title: Mechanism-based affinity capture of sirtuins.
abstract: The ability to probe for catalytic activities of enzymes and to detect their abundance in complex biochemical contexts has traditionally relied on a combination of kinetic assays and techniques such as western blots that use expensive reagents such as antibodies. The ability to simultaneously detect activity and isolate a protein catalyst from a mixture is even more difficult and currently impossible in most cases. In this manuscript we describe a chemical approach that achieves this goal for a unique family of enzymes called sirtuins using novel chemical tools, enabling rapid detection of activity and isolation of these protein catalysts. Sirtuin deacetylases are implicated in the regulation of many physiological functions including energy metabolism, DNA-damage response, and cellular stress resistance. We synthesized an aminooxy-derivatized NAD(+) and a pan-sirtuin inhibitor that reacts on sirtuin active sites to form a chemically stable complex that can subsequently be crosslinked to an aldehyde-substituted biotin. Subsequent retrieval of the biotinylated sirtuin complexes on streptavidin beads followed by gel electrophoresis enabled simultaneous detection of active sirtuins, isolation and molecular weight determination. We show that these tools are cross reactive against a variety of human sirtuin isoforms including SIRT1, SIRT2, SIRT3, SIRT5, SIRT6 and can react with microbial derived sirtuins as well. Finally, we demonstrate the ability to simultaneously detect multiple sirtuin isoforms in reaction mixtures with this methodology, establishing proof of concept tools for chemical studies of sirtuins in complex biological samples.
authors.affiliation: Department of Pharmacology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10065, USA.
authors.forename: Yana
authors.initials: Y
authors.lastname: Cen
authors.forename: Jessica N
authors.initials: JN
authors.lastname: Falco
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
authors.forename: Dou Yeon
authors.initials: DY
authors.lastname: Youn
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Sauve
mesh_terms: D000107:Acetylation; D002384:Catalysis; D006801:Humans; D015394:Molecular Structure; D037761:Sirtuins / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
chemical_list: D037761:Sirtuins