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

Abstract

Lysine demethylation of proteins such as histones is catalyzed by several classes of enzymes, including the FAD-dependent amine oxidases KDM1A/B. The KDM1 family is homologous to the mitochondrial monoamine oxidases MAO-A/B and produces hydrogen peroxide in the nucleus as a byproduct of demethylation. Here, we show KDM1A is highly thiol-reactive in vitro and in cellular models. Enzyme activity is potently and reversibly inhibited by the drug disulfiram and by hydrogen peroxide. Hydrogen peroxide produced by KDM1A catalysis reduces thiol labeling and inactivates demethylase activity over time. MALDI-TOF mass spectrometry indicates that hydrogen peroxide blocks labeling of cysteine 600, which we propose forms an intramolecular disulfide with cysteine 618 to negatively regulate the catalytic activity of KDM1A. This activity-dependent regulation is unique among histone-modifying enzymes but consistent with redox sensitivity of epigenetic regulators.

Keywords: chemical biology; disulfide; drug screening; enzyme inactivation; flavoprotein; histone demethylase; hydrogen peroxide; reactive oxygen species (ROS); redox regulation; thiol.

FIGURE 1.

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.

FIGURE 2.

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.

FIGURE 3.

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.

FIGURE 4.

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.

FIGURE 5.

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.

FIGURE 6.

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.

FIGURE 7.

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.

FIGURE 8.

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