Determination of Protein Lysine Deacetylation

Danielle J.P. Ellis1, Zhigang Yuan1, Edward Seto1

1 H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.12
DOI:  10.1002/0471140864.ps1412s54
Online Posting Date:  November, 2008
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Histone deacetylases (HDACs) are members of a diverse family of enzymes that catalyze the removal of an acetyl moiety from an acetyl‐lysine‐containing substrate. HDACs target a variety of substrates, including histone and nonhistone proteins, to mediate alterations in protein localization, stability, and activity. In addition, HDACs have been shown to modulate changes in gene expression, primarily through the recruitment of transcriptional cofactors to promoter regions. Mammalian HDACs are organized into distinct classes based on their homology to yeast HDACs. Classes I, II and IV HDACs are structurally and catalytically similar, whereas, class III HDACs require NAD+ as a cofactor in the deacetylation reaction. This unit provides guidance for choosing and preparing a substrate suitable for assaying an HDAC of interest and describes key protocols necessary for assaying HDAC activity. Curr. Protoc. Protein Sci. 54:14.12.1‐14.12.14. © 2008 by John Wiley & Sons, Inc.

Keywords: HDAC; protein deacetylation; histone deacetylation

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Histone Deacetylase Assay Using Radiolabeled Core Histones
  • Alternate Protocol 1: Histone Deacetylase Assay Using Radiolabeled Peptides
  • Alternate Protocol 2: HDAC Assay Using a Colorimetric Substrate
  • Support Protocol 1: Radiolabeling of Core Histones as Substrates for Deacetylation Assays
  • Support Protocol 2: Radiolabeling Peptides for use as Substrates for HDAC Deacetylation Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Histone Deacetylase Assay Using Radiolabeled Core Histones

  • 5× HDAC buffer: 50 mM Tris⋅Cl, pH 8.0 (see appendix 2E)/750 mM NaCl/50% glycerol; store up to 1 year at −20°C
  • [3H]‐labeled core histones ( protocol 4)
  • HDAC sample (see )
  • HDAC inhibitor stock solutions, optional: 3.3 mM trichostatin A (TSA; for class I and II HDACs) or 2 M nicotinamide (for class III HDACs, also known as sirtuins)
  • 50 mM nicotinamide adenine dinucleotide (NAD+) cofactor stock solution (for class III HDAC assay only)
  • Stop solution: 1 M HCl/0.4 M acetic acid
  • Ethyl acetate
  • Scintillation fluid
  • Scintillation vials

Alternate Protocol 1: Histone Deacetylase Assay Using Radiolabeled Peptides

  • [3H]‐labeled peptide ( protocol 5)
  • GST‐SIRT1 or control GST protein (e.g., see unit 6.6)

Alternate Protocol 2: HDAC Assay Using a Colorimetric Substrate

  • HDAC sample (see ) and (optional) heat‐inactivated (5 min at 95°C) HDAC sample
  • HDAC inhibitor stock solutions, optional: 3.3 mM trichostatin A (TSA; for class I and II HDACs) or 2 M nicotinamide (for class III HDACs, also known as sirtuins)
  • 10× HDAC assay buffer (Biovision)
  • HDAC substrate solution: 10 mM Boc‐Lys(Ac)‐pNA (Biovision)
  • Lysine developer (Biovision)
  • U‐bottom 96‐well plates
  • Microplate spectrophotometer (ELISA reader)

Support Protocol 1: Radiolabeling of Core Histones as Substrates for Deacetylation Assays

  • HeLa cells, confluent on 150‐mm plate
  • 1× phosphate‐buffered saline (PBS; appendix 2E), 37°C
  • Mix A (see recipe)
  • Mix B (see recipe)
  • 2 M sodium butyrate (NaB)
  • NIB buffer: 1% Nonidet P‐40 in IB buffer (see recipe)
  • IB Buffer (see recipe)
  • Storage buffer (see recipe)
  • 5 M NaCl
  • 0.2 M H 2SO 4
  • 50‐ml tubes
  • 3500 MWCO SnakeSkin pleated dialysis tubing (Pierce)

Support Protocol 2: Radiolabeling Peptides for use as Substrates for HDAC Deacetylation Assay

  • 2.95 kDa p53 peptide (364‐389; AHSSHLKSKKGQSTSRHKKLMFKTEG): synthetic peptide purified by HPLC to >90% purity by the supplier
  • [3H]acetic acid (NEN, 2‐5 Ci/mmol in ethanol)
  • BOP solution: 0.24 M (benzotriazol‐1‐yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (Aldrich) /0.2 M triethylamine (Aldrich) in acetonitrile; prepare fresh
  • 10 mM HCl in methanol
  • 10 mM HCl in 10% (v/v) methanol
  • 3 N HCl in 50% (v/v) isopropanol
  • Microcon‐SCX spin column (Millipore)
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Literature Cited

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