Assays for Investigating deSUMOylation Enzymes

Ikenna G. Madu1, Yuan Chen1

1 Department of Molecular Medicine, Beckman Research Institute of the City of Hope, Duarte, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.30
DOI:  10.1002/0471142727.mb1030s99
Online Posting Date:  July, 2012
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Abstract

Post‐translational modifications by the SUMO (Small Ubiquitin‐like MOdifier) family of proteins are recently discovered essential regulatory mechanisms. All SUMO proteins are synthesized as larger precursors that are matured by SUMO‐specific proteases, known as SENPs, which remove several C‐terminal amino acids of SUMO to expose the Gly‐Gly motif. SENPs also remove SUMO modifications from target proteins, making this modification highly dynamic. At least six deSUMOylation enzymes, all of which are encoded by essential genes, have been identified in mammals. SENP1 has been shown to play an important role in the development of prostate cancer and in angiogenesis. This unit describes and discusses methods for characterizing the deSUMOylation enzymes. These assays enable the identification of inhibitors of these enzymes and investigation of their mechanism of inhibition in order to develop research tools and future therapeutics. Curr. Protoc. Mol. Biol. 99:10.30.1‐10.30.13. © 2012 by John Wiley & Sons, Inc.

Keywords: SUMO; SENP; vinyl sulfone; FRET; bioluminescence

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determination of SENP Activity by FRET
  • Alternate Protocol 1: Determination of SENP Activity Using a Gel‐Based Assay
  • Basic Protocol 2: In Vitro Assay of SENP Activities Using SUMO‐7‐Amino‐4‐Methylcoumarin (SUMO‐AMC) as a Substrate
  • Basic Protocol 3: Quantitative Determination of SENP Activities Using a Bioluminescence‐Based Assay
  • Basic Protocol 4: SENP Labeling with Hemagglutinin (HA)‐Tagged SUMO Vinyl Sulfone (VS)
  • Support Protocol 1: YSE and SENP Protein Expression and Purification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determination of SENP Activity by FRET

  Materials
  • YSE (S=SUMO1 or SUMO2) fusion proteins ( protocol 6)
  • SENP1 or SENP2 ( protocol 6)
  • SENP buffer (see recipe)
  • 50 mM Tris⋅Cl, pH 7.5 ( appendix 22) containing 0.02% (w/v) NaN 3
  • 6.25% (v/v) DMSO (only needed as vehicle control for studies involving inhibitors, which are typically dissolved in 99.9% pure DMSO)
  • 384‐well microtiter plates: flat, clear bottom, and black sides (Corning)
  • Plate reader with FRET capability: excitation wavelength 400 to 410, emission detection at 480 and 530 nm (e.g., PHERAstar Plus; BMG LABTECH, http://www.bmglabtech.com/)

Alternate Protocol 1: Determination of SENP Activity Using a Gel‐Based Assay

  Materials
  • 320 nM stock of human SENP1 or 2 catalytic domain ( protocol 6)
  • SENP buffer (see recipe)
  • 50 µg/ml YSE (S=SUMO1 or SUMO2) fusion proteins ( protocol 6)
  • 10× reaction buffer (see recipe)
  • 3× sample buffer (see recipe)
  • Instant Blue Coomassie stain (Expedeon, http://www.expedeon.com/)
  • 0.5‐ml microcentrifuge tubes
  • 37°C heat block or water bath
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2)

Basic Protocol 2: In Vitro Assay of SENP Activities Using SUMO‐7‐Amino‐4‐Methylcoumarin (SUMO‐AMC) as a Substrate

  Materials
  • SUMO‐AMC (SUMO1 and SUMO2 variations; Boston Biochem, 50‐µg unit; http://www.bostonbiochem.com/)
  • Assay buffer (see recipe)
  • SENP1 or SENP2 ( protocol 6)
  • 96‐ or 384‐well microtiter plates
  • Fluorometer (380 nm excitation and 460 nm emission wavelengths)

Basic Protocol 3: Quantitative Determination of SENP Activities Using a Bioluminescence‐Based Assay

  Materials
  • DUB‐Glo Protease Assay kit (containing the substrate at 4 mM and sufficient material for 1000 assays at 50 µl/assay in 96‐well plates; Promega)
  • Human SENP1 or SENP2 catalytic domain expressed and purified from E.coli (50 nM final concentration from a 3.2 µM stock; protocol 6)
  • SENP buffer (see recipe)
  • Inhibitors to be evaluated
  • Well‐plate luminometer (Spectra max M5 from Molecular Devices)
  • 1.5‐ml pop‐top microcentrifuge tubes (Denville, http://www.denvillescientific.com/)
  • Opaque 96‐well microtiter plate (Costar, Corning)
  • Softmax Pro software 5.4 (Molecular Devices)
  • GraphPad Prism 5.04 (GraphPad Software Inc.)

Basic Protocol 4: SENP Labeling with Hemagglutinin (HA)‐Tagged SUMO Vinyl Sulfone (VS)

  Materials
  • Lysate (see, e.g., unit 10.16) of HeLa cells (ATCC no. CCL2)
  • Human SENP1 or SENP2 catalytic domain expressed and purified from E.coli (50 nM final concentration from a 3.2 µM stock; protocol 6)
  • HA‐SUMO1‐VS (Boston Biochem, http://www.bostonbiochem.com/)
  • HA‐SUMO2‐VS (Boston Biochem, http://www.bostonbiochem.com/)
  • 3× sample buffer (see recipe)
  • Blocking buffer (Odyssey from Li‐Cor; http://www.licor.com/)
  • Anti‐SENP1 rabbit monoclonal primary antibodies (Epitomics, http://www.epitomics.com/)
  • Anti‐SENP2 rabbit monoclonal primary antibodies (Epitomics, http://www.epitomics.com/)
  • Anti‐SENP3 rabbit monoclonal primary antibodies (Epitomics, http://www.epitomics.com/)
  • Anti‐SENP5 mouse monoclonal primary antibodies (Epitomics, http://www.epitomics.com/)
  • Anti‐HA mouse monoclonal primary antibodies (Sigma)
  • Phosphate‐buffered saline with Tween 20 (PBST; see recipe)
  • Anti‐mouse secondary antibodies [IgG (H+L) HRP‐conjugated; Promega]
  • Anti‐rabbit secondary antibodies [IgG (H+L) HRP‐conjugated; Promega)]
  • Super signal west Pico chemiluminescent substrate (Thermo Scientific)
  • 100‐mm tissue culture plates (Corning)
  • 1.5‐ml pop‐top microcentrifuge tubes (Denville)
  • Cell scraper (BD Biosciences)
  • Sonicator XL with a micro tip probe (Misonix)
  • Tabletop microcentrifuge at 4°C (Eppendorf, model No. 5417R)
  • Boiling water bath
  • Trans blot turbo transfer system apparatus (Bio‐Rad)
  • 0.2‐µm PVDF trans blot turbo membrane (Bio‐Rad)
  • Rocker
  • Blue X‐ray film (Bioland, http://www.bioland‐sci.com/)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2) and immunoblotting (unit 10.8)
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Figures

Videos

Literature Cited

Literature Cited
   Bailey, D. and O'Hare, P. 2004. Characterization of the localization and proteolytic activity of the SUMO‐specific protease, SENP1. J. Biol. Chem. 279:692‐703.
   Bawa‐Khalfe, T., Cheng, J., Lin, S.H., Ittmann, M.M., and Yeh, E.T. 2010. SENP1 induces prostatic intraepithelial neoplasia through multiple mechanisms. J. Biol. Chem. 285:25859‐25866.
   Cheng, J., Bawa, T., Lee, P., Gong, L., and Yeh, E.T. 2006. Role of desumoylation in the development of prostate cancer. Neoplasia 8:667‐676.
   Cheng, J., Kang, X., Zhang, S., and Yeh, E.T. 2007. SUMO‐specific protease 1 is essential for stabilization of HIF1alpha during hypoxia. Cell 131:584‐595.
   Gong, L. and Yeh, E.T. 2006. Characterization of a family of nucleolar SUMO‐specific proteases with preference for SUMO‐2 or SUMO‐3. J. Biol. Chem. 281:15869‐15877.
   Kaikkonen, S. Jääskeläinen, T., Karvonen, U., Rytinki, M.M., Makkonen, H., Gioeli, D., Paschal, B.M., and Palvimo, J.J. 2009. SUMO‐specific protease 1 (SENP1) reverses the hormone‐augmented SUMOylation of androgen receptor and modulates gene responses in prostate cancer cells. Mol. Endocrinol. 23:292‐307.
   Kolli, N., Mikolajczyk, J., Drag, M., Mukhopadhyay, D., Moffatt, N., Dasso, M., Salvesen, G., and Wilkinson, K.D. 2010. Distribution and paralogue specificity of mammalian deSUMOylating enzymes. Biochemical J. 430:335‐344.
   Mukhopadhyay, D., Ayaydin, F., Kolli, N., Tan, S.H., Anan, T., Kametaka, A., Azuma, Y., Wilkinson, K.D., and Dasso, M. 2006. SUSP1 antagonizes formation of highly SUMO2/3‐conjugated species. J. Cell Biol. 174:939‐949.
   Nishida, T., Tanaka, H., and Yasuda, H. 2000. A novel mammalian Smt3‐specific isopeptidase 1 (SMT3IP1) localized in the nucleolus at interphase. Eur. J. Biochem. 267:6423‐6427.
   Reverter, D. and Lima, C. 2006a. Structural basis for SENP2 protease interactions with SUMO precursors and conjugated substrates. Nat. Struct. Mol. Biol. 13:1060‐1068.
   Reverter, D. and Lima, C.D. 2006b. Structural basis for SENP2 protease interactions with SUMO precursors and conjugated substrates. Nat. Struct. Mol. Biol. 13:1060‐1068.
   Shen, L.N. Tatham, M.H., Dong, C., Zagórska, A., Naismith, J.H., and Hay, R.T. 2006a. SUMO protease SENP1 induces isomerization of the scissile peptide bond. Nat. Struct. Mol. Biol. 13:1069‐1077.
   Shen, L.N., Dong, C., Liu, H., Naismith, J.H., and Hay, R.T. 2006b. The structure of SENP1‐SUMO‐2 complex suggests a structural basis for discrimination between SUMO paralogues during processing. Biochem. J. 397:279‐288.
   Shen, L.N., Tatham, M.H., Dong, C., Zagórska, A., Naismith, J.H., and Hay, R.T. 2006c. SUMO protease SENP1 induces isomerization of the scissile peptide bond. Nat. Struct. Mol. Biol. 13:1069‐1077.
   Shen, L.N., Geoffroy, M.C., Jaffray, E.G., and Hay, R.T. 2009. Characterization of SENP7, a SUMO‐2/3‐specific isopeptidase. Biochem. J. 421:223‐230.
   Tatham, M.H. and Hay, R.T. 2009. FRET‐based in vitro assays for the analysis of SUMO protease activities. Methods Mol. Biol. 497:253‐268.
   Xu, Z. and Au, S.W. 2005. Mapping residues of SUMO precursors essential in differential maturation by SUMO‐specific protease, SENP1. Biochem. J. 386:325‐330.
   Xu, Y., Zuo, Y., Zhang, H., Kang, X., Yue, F., Yi, Z., Liu, M., Yeh, E.T., Chen, G., and Cheng, J. 2010. Induction of SENP1 in endothelial cells contributes to hypoxia‐driven VEGF expression and angiogenesis. J. Biol. Chem. 285:36682‐36688.
   Yeh, E.T. 2009. SUMOylation and De‐SUMOylation: Wrestling with life's processes. J. Biol. Chem. 284:8223‐8227.
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