Analysis of Protein Prenylation In Vitro and In Vivo Using Functionalized Phosphoisoprenoids

Uyen T.T. Nguyen1, Yaowen Wu2, Andrew Goodall3, Kirill Alexandrov3

1 Max Planck Institute, Dortmund, Germany, 2 Department of Physical Biochemistry, Max‐Planck Institute of Molecular Physiology, Dortmund, Germany, 3 Department of Cell and Molecular Biology, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Australia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.3
DOI:  10.1002/0471140864.ps1403s62
Online Posting Date:  November, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Post‐translational modifications (PTMs) expand the number of protein isoforms in eukaryotic proteome by orders of magnitude. Protein modification with isoprenoid lipids is a common PTM affecting hundreds of proteins controlling the transport of information and materials into, through, and out of the eukaryotic cell. In this modification, a soluble phosphoisoprenoid such as farnesyl (C15) or geranylgeranyl (C20) pyrophosphate moiety is recruited by one of three protein prenyltransferases to covalently modify a C‐terminal cysteine(s) in a target protein. The three mammalian prenyltransferases are farnesyltransferase (FTase), geranylgeranyltransferase type I (GGTase I), and Rab geranylgeranyl transferase (also termed geranylgeranyltransferase type II ‐ GGTase II). In this unit, synthetic isoprenoids conjugated to either a fluorophore or biotin group are used to assay the activity of protein prenyltransferases in vitro or to affinity tag prenylatable proteins in cell lysates. These protocols and their modifications can be used to study the mechanisms of protein prenylation, identify prenylation targets, and characterize inhibitors of protein prenyltransferases in vitro and in vivo. Curr. Protoc. Protein Sci. 62:14.3.1‐14.3.15. © 2010 by John Wiley & Sons, Inc.

Keywords: protein prenylation; isoprenoids; protein prenyltransferases

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: SDS‐PAGE End‐Point Assay for Evaluation of Potential Inhibitors of Mammalian Prenyltransferases
  • Support Protocol 1: Preparation of Lysates for Protein Prenyltransferase Assays
  • Basic Protocol 2: In Vitro Prenylation of Lysates with Biotin‐Geranyl
  • Basic Protocol 3: Prenylation and Sample Preparation for MS/MS Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: SDS‐PAGE End‐Point Assay for Evaluation of Potential Inhibitors of Mammalian Prenyltransferases

  Materials
  • Fluorescent lipid donors: 4 mM NBD‐GPP and 4 mM NBD‐FPP stocks in 20 mM (NH 4) 2CO 3 (JenaBioscience, cat. no. LI‐014 and LI‐013, respectively)
  • FTase wild‐type (93 kDa) and substrate GST‐Ki‐Ras protein (51 kDa) (JenaBioscience, cat. no. PR‐102 and PR‐204, respectively)
  • GGTase‐I wild‐type and substrate GST‐RhoA protein (54 kDa) (JenaBioscience, cat. no. PR‐101 and PR‐363, respectively)
  • RabGGTase wild‐type (100 kDa) and substrate Rab7 protein (23 kDa) (JenaBioscience, cat. no. PR‐103 and PR‐108, respectively)
  • Prenylation buffer (see recipe)
  • 0.5 M dithioerythritol (DTE; Gebu Biotechnik GmbH, cat. no. 1007)
  • 10 mM test inhibitor stock in DMSO
  • REP‐1 (Rab escort protein 1; 76 kDa; JenaBioscience, cat. no. PR‐105)
  • 2× SDS sample buffer (see recipe), ice cold
  • Low‐molecular‐weight SDS‐PAGE markers
  • 15% SDS‐PAGE gels (unit 10.1)
  • Coomassie blue staining solution (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • 37°C water bath
  • 95°C heating block
  • Fluorescent Image Reader FLA‐5000 (Fujifilm) or similar type of device (excitation laser at 473 nm and emission cut‐off filter at 510 nm)
  • Densitometry software (AIDA, Fujifilm or similar)
  • Additional reagents and equipment for SDS‐polyacrylamide gels (unit 10.1)

Support Protocol 1: Preparation of Lysates for Protein Prenyltransferase Assays

  Materials
  • Cultured cell line
  • Complete tissue culture medium (cell line specific)
  • 1 mM compactin (mevastatin) (Sigma‐Aldrich, cat. no. 27696) stock dissolved in 8% ethanol and 25 mM NaOH, pH 8.0, or expected inhibitory concentration of protein prenyltransferase inhibitor (dissolved in DMSO)
  • PBS, pH 7.2, ice‐cold (see recipe)
  • Lysis/prenylation buffer (see recipe)
  • Aspirator
  • 1.5‐ml polyallomer microcentrifuge tubes (Beckman, cat. no. 357448)
  • 23‐G needles and 1‐ml syringes
  • Refrigerated bench‐top centrifuge
  • Refrigerated ultracentrifuge
NOTE: all reagents and equipment that come into contact with live cells must be sterile, and proper sterile technique should be applied accordingly.NOTE: All cell culture incubations are performed in a humidified 37°C, 5% CO 2 incubator

Basic Protocol 2: In Vitro Prenylation of Lysates with Biotin‐Geranyl

  Materials
  • Lysate (see protocol 2)
  • 1 M DTE (if frozen lysates are used)
  • 1 mM ZnCl 2
  • 20 mM GDP
  • Recombinant rat FTase W102T_Y154T and FTase W102T_Y154T_Y205T mutants (JenaBioscience)
  • Recombinant rat GGTase‐I F53Y_Y126T mutant (JenaBioscience)
  • Recombinant rat wild‐type RabGGTase (JenaBioscience, cat. no. PR‐103)
  • Recombinant rat REP‐1 (JenaBioscience, cat. no. PR‐105)
  • 5 mM biotin‐geranyl pyrophosphate (BGPP) stock in 25 mM (NH 4) 2CO 3 (JenaBioscience)
  • 6× SDS sample buffer (see recipe)
  • NuPAGE 4% to 12% Tris‐Bis SDS PAGE gels (Invitrogen, cat. no. NP0342BOX)
  • ECL western blotting solution (GE Healthcare, cat. no. RPN2209)
  • Blocking solution (see recipe)
  • 1 mg/ml streptavidin‐horseradish peroxidase (STR‐HRP) stock (Invitrogen, cat. no. 43‐4323)
  • TBST (see recipe)
  • ECL solutions (Amersham ECL Advance western blotting detection kit, GE Healthcare, cat. no. RPN2135)
  • 90°C heating block
  • Gel‐blotting Whatman filters
  • PVDF Immobilon‐P transfer membrane (Millipore, cat. no. IPVH00010)
  • Film or appropriate chemiluminescent detection equipment (e.g., Lumi‐Imager F1, Roche)
  • Software package for scanning densitometry (e.g., AIDA, Fujifilm, or ImageJ)
  • Additional reagents and equipment for gel blotting (unit 10.7)

Basic Protocol 3: Prenylation and Sample Preparation for MS/MS Analysis

  Materials
  • Cell lysate (see protocol 2)
  • Ammonium sulfate
  • Lysis/prenylation buffer (see recipe)
  • ZnCl 2 for FTase and GGTase‐I
  • 20 mM GDP
  • 5 mM BGPP
  • FTase W102T_Y154T and FTase W102T_Y154T_Y205T, GGTase‐I F53Y_Y126T, or wild‐type RabGGTase/REP1
  • PBS (see recipe)
  • Illusta NAP‐5 columns (Sephadex G‐25, DNA‐grade; GE Healthcare, cat. no. 17085302)
  • Magnetic streptavidin beads (New England Biolabs, cat. no. S1420S)
  • PBS with 1% NP‐40 (see recipe for PBS)
  • 4 M urea in PBS
  • 62.5 mM ammonium bicarbonate
  • Acetonitrile
  • 100 ng/µl modified, sequencing‐grade trypsin (Roche, cat. no. 11418033001) stock in 1 mM HCl
  • Methanol
  • 100 ng/µl sequencing‐grade chymotrypsin (Roche, cat. no. 11418467001) stock in 1 mM HCl
  • 5% formic acid
  • 1000‐µm i.d. × 15‐cm Scx column (Dionex, cat. no. 164262) packed with MIC‐15‐polysulfoethyl‐Asp resin
  • Scx buffer A (see recipe)
  • Scx buffer B (see recipe)
  • 150 × 0.3–mm reversed‐phase nano‐column (Alltech, cat. no. 218MS5.315) packed with Vydac MS 5µm C18 300A resin
  • RP buffer A: 0.1% formic acid
  • RP buffer B: 90:10 (v/v) acetonitrile/0.1% formic acid
  • 8‐kDa cut‐off mini dialysis spin columns (GE Healthcare, cat. no. 80648413)
  • Dynal magnetic separation rack (Invitrogen, cat. no. 123‐21D)
  • 1.5‐ml microcentrifuge tubes
  • 37°C thermo‐mixer
  • SpeedVac rotary evaporator
  • 4000 QTRAP MS/MS system equipped with a TurboIonSpray Source (Applied Biosystems)
  • Additional reagents and equipment for precipitation protocol (unit 4.5)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Alexandrov, K., Horiuchi, H., Steele‐Mortimer, O., Seabra, M.C., and Zerial, M. 1994. Rab escort protein‐1 is a multifunctional protein that accompanies newly prenylated Rab proteins to their target membranes. EMBO J. 13:5262‐5273.
   Andres, D.A., Seabra, M.C., Brown, M.S., Armstrong, S.A., Smeland, T.E., Cremers, F.P., and Goldstein, J.L. 1993. cDNA cloning of component A of Rab geranylgeranyl transferase and demonstration of its role as a Rab escort protein. Cell 73:1091‐1099.
   Casey, P.J. and Seabra, M.C. 1996. Protein prenyltransferases. J. Biol. Chem. 271:5289‐5292.
   Cassidy, P.B., Dolence, J.M., and Poulter, C.D. 1995. Continuous fluorescence assay for protein prenyltransferases. Methods Enzymol. 250:30‐43.
   Denoyelle, C., Albanese, P., Uzan, G., Hong, L., Vannier, J.P., Soria, J., and Soria, C. 2003. Molecular mechanism of the anti‐cancer activity of cerivastatin, an inhibitor of HMG‐CoA reductase, on aggressive human breast cancer cells. Cell Signal. 15:327‐338.
   Dursina, B., Reents, R., Delon, C., Wu, Y., Kulharia, M., Thutewohl, M., Veligodsky, A., Kalinin, A., Evstifeev, V., Ciobanu, D., Szedlacsek, S.E., Waldmann, H., Goody, R.S., and Alexandrov, K. 2006. Identification and specificity profiling of protein prenyltransferase inhibitors using new fluorescent phosphoisoprenoids. J. Am. Chem. Soc. 128:2822‐2835.
   Gelb, M.H., Brunsveld, L., Hrycyna, C.A., Michaelis, S., Tamanoi, F., Van Voorhis, W.C., and Waldmann, H. 2006. Therapeutic intervention based on protein prenylation and associated modifications. Nat. Chem. Biol. 2:518‐528.
   Hartman, H.L., Hicks, K.A., and Fierke, C.A. 2005. Peptide specificity of protein prenyltransferases is determined mainly by reactivity rather than binding affinity. Biochemistry 44:15314‐15324.
   Kale, T.A., Hsieh, S.A., Rose, M.W., and Distefano, M.D. 2003. Use of synthetic isoprenoid analogues for understanding protein prenyltransferase mechanism and structure. Curr. Top. Med. Chem. 3:1043‐1074.
   Kho, Y., Kim, S.C., Jiang, C., Barma, D., Kwon, S.W., Cheng, J., Jaunbergs, J., Weinbaum, C., Tamanoi, F., Falck, J., and Zhao, Y. 2004. A tagging‐via‐substrate technology for detection and proteomics of farnesylated proteins. Proc. Natl. Acad. Sci. U.S.A. 101:12479‐12484.
   Konstantinopoulos, P.A., Karamouzis, M.V., and Papavassiliou, A.G. 2007. Post‐translational modifications and regulation of the RAS superfamily of GTPases as anticancer targets. Nat. Rev. Drug Discov. 6:541‐555.
   Liu, X.H. and Prestwich, G.D. 2002. Didehydrogeranylgeranyl (Delta Delta GG): A fluorescent probe for protein prenylation. J. Am. Chem. Soc. 124:20‐21.
   Magee, A.I. 2001. Analysis of protein prenylation and carboxyl‐methylation. Curr. Protoc. Protein Sci. 5:14.13.1‐14.13.11.
   Maurer‐Stroh, S., Washietl, S., and Eisenhaber, F. 2003. Protein prenyltransferases. Genome Biol. 4:212.1‐212.9.
   Nguyen, U.T., Guo, Z., Delon, C., Wu, Y., Deraeve, C., Franzel, B., Bon, R.S., Blankenfeldt, W., Goody, R.S., Waldmann, H., Wolters, D., and Alexandrov, K. 2009. Analysis of the eukaryotic prenylome by isoprenoid affinity tagging. Nat. Chem. Biol. 5:227‐235.
   Owen, D.J., Alexandrov, K., Rostkova, E., Scheidig, A.J., Goody, R.S., and Waldmann, H. 1999. Chemo‐enzymatic synthesis of fluorescent Rab 7 proteins: Tools to study vesicular trafficking in cells. Angewandte Chemie‐Int. Ed. 38:509‐512.
   Pais, J.E., Bowers, K.E., Stoddard, A.K., and Fierke, C.A. 2005. A continuous fluorescent assay for protein prenyltransferases measuring diphosphate release. Anal. Biochem. 345:302‐311.
   Pompliano, D.L., Gomez, R.P., and Anthony, N.J. 1992. Intramolecular fluorescence enhancement—A continuous assay of ras farnesyl‐protein transferase. J. Am. Chem. Soc. 114:7945‐7946.
   Seabra, M.C., Reiss, Y., Casey, P.J., Brown, M.S., and Goldstein, J.L. 1991. Protein farnesyltransferase and geranylgeranyltransferase share a common alpha subunit. Cell 65:429‐434.
   Seabra, M.C., Brown, M.S., Slaughter, C.A., Sudhof, T.C., and Goldstein, J.L. 1992. Purification of component A of Rab geranylgeranyl transferase: Possible identity with the choroideremia gene product. Cell 70:1049‐1057.
   Troutman, J.M., Roberts, M.J., Andres, D.A., and Spielmann, H.P. 2005. Tools to analyze protein farnesylation in cells. Bioconjug. Chem. 16:1209‐1217.
   Walsh, C.T., Garneau‐Tsodikova, S., and Gatto, G.J. Jr. 2005. Protein posttranslational modifications: The chemistry of proteome diversifications. Angew Chem Int. Ed. Engl. 44:7342‐7372.
   Wu, Y.W., Alexandrov, K., and Brunsveld, L. 2007. Synthesis of a fluorescent analogue of geranylgeranyl pyrophosphate and its use in a high‐throughput fluorometric assay for Rab geranylgeranyltransferase. Nat. Protoc. 2:2704‐2711.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library