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November 01, 2011 (Vol. 31, No. 19)

Detection of Lysine Deacetylase Activities

Bioluminescent Assays Offer Distinct Advantages over Fluorescent Approaches

  • Histone deacetylase (HDAC) enzymes play a critical role in normal gene-regulation events and the maintenance of homeostasis. However, their dysregulation has been implicated in a variety of disease states, including several forms of cancer, congestive heart failure, diabetes, inflammation, and neurological disorders. Because of the early success of first-generation HDAC inhibitors in both basic research and clinical applications, there is strong continuing interest in developing more potent and selective compounds.

    Unfortunately, existing research tools for characterization of HDAC function suffer from poor sensitivity, detection interferences, or are time-consuming and expensive to apply. In this article, we describe a simple, robust, and sensitive bioluminescent assay method that measures the activity of various HDAC enzyme isoforms from recombinant or cell-based sources.

  • Two Enzymatic Mechanisms

    The histone deacetylase family of enzymes consists of 18 members that are organized based upon homology with their yeast counterparts. This family is divided into four main groups: the zinc-dependent class I (HDACs 1, 2, 3, and 8), class IIa (HDACs 4, 5, 7, and 9) and class IIb (HDACs 6 and 10) enzymes; and the NAD+-dependent class III sirtuins (Sirts 1–7).

    HDAC 11 shares features from both class I and II HDACs and is often grouped into a separate class IV. Although mechanistically distinct, both the zinc- and NAD+-dependent enzymes can remove ε-acetyl moieties on lysine residues from a diverse set of cellular proteins. Nuclear HDACs and sirtuins are capable of deacetylating N-terminal histone tails, resulting in a condensed chromatin structure and typically repressed gene transcription. However, cytosolic and mitochondrial deacetylases are also known to regulate numerous nonhistone proteins, leading to diverse biological effects.

  • Assay Design and Procedure

    Promega’s HDAC-Glo™ I/II and SIRT-Glo™ Assays are based on optimized, acetylated peptide substrates derived from sequences found in histone 4 and p53 proteins, respectively, which are conjugated to aminoluciferin. These chemically engineered peptides do not react with the developer or luciferase enzymes prior to the specific HDAC or sirtuin deacetylation event. Upon deacetylation, the de-protected substrate undergoes specific proteolytic cleavage by the developer reagent to yield aminoluciferin and the peptide.

    Liberated aminoluciferin product is then quantified using Ultra-Glo™ recombinant firefly luciferase. These enzymatic reactions occur virtually simultaneously to produce “glow-type” luminescence upon addition of a single reagent.

    The “add-mix-measure” reagents are created by simply rehydrating the lyophilized HDAC-Glo I/II or SIRT-Glo Substrate with buffer, then adding the developer reagent. The SIRT-Glo Substrate also contains the necessary NAD+ co-factor. These complete reagents can be added directly to the deacetylase source in a 1:1 ratio, followed by a brief incubation (15–45 minutes) to achieve signal steady state. This steady-state signal is stable for several hours, allowing additional flexibility for automation and processing multiple assay plates.

  • Pan-Enzyme Utility

    Click Image To Enlarge +
    Figure 1. Broad utility of HDAC-Glo I/II and SIRT-Glo deacetylase activity assays: Recombinant HDAC or sirtuin was diluted to 400 or 800 ng/mL, respectively, in either HDAC-Glo I/II or SIRT-Glo Buffer, then twofold serially diluted in a 96-well plate. An equal volume of either HDAC-Glo I/II or SIRT-Glo Reagent was added, and luminescence was measured after 15 minutes at room temperature. The recombinant enzymes were obtained from various vendors (HDACs 1–3, 6, and 8 from Enzo; HDAC 4 from Cayman Chemical; HDAC 7 from EMD Millipore; HDACs 5, 9–11 and Sirts 1–3 from SignalChem). Data was plotted using GraphPad Prism® software. Data represents the mean ± standard deviation of four samples.

    To demonstrate broad isozyme responsiveness, recombinant deacetylases were titrated and analyzed with either the HDAC-Glo I/II or SIRT-Glo Assay (Figure 1). The HDAC-Glo I/II Assay was responsive to all members of the class I and II HDACs, including HDAC 11. Subsequent experiments using pharmacologically relevant HDAC inhibitors produced IC50 profiles consistent with values reported in the literature.

    The SIRT-Glo Assay measured activity from sirtuins 1, 2, and 3 that was NAD+-dependent and nicotinamide-inhibitable (data not shown). Both assays achieved dynamic signal-to-noise ratios that remained linear over several orders of magnitude of enzyme concentration, thus reducing the need for extensive enzyme titrations or high enzyme content per well.

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