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001-es BibID:	BIBFORM081231
035-os BibID:	(WoS)000481600100003 (Scopus)85070484588
Első szerző:	Trümper, Verena
Cím:	Flow cytometry-based FRET identifies binding intensities in PPAR[gamma]1 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, György Vereb, László Ujlaky-Nagy, Bernhard Brüne, Beate Röder, Michael Schindler, Michael J. Parnham, Tilo Knape
Dátum:	2019
ISSN:	1838-7640
Megjegyzések:	PPAR gamma is a pharmacological target in inflammatory and metabolic diseases. Upon agonistic treatment or following antagonism, binding of co-factors is altered, which consequently affects PPAR gamma-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 PPAR gamma 1 binding to its heterodimerization partner RXR alpha. Analyses of PPAR gamma-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 PPAR gamma-binding to the co-repressor N-CoR2. Results: We observed high FRET in cells expressing Clover-PPAR gamma 1 and mRuby2-RXR alpha, but no FRET when cells express a mRuby2-RXR alpha deletion mutant, lacking the PPAR gamma interaction domain. Focusing on the co-repressor N-CoR2, we identified in HEK293T cells the new splice variant N-CoR2-Delta ID1-exon. Overexpressing this isoform tagged with mRuby2, revealed no binding to Clover-PPAR gamma 1, 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 PPAR gamma binding are deleted. These data suggest a possible role of N-CoR2-Delta ID1-exon as a dominant negative variant. Because binding to N-CoR2-mRuby2 was not altered following activation or antagonism of Clover-PPAR gamma 1, 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 PPAR gamma antagonism, PPAR gamma 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.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk PPAR[gamma] FRET
Megjelenés:	Theranostics. - 9 : 19 (2019), p. 5444-5463. -
További szerzők:	von Knethen, Andreas Preuß, Annegret Ermilov, Eugeny Hackbarth, Steffen Kuchler, Laura Gunne, Sandra Schäfer, Anne Bornhütter, Tobias Vereb György (1965-) (biofizikus, orvos) Ujlaky-Nagy László (1977-) (biofizikus) Brüne, Bernhard Röder, Beate Schindler, Michael Parnham, Michael J. Knape, Tilo
Pályázati támogatás:	German Research Foundation (DFG) KN493/13-1 Egyéb
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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