error bars indicate S.D.; *, p < 0.05 by 2-tailed t test with correction for multiple comparisons. C, 6×His-tagged KDM1A is inhibited by thiol-reactive inhibitors. Enzyme activity was monitored by detection of catalytically generated H2O2 with an HRP-coupled assay. D–F, thiol-reactive compounds inhibit activity of full-length KDM1A immunoprecipitated from HeLa cells. D, thiol-reactive inhibitors reduce catalytically-generated H2O2 produced by immunoprecipitated KDM1A as measured by HRP-coupled detection. Error bars indicate S.D. in n = 2 replicate reactions; ***, p < 0.001 by 1-way ANOVA with correction for multiple comparisons. E, Western blot analysis of the depletion of H3K4me2 starting material by immunoprecipitated KDM1A. For both cell-free assays, immunoprecipitated KDM1A was pre-incubated with inhibitor for 10 min prior to demethylation of H3K4me2 peptide substrate for 1 h. F, quantification of Western blot data of immunoprecipitated KDM1A inhibition by thiol-reactive compounds (10 μm) in n = 4 assays from biological duplicates. Error bars indicate S.D.; **, p < 0.01 by one-way ANOVA with correction for multiple comparisons.

Article Snippet: Membranes were probed for KDM1A (1:5000, Cell Signaling, catalogue no. 2184S, lot no. 1), KDM1B (1:5000, Abcam catalogue no. ab52001, lot no. GR81275-1), MAO-A (1:1000, Santa Cruz, catalogue no. sc-20156, lot no. D2710) or MAO-B (1:1000, Abcam catalogue no. ab125010, lot no. GR188691-2).

Techniques: In Vitro, Recombinant, Inhibition, Modification, Incubation, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Generated, Immunoprecipitation, Produced, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Activity-dependent Regulation of Histone Lysine Demethylase KDM1A by a Putative Thiol/Disulfide Switch *

doi: 10.1074/jbc.M116.734426

Figure Lengend Snippet: MALDI-TOF analysis of KDM1A tryptic digests identifies Cys-600 as a site of Biotin-Mal labeling. A, recombinant KDM1A (5 μm) was pre-reduced, desalted with buffer exchange, and labeled with various equivalents of Biotin-Mal for 30, 60, or 90 min. The extent of thiol labeling was monitored by blotting with a streptavidin-HRP conjugate. B, inhibition of recombinant KDM1A by Biotin-Mal labeling was determined by Western blot analysis of the depletion of H3K4me2 starting material. Recombinant KDM1A was pre-incubated with Biotin-Mal for 30 min prior to demethylation of H3K4me2 peptide substrate for 1 h. C, pre-treatment of recombinant KDM1A with H2O2 or disulfiram followed by desalting with buffer exchange blocks labeling with Biotin-Mal, as measured by blotting with a streptavidin-HRP conjugate. D, crystal structure (PDB ID: 2HKO) indicating the locations of the nine cysteine residues of KDM1A. Cys-195 is in the SWIRM domain (pink), Cys-573, -600, -618, and -623 are in the FAD-binding amine oxidase domain (light blue), Cys-360, -665, and -727 are in the substrate-binding amine oxidase domain (dark blue), and Cys-491 is in the tower domain (teal). E, tabular summary of cysteine-containing tryptic KDM1A peptides identified by MALDI-TOF. Recombinant KDM1A (5 μm) was pre-reduced and desalted with buffer exchange, then labeled with 10 μm Biotin-Mal for 20 min prior to in-gel alkylation and digestion.

Article Snippet: Membranes were probed for KDM1A (1:5000, Cell Signaling, catalogue no. 2184S, lot no. 1), KDM1B (1:5000, Abcam catalogue no. ab52001, lot no. GR81275-1), MAO-A (1:1000, Santa Cruz, catalogue no. sc-20156, lot no. D2710) or MAO-B (1:1000, Abcam catalogue no. ab125010, lot no. GR188691-2).

Techniques: Labeling, Recombinant, Buffer Exchange, Inhibition, Western Blot, Incubation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Activity-dependent Regulation of Histone Lysine Demethylase KDM1A by a Putative Thiol/Disulfide Switch *

doi: 10.1074/jbc.M116.734426

Figure Lengend Snippet: KDM1A forms a putative intramolecular disulfide bond. A, crystal structures of the FAD-binding amine oxidase domains of KDM1A/B and MAO-A/B indicate a unique pair of proximal cysteine residues in KDM1A which may be capable of disulfide bond formation (respective PDB accession codes: 2HKO, 4GUU, 2BXR, and 2XFU, only residues in the amine oxidase domains are displayed). Cys-600 is ∼5 Å away from Cys-618 in the crystal structure of KDM1A, and this pair of cysteines abuts the Rossmann fold responsible for FAD cofactor binding. B, MALDI-TOF analysis of KDM1A tryptic digests reveals Biotin-Mal labeling of Cys-600 is blocked when KDM1A is pre-treated with H2O2. C, KDM1A is readily labeled with 10 μm Biotin-Mal in SH-SY5Y cell lysate and pulled down on streptavidin-agarose beads, and labeling is blocked by pulse pre-treatment (10 min) of intact cells with 1 mm H2O2. Other FAD-dependent amine oxidases do not appear to be as thiol-reactive. D, dose response curve of pulse pre-treatment (10 min) of intact SH-SY5Y cells with H2O2. E, quantification of KDM1A Biotin-Mal labeling, n = 2 assays from biological duplicates. Error bars indicate S.D.

Article Snippet: Membranes were probed for KDM1A (1:5000, Cell Signaling, catalogue no. 2184S, lot no. 1), KDM1B (1:5000, Abcam catalogue no. ab52001, lot no. GR81275-1), MAO-A (1:1000, Santa Cruz, catalogue no. sc-20156, lot no. D2710) or MAO-B (1:1000, Abcam catalogue no. ab125010, lot no. GR188691-2).

Techniques: Binding Assay, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Activity-dependent Regulation of Histone Lysine Demethylase KDM1A by a Putative Thiol/Disulfide Switch *

doi: 10.1074/jbc.M116.734426

Figure Lengend Snippet: Recombinant KDM1A is inhibited by hydrogen peroxide. A, exogenously applied H2O2 inhibits recombinant KDM1A with an apparent IC50 value of 630 nm, error bars indicate S.D. B, inhibition of recombinant KDM1A by H2O2 or dimedone plus H2O2 is reversible by the addition of DTT. KDM1A was pre-incubated with dimedone (100 μm) or vehicle for 10 min, then treated with H2O2 (100 μm or 250 μm) for 10 min prior to reduction. Error bars indicate S.D.; ***, p < 0.001 by 2-tailed t test with correction for multiple comparisons. For both A and B, demethylation of H3K4me2 starting material was detected by LC-MS after 1-h reaction.

Article Snippet: Membranes were probed for KDM1A (1:5000, Cell Signaling, catalogue no. 2184S, lot no. 1), KDM1B (1:5000, Abcam catalogue no. ab52001, lot no. GR81275-1), MAO-A (1:1000, Santa Cruz, catalogue no. sc-20156, lot no. D2710) or MAO-B (1:1000, Abcam catalogue no. ab125010, lot no. GR188691-2).

Techniques: Recombinant, Inhibition, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Activity-dependent Regulation of Histone Lysine Demethylase KDM1A by a Putative Thiol/Disulfide Switch *

doi: 10.1074/jbc.M116.734426

Figure Lengend Snippet: Activity-dependent regulation of KDM1A. A, demethylation of H3K4me2 peptide substrate by recombinant KDM1A is enhanced in the presence of DTT (1 mm), or B, the enzyme catalase (10 μg/ml). For both A and B, demethylation of H3K4me2 starting material was detected by LC-MS. Error bars indicate S.D.; *, p < 0.05; ***, p < 0.001 by one-way ANOVA with correction for multiple comparisons. C, labeling of recombinant KDM1A with Biotin-Mal (25 μm) is reduced with extended demethylation reaction durations. Addition of the FAD-directed inhibitor RN1 (25 μm) blocks the time-dependent reduction in labeling. D, quantification of Biotin-Mal labeling as measured by blotting with a streptavidin-HRP conjugate and normalized to total recombinant KDM1A over three replicate experiments. Error bars indicate S.D.; *, p < 0.05; **, p < 0.01, by 2-tailed t test with correction for multiple comparisons. E, labeling of KDM1A in SH-SY5Y cells by Biotin-Mal (25 μm) is reduced with extended incubation in PBS. Addition of the FAD-directed inhibitor RN1 (25 μm) blocks the time-dependent reduction in labeling. F, quantification of Biotin-Mal labeling as measured by pull down with streptavidin-agarose beads and Western conjugate and normalized to total recombinant KDM1A over three replicate experiments. Error bars indicate S.D.; *, p < 0.05 by 2-tailed t test with correction for multiple comparisons.

Article Snippet: Membranes were probed for KDM1A (1:5000, Cell Signaling, catalogue no. 2184S, lot no. 1), KDM1B (1:5000, Abcam catalogue no. ab52001, lot no. GR81275-1), MAO-A (1:1000, Santa Cruz, catalogue no. sc-20156, lot no. D2710) or MAO-B (1:1000, Abcam catalogue no. ab125010, lot no. GR188691-2).

Techniques: Activity Assay, Recombinant, Liquid Chromatography with Mass Spectroscopy, Labeling, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Activity-dependent Regulation of Histone Lysine Demethylase KDM1A by a Putative Thiol/Disulfide Switch *

doi: 10.1074/jbc.M116.734426

Figure Lengend Snippet: Working model of KDM1A activity-dependent regulation by a thiol/disulfide redox switch.

Article Snippet: Membranes were probed for KDM1A (1:5000, Cell Signaling, catalogue no. 2184S, lot no. 1), KDM1B (1:5000, Abcam catalogue no. ab52001, lot no. GR81275-1), MAO-A (1:1000, Santa Cruz, catalogue no. sc-20156, lot no. D2710) or MAO-B (1:1000, Abcam catalogue no. ab125010, lot no. GR188691-2).

Techniques: Activity Assay