A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes.
Yunlong JiaJonathan RebouletBenjamin GilletSandrine HughesChristelle ForcetViolaine TribolletNawal Hajj SleimanCindy KundlaczJean-Marc VanackerFrançoise BleicherSamir MerabetPublished in: Cells (2023)
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.
Keyphrases
- single cell
- amino acid
- cell therapy
- endothelial cells
- high throughput
- single molecule
- mesenchymal stem cells
- binding protein
- stem cells
- wild type
- mass spectrometry
- brain injury
- bone marrow
- blood brain barrier
- high throughput sequencing
- quantum dots
- molecular dynamics simulations
- energy transfer
- subarachnoid hemorrhage
- bioinformatics analysis