title: Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor.
abstract: Dynamic methylations and demethylations of histone lysine residues are important for gene regulation and are facilitated by histone methyltransferases and histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2-specific lysine demethylase belonging to the JmjC domain-containing family of histone demethylases (JHDMs). Several studies have linked KDM5B to breast, prostate and skin cancer, highlighting its potential as a drug target. However, most inhibitor studies have focused on other JHDMs, and inhibitors for KDM5B remain to be explored. Here, we report the expression, purification and characterization of the catalytic core of recombinant KDM5B (ccKDM5B, residues 1-769). We show that ccKDM5B, recombinantly expressed in insect cells, demethylates H3K4me3 and H3K4me2 in vitro. The kinetic characterization showed that ccKDM5B has an apparent Michaelis constant (K(m) (app) ) value of 0.5 μm for its trimethylated substrate H3(1-15)K4me3, a considerably increased apparent substrate affinity than reported for related HDMs. Despite the presence of a PHD domain, the catalytic activity was not affected by additional methylation at the H3K9 position, suggesting that in vitro chromatin cross-talk between H3K4 and H3K9 does not occur for ccKDM5B. Inhibition studies of ccKDM5B showed both in vitro and in cell inhibition of ccKDM5B by 2,4-pyridinedicarboxylic acid (2,4-PDCA) with a potency similar to that reported for the HDM KDM4C. Structure-guided sequence alignment indicated that the binding mode of 2,4-PDCA is conserved between KDM4A/C and KDM5B.
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
authors.forename: Line H
authors.initials: LH
authors.lastname: Kristensen
authors.forename: Anders L
authors.initials: AL
authors.lastname: Nielsen
authors.forename: Charlotte
authors.initials: C
authors.lastname: Helgstrand
authors.forename: Michael
authors.initials: M
authors.lastname: Lees
authors.forename: Paul
authors.initials: P
authors.lastname: Cloos
authors.forename: Jette S
authors.initials: JS
authors.lastname: Kastrup
authors.forename: Kristian
authors.initials: K
authors.lastname: Helin
authors.forename: Lars
authors.initials: L
authors.lastname: Olsen
authors.forename: Michael
authors.initials: M
authors.lastname: Gajhede
mesh_terms: D000595:Amino Acid Sequence; D016367:Baculoviridae; D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012516:Osteosarcoma / Q000378:metabolism / Q000473:pathology; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D002843:Chromatin; D004791:Enzyme Inhibitors; D006657:Histones; D009687:Nuclear Proteins; D020033:Protein Isoforms; D011725:Pyridines; D011994:Recombinant Proteins; D012097:Repressor Proteins; C055293:2,4-pyridinedicarboxylic acid; D056484:Jumonji Domain-Containing Histone Demethylases; C119611:KDM5B protein, human; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D008239:Lysine