Monitoring regulated protein-protein interactions using split TEV

Wehr, Michael C.; Laage, Rico; Bolz, Ulrike; Fischer, Tobias M.; Grünewald, Sylvia; Scheek, Sigrid; Bach, A.; Nave, Klaus‐Armin; Rossner, Moritz J.

doi:10.1038/nmeth967

Cited by 256 publications

(286 citation statements)

References 28 publications

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“…Recently, a protein interaction technique based on the complementation of split TEV protease fragments has been described (1). Like the Tango method described here, this split TEV approach involves a cleavage step catalyzed by TEV protease, thereby converting transient interactions into a long-lasting signaling readout.…”

Section: Discussionmentioning

confidence: 99%

The genetic design of signaling cascades to record receptor activation

Barnea

Strapps

Herrada

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

595

603

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We have developed an experimental strategy to monitor protein interactions in a cell with a high degree of selectivity and sensitivity. A transcription factor is tethered to a membrane-bound receptor with a linker that contains a cleavage site for a specific protease. Activation of the receptor recruits a signaling protein fused to the protease that then cleaves and releases the transcription factor to activate reporter genes in the nucleus. This strategy converts a transient interaction into a stable and amplifiable reporter gene signal to record the activation of a receptor without interference from endogenous signaling pathways. We have developed this assay for three classes of receptors: G protein-coupled receptors, receptor tyrosine kinases, and steroid hormone receptors. Finally, we use the assay to identify a ligand for the orphan receptor GPR1, suggesting a role for this receptor in the regulation of inflammation.cellular assays ͉ G protein-coupled receptor ͉ protein interaction A ll cells have evolved mechanisms to respond to rapid changes in the environment. Extracellular signals are detected by transmembrane receptors that translate binding into intracellular signaling events. Most signaling systems that respond to environmental cues exhibit adaptation mechanisms that afford the cell a facile response to rapid changes in their surroundings. Mechanisms to assure the rapid but transient response to environmental cues are of obvious advantage to the cell but seriously limit most assays for receptor function. We have genetically modified receptors such that transient responses to ligand result in the stable transcription of a reporter gene. The transformation of a transient intracellular response to a stable amplifiable readout provides a sensitive and quantitative assay for receptor function.We have developed an assay for receptor activation and more generally for protein-protein interaction that involves the fusion of a membrane receptor with a transcriptional activator. The membrane-bound receptor and transcription factor sequences are separated by a cleavage site for a highly specific viral protease. A second gene encodes a fusion of the viral protease with a cellular protein that interacts only with activated receptor. Ligand binding to the receptor will stimulate this proteinprotein interaction, recruiting the protease to its cleavage site. Site-specific cleavage will release the transcriptional regulator that can now enter the nucleus and activate reporter genes. Recently, a similar principle, based on the complementation of split tobacco etch virus (TEV) protease fragments, has been used to monitor protein interactions (1). Our experimental scheme derives conceptually from the mechanism of action of the Notch receptor in which ligand binding elicits proteolytic cleavage events in the receptor to release a Notch intracellular domain that translocates to the nucleus and modulates transcription of downstream target genes (2, 3) (Fig. 1A).The assay we have developed relies solely on exogenous genes in...

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Section: Discussionmentioning

confidence: 99%

The genetic design of signaling cascades to record receptor activation

Barnea

Strapps

Herrada

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

595

603

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“…In both constructs, TEV protease is co-expressed from the same plasmid employing an internal ribosome entry site (IRES) mediating release of the CD74 ICD specifically from the fusion protein with the TEV cleavage site, whereas the CD74 ICD remains membrane bound in the control. Because the TEV protease is highly specific, heterologous expression of this protease is well tolerated in different experimental systems, including mammalian cells (30).…”

Section: A Potential Role Of the Cd74 Icd In Transcriptional Regulationmentioning

confidence: 99%

A Cell‐Based Assay Reveals Nuclear Translocation of Intracellular Domains Released by SPPL Proteases

Mentrup

Häsler

Fluhrer

et al. 2015

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“…Over the last decade, we could observe significant progress in the experimental techniques for the identification of interactions between proteins. Several types of experimental assay, such as the yeast two-hybrid assay [37][38][39][40] or tandem affinity purification [41], allow the high efficiency experimental analysis of protein-protein interactions on the whole proteome scale (for a review of the experimental methods, please refer to [11,12,[42][43][44][45][46][47][48][49][50][51][52]). Those advances in experimental methodology quickly led to progress in theoretical approaches, such as those based on homology [53], protein pathways analysis [54], multimeric threading [55], or the prediction of interaction sites by docking methods [56].…”

Section: Algorithmsmentioning

confidence: 99%

The interactome: Predicting the protein-protein interactions in cells

Plewczyński

Ginalski

2009

Cellular and Molecular Biology Letters

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Abstract:The term Interactome describes the set of all molecular interactions in cells, especially in the context of protein-protein interactions. These interactions are crucial for most cellular processes, so the full representation of the interaction repertoire is needed to understand the cell molecular machinery at the system biology level. In this short review, we compare various methods for predicting protein-protein interactions using sequence and structure information. The ultimate goal of those approaches is to present the complete methodology for the automatic selection of interaction partners using their amino acid sequences and/or three dimensional structures, if known. Apart from a description of each method, details of the software or web interface needed for high throughput prediction on the whole genome scale are also provided. The proposed validation of the theoretical methods using experimental data would be a better assessment of their accuracy.

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Monitoring regulated protein-protein interactions using split TEV

Cited by 256 publications

References 28 publications

The genetic design of signaling cascades to record receptor activation

The genetic design of signaling cascades to record receptor activation

A Cell‐Based Assay Reveals Nuclear Translocation of Intracellular Domains Released by SPPL Proteases

The interactome: Predicting the protein-protein interactions in cells

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