Protein‐Lipid Interaction by Fluorescence (PLIF) to Characterize and Screen for Inhibitors of Protein‐Phosphoinositide Interactions

Laurie Ceccato1, Mélanie Mansat1, Bernard Payrastre2, Frédérique Gaits‐Iacovoni1, Julien Viaud1

1 INSERM U1048 and Université Toulouse 3, Toulouse, 2 CHU (Centre Hospitalier Universitaire) de Toulouse, Laboratoire d'Hématologie
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.31
DOI:  10.1002/cpps.35
Online Posting Date:  August, 2017
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Abstract

Phosphoinositides are key signaling and regulatory phospholipids that mediate important pathophysiological processes. This is achieved through the interaction of their phosphorylated inositol head group with a wide range of protein domains. Therefore, being able to determine the phosphoinositide specificity for effector protein is essential to the understanding of its cellular function. This unit describes a novel method named Protein‐Lipid Interaction by Fluorescence, or PLIF. PLIF is a fast, reliable and high throughput assay that allows determination of the phosphoinositide specificity of proteins, simultaneously providing relative affinities. In addition, PLIF is suitable for screening inhibitors of protein‐ phosphoinositide interaction, allowing identification of potential pharmacological compounds. © 2017 by John Wiley & Sons, Inc.

Keywords: phosphoinositide; protein‐lipid interaction; PH domain; liposome; screening

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Protein‐Lipid Interaction by Fluorescence (PLIF)
  • Basic Protocol 2: Relative Affinity Determination with PLIF
  • Basic Protocol 3: Inhibitor Screening with PLIF
  • Support Protocol 1: Preparation of Fluorescent Phosphoinositide‐Containing Liposomes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Protein‐Lipid Interaction by Fluorescence (PLIF)

  Materials
  • Phosphate‐buffered saline (PBS; Sigma Aldrich, cat. no. D8537)
  • Liposome binding buffer: PBS (Sigma Aldrich, cat. no. D8537), 150 mM potassium acetate (Sigma Aldrich, cat. no. P1147), and 1 mM MgCl 2 (Sigma Aldrich, cat. no. M8266)
  • Liposome lysis buffer: PBS (Sigma Aldrich, cat. no. D8537), 1% (v/v) Triton x‐100 (Sigma Aldrich, cat. no. X‐100)
  • GST‐tagged recombinant protein, thoroughly dialyzed against PBS buffer (to remove the glutathione used to eluate the protein during the purification): 8 µg are necessary for n = 1
  • Ice
  • 1 mM Fluorescent phosphosphoinositide‐containing liposomes (see protocol 4Support Protocol)
  • Reagent reservoir for multichannel pipette
  • Glutathione‐coated plates, clear, 8‐well strip (Thermo Scientific Pierce, cat. no. 15140)
  • Multichannel pipette (for 100 and 200 µl pipetting)
  • Microplate shaker (heidolph rotamax 120, heidolph, cat. no. 544‐41200‐00)
  • Paper towels
  • Microtiter plate, white, 96 wells, nonsterile (Nunc, cat. no. 12‐566‐04)
  • Plate reader for 96‐well microtiter plate (Thermo Scientific, Varioskan Flash Type 3001, cat. no. 5250040)
  • GraphPad Prism software (GraphPad Software) or Excel

Basic Protocol 2: Relative Affinity Determination with PLIF

  Additional Materials (see protocol 1)
  • Compound library
  • Disposable 96‐pin replicators (e.g., Fisher scientific, cat. no. NC9584102)

Basic Protocol 3: Inhibitor Screening with PLIF

  Materials
  • 10 mg/ml 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC; Avanti, cat. no. 850457; see recipe)
  • 10 mg/ml 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE; Avanti, cat. no. 850757; see recipe)
  • 1 mg/ml carboxyfluorescein‐phosphoethanolamine (carboxyfluorescein‐PE or CF‐PE) (Avanti, cat. 810332; see recipe)
  • Chloroform
  • 1 mM PtdIns3P (Echelon, cat. no. P‐3016; see recipe)
  • 1 mM PtdIns4P (Avanti, cat. no. 840045; see recipe)
  • 1 mM PtdIns5P (Echelon, cat. no. P‐5016; see recipe)
  • 1 mM PtdIns(3,4)P 2 (Echelon, cat. no. P‐3416; see recipe)
  • 1 mM PtdIns(3,5)P 2 (Echelon, cat. no. P‐3516; see recipe)
  • 1 mM PtdIns(4,5)P 2 (Avanti, cat. no. 840046; see recipe)
  • 1 mM PtdIns(3,4,5)P 3 (Echelon, cat. no. P‐3916; see recipe)
  • Liquid nitrogen
  • Liposome resuspension buffer: PBS (Sigma Aldrich, cat. no. D8537) and 150 mM Potassium acetate (Sigma Aldrich, cat. no. P1147)
  • Glass tubes (VSM, cat. no. 621.1225075080.7R000)
  • Evaporator N2 (Organomation, The Meyer N‐EVAP analytical evaporator, model 111)
  • Vacuum desiccator (Dutscher, cat. no. 029364)
  • Water bath sonicator (VWR, Ultrasonic Cleaner, cat. no. 142‐6004)
  • 1.5‐ml microcentrifuge tubes
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Figures

Videos

Literature Cited

  Berrie, C. P., & Falasca, M. (2000). Patterns within protein/polyphosphoinositide interactions provide specific targets for therapeutic intervention. FASEB Journal, 14(15), 2618–2622. doi: 10.1096/fj.00‐0096hyp.
  Bigay, J., & Antonny, B. (2005). Real‐time assays for the assembly‐disassembly cycle of COP coats on liposomes of defined size. Methods in Enzymology, 404, 95–107. doi: 10.1016/S0076‐6879(05)04010‐3.
  Ceccato, L., Chicanne, G., Nahoum, V., Pons, V., Payrastre, B., Gaits‐Iacovoni, F., & Viaud, J. (2016). PLIF: A rapid, accurate method to detect and quantitatively assess protein‐lipid interactions. Science Signaling, 9(421), rs2. doi: 10.1126/scisignal.aad4337.
  Dowler, S., Kular, G., & Alessi, D. R. (2002). Protein lipid overlay assay. Science's STKE, 2002(129), pl6. doi: 10.1126/stke.2002.129.pl6.
  Furutani, M., Tsujita, K., Itoh, T., Ijuin, T., & Takenawa, T. (2006). Application of phosphoinositide‐binding domains for the detection and quantification of specific phosphoinositides. Analytical Biochemistry, 355, 8–18. doi: 10.1016/j.ab.2006.05.014.
  Kavran, J. M., Klein, D. E., Lee, A., Falasca, M., Isakoff, S. J., Skolnik, E. Y., & Lemmon, M. A. (1998). Specificity and promiscuity in phosphoinositide binding by pleckstrin homology domains. The Journal of Biological Chemistry, 273, 30497–30508. doi: 10.1074/jbc.273.46.30497.
  Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., & Czech, M. P. (1997). Signaling by phosphoinositide‐3,4,5‐trisphosphate through proteins containing pleckstrin and Sec7 homology domains. Science, 275, 1927–1930. doi: 10.1126/science.275.5308.1927.
  Lemmon, M. A. (2003). Phosphoinositide recognition domains. Traffic, 4(4), 201–213. doi: 10.1034/j.1600‐0854.2004.00071.x.
  Narayan, K., & Lemmon, M. A. (2006). Determining selectivity of phosphoinositide‐binding domains. Methods, 39(2), 122–133. doi: 10.1016/j.ymeth.2006.05.006.
  Pendaries, C., Tronchere, H., Plantavid, M., & Payrastre, B. (2003). Phosphoinositide signaling disorders in human diseases. FEBS Letters, 546(1), 25–31. doi: 10.1016/S0014‐5793(03)00437‐X.
  Viaud, J., Mansour, R., Antkowiak, A., Mujalli, A., Valet, C., Chicanne, G., … Payrastre, B. (2016). Phosphoinositides: Important lipids in the coordination of cell dynamics. Biochimie, 125, 250–258. doi: 10.1016/j.biochi.2015.09.005.
Key References
  Ceccato, L., Chicanne, G., Nahoum, V., Pons, V., Payrastre, B., Gaits‐Iacovoni, F., & Viaud, J. (2016). PLIF: A rapid, accurate method to detect and quantitatively assess protein‐lipid interactions. Science Signaling, 9(421), rs2. doi: 10.1126/scisignal.aad4337.
  This article describes the validation of the PLIF assay using well characterized protein domains as well as its use for high‐throughput screening of protein‐lipid interaction inhibitors.
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