Biochemical Analysis of Protein SUMOylation

Aileen Y. Alontaga1, Ekaterina Bobkova2, Yuan Chen1

1 Department of Molecular Medicine, Beckman Research Institute of the City of Hope, Duarte, California, 2 Conrad Prebys Center for Chemical Genomics, Sanford‐Burnham Medical Research Institute, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.29
DOI:  10.1002/0471142727.mb1029s99
Online Posting Date:  July, 2012
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Abstract

SUMOylation, the covalent attachment of Small Ubiquitin‐like MOdifier (SUMO) polypeptides to other proteins, is among the most important post‐translational modifications that regulate the functional properties of a large number of proteins. SUMOylation is broadly involved in cellular processes such as gene transcription, hormone response, signal transduction, DNA repair, and nuclear transport. SUMO modification has also been implicated in the pathogenesis of human diseases, such as cancer, neurodegenerative disorders, and viral infection. Attachment of a SUMO protein to another protein is carried out in multiple steps catalyzed by three enzymes. This unit describes and discusses the in vitro biochemical methods used for investigating each step of the SUMOylation process. In addition, a high‐throughput screening protocol is included for the identification of inhibitors of SUMOylation. Curr. Protoc. Mol. Biol. 99:10.29.1‐10.29.29. © 2012 by John Wiley & Sons, Inc.

Keywords: SUMOylation; SUMO; E1; E2; E3; Ubc9; SAE1; SAE2; RanGAP1; RanBP2; Sp100; HTS

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Quantitative Examination of SUMO Adenylation by ATP:PPi Isotope Exchange Assay
  • Alternate Protocol 1: Determination of SUMO Adenylation by Quantitative ATP:PPi Isotope Exchange Assay Using Phosphor Imaging
  • Basic Protocol 2: Determination of E1‐Catalyzed ATP:AMP Exchange Rates Using Thin‐Layer Chromatography
  • Basic Protocol 3: Determination of E1⋅SUMO Conjugates by Gel‐Based Assay
  • Basic Protocol 4: Detection of SUMO Thioester Transfer from E1 to E2 (UBC9)
  • Basic Protocol 5: Determination of E2⋅SUMO Thioester Formation
  • Basic Protocol 6: Detection of Substrate‐SUMO Isopeptide Formation
  • Basic Protocol 7: Determination of Substrate SUMOylation in the Presence of E3
  • Basic Protocol 8: High‐Throughput Screening Assay to Monitor SUMOylation
  • Support Protocol 1: Expression and Purification of His‐Tagged Proteins E1, SUMO, Ubc9, RanGAP1, and RanBP2 for Sumoylation Studies
  • Support Protocol 2: Expression and Purification of GST‐Tagged Proteins (GST‐SUMO and GST‐Sp100)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quantitative Examination of SUMO Adenylation by ATP:PPi Isotope Exchange Assay

  Materials
  • 5× Buffer A (see recipe)
  • 50 mM ATP (Sigma)
  • 50 mM [32P]PPi (Perkin Elmer Life Sciences)
  • 100 µM SUMO (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5
  • 10 µM E1 enzyme (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5
  • 5% (w/v) trichloroacetic acid (TCA) containing 4 mM carrier (nonradioactive) PPi (pyrophosphate sodium salt; Sigma)
  • 10% (w/v) slurry of activated charcoal (Sigma) in 2% (w/v) TCA
  • 2% (w/v) trichloroacetic acid (TCA)
  • 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 37°C heat block
  • Cherenkov counter (Beckman LS6500 scintillation counter)

Alternate Protocol 1: Determination of SUMO Adenylation by Quantitative ATP:PPi Isotope Exchange Assay Using Phosphor Imaging

  • 2% (w/v) trichloroacetic acid (TCA) containing 10 mM carrier PPi (pyrophosphate sodium salt; Sigma)
  • B‐safe activated charcoal paper (Whatman)
  • Dot/slot blotting manifold (BioRad Bio‐Dot SF or Schleicher and Schuell Minifold‐II)
  • Vacuum pump (GAST Manufacturing, http://www.gastmfg.com)
  • Flat bottom dish (Pyrex)
  • Rocking shaker (Reliable Scientific, Inc., http://www.reliablescientific.com/)
  • Phosphor imaging plate (20 × 25 cm; GE Life Sciences)
  • Typhoon phosphor imager (GE Life Sciences)
  • Image Reader 1.8 and Multi Gauge 3.1 software (Fuji Film Life Sciences)

Basic Protocol 2: Determination of E1‐Catalyzed ATP:AMP Exchange Rates Using Thin‐Layer Chromatography

  Materials
  • 5× Buffer A (see recipe)
  • 10 mM ATP (Sigma)
  • 1 mM AMP (Sigma)
  • 200 µM PPi (pyrophosphate sodium salt, not radioactive; Sigma)
  • 20 µM SUMO (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 10 µM E1 enzyme (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 1 mM [14C]AMP (Perkin Elmer Life Sciences)
  • 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 8 M urea (Sigma)
  • 0.5 M LiCl/1 M formic acid solution
  • 37°C Heat block
  • Polyethyleneimine (PEI) cellulose thin‐layer chromatography (TLC) plate, 10 × 20 cm (Sigma)
  • Glass developing chamber for TLC
  • Phosphor imaging plate (20 × 25 cm) (GE Life Sciences)
  • Typhoon phosphor imager (GE Life Sciences)

Basic Protocol 3: Determination of E1⋅SUMO Conjugates by Gel‐Based Assay

  Materials
  • 10× ATP regeneration system (see recipe)
  • 100 µM SUMO (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 50 µM E1 enzyme (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 2× SDS sample buffer (with and without DTT and with no urea; see recipe)
  • 4%‐12% Bis‐Tris gradient gel (Invitrogen)
  • NuPAGE MOPS SDS running buffer (Invitrogen)
  • SimplyBlue Stain (Invitrogen) or Coomassie‐based stain (see unit 10.6)
  • 0.5‐ml sterile microcentrifuge tubes
  • 37° and 95°C heat blocks
  • Plastic gel box with cover
  • Rocking shaker (Reliable Scientific, Inc., http://www.reliablescientific.com/)
  • GS‐800 Densitometer (BioRad)
  • Additional reagents and equipment for electrophoresis (unit 10.2) and immunoblotting (unit 10.8)

Basic Protocol 4: Detection of SUMO Thioester Transfer from E1 to E2 (UBC9)

  Materials
  • Alexa680 fluorescent‐labeled SUMO (Invitrogen)
  • 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 10× ATP regeneration system (see recipe)
  • 10 µM E1 enzyme (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 500 mM disodium or tetrasodium EDTA, pH 8.0
  • 10 µM E2 (Ubc9) (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 2× SDS sample buffer with 8 M urea and no DTT (see recipe)
  • 4% to 12% Bis‐Tris gradient gel (Invitrogen)
  • NuPAGE MOPS SDS running buffer (Invitrogen)
  • 37°C heat block
  • 0.5‐ml sterile microcentrifuge tubes
  • Rocking shaker (Reliable Scientific, Inc., http://www.reliablescientific.com/)
  • Odyssey infrared imaging system (Li‐Cor Biosciences, http://www.licor.com)
  • ImageQuant5.2 software (GE Life Sciences)
  • Additional reagents and equipment for electrophoresis (unit 10.2)

Basic Protocol 5: Determination of E2⋅SUMO Thioester Formation

  Materials
  • 10× ATP regeneration system (see recipe)
  • 100 µM SUMO (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 1 µM E1 enzyme (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 100 µM Ubc9 (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 2× SDS sample buffer with no urea and no DTT
  • 4%‐12% Bis‐Tris gradient gel (Invitrogen)
  • NuPAGE MOPS SDS running buffer (Invitrogen)
  • SimplyBlue Stain (Invitrogen)
  • 0.5‐ml sterile microcentrifuge tubes
  • 37°C heat block
  • Rocking shaker (Reliable Scientific, Inc., http://www.reliablescientific.com/)
  • GS‐800 densitometer (Bio‐Rad)
  • Additional reagents and equipment for electrophoresis (unit 10.2) and immunoblotting (unit 10.8)

Basic Protocol 6: Detection of Substrate‐SUMO Isopeptide Formation

  Materials
  • 250, 400, and 625 nM Ubc9 (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 10× ATP regeneration system (see recipe)
  • 100 µM SUMO (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 1 µM E1 enzyme (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 100 µM RanGAP1 (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 2× SDS sample buffer with DTT and no urea (see recipe)
  • 4% to 12% Bis‐Tris gradient gel (Invitrogen)
  • NuPAGE MOPS SDS running buffer (Invitrogen)
  • Blocking buffer (Odyssey from Li‐Cor Biosciences, http://www.licor.com)
  • Mouse monoclonal primary antibody against SUMO‐1 (Abgent, http://www.abgent.com/)
  • Phosphate‐buffered saline with Tween‐20 (PBST; see recipe)
  • IRDye 680‐labeled secondary antibody (Li‐Cor Biosciences, http://www.licor.com)
  • 0.5‐ml sterile microcentrifuge tubes
  • 37°C heat block
  • 0.2 µm PVDF trans‐blot turbo membrane (BioRad)
  • Trans‐blot turbo transfer system apparatus (BioRad)
  • Rocking shaker (Reliable Scientific, Inc., http://www.reliablescientific.com/)
  • Odyssey infrared imaging system (Li‐Cor Biosciences)
  • ImageQuant 5.2 software (GE Life Sciences)
  • Additional reagents and equipment for electrophoresis (unit 10.2) and immunoblotting (unit 10.8)

Basic Protocol 7: Determination of Substrate SUMOylation in the Presence of E3

  Materials
  • 5× assay buffer (see recipe)
  • 250 µM SUMO (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 8 µM E1 enzyme (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 3.5 µM E2 (Ubc9) (see protocol 10) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 25 µM RanBP2 (see protocol 10) in phosphate‐buffered saline (PBS; appendix 22)
  • 70 mM ATP (Sigma)
  • 21, 42, 56, and 84 µM GST‐Sp100 (see protocol 11) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 22)
  • 2× SDS sample buffer with DTT and no urea (see recipe)
  • 4%‐12% Bis‐Tris gradient gel (Invitrogen)
  • NuPAGE MOPS SDS running buffer (Invitrogen)
  • Blocking buffer (Odyssey from Li‐Cor Biosciences, http://www.licor.com)
  • Mouse monoclonal primary antibody against SUMO‐1 (Abgent, http://www.abgent.com/)
  • Phosphate‐buffered saline with Tween‐20 (PBST; see recipe)
  • Donkey anti‐mouse Ig secondary antibody (Abgent, http://www.abgent.com/)
  • 0.5‐ and 1.0‐ml sterile microcentrifuge tubes
  • Heat blocks at 25°, 28°, 31°, and 37°C
  • Rocking shaker (Reliable Scientific, Inc.)
  • 0.2‐µm PVDF trans‐blot turbo membrane (BioRad)
  • Trans‐blot turbo transfer system apparatus (BioRad)
  • Odyssey infrared imaging system (Li‐Cor Biosciences, http://www.licor.com/)
  • ImageQuant 5.2 software (GE Life Sciences)
  • Additional reagents and equipment for electrophoresis (unit 10.2) and immunoblotting (unit 10.8)

Basic Protocol 8: High‐Throughput Screening Assay to Monitor SUMOylation

  Materials
  • 35.5 µM E1 enzyme (see protocol 10)
  • 30.8 µM His‐RanGAP1 (see protocol 10)
  • Anti‐GST XL665‐labeled antibody (Cis‐Bio Bioassays)
  • HTS assay buffer (see recipe)
  • ATP (Sigma)
  • 222 µM E2 (Ubc9) (see protocol 10)
  • 368 µM GST‐SUMO (see protocol 10)
  • Anti‐His Europium‐labeled antibody (Cis‐Bio Bioassays, http://www.ria‐cis.com/)
  • 20 mM test compounds in DMSO
  • 500 mM potassium fluoride (KF)
  • DMSO (Sigma)
  • 1536‐well white plate (Corning)
  • Multidrop Combi dispenser (Thermo Scientific)
  • HighRes biosolutions pintool (Thermo Scientific)
  • Microplate reader (e.g., BMG Labtech Pherastar, http://www.bmglabtech.com/)
  • CBIS software (ChemInnovations, Inc., http://www.cheminnovation.com/)

Support Protocol 1: Expression and Purification of His‐Tagged Proteins E1, SUMO, Ubc9, RanGAP1, and RanBP2 for Sumoylation Studies

  Materials
  • Recombinant plasmid harboring the gene of each enzyme: SUMO, Ubc9, RanGAP1, RanGAP2 (available from the authors; YChen@coh.org)
  • E. coli BL21‐CodonPlus(DE‐3) (Stratagene)
  • Luria‐Bertani (LB) broth (Sigma): 20 g LB powder/liter H 2O, autoclaved
  • 500 mg/ml kanamycin stock solution (Fisher Scientific), sterilized using 0.22‐µm filter
  • 100 mg/ml ampicillin stock solution (Fisher Scientific), sterilized using 0.22‐µm filter
  • 50 mg/ml chloramphenicol stock solution (Fisher Scientific) in ethanol, sterilized using 0.22‐µm filter
  • 1 M isopropyl‐β‐D‐thiogalactopyranoside (IPTG; Fisher Scientific)
  • 2× binding buffer (see recipe)
  • 10× BugBuster protein extraction reagent (Novagen)
  • Protease inhibitor cocktail tablets (Roche)
  • Benzonase (Novagen)
  • Ni‐NTA resin (Qiagen)
  • Wash buffer (see recipe)
  • Elution buffer (see recipe)
  • 2× SDS sample buffer with DTT (no urea)
  • 4%‐12% Bis‐Tris gradient gel (Invitrogen)
  • NuPAGE MOPS SDS running buffer (Invitrogen)
  • SimplyBlue Stain (Invitrogen)
  • Dithiothreitol (DTT) (Sigma)
  • Dialysis buffer (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Bradford protein assay kit (BioRad)
  • 50 mM phenylmethylsulfonyl fluoride (PMSF)
  • Shaker incubator (Thermo‐Electron Corp.)
  • Ultracentrifuge (Beckman Avanti J‐25I)
  • Chromatography column (10 × 100 mm; Sigma)
  • Column stand with clampGS‐800 densitometer (BioRad)
  • Dialysis membrane or Centricon concentrators (Millipore)
  • Spectramax M5 spectrophotometer (Molecular Devices)
  • Additional reagents and equipment for transformation of E. coli (unit 1.8) culture of E. coli (unit 1.2), electrophoresis (unit 10.2), and dialysis ( appendix 3C)

Support Protocol 2: Expression and Purification of GST‐Tagged Proteins (GST‐SUMO and GST‐Sp100)

  • 2× GST binding buffer (see recipe)
  • Glutathione agarose resin (Amersham Biosciences)
  • Reduced glutathione (Sigma)
  • 1× GST wash buffer (see recipe)
  • GST elution buffer (see recipe)
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Figures

Videos

Literature Cited

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Key References
   Wang and Chen, 2010. See above.
   (ATP:PPi exchange assay) is derived from this paper.
   Wang et al., 2009. See above.
   is derived from this paper.
   Wang et al., 2007. See above.
  Basic Protocols 3 and 6 are derived from this paper.
   Hay, 2005. See above.
  This review article clearly illustrates the overall picture of protein SUMOylation.
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