Insights into the mechanism and substrate specificity of human lysine -specific demethylase-1

Abstract

Histones are small basic proteins that function to organize DNA in cells. The nucleosomal core particle, the fundamental unit of chromatin, consists of 146-147 base pairs of DNA wound around a complex of two H2A-H2B histone dimers and an H3-H4 tetramer. Histones are subject to a myriad of post-translational modifications, including methylation, phosphorylation, acetylation, and ubiquitination, which function in the regulation of various cell processes, including gene transcription. Lysine-Specific Demethylase-1 (LSD1), a member of the monoamine oxidase (MAO) family of flavoprotein amine oxidases, has been shown to remove methyl groups from lysine residues four and nine of histone H3 (H3K4 and H3K9), as well as lysine 370 of the tumor suppressor protein p53. The enzyme has been shown to play a role in controlling cell differentiation, and its expression correlates with neuroblastoma and prostate cancer progression; thus, selective inhibitors of LSD1 may be beneficial for treatment of various diseases. Thorough characterization of the mechanism and substrate specificity of LSD1 are essential for the development of such inhibitors, as well as for increasing the understanding of transcriptional regulation and cancer proliferation. The substrate specificity of LSD1 has been studied in vitro by utilizing a series of peptide substrates corresponding to the amino acid sequence of the N-terminal tail of histone H3. Recombinant human LSD1 was shown to act specifically on the dimethylated H3K4 residue in vitro, with arginine residues in the peptide substrate being essential for recognition of the substrate by the enzyme. Steady-state and transient kinetic studies have shown that C-H bond cleavage is rate-limiting in oxidation of a peptide substrate by LSD1. Furthermore, the redox potential of LSD1, significantly higher than that of free flavin, demonstrates that the enzyme provides a more favorable environment for flavin-catalyzed oxidation. Finally, evidence suggests that a lysine residue conserved in the MAO family, Lys661 in LSD1, may play a role in enzyme stability. These studies contribute to the overall understanding of the mechanism and substrate-specificity of LSD1