A Binary Quantitative Interaction Mapping Approach: Elucidating Multiprotein Complexes in...

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Automated yeast two‑hybrid screens test ~70,000 pairings per week from a 17,000 human cDNA array but detect only ~20–25% of known interactions; a new LUCY quantitative BRET assay raises sensitivity to ~71% against a random set and near 100% on structure‑supported pairs while running ~1,000 pairs/week in mammalian cells. Binary screens map huntingtin:HAP40 dependency, show ASPL disassembles p97 hexamers linking p97 to splicing, and identify an NSP10:NSP16 interface pursued by in silico docking of ~200 million compounds. CELFI uses CRISPR knock‑in endogenous reporters to confirm interactions without overexpression.

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" chapters (15)

1. 0:00Intro
2. 1:19The activity of the cell is determined by complex protein interactions
3. 4:26Our focus: Find new ways of identifying interactions in health and disease
4. 5:33An automated yeast two-hybrid system
5. 6:50Y2H system: Automated interaction mating
6. 9:04Prediction of disease modules from NeuroNet PPI data with network propagation and clustering
7. 10:59Alzheimer's disease: a multiprotein aggregation disease?
8. 12:10Quality assessment of Y2H interactions with positive and negative reference sets
9. 13:34LuTHy: a quantitative bioluminescence-based double- readout two-hybrid technology
10. 16:16Automated validation of human protein interactions
11. 16:52Mapping binary interactions within multiprotein complexes using LUTHy and mN2H assays
12. 18:38Systematic protein-protein interaction mapping
13. 19:30Knock-out of HTT decreases HAP40 protein levels in HEK293 cells
14. 22:15ASPL promotes the interaction between U2AF2 and p97 and influences mRNA splicing
15. 24:13Mapping interactions between endogenously expressed hybrid fusion proteins: CellFIE