title: Purification, Biochemical Analysis, and Structure Determination of JmjC Lysine Demethylases.
abstract: Jumonji C (JmjC) lysine demethylases (KDMs) catalyze the site- and state-specific demethylation of lysine residues in histone and nonhistone protein substrates. These enzymes have been implicated in diverse genomic processes, including epigenetic gene regulation, DNA damage response, DNA replication, and regulation of heterochromatin structure. In addition, a number of JmjC KDMs contribute to the incidence of numerous cancers, rendering them targets for the development of novel chemotherapeutic drugs. Using the JMJD2 KDM subfamily as representative examples, this chapter outlines strategies for purifying highly active, recombinant JmjC KDMs lacking inhibitory transition metal ions, characterizing kinetic parameters of these enzymes using a coupled fluorescent assay, and determining crystal structures of the enzymes in complex with methylated histone peptides. Together, these approaches provide a foundation for structural and biochemical characterization of the JmjC KDMs and facilitate efforts to identify small molecule inhibitors through high-throughput screening and structure-guided design.
authors.affiliation: University of Michigan, Ann Arbor, MI, United States.
authors.forename: S
authors.initials: S
authors.lastname: Krishnan
authors.affiliation: University of Michigan, Ann Arbor, MI, United States. Electronic address: rtrievel@umich.edu.
authors.forename: R C
authors.initials: RC
authors.lastname: Trievel
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D006657:Histones / Q000378:metabolism; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D006657:Histones; D011994:Recombinant Proteins; D056484:Jumonji Domain-Containing Histone Demethylases; C521694:KDM4D protein, human; C494424:KDM4A protein, human
keywords: Chromatin; Epigenetics; Histones; Lysine methylation; Posttranslational modifications; Protein lysine demethylase