基于PUP-IT技术的Smad3稳转细胞系构建及其蛋白互作生物素标记分析OA

Aim To develop and evaluate the application value of the PUP-IT proximity labeling technique in identifying Smad3-interacting proteins and constructing interaction net-works,aiming to provide an efficient and reproducible method for pharmacological research.Methods A PafA-linker-Smad3 fusion construct and a carboxylase-fused Pup(E)(Bccp)ex-pression cassette were generated via homologous recombination and cloned into transposon vectors.These constructs were sta-bly integrated into TCMK1 cells using transposase-mediated in-sertion to establish stable overexpression cell lines.Upon opti-mization 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 co-immunoprecipitation(Co-IP)assays.Results A stable cell model expressing the PUP-IT system was successfully estab-lished.Multiple Smad3-interacting proteins were identified,in-cluding 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 speci-ficity.Conclusions As an innovative approach for probing protein-protein interactions,PUP-IT offers high efficiency,re-producibility,and practical applicability.It serves as a power-ful complement to conventional Co-IP methods and provides new insights into the Smad3 interaction network,its associated signaling pathways,and disease mechanisms.