Class III HDACs, also known as sirtuins, couple deacetylation of the peptide/protein substrate to cleavage of NAD+ to form nicotinamide and O-acetyl-ADP-ribose. The mammalian sirtuins comprise 7 members, termed Sirt1-7, which share the NAD+-binding catalytic domain, but differ in N- and C-termini, subcellular localization, substrate preference, and ultimately in biological function. Poly(ADP-ribose) polymerases (PARPs) also cleave NAD+ to generate nicotinamide, but instead covalently attach the ADP-ribose moiety either to a peptide substrate or to other preexisting mono- or poly(ADP-ribose) units. The assays most commonly used for measuring sirtuin and PARP activities in high throughput employ chemically modified substrates. The most widely used method for quantifying sirtuin activity utilizes a fluorophore-modified acetylated peptide substrate that, upon deacetylation, is cleaved by a protease to liberate the fluorophore to generate a fluorescent signal. Although this method has been used to discover activators of Sirt1, such as resveratrol and related compounds, interpretation of the physiological relevance of this activation is complicated by the observation that these compounds fail to stimulate Sirt1 activity on most native acetylated peptide substrates. A potential explanation for these contradictory results is that stimulation of Sirt1 activity by resveratrol-like compounds is dependent upon the sequence of the acetylated substrate, but a high-throughput assay with unlabeled acetylated peptide substrate is needed to define such substrate selectivity.Here we describe assays for Sirtuin and PARP family members that employ unmodified substrates. The assays employ purified recombinant PNC1 nicotinamidase to measure nicotinamide generated upon cleavage of NAD+ during sirtuin-mediated deacetylation or PARP-mediated ADP-ribosylation. The nicotinamidase converts nicotinamide to nicotinic acid and ammonia, and the ammonia is detected with a fluorogenic reagent, o-phthalaldehyde, to produce a fluorescent signal. We show that the Sirtuin assay method 1) detects activity of multiple Sirt family members, 2) distinguishes sirtuin activity on different acetylated substrates, and 3) does not detect activation of Sirt1 by resveratrol on an acetylated H3K9 peptide substrate, in contrast to the stimulation observed with fluorophore-modified peptide substrates. Thus, this approach enables flexibility in choice of sirtuin isoform and peptide substrate, and enables extensive characterization of substrate dependence of sirtuin modulators. In addition, we demonstrate that the utility of the PARP assay method for sensitive detection of DNA damage-dependent PARP1 and PARP2 auto-PARylation activities.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 216. doi:1538-7445.AM2012-216

©2012 American Association for Cancer Research
2012