A growth-based platform for detecting domain-peptide interactions in the cytoplasm of mammalian cells.
Sci Rep
; 12(1): 18028, 2022 10 27.
Article
in En
| MEDLINE
| ID: mdl-36302843
ABSTRACT
Development of a method for detecting protein-protein interactions (PPIs) in living cells is important for therapeutic drug screening against various diseases including infectious diseases. We have recently developed a method named SOS localization-based interaction screening (SOLIS), in which we designed membrane-anchored and SOS-fused chimeric proteins, whose PPI-dependent association triggers membrane localization of the SOS-fused chimeric protein, activates the Ras/MAPK pathway, and induces cell growth. While SOLIS was able to detect relatively strong PPIs, further sensitivity was required for detecting intracellular endogenous PPIs typically having a micromolar order of dissociation constant (Kd). Here we develop high-sensitive SOLIS (H-SOLIS) that could universally detect PPIs with lower affinities. In order to improve the sensitivity, H-SOLIS introduces a heterodimeric helper interaction, in which addition of a small-molecule helper ligand could accommodate association of the two chimeric proteins and regulate the sensitivity. Four types of domain-peptide interactions having known Kd values are employed to examine the versatility and detection limit of H-SOLIS. Consequently, the heterodimer-inducible helper ligand dramatically enhances detection sensitivity, lowering the detection limit to a ten-micromolar order of Kd. Thus, H-SOLIS could be a platform to detect disease-related domain-peptide interactions for drug discovery screening.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Peptides
/
Protein Interaction Mapping
Limits:
Animals
Language:
En
Journal:
Sci Rep
Year:
2022
Document type:
Article
Affiliation country:
Japón