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

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.

Redox Regulation of PPARγ in Polarized Macrophages

“…Expression vectors for stable overexpression of the proteins Clover-PPAR γ , roGFP2, and Grx1-roGFP2 were generated by replacing EGFP of pHR′SIN-cPPT-SE [ 22 ] by the appropriate coding sequences due to In-Fusion® (Clontech, Takara, Japan) recombination as previously described [ 23 ]. In brief, the plasmids pcDNA3-Clover [ 24 ], pDsRed-Monomer-C1-hPPAR γ 1 [ 25 ], and pQE-60_Grx1-roGFP2 [ 26 ] were used as templates.…”

“…PPAR γ -dependent transactivation assays were performed after cotransfection of Clover-hPPARγ encoding vectors together with the Firefly and Renilla luciferase genes containing vectors p(AOX) 3 -TK-Luc [ 27 , 28 ] and pRL-CMV (Promega GmbH) as previously described [ 23 ]. In parallel to the Clover-hPPAR γ wild type construct, also cysteine to alanine mutants regarding hPPAR γ (C109A, C112A, C126A, C129A, C146A, C150A, C160A, C163A, C168A, and C284A) was used.…”

“…To produce stably overexpressing cell lines, lentiviral particles were used which were generated by JetPRIME® (PEQLAB Biotechnologie GmbH, Erlangen, Germany) transfection of HEK293T with the lentiviral vectors psPAX2 (Addgene#12260), pMD2.G (Addgene#12259), and cloned vectors as previously described [23]. Transduced J774A.1 cells were sorted for green positive cells using BD FACSAria™ III cell sorter (BD Biosciences, Heidelberg, Germany).…”

Flow cytometry based-FRET: basics, novel developments and future perspectives

“…As an example, CFP and YFP which have been the most popular fluorophores for long [ 29 ] are less utilized nowadays. The reason is that CFP and YFP have some limitations in their applicability [ 30 ], such as their dependence on pH and the relatively low quantum yield of CFP, which renders it non-optimal as a FRET donor. A feasible alternative, therefore, could be the use of Clover/mRuby2 as FRET-pair, a green and a red fluorophore, which show increased Förster distance and yield better possibilities for the detection of fast molecular interactions [ 4 , 31 ].…”

Development of a Fluorescent Protein Based FRET Biosensor for Determination of Protease Activity

“…Fluorescent proteins are in most cases nontoxic to host cells and can remain stable in cells for a long time thanks to their high intracellular stability [2,6]. In this context, fluorescent proteins and FRET-based biosensors have become powerful tools for in vitro and in vivo applications [6][7][8]. In FP-FRET sensors, the selection of the FRET pair (donor and acceptor fluorophores) is the most important parameter for the performance of the biosensors to be designed [9].…”