A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes
Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein–protein interactions (PPIs) in live conditions. Here we p...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-01-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/12/1/200 |
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| author | Yunlong Jia Jonathan Reboulet Benjamin Gillet Sandrine Hughes Christelle Forcet Violaine Tribollet Nawal Hajj Sleiman Cindy Kundlacz Jean-Marc Vanacker Françoise Bleicher Samir Merabet |
| author_facet | Yunlong Jia Jonathan Reboulet Benjamin Gillet Sandrine Hughes Christelle Forcet Violaine Tribollet Nawal Hajj Sleiman Cindy Kundlacz Jean-Marc Vanacker Françoise Bleicher Samir Merabet |
| author_sort | Yunlong Jia |
| collection | DOAJ |
| description | Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein–protein interactions (PPIs) in live conditions. Here we presented an experimental strategy: the Cell-PCA (cell-based protein complementation assay), which was based on bimolecular fluorescence complementation (BiFC) for ORFeome-wide screening of proteins that interact with different bait proteins in the same live cell context, by combining high-throughput sequencing method. The specificity and sensitivity of the Cell-PCA was established by using a wild-type and a single-amino-acid-mutated HOXA9 protein, and the approach was subsequently applied to seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 that promoted its proliferative activity in a cancer cell context. Taken together, our work demonstrated that the Cell-PCA was pertinent for revealing and, importantly, comparing the interactomes of different or highly related bait proteins in the same cell context. |
| first_indexed | 2024-03-11T10:04:41Z |
| format | Article |
| id | doaj.art-2cfdbbf8254f4fbb96a7fa981fb88c90 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-11T10:04:41Z |
| publishDate | 2023-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-2cfdbbf8254f4fbb96a7fa981fb88c902023-11-16T15:07:27ZengMDPI AGCells2073-44092023-01-0112120010.3390/cells12010200A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX InteractomesYunlong Jia0Jonathan Reboulet1Benjamin Gillet2Sandrine Hughes3Christelle Forcet4Violaine Tribollet5Nawal Hajj Sleiman6Cindy Kundlacz7Jean-Marc Vanacker8Françoise Bleicher9Samir Merabet10IGFL, CNRS UMR5242, ENS-Lyon, UCBL-1, INRA USC1370, 32 Av. Tony Garnier, 69007 Lyon, FranceIGFL, CNRS UMR5242, ENS-Lyon, UCBL-1, INRA USC1370, 32 Av. Tony Garnier, 69007 Lyon, FranceIGFL, CNRS UMR5242, ENS-Lyon, UCBL-1, INRA USC1370, 32 Av. Tony Garnier, 69007 Lyon, FranceIGFL, CNRS UMR5242, ENS-Lyon, UCBL-1, INRA USC1370, 32 Av. Tony Garnier, 69007 Lyon, FranceIGFL, CNRS UMR5242, ENS-Lyon, UCBL-1, INRA USC1370, 32 Av. Tony Garnier, 69007 Lyon, FranceIGFL, CNRS UMR5242, ENS-Lyon, UCBL-1, INRA USC1370, 32 Av. Tony Garnier, 69007 Lyon, FranceIGFL, CNRS UMR5242, ENS-Lyon, UCBL-1, INRA USC1370, 32 Av. Tony Garnier, 69007 Lyon, FranceIGFL, CNRS UMR5242, ENS-Lyon, UCBL-1, INRA USC1370, 32 Av. Tony Garnier, 69007 Lyon, FranceIGFL, CNRS UMR5242, ENS-Lyon, UCBL-1, INRA USC1370, 32 Av. Tony Garnier, 69007 Lyon, FranceIGFL, CNRS UMR5242, ENS-Lyon, UCBL-1, INRA USC1370, 32 Av. Tony Garnier, 69007 Lyon, FranceIGFL, CNRS UMR5242, ENS-Lyon, UCBL-1, INRA USC1370, 32 Av. Tony Garnier, 69007 Lyon, FranceBiological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein–protein interactions (PPIs) in live conditions. Here we presented an experimental strategy: the Cell-PCA (cell-based protein complementation assay), which was based on bimolecular fluorescence complementation (BiFC) for ORFeome-wide screening of proteins that interact with different bait proteins in the same live cell context, by combining high-throughput sequencing method. The specificity and sensitivity of the Cell-PCA was established by using a wild-type and a single-amino-acid-mutated HOXA9 protein, and the approach was subsequently applied to seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 that promoted its proliferative activity in a cancer cell context. Taken together, our work demonstrated that the Cell-PCA was pertinent for revealing and, importantly, comparing the interactomes of different or highly related bait proteins in the same cell context.https://www.mdpi.com/2073-4409/12/1/200Cell-PCABiFCORFeome-widehuman HOX proteinsprotein-protein interactionslive cells |
| spellingShingle | Yunlong Jia Jonathan Reboulet Benjamin Gillet Sandrine Hughes Christelle Forcet Violaine Tribollet Nawal Hajj Sleiman Cindy Kundlacz Jean-Marc Vanacker Françoise Bleicher Samir Merabet A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes Cells Cell-PCA BiFC ORFeome-wide human HOX proteins protein-protein interactions live cells |
| title | A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes |
| title_full | A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes |
| title_fullStr | A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes |
| title_full_unstemmed | A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes |
| title_short | A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes |
| title_sort | live cell protein complementation assay for orfeome wide probing of human hox interactomes |
| topic | Cell-PCA BiFC ORFeome-wide human HOX proteins protein-protein interactions live cells |
| url | https://www.mdpi.com/2073-4409/12/1/200 |
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