A High‐Throughput Screening Method for Identification of Inhibitors of the Deubiquitinating Enzyme USP14

Byung‐Hoon Lee1, Daniel Finley1, Randall W. King1

1 Department of Cell Biology, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch120078
Online Posting Date:  December, 2012
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Abstract

Deubiquitinating enzymes (DUBs) reverse the process of ubiquitination, and number nearly 100 in humans. In principle, DUBs represent promising drug targets, as several of the enzymes have been implicated in human diseases. The isopeptidase activity of DUBs can be selectively inhibited by targeting the catalytic site with drug‐like compounds. Notably, the mammalian 26S proteasome is associated with three major DUBs: RPN11, UCH37, and USP14. Because the ubiquitin ‘chain‐trimming’ activity of USP14 can inhibit proteasome function, inhibitors of USP14 can stimulate proteasomal degradation. We recently established a high‐throughput screening (HTS) method to identify small‐molecule inhibitors specific for USP14. The protocols in this article cover the necessary procedures for preparing assay reagents, performing HTS for USP14 inhibitors, and carrying out post‐HTS analysis. Curr. Protoc. Chem. Biol. 4:311‐330 © 2012 by John Wiley & Sons, Inc.

Keywords: 26S proteasome; USP14; deubiquitinating enzyme; high‐throughput screening; small‐molecule inhibitor

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Deubiquitination Activity of USP14
  • Basic Protocol 2: High‐Throughput Screening of Small‐Molecule Inhibitors of Proteasome‐Associated USP14
  • Basic Protocol 3: Secondary Screening and Analysis of Primary Hit Compounds of Proteasome‐Associated USP14
  • Support Protocol 1: Purification of Human 26S Proteasomes that Lack Endogenous USP14 and are Devoid of UB‐AMC Hydrolysis Activity
  • Support Protocol 2: Purification of Recombinant Human USP14 from E. coli
  • Support Protocol 3: Synthesis and Purification of Ubiquitin‐AMC
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of Deubiquitination Activity of USP14

  Materials
  • Ub‐VS‐treated human proteasomes (VS‐26S; approximately 200 to 400 nM) (see protocol 4)
  • Recombinant USP14 (approximately 15 to 30 µM) (see protocol 5 or purchase commercially from Boston Biochem or Enzo Life Sciences)
  • Purified Ub‐AMC (approximately 150 to 250 µM) (see protocol 6 or commercially from Boston Biochem)
  • Ub‐AMC buffer (see recipe)
  • Dithiothreitol (DTT)
  • 0.25 M ATP‐MgCl 2 (see recipe)
  • 384‐well microplate, low‐volume, flat‐bottom, non‐binding, black (Corning, cat. no. 3820)
  • Envision plate reader (Perkin Elmer)

Basic Protocol 2: High‐Throughput Screening of Small‐Molecule Inhibitors of Proteasome‐Associated USP14

  Materials
  • Ub‐VS‐treated human proteasomes (VS‐26S; see protocol 4)
  • Recombinant USP14 (see protocol 5 or commercial)
  • Purified Ub‐AMC (see protocol 6 or commercial)
  • Ub‐AMC buffer (see recipe)
  • 0.25 M ATP‐MgCl 2 (see recipe)
  • Compound libraries dissolved in dimethyl sulfoxide (DMSO) in 384‐well plate format (various sources, see http://iccb.med.harvard.edu)
  • Desiccated container
  • 384‐well microplate, low‐volume, flat bottom, non‐binding, black (Corning, cat. no. 3820)
  • Matrix WellMate liquid dispenser (Thermo Scientific; Rudnicki and Johnston, )
  • WellMate, 8‐channel, small‐bore disposable tubing cartridge, pre‐sterilized (Thermo Scientific; Rudnicki and Johnston, )
  • Compound Pin Transfer Robot (Seiko, see http://iccb.med.harvard.edu; Rudnicki and Johnston, )
  • Envision plate reader (Perkin Elmer)

Basic Protocol 3: Secondary Screening and Analysis of Primary Hit Compounds of Proteasome‐Associated USP14

  Materials
  • AMC amine (Sigma)
  • Dimethylsulfoxide (DMSO)
  • Primary hit compounds ( protocol 2) at 10 mM in DMSO
  • IsoT/USP5, human (Boston Biochem)
  • Recombinant USP14 (see protocol 5 or purchase commercially from Boston Biochem)
  • Ubiquitin, bovine (Sigma)
  • 384‐well microplates, low‐volume, flat bottom, non‐binding, black (Corning, cat. no. 3820)
  • Envision plate reader (Perkin Elmer)
  • Scientific curve fitting software (GraphPad Prism or SigmaPlot)
  • Additional reagents and equipment for measurement of deubiquitination activity of USP14 ( protocol 1)

Support Protocol 1: Purification of Human 26S Proteasomes that Lack Endogenous USP14 and are Devoid of UB‐AMC Hydrolysis Activity

  Materials
  • 293T cells stably expressing RPN11 appended with C‐terminal TEV cleavage and biotinylation sites (Lan Huang, UC Irvine, )
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010), ice cold
  • Proteasome lysis buffer (see recipe)
  • Dithiothreitol (DTT)
  • 0.25 M ATP‐MgCl 2 solution (see recipe)
  • Immobilized NeutrAvidin resin (Thermo Scientific)
  • Protease inhibitor cocktail, complete tablet (Roche) or equivalent
  • Proteasome wash buffer (see recipe)
  • Proteasome elution buffer: 50 mM Tris⋅Cl, pH 7.5 (store up to several months at 4°C)
  • Ubiquitin‐vinyl sulfone (Ub‐VS) (Boston Biochem)
  • TEV protease (Invitrogen)
  • Glycerol
  • 150 × 25–mm tissue culture plates
  • 50‐ml conical polypropylene centrifuge tubes (Corning and Thermo Scientific)
  • Refrigerated centrifuge (Sorvall RC‐5B or equivalent)
  • Cell scraper (BD Falcon)
  • Econo‐Column for chromatography (Bio‐Rad)
  • 30°C incubator or water bath
  • Additional reagents and equipment for measuring protein concentration (Simonian and Smith, )
NOTE: All purification steps are conducted on ice or in the cold room at 4°C, unless otherwise noted.

Support Protocol 2: Purification of Recombinant Human USP14 from E. coli

  Materials
  • Competent E. coli strains suitable for GST‐USP14 expression (e.g., BL21 or Rosetta 2 series from Novagen)
  • Expression vector (e.g., pGEX‐USP14; Lee et al., )
  • Selection antibiotic (ampicillin for pGEX‐USP14)
  • Protease inhibitor cocktail, complete tablet (Roche) or equivalent
  • Isopropyl‐β‐D‐1‐thiogalactopyranoside (IPTG) (various commercial sources): prepare 1 M solution in sterilized water, and store it at −20°C
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010), ice cold
  • Protease inhibitor cocktail, complete tablet (Roche) or equivalent
  • Dithiothreitol (DTT)
  • Lysozyme (various commercial sources): prepare 10 mg/ml in water or add directly to lysis buffer at 1 mg/ml as powder
  • Glutathione‐Sepharose 4 Fast Flow resin (GE Healthcare)
  • NaCl
  • Thrombin cleavage buffer (see recipe)
  • Thrombin (R&D Systems)
  • Benzamidine‐Sepharose 6B resin (GE Healthcare)
  • GST elution buffer: 50 mM Tris⋅Cl, pH 8.0 (store up to several months at 4°C)
  • Reduced glutathione (Sigma)
  • Glycerol
  • 2‐ to 2.6‐liter Erlenmeyer flask
  • Temperature‐adjustable shaking incubator that can hold 2‐ to 2.6‐liter flasks (e.g., I 26 series, Fisher Scientific)
  • Spectrophotometer
  • Refrigerated centrifuge (Sorvall RC‐5B or equivalent)
  • French press or sonicator with microtip for cell lysis
  • Polypropylene centrifuge tubes and bottles (15 ml, 50 ml, and 1 liter; Corning and Thermo Scientific)
  • Econo‐Column for chromatography (Bio‐Rad)
  • Disposable Micro Bio‐Spin column (Bio‐Rad)
  • Amicon Ultra centrifugation filter (Millipore)
  • Additional reagents and equipment for transformation of E. coli (Seidman et al., ) and measurement of protein concentration (Simonian and Smith, )
NOTE: All purification steps are conducted on ice or in the cold room at 4°C, unless otherwise noted.

Support Protocol 3: Synthesis and Purification of Ubiquitin‐AMC

  Materials
  • Competent E. coli strains, BL21 cells or Rosetta 2 cells (from Novagen), suitable for expressing pTYB2‐Ub‐Intein‐chitin binding domain (CBD) or equivalent plasmid
  • pTYB2 vector (New England Biolabs, cat. no. N6702S): Intein‐fusion protein expressing pTYB vector series
  • Isopropyl‐β‐D‐1‐thiogalactopyranoside (IPTG) (various commercial sources): prepare 1 M solution in sterilized water, and store it at −20°C
  • Ub‐intein lysis buffer (see recipe)
  • Protease inhibitor cocktail, complete tablet (Roche) or equivalent
  • Chitin binding beads (New England Biolabs)
  • 2‐mercaptoethanesulfonic acid, sodium salt (MESNa; Sigma)
  • Liquid N 2
  • Glycyl‐7‐amido‐4‐methyl coumarin (Gly‐AMC), hydrobromide salt (Glycosynth or Golden Biotechnology)
  • N,N‐dimethylformamide (DMF), HPLC grade (Sigma)
  • N‐hydroxysuccinimide (NHS; Sigma)
  • HEPES base, sodium salt (Sigma)
  • 5% hydrochloric acid (HCl)
  • Dialysis buffer: 50 mM sodium acetate, pH 4.5 (prepare fresh)
  • NaCl
  • 2‐ to 2.6‐liter Erlenmeyer flask
  • Temperature‐adjustable shaking incubator that can hold 2‐ to 2.6‐liter flasks (e.g., I 26 series, Fisher Scientific)
  • Spectrophotometer
  • Refrigerated centrifuge (Sorvall RC‐5B or equivalent)
  • French press or sonicator with microtip for cell lysis
  • Polypropylene centrifuge tubes and bottles (15 ml, 50 ml, and 1 liter: Corning and Thermo Scientific)
  • Econo‐Column for chromatography (Bio‐Rad)
  • Amicon Ultra centrifugation filter (5‐kDa cut‐off; Millipore)
  • 15‐ml or 50‐ml borosilicate glass tubes (Kimble Glass)
  • SnakeSkin Pleated Dialysis Tubing, 3.5 kDa cut‐off (Thermo Scientific), or equivalent
  • ÄKTA fast protein liquid chromatography (FPLC) system (GE Healthcare)
  • HiTrap‐SPHP ionic exchange column, 1 ml (GE Healthcare), or equivalent
  • Additional reagents and equipment for transformation of E. coli (Seidman et al., )
NOTE: All purification steps were conducted on ice or in the cold room at 4°C, unless otherwise noted.
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Figures

Videos

Literature Cited

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Internet Resources
  http://iccb.med.harvard.edu
  The Web page of the ICCB screening facility at Harvard Medical School. This site provides general guidance and detailed information of small‐molecule inhibitor screening.
  http://www.ncbi.nlm.nih.gov/books/NBK53196/
  Assay Guidance Manual by Eli Lilly & Company and the National Center for Advancing Translational Sciences at the NIH.
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