A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes

Area of Science:

• Molecular Biology
• Proteomics
• Genetics

Background:

• Biological pathways depend on complex protein interaction networks (interactomes).
• Capturing transient and low-affinity protein-protein interactions (PPIs) in live cells requires advanced tools.
• Understanding interactomes is crucial for deciphering cellular functions and disease mechanisms.

Purpose of the Study:

• To present Cell-PCA (cell-based protein complementation assay), a novel experimental strategy for ORFeome-wide screening of PPIs in live cells.
• To validate the specificity and sensitivity of Cell-PCA using HOXA9 protein variants.
• To apply Cell-PCA for exploring HOX protein interactomes and identifying novel interaction partners.

Main Methods:

• Development of the Cell-PCA strategy, integrating bimolecular fluorescence complementation (BiFC) with high-throughput sequencing.
• Validation of Cell-PCA using wild-type and mutated HOXA9 proteins.
• Application of Cell-PCA to screen interactors for seven human HOX proteins in a live-cell context.

Main Results:

• Demonstrated the specificity and sensitivity of the Cell-PCA method.
• Revealed novel molecular properties of HOX protein interactomes.
• Identified a new cofactor for HOXB13 that enhances its proliferative activity in cancer cells.

Conclusions:

• Cell-PCA is a robust and versatile method for identifying and comparing protein interactomes in live cells.
• The study provides new insights into HOX protein interactions and their roles in cellular processes.
• The identified HOXB13 cofactor presents a potential target for cancer therapeutics.