Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells

Verena Trümper, Andreas von Knethen, Annegret Preuß, Eugeny Ermilov, Steffen Hackbarth, Laura Kuchler, Sandra Gunne, Anne Schäfer, Tobias Bornhütter, G. Vereb, Lázló Ujlaky-Nagy, Bernhard Brüne, Beate Röder, Michael Schindler, Michael J. Parnham, Tilo Knape

Research output: Contribution to journalArticle

Abstract

PPARγ is a pharmacological target in inflammatory and metabolic diseases. Upon agonistic treatment or following antagonism, binding of co-factors is altered, which consequently affects PPARγ-dependent transactivation as well as its DNA-independent properties. Therefore, establishing techniques to characterize these interactions is an important issue in living cells. Methods: Using the FRET pair Clover/mRuby2, we set up a flow cytometry-based FRET assay by analyzing PPARγ1 binding to its heterodimerization partner RXRα. Analyses of PPARγ-reporter and co-localization studies by laser-scanning microscopy validated this system. Refining the system, we created a new readout to distinguish strong from weak interactions, focusing on PPARγ-binding to the co-repressor N-CoR2. Results: We observed high FRET in cells expressing Clover-PPARγ1 and mRuby2-RXRα, but no FRET when cells express a mRuby2-RXRα deletion mutant, lacking the PPARγ interaction domain. Focusing on the co-repressor N-CoR2, we identified in HEK293T cells the new splice variant N-CoR2-ΔID1-exon. Overexpressing this isoform tagged with mRuby2, revealed no binding to Clover-PPARγ1, nor in murine J774A.1 macrophages. In HEK293T cells, binding was even lower in comparison to N-CoR2 constructs in which domains established to mediate interaction with PPARγ binding are deleted. These data suggest a possible role of N-CoR2-ΔID1-exon as a dominant negative variant. Because binding to N-CoR2-mRuby2 was not altered following activation or antagonism of Clover-PPARγ1, we determined the effect of pharmacological treatment on FRET intensity. Therefore, we calculated flow cytometry-based FRET efficiencies based on our flow cytometry data. As with PPARγ antagonism, PPARγ agonist treatment did not prevent binding of N-CoR2. Conclusion: Our system allows the close determination of protein-protein interactions with a special focus on binding intensity, allowing this system to characterize the role of protein domains as well as the effect of pharmacological agents on protein-protein interactions.

Original languageEnglish
Pages (from-to)5444-5463
Number of pages20
JournalTheranostics
Volume9
Issue number19
DOIs
Publication statusPublished - Jan 1 2019

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Peroxisome Proliferator-Activated Receptors
Flow Cytometry
Medicago
Proteins
Co-Repressor Proteins
Pharmacology
Exons
Metabolic Diseases
Confocal Microscopy
Transcriptional Activation
Protein Isoforms
Macrophages
DNA

Keywords

  • Binding affinity and intensity
  • Co-localization analysis
  • Flow cytometry-based FRET assay
  • FRET
  • N-CoR2
  • NHR co-factors protein-protein interactions
  • PPARγ1
  • RXRα

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Pharmacology, Toxicology and Pharmaceutics (miscellaneous)

Cite this

Trümper, V., von Knethen, A., Preuß, A., Ermilov, E., Hackbarth, S., Kuchler, L., ... Knape, T. (2019). Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells. Theranostics, 9(19), 5444-5463. https://doi.org/10.7150/thno.29367

Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells. / Trümper, Verena; von Knethen, Andreas; Preuß, Annegret; Ermilov, Eugeny; Hackbarth, Steffen; Kuchler, Laura; Gunne, Sandra; Schäfer, Anne; Bornhütter, Tobias; Vereb, G.; Ujlaky-Nagy, Lázló; Brüne, Bernhard; Röder, Beate; Schindler, Michael; Parnham, Michael J.; Knape, Tilo.

In: Theranostics, Vol. 9, No. 19, 01.01.2019, p. 5444-5463.

Research output: Contribution to journalArticle

Trümper, V, von Knethen, A, Preuß, A, Ermilov, E, Hackbarth, S, Kuchler, L, Gunne, S, Schäfer, A, Bornhütter, T, Vereb, G, Ujlaky-Nagy, L, Brüne, B, Röder, B, Schindler, M, Parnham, MJ & Knape, T 2019, 'Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells', Theranostics, vol. 9, no. 19, pp. 5444-5463. https://doi.org/10.7150/thno.29367
Trümper V, von Knethen A, Preuß A, Ermilov E, Hackbarth S, Kuchler L et al. Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells. Theranostics. 2019 Jan 1;9(19):5444-5463. https://doi.org/10.7150/thno.29367
Trümper, Verena ; von Knethen, Andreas ; Preuß, Annegret ; Ermilov, Eugeny ; Hackbarth, Steffen ; Kuchler, Laura ; Gunne, Sandra ; Schäfer, Anne ; Bornhütter, Tobias ; Vereb, G. ; Ujlaky-Nagy, Lázló ; Brüne, Bernhard ; Röder, Beate ; Schindler, Michael ; Parnham, Michael J. ; Knape, Tilo. / Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells. In: Theranostics. 2019 ; Vol. 9, No. 19. pp. 5444-5463.
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AU - Trümper, Verena

AU - von Knethen, Andreas

AU - Preuß, Annegret

AU - Ermilov, Eugeny

AU - Hackbarth, Steffen

AU - Kuchler, Laura

AU - Gunne, Sandra

AU - Schäfer, Anne

AU - Bornhütter, Tobias

AU - Vereb, G.

AU - Ujlaky-Nagy, Lázló

AU - Brüne, Bernhard

AU - Röder, Beate

AU - Schindler, Michael

AU - Parnham, Michael J.

AU - Knape, Tilo

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