Construction of a stable Smad3-overexpressing cell line and biotinylation-based protein interaction analysis using PUP-IT technology

AimTo develop and evaluate the application value of the PUP-IT proximity labeling technique in identifying Smad3-interacting proteins and constructing interaction networks, aiming to provide an efficient and reproducible method for pharmacological research.MethodsA PafA-linker-Smad3 fusion construct and a carboxylase-fused Pup(E)(Bccp)expression cassette were generated via homologous recombination and cloned into transposon vectors. These constructs were stably integrated into TCMK1 cells using transposase-mediated insertion to establish stable overexpression cell lines. Upon optimization of Bccp induction conditions, biotinylated proteins were enriched using streptavidin magnetic beads and subjected to mass spectrometry-based identification of Smad3-interacting proteins. Results were further compared with conventional coimmunoprecipitation (Co-IP) assays.ResultsA stable cell model expressing the PUP-IT system was successfully established. Multiple Smad3-interacting proteins were identified, including both previously reported disease-associated proteins and novel candidates. Compared with Co-IP, the PUP-IT technique demonstrated broader coverage, enabling detection of transient or low-affinity interactions with improved sensitivity and specificity.ConclusionsAs an innovative approach for probing protein-protein interactions, PUP-IT offers high efficiency, reproducibility, and practical applicability. It serves as a powerful complement to conventional Co-IP methods and provides new insights into the Smad3 interaction network, its associated signaling pathways, and disease mechanisms.