Dataset Information

Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay.

ABSTRACT: The interferon-induced transmembrane proteins 1-3 (IFITM1-3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1-3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein-protein interactions. Coexpression of IFITM1-3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

SUBMITTER: Winkler M

PROVIDER: S-EPMC6719045 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay.

Winkler Michael M Wrensch Florian F Bosch Pascale P Knoth Maike M Schindler Michael M Gärtner Sabine S Pöhlmann Stefan S

International journal of molecular sciences 20190808 16

The interferon-induced transmembrane proteins 1-3 (IFITM1-3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1-3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy tr ...[more]

PMID: 31398796

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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

Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay.

Publications

Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay.

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