title: Azalysine analogues as probes for protein lysine deacetylation and demethylation.
abstract: Reversible lysine acetylation and methylation regulate the function of a wide variety of proteins, including histones. Here, we have synthesized azalysine-containing peptides in acetylated and unacetylated forms as chemical probes of the histone deacetylases (HDAC8, Sir2Tm, and SIRT1) and the histone demethylase, LSD1. We have shown that the acetyl-azalysine modification is a fairly efficient substrate for the sirtuins, but a weaker substrate for HDAC8, a classical HDAC. In addition to deacetylation by sirtuins, the acetyl-azalysine analogue generates a novel ADP-ribose adduct that was characterized by mass spectrometry, Western blot analysis, and nuclear magnetic resonance spectroscopy. This peptide-ADP-ribose adduct is proposed to correspond to a derailed reaction intermediate, providing unique evidence for the direct 2'-hydroxyl attack on the O-alkylimidate intermediate that is formed in the course of sirtuin catalyzed deacetylation. An unacetylated azalysine-containing H3 peptide proved to be a potent inhibitor of the LSD1 demethylase, forming an FAD adduct characteristic of previously reported related structures, providing a new chemical probe for mechanistic analysis.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, United States.
authors.forename: Blair C R
authors.initials: BC
authors.lastname: Dancy
authors.forename: Shonoi A
authors.initials: SA
authors.lastname: Ming
authors.forename: Romeo
authors.initials: R
authors.lastname: Papazyan
authors.forename: Christine A
authors.initials: CA
authors.lastname: Jelinek
authors.forename: Ananya
authors.initials: A
authors.lastname: Majumdar
authors.forename: Yan
authors.initials: Y
authors.lastname: Sun
authors.forename: Beverley M
authors.initials: BM
authors.lastname: Dancy
authors.forename: William J
authors.initials: WJ
authors.lastname: Drury
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Cotter
authors.forename: Sean D
authors.initials: SD
authors.lastname: Taverna
authors.forename: Philip A
authors.initials: PA
authors.lastname: Cole
mesh_terms: D000107:Acetylation; D001372:Aza Compounds / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006655:Histone Deacetylases / Q000737:chemistry / Q000378:metabolism*; D056466:Histone Demethylases / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D008745:Methylation; D015394:Molecular Structure; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001372:Aza Compounds; D005456:Fluorescent Dyes; D010455:Peptides; D056466:Histone Demethylases; D006655:Histone Deacetylases; D008239:Lysine