title: Development of substrate-selective probes for affinity pulldown of histone demethylases.
abstract: JmjC-domain containing histone demethylases (JHDMs) play critical roles in many key cellular processes and have been implicated in multiple disease conditions. Each enzyme within this family is known to have a strict substrate scope, specifically the position of the lysine within the histone and its degree of methylation. While much progress has been made in determining the substrates of each enzyme, new methods with which to systematically profile each histone mark are greatly needed. Novel chemical tools have the potential to fill this role and, furthermore, can be used as probes to answer fundamental questions about these enzymes and serve as potential therapeutic leads. In this work, we first investigated three small-molecule probes differing in the degree of "methylation state" and their differential bindings to JHDM1A (an H3K36me1/2 demethylase) using a fluorescence polarization-based competition assay. We then applied this specificity toward the "methylation state" and combined it with specificity toward lysine position in the design and synthesis of a peptidic probe targeting H3K36me2 JHDMs. The probe is further functionalized with a benzophenone cross-linking moiety and a biotin for affinity purification. Results showed binding of the peptidic probe to JHDM1A and specific enrichment of this protein in the presence of its native histone substrates. Affinity purification pulldown experiments from nuclear lysate coupled with mass spectrometry revealed the capability of the probe to pull out and enrich JHDMs along with other epigenetic proteins and transcriptional regulators.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Marholz
authors.forename: Le
authors.initials: L
authors.lastname: Chang
authors.forename: William M
authors.initials: WM
authors.lastname: Old
authors.forename: Xiang
authors.initials: X
authors.lastname: Wang
mesh_terms: D000345:Affinity Labels; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D005454:Fluorescence Polarization; D056466:Histone Demethylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D008745:Methylation; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D000345:Affinity Labels; D004791:Enzyme Inhibitors; D006657:Histones; D015335:Molecular Probes; D056466:Histone Demethylases