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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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‐MgCl₂ (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‐MgCl₂ (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‐MgCl₂ 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 N₂
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

Figure 1. DUB assays with Ub‐VS‐untreated 26 proteasome (A , 26S) or Ub‐VS‐treated 26 proteasome (B , VS‐26S) in the USP14 reconstitution system. Ub‐VS is a commercially available active‐site‐directed inhibitor that irreversibly inactivates thiol protease DUBs. 26S (1 nM) or VS‐26S (1 nM) in the absence or presence of indicated concentrations of recombinant USP14 wild‐type (WT) or catalytic inactive mutant (CA) was analyzed for the Ub‐AMC hydrolysis in a given time. Ub‐AMC (1 µM) was added as substrate. Note that most of the DUB activity of 26S in (A) comes from proteasome‐associated UCH37 since RPN11 does not cleave Ub‐AMC. RFU indicates relative fluorescence unit. Detailed descriptions for the assay reagents and the DUB assay can be found in the protocols.

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Figure 2. Workflow of the HTS for small‐molecule inhibitors of proteasome‐associated USP14. The screening procedures were based on the equipment at the ICCB‐Longwood Screening Facility, Harvard Medical School (http://iccb.med.harvard.edu).

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Figure 3. Representative preparations of VS‐26S (A ), Ub‐AMC (B ), and recombinant USP14 (C ). (A ) Native gel analysis of 26S and VS‐26S. The proteasome (7 µg) was analyzed for in‐gel peptidase activity with LLVY‐AMC as the substrate (left) or for Coomassie Brilliant Blue staining (middle, CBB) on a 3.6% native gel. SDS‐PAGE analysis of 7 µg of purified proteasome is shown (right, CBB). (B ) SDS‐PAGE analysis and CBB staining of purified Ub thiol esters (2 µg), and 1 µg of purified Ub‐AMC and HA‐tagged Ub‐AMC (HA‐Ub‐AMC). Free Ub (1 µg) was loaded for comparison. (C ) SDS‐PAGE analysis and CBB staining of purified USP14 WT and CA. A known amount of BSA was loaded for comparison. RP₂ CP indicates doubly capped proteasome holoenzyme.

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Figure 4. Schematic flowchart of the required amount of assay reagents for a given scale of screen.

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Videos

Literature Cited

	Altun, M., Kramer, H.B., Willems, L.I., McDermott, J.L., Leach, C.A., Goldenberg, S.J., Kumar, K.G., Konietzny, R., Fischer, R., Kogan, E., Mackeen, M.M., McGouran, J., Khoronenkova, S.V., Parsons, J.L., Dianov, G.L., Nicholson, B., and Kessler, B.M. 2011. Activity‐based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes. Chem. Biol. 18:1401‐1412.
	Chen, J., Dexheimer, T.S., Ai, Y., Liang, Q., Villamil, M.A., Inglese, J., Maloney, D.J., Jadhav, A., Simeonov, A., and Zhuang, Z. 2011. Selective and cell‐active inhibitors of the USP1/UAF1 deubiquitinase complex reverse cisplatin resistance in non‐small cell lung cancer cells. Chem. Biol. 18:1390‐1400.
	Cohen, P. and Tcherpakov, M. 2010. Will the ubiquitin system furnish as many drug targets as protein kinases? Cell 143:686‐693.
	Colland, F. 2010. The therapeutic potential of deubiquitinating enzyme inhibitors. Biochem. Soc. Trans. 38:137‐143.
	D'Arcy, P., Brnjic, S., Olofsson, M.H., Fryknäs, M., Lindsten, K., Cesare, M.D., Perego, P., Sadeghi, B., Hassan, M., Larsson, R., and Linder, S. 2011. Inhibition of proteasome deubiquitinating activity as a new cancer therapy. Nat. Med. 17:1636‐1641.
	Elsasser, S., Schmidt, M., and Finley, D. 2005. Characterization of the proteasome using native gel electrophoresis. Methods Enzymol. 398:353‐363.
	Finley, D. 2009. Recognition and processing of ubiquitin‐protein conjugates by the proteasome. Annu. Rev. Biochem. 78:477‐513.
	Inglese, J., Shamu, C.E., and Guy, R.K. 2007a. Reporting data from high‐throughput screening of small‐molecule libraries. Nat. Chem. Biol. 3:438‐441.
	Inglese, J., Johnson, R.L., Simeonov, A., Xia, M., Zheng, W., Austin, C.P., and Auld, D.S. 2007b. High‐throughput screening assays for the identification of chemical probes. Nat. Chem. Biol. 3:466‐479.
	Lee, B.H., Lee, M.J., Park, S., Oh, D.C., Elsasser, S., Chen, P.C., Gartner, C., Dimova, N., Hanna, J., Gygi, S.P., Wilson, S.M., King, R.W., and Finley, D. 2010. Enhancement of proteasome activity by a small‐molecule inhibitor of USP14. Nature 467:179‐184.
	Lee, M.J., Lee, B.H., Hanna, J., King, R.W., and Finley, D. 2011. Trimming of ubiquitin chains by proteasome‐associated deubiquitinating enzymes. Mol. Cell. Proteomics 10:R110.003871.
	Liu, Y., Lashuel, H.A., Choi, S., Xing, X., Case, A., Ni, J., Yeh, L.A., Cuny, G.D., Stein, R.L., and Lansbury, P.T. 2003. Discovery of inhibitors that elucidate the role of UCH‐L1 activity in the H1299 lung cancer cell line. Chem. Biol. 10:837‐846.
	Malo, N., Hanley, J., Cerquozzi, S., Pelletier, J., and Nadon, R. 2006. Statistical practice in high‐throughput screening data analysis. Nat. Biotechnol. 24:167‐175.
	Ovaa, H., Galardy, P.J., and Ploegh, H.L. 2005. Mechanism‐based proteomics tools based on ubiquitin and ubiquitin‐like proteins: Synthesis of active site‐directed probes. Methods Enzymol. 399:468‐478.
	Reyes‐Turcu, F.E., Ventii, K.H., and Wilkinson, K.D. 2009. Regulation and cellular roles of ubiquitin‐specific deubiquitinating enzymes. Annu. Rev. Biochem. 78:363‐397.
	Rudnicki, S. and Johnston, S. 2009. Overview of liquid handling instrumentation for high‐throughput screening applications. Curr. Protoc. Chem. Biol. 1:43‐54.
	Sacco, J.J., Coulson, J.M., Clague, M.J., and Urbe', S. 2010. Emerging roles of deubiquitinases in cancer‐associated pathways. IUBMB Life 62:140‐157.
	Seidman, C.E., Struhl, K., Sheen, J., and Jessen, T. 1997. Introduction of plasmid DNA into cells. Curr. Protoc. Mol. Biol. 37:1.8.1‐1.8.10.
	Simonian, M.H. and Smith, J.A. 2006. Spectrophotometric and colorimetric determination of protein concentration. Curr. Protoc. Mol. Biol. 76:10.1.1‐10.1A.9.
	Wang, X., Chen, C.F., Baker, P.R., Chen, P.I., Kaiser, P., and Huang, L. 2007. Mass spectrometric characterization of the affinity‐purified human 26S proteasome complex. Biochemistry 46:3553‐3565.
	Wilkinson, KD, Gan‐Erdenem, T, Kolli, N. 2005. Derivitization of the C‐terminus of ubiquitin and ubiquitin‐like proteins using intein chemistry: Methods and uses. Methods Enzymol. 399:37‐51.
	Zhang, J.H., Chung, T.D., and Oldenburg, K.R. 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Scr. 4:67‐73.
	Zeng, X., Sigoillot, F., Gaur, S., Choi, S., Pfaff, K.L., Oh, D.C., Hathaway, N., Dimova, N., Cuny, G.D., and King, R.W. 2010. Pharmacologic inhibition of the anaphase‐promoting complex induces a spindle checkpoint‐dependent mitotic arrest in the absence of spindle damage. Cancer Cell 18:382‐395.
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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A High‐Throughput Screening Method for Identification of Inhibitors of the Deubiquitinating Enzyme USP14

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited