Two-hybrid and its recent adaptations

Lievens, Sam; Lemmens, Irma; Montoye, Tony; Eyckerman, Sven; Tavernier, Jan

doi:10.1016/j.ddtec.2006.09.006

Cited by 6 publications

(4 citation statements)

References 47 publications

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“…Y2H was reported to not work for a majority of baits due to auto-activation or failure of nuclear localization of bait and/or prey, and typically reports strong PPIs [40,71]. Nevertheless, we were able to validate the interaction of PDCD4 with the full-length EIF4A1 protein, reported by Virotrap.…”

Section: Discussionmentioning

confidence: 62%

N-terminal proteoforms may engage in different protein complexes

Bogaert

Fijałkowska

Staes

et al. 2023

Preprint

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Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared to their canonical counterparts. Such proteoforms can have altered localizations, stabilities and functions. While proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. Additionally, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues as well as tissue-specific expression, highlighting their biological relevance. Protein-protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared to their canonical counterpart, thus further expanding the functional diversity of proteomes.

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Section: Discussionmentioning

confidence: 62%

N-terminal proteoforms may engage in different protein complexes

Bogaert

Fijałkowska

Staes

et al. 2023

Preprint

Self Cite

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show abstract

“…The lower number of PPIs found by Y2H could partially be explained by the technology’s limitations and the considered approach, as baits were only tested in one setup (bait coupled to AD and tested for interaction with prey coupled to DB, and not in other setups such as bait coupled C-terminally to AD or bait coupled to DB). Therefore, we found that Y2H did not work for most of the baits, also likely, because of auto-activation or failure of nuclear localization of bait and/or prey ( 8 , 54 ). Nevertheless, we were able to validate the interaction of PDCD4 with the full-length EIF4A1 protein, reported by Virotrap.…”

Section: Discussionmentioning

confidence: 86%

N-terminal proteoforms may engage in different protein complexes

et al. 2023

Self Cite

View full text Add to dashboard Cite

Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared with their canonical counterparts. Such proteoforms can have altered localizations, stabilities, and functions. Although proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. In addition, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues, as well as tissue-specific expression, highlighting their biological relevance. Protein–protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared with their canonical counterparts, thus further expanding the functional diversity of proteomes.

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“…It binds as a dimer to 17-bp palindromic binding sites (17), and typical reporter constructs contain 5 or 6 dimeric binding sites (18). To test whether this represents an optimized number for reporter sensitivity, we introduced up to 48 Gal4 binding sites into the firefly luciferase (Fluc) reporter construct (corresponding to 24 dimeric consensus binding sites).…”

Section: Resultsmentioning

confidence: 99%

A Sensitive, Semi-Quantitative Mammalian Two-Hybrid Assay

2017

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Protein-protein interactions critically determine the function of a protein within the cell. Several methods have been developed for the analysis of protein interactions, including two-hybrid assays in yeast and mammals. Mammalian two-hybrid systems provide the ideal physiological environment to study the interactions of mammalian proteins; however, these approaches are limited in sensitivity and their ability to quantify interaction strength. Here, we present an inducible mammalian two-hybrid (iM2H) system using the small-molecule dimerizer rapalog for recruitment of multiple transactivation domains into the M2H system. This inducibility, combined with additional improvements of the iM2H components, results in an up to 100-fold increase in sensitivity compared with conventional M2H approaches. In addition, we include a number of reference interactions in our iM2H approach, which enable semiquantitative assessment of protein interactions. Using Groucho/Tle proteins and their binding partners, we demonstrate the applicability of our iM2H to established protein networks. Finally, to test the applicability of our system for drug screening, the interference of a small-molecule inhibitor on a known protein-protein interaction was tested, and the particular advantages of the internal reference interactions were shown.

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Two-hybrid and its recent adaptations

Cited by 6 publications

References 47 publications

N-terminal proteoforms may engage in different protein complexes

N-terminal proteoforms may engage in different protein complexes

N-terminal proteoforms may engage in different protein complexes

A Sensitive, Semi-Quantitative Mammalian Two-Hybrid Assay

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