中国药理学通报2026，Vol.42Issue(2)：393-398,6.DOI:10.12360/CPB202504013
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
Construction of a stable Smad3-overexpressing cell line and biotinylation-based protein interaction analysis using PUP-IT technology
摘要
Abstract
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.关键词
邻近标记/PUP-IT系统/Smad3/肾小管上皮细胞/蛋白质互作网络/肾纤维化Key words
proximity labeling/PUP-IT system/Smad3/renal tubular epithelial cells/protein-protein interaction network/renal fibrosis分类
医药卫生引用本文复制引用
李倩倩,张璐娜,倪玉芳,王洪连,王丽,李健春..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[J].中国药理学通报,2026,42(2):393-398,6.基金项目
国家自然科学基金资助项目(No 82104665) （No 82104665）
泸州市人民政府-西南医科大学科技战略合作面上项目(No 2024LZXNYDJ020) （No 2024LZXNYDJ020）
西南医科大学中西医结合专项(No 2024ZXYZX05) （No 2024ZXYZX05）
