Overview of glutathione-dependent formaldehyde metabolism.

<p>(A) Summary scheme of glutathione-dependent formaldehyde metabolism. Formaldehyde (HCHO) reacts with glutathione (GSH) via its nucleophilic thiol group to form <i>S</i>-hydroxymethylglutathione (HMG), which is a substrate of glutathione-dependent alcohol dehydrogenase (ADH, ADH5 in humans). The product, <i>S</i>-formylglutathione, is then further metabolised by <i>S</i>-formylglutathione hydrolase to give formate and GSH. The reaction of HCHO and GSH, i.e. the first step in GSH-dependent metabolism, occurs spontaneously in aqueous solution; however, the reaction might also be catalysed by GFA (and homologues in other organisms, e.g. CENPV in humans[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145085#pone.0145085.ref013" target="_blank">13</a>]). There is also evidence, at least <i>in vitro</i>, that GSH can react with HCHO to form cyclised adducts[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145085#pone.0145085.ref010" target="_blank">10</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145085#pone.0145085.ref012" target="_blank">12</a>]. (B) Views of X-ray crystal structures of GFA from <i>Paracoccus denitrificans</i> (PDB IDs: 1X6M and 1XA8[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145085#pone.0145085.ref014" target="_blank">14</a>]). The GFA domain contains two zinc binding sites; one zinc ion is coordinated by four cysteinyl thiols (C31, C33, C99 and C102) in a tetrahedral geometry, whereas the other zinc ion is coordinated by three cysteinyl thiols (C52, C54 and C57) in a trigonal planar geometry. Crystallographic studies have proposed that GSH binding induces translocation of the second zinc ion (circles)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145085#pone.0145085.ref014" target="_blank">14</a>].</p

GFA is a GSH-binding protein that induces an increase in EXSY correlation intensities between GSH and HMG, but does not catalyse HMG formation / fragmentation.

<p>(A) 2D EXSY spectra of equilibrium mixtures of GSH (initial concentration 15 mM) and HMG in the absence (left) and presence (right) of GFA (20 μM). Mixing time (τ_m) = 400 ms. EXSY-correlation intensities between GSH and HMG are increased in the presence of GFA. (B) 1D EXSY spectra of equilibrium mixtures of GSH (initial concentration 15 mM) and HMG in the presence of GFA (20 μM) conducted at different mixing times (τ_m = 32–300 ms). Irradiation (inversion) of a β-cysteinyl resonance of HMG (δ_H 2.95 ppm) induced an exchange correlation at δ_H 2.87 ppm, corresponding to the β-cysteinyl resonance of GSH, which increased in intensity at longer mixing times. (C) Graph showing the intensity of the GSH cross-peak relative to the inverted HMG resonance in the absence and presence of GFA at different mixing times, using either NOESY or ROESY pulse sequences. τ_m = 4–400 ms. (D) Bar graph showing the intensity of the GSH 1D EXSY-correlation relative to the irradiated HMG resonance in the absence (blue) and presence (green) of GFA (τ_m = 80 ms). The build-up rates of the 1D EXSY analyses (note: a τ_m of 80 ms is within the linear range of the EXSY build-up curves, Fig 2C) correlate with the rates of GSH/HMG exchange at equilibrium. Therefore, the observed increase in correlation intensity in the presence of GFA implies an increase in GSH/HMG inter-conversion rate. (E) Non-denaturing MS analyses of GSH binding to GFA. Two new peaks corresponding to the masses of monomeric GFA (with two zinc ions in complex) bound to one and two GSH molecules respectively were observed upon incubation with GSH (4 equivalents, right). (F) Binding curve of GSH binding to GFA obtained using waterLOGSY. Selective irradiation of the solvent H₂O ¹H resonance results in magnetisation transfer to GSH, resulting in the emergence of GSH ¹H resonances with opposite sign to the irradiated H₂O resonance. The (negative) intensities of the GSH resonances are linearly dependent on the GSH concentration (blue). Addition of GFA results in a slower net tumbling rate for GSH in solution due to binding with GFA. The slower tumbling rate leads to ‘(more) positive’ GSH resonance intensities as a function of the extent of ligand binding (green). Subtraction of the intensities in the absence (blue) and presence (green) of GFA gives a normalised binding curve (orange, K_D value of roughly 500 μM assuming binding of one GSH molecule per GFA subunit). The experiments were carried out at 280 K. τ_m = 1 s. (G) Graph showing production of HMG from mixtures of GSH (13.3 mM) and HCHO (13.3 mM) in the absence (blue) and presence (green) of GFA (16 μM) in BisTris buffer pH 6.0. GFA does not affect the initial HMG formation rate.</p

Selective Inhibitors of the JMJD2 Histone Demethylases: Combined Nondenaturing Mass Spectrometric Screening and Crystallographic Approaches

Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of <i>N</i>^ε-methylated lysine residues in histones. Here we report studies on the inhibition of the JMJD2 subfamily of histone demethylases, employing binding analyses by nondenaturing mass spectrometry (MS), dynamic combinatorial chemistry coupled to MS, turnover assays, and crystallography. The results of initial binding and inhibition assays directed the production and analysis of a set of <i>N</i>-oxalyl-d<i>-</i>tyrosine derivatives to explore the extent of a subpocket at the JMJD2 active site. Some of the inhibitors were shown to be selective for JMJD2 over the hypoxia-inducible factor prolyl hydroxylase PHD2. A crystal structure of JMJD2A in complex with one of the potent inhibitors was obtained; modeling other inhibitors based on this structure predicts interactions that enable improved inhibition for some compounds

Selective Small Molecule Probes for the Hypoxia Inducible Factor (HIF) Prolyl Hydroxylases

The hypoxia inducible factor (HIF) system is central to the signaling of low oxygen (hypoxia) in animals. The levels of HIF-α isoforms are regulated in an oxygen-dependent manner by the activity of the HIF prolyl-hydroxylases (PHD or EGLN enzymes), which are Fe­(II) and 2-oxoglutarate (2OG) dependent oxygenases. Here, we describe biochemical, crystallographic, cellular profiling, and animal studies on PHD inhibitors including selectivity studies using a representative set of human 2OG oxygenases. We identify suitable probe compounds for use in studies on the functional effects of PHD inhibition in cells and in animals