Characterization of Nuclear Receptor Ligands by Multiplexed Peptide Interactions

互联网2013-12-31

1486

Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit describes a method to evaluate the effect that small molecules have on the binding interactions of a nuclear receptor protein with a series of peptides. The multiplexed microsphere?based system employs peptide?coupled microsphere populations that are fluorescently unique and thereby identifiable by flow cytometric analysis. Up to 100 different peptide?nuclear receptor interactions may be analyzed in a single well of a 96?well microtiter plate. This approach allows rapid and sensitive characterization of nuclear receptor ligands based on nuclear receptor protein?peptide interaction profiles. Since nuclear receptor binding interactions are dynamically related to protein conformation, the approach allows rapid evaluation of nuclear receptor ligands that may impart unique protein structure. The no?wash format and the high surface density of the microsphere?coupled interaction partner offer a moderately high?throughput system to examine low? to high?affinity interactions with excellent sensitivity. This approach, although described for nuclear receptors, may also be applied to other types of molecular interactions.

Keywords: Microsphere; flow cytometry; nuclear receptor; coactivator; corepressor; cofactor; agonist; antagonist; ligand; ligand binding domain; protein conformation

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Basic Protocol 1: Multiplexed Binding Assay
Support Protocol 1: Coupling of Biotinylated Peptides to Lumavidin‐Coated Microspheres
Support Protocol 2: Coupling of Biotinylated Nuclear Receptor LBD to Streptavidin‐Alexa fluor 532
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Multiplexed Binding Assay

Materials

Peptide‐coupled microsphere suspension (see protocol 2 )
PBS‐TBN/DTT (see recipe )
DMSO with or without ligand
Alexa Fluor 532–labeled NR LBD (see protocol 3 )

96‐well flat‐bottom assay plates, non‐binding surface, non‐sterile (Corning)
Multichannel pipet
LX‐100 flow cytometer with x‐y plate sampler for automated sampling from 96‐well microtiter plates (Luminex Corporation)

Support Protocol 1: Coupling of Biotinylated Peptides to Lumavidin‐Coated Microspheres

Materials

Phosphate buffered saline without calcium or magnesium (PBS; appendix 2A )
xMAP LumAvidin‐coated polystyrene microsphere populations (5.6‐µm diameter; Luminex Corporation); store at 4°C in the dark
PBS‐TBN/DTT (see recipe )
Lyophilized biotinylated peptide (Synpep or American Peptide)
DMSO
5 mM D‐biotin (see recipe )
96‐well filter‐bottom plates, MultiScreen BV 1.2‐µm, clear, non‐sterile (Millipore)
MultiScreen vacuum manifold for 96‐well plates (Millipore)

Support Protocol 2: Coupling of Biotinylated Nuclear Receptor LBD to Streptavidin‐Alexa fluor 532

Materials

Biotinylated nuclear receptor ligand binding domain (NR LBD; non‐biotinylated receptors are commercially available from Invitrogen, Active Motif, Jena Bioscience, Protein One, as well as other manufacturers)
16 µM streptavidin–Alexa Fluor 532 conjugate (see recipe )
5 mM free D‐biotin (see recipe )
PBS‐TBN/DTT (see recipe )

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 13.6.1 Luminex system for multiplexed microsphere‐based analysis. (A ) Dot plot of 70 microsphere populations is shown represented in terms of red fluorescence (microsphere classification 1) versus near‐infrared fluorescence (microsphere classification 2). Each individual dot represents the fluorescence measured from a single microsphere, and each microsphere population will fall into a single default fluorescent zone indicated by the white circular region. (B ) Histogram of microsphere side‐scatter measurement. In order to restrict analysis to single microspheres and exclude debris, doublets, and aggregates, the microspheres are gated based on side scatter. (C ) Alexa Fluor 532 reporter fluorescence as mean fluorescence intensity units (MFI). Reporter fluorescence is measured at ∼575 nm. MFIs for each population are reported in spreadsheet format for each well.

View Image

Figure 13.6.2 Schematic representation of the multiplexed peptide–nuclear receptor interaction assay at a fixed concentration of subsaturating fluorochrome‐coupled nuclear receptor and a receptor‐saturating concentration of ligand.

View Image

Figure 13.6.3 Glucocorticoid receptor [F602S] LBD binding to the coactivator peptide PGC‐1 alpha (130‐154) (biotin‐DGTPPPQEAEEPSLLKKLLLAPANT‐CONH2) (A ) or the corepressor peptide NCoR (2251‐2275) (biotin‐GHSFADPASNLGLEDIIRKALMGSF‐CONH2) (B ) in the absence or presence of 10 µM dexamethasone or mifepristone. The GR [F602S] LBD was labeled with Alexa Fluor 532. Incubation was 4 hr at room temperature.

View Image

Figure 13.6.4 ERα LBD binding to the coactivator peptide SRC‐1(2) (676‐700) (biotin‐CPSSHSSLTERHKILHRLLQEGSPS‐CONH2) in the absence or presence of 10 µM estradiol or raloxifene. The ERα LBD was labeled with Alexa Fluor 532. Incubation was 2 hr at room temperature. See Figure for the chemical structures of estradiol and raloxifene.

View Image

Figure 13.6.5 Example of ERα LBD peptide‐profiling data and cluster analysis using data from a microsphere‐based flow cytometric assay. Two example profile plots for 17‐β estradiol and raloxifene are shown. Each plot represents binding data for a single concentration of fluorescently labeled ERα LBD under saturating ligand conditions. To show the effect of ligand on peptide binding, the basal fluorescence is subtracted from the fluorescence in the presence of ligand. A value of zero indicates no effect on peptide binding relative to unliganded receptor. Each vertical grey line shows data for a single peptide, and the histogram data points are connected at the maximum or minimum to yield a profile plot. After acquiring data from a large set, principal components analysis selects the most statistically relevant peptides and the profiles are then clustered based on overall profile shape. The bottom of the figure shows an example of a cluster analysis for ERα data, where a few key tool compounds are highlighted as shaded profile plots. Each grey profile plot represents data for a single compound. Reproduced with permission from Drug Discovery Today .

View Image

Videos

Literature Cited

Literature Cited
	Brzozowski, A.M., Pike, A.C.W., Dauter, Z., Hubbard, R.E., Bonn, T., Engström, O., Ohman, L., Greene, G.L., Gustafsson. J.‐A., and Carlquist, M. 1997. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753‐758.
	Glickman, J.F., Wu, X., Mercuri, R., Illy, C., Bowen, B.R., He, Y., and Sills, M. 2002. A comparison of ALPHAScreen, TR‐FRET, and TRF as assay methods for FXR nuclear receptors. J. Biomol. Screen. 7:3‐10.
	Iannone, M.A., Consler, T.G., Pearce, K.H., Stimmel, J.B., Parks, D.J., and Gray, J.G. 2001. Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres. Cytometry 44:326‐337.
	Iannone, M.A., Simmons, C.A., Kadwell, S.H., Svoboda, D.L., Vanderwall, D.E., Deng, S.‐J., Consler, T.G., Shearin, J., Gray, J.G. and Pearce, K.H. 2004. Correlation between in vitro peptide binding profiles and cellular activities for estrogen receptor modulating compounds. Mol. Endocrinol. 18:1064‐1081.
	Lee, J.W., Lee, Y.C., Na, S.‐Y., Jung, D.‐J. and Lee, S.‐K. 2001. Transcriptional coregulators of the nuclear receptor superfamily: Coactivators and corepressors. Cell. Molec. Life Sci. 58:287‐297.
	Lonard, D.M. and O'Malley, B.W. 2005. Expanding functional diversity of the coactivators. Trends Biochem. Sci. 30:126‐132.
	Myszka, D.G. 2000. Kinetic, equilibrium, and thermodynamic analysis of macromolecular interactions with BIACORE. Methods Enzymol. 323:325‐340.
	Nettles, K.W. and Greene, G.L. 2005. Ligand control of coregulator recruitment to nuclear receptors. Annu. Rev. Physiol. 67:309‐333.
	Nilsson, M., Dahlman‐Wright, K., and Gustafsson, J.A. 2004. Nuclear receptors in disease: The oestrogen receptors. Essays Biochem. 40:157‐167.
	Norris, J.D., Paige, L.A., Christensen, D.J., Chang, C.Y., Huacani, M.R., Fan, D., Hamilton, P.T., Fowlkes, D.M., and McDonnell, D.P. 1999. Peptide antagonists of the human estrogen receptor. Science 285:744‐746.
	Northrop, J.P., Nguyen, D., Piplani, S., Olivan, S.E., Kwan, S.T., Go, N.F., Hart, C.P., and Schatz, P.J. 2000. Selection of estrogen receptor beta‐ and thyroid hormone receptor beta‐specific coactivator‐mimetic peptides using recombinant peptide libraries. Mol. Endocrinol. 14:605‐622.
	Novac, N. and Heinzel, T. 2005. Nuclear receptors: Overview and classification. Curr. Drug Targets ‐ Inflammation & Allergy 3:335‐346.
	Paige, L.A., Christensen, D.J., Gron, H., Norris, J.D. Gottlin, E.B., Padilla, K.M., Chang, C.Y., Ballas, L.M., Hamilton, P.T., McDonnell, D.P., and Fowlkes, D.M. 1999. Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta. Proc. Natl. Acad. Sci. U.S.A. 96:3999‐4004.
	Parker, G.J., Law, T.L., Lenoch, F.J., and Bolger, R.E. 2000. Development of high throughput screening assays using fluorescence polarization: Nuclear receptor‐ligand‐binding and kinase/ phosphatase assays. J. Biomol. Screen. 5:77‐88.
	Pearce, K.H., Iannone, M.A., Simmons, C.A., and Gray, J.G. 2004. Discovery of novel nuclear receptor modulating ligands: An integral role for peptide interaction profiling. Drug Discov. Today 9:741‐751.
	Savkur, R.S., Bramlett, K.S., Clawson, D., and Burris, T.P. 2004. Pharmacology of nuclear receptor‐coregulator recognition. Vitam. Horm. 68:145‐183.
	Schulman, I.G. and Heyman, R.A. 2004. The flip side: Identifying small molecule regulators of nuclear receptors. Chem. Biol. 11:639‐646.
	Shiau, A.K., Barstad, D., Loria, P.M., Cheng, L., Kushner, P.J., Agard, D.A., and Greene, G.L. 1998. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927‐937.
	Sidhu, S.S., Lowman, H.B., Cunningham, B.C., and Wells, J.A. 2000. Phage display for selection of novel binding peptides. Methods Enzymol. 328:333‐363.
	Sladek, R. and Giguere, V. 2000. Orphan nuclear receptors: An emerging family of metabolic regulators. Adv. Pharmacol. 47:23‐87.
	Spencer, T.E., Jenster, G., Burcin, M.M., Allis, C.D., Zhou, J., Mizzen, C.A., McKenna, N.J., Onate, S.A., Tsai, S.Y., Tsai, M.J., and O'Malley, B.W. 1997. Steroid receptor coactivator‐1 is a histone acetyl‐transferase. Nature 389:194‐198.
	Zhou, G., Cummings, R., Hermes, J., and Moller, D.E. 2001. Use of homogeneous time‐resolved fluorescence energy transfer in the measurement of nuclear receptor activation. Methods 25:54‐61.