Dataset Information

A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells.

ABSTRACT:

Background

Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-formation.

Results

Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format.

Conclusion

The presented combination of FRET and FACS offers the precious possibility to discover and define protein interactions in living cells and is expected to contribute to the identification of novel therapeutic targets for treatment of human diseases.

SUBMITTER: Banning C

PROVIDER: S-EPMC2825263 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Publications

A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells.

Banning Carina C Votteler Jörg J Hoffmann Dirk D Koppensteiner Herwig H Warmer Martin M Reimer Rudolph R Kirchhoff Frank F Schubert Ulrich U Hauber Joachim J Schindler Michael M

PloS one 20100222 2

<h4>Background</h4>Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-format ...[more]

PMID: 20179761

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Project description:PPAR? 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?-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?1 binding to its heterodimerization partner RXR?. Analyses of PPAR?-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?-binding to the co-repressor N-CoR2. Results: We observed high FRET in cells expressing Clover-PPAR?1 and mRuby2-RXR?, but no FRET when cells express a mRuby2-RXR? deletion mutant, lacking the PPAR? interaction domain. Focusing on the co-repressor N-CoR2, we identified in HEK293T cells the new splice variant N-CoR2-?ID1-exon. Overexpressing this isoform tagged with mRuby2, revealed no binding to Clover-PPAR?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? binding are deleted. These data suggest a possible role of N-CoR2-?ID1-exon as a dominant negative variant. Because binding to N-CoR2-mRuby2 was not altered following activation or antagonism of Clover-PPAR?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? antagonism, PPAR? 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.

Dataset Information

A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells.

Background

Results

Conclusion

Publications

A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells.

Similar Datasets

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