Gomes-Junior Rubens, Moreira Claudia Maria do Nascimento, Dallagiovanna Bruno
Basic Stem Cell Biology Laboratory, Carlos Chagas Institute, Fiocruz Paraná, Curitiba, Brazil.
Gene Expression Regulation Laboratory, Carlos Chagas Institute, Fiocruz Paraná, Curitiba, Brazil.
PLoS One. 2025 May 30;20(5):e0324779. doi: 10.1371/journal.pone.0324779. eCollection 2025.
Identification of protein-protein interactions is essential for understanding protein functions in biological processes. While immunoprecipitation has traditionally been used to isolate proteins and their partners, it faces limitations in capturing transient interactions. Proximity labeling, particularly with the biotin ligase TurboID, addresses this challenge by enabling rapid and efficient identification of interacting proteins in vivo. Human induced pluripotent stem cells are valuable models for studying human development, however certain biological processes, such as differentiation, can be difficult to analyze because conventional transfection methods are challenging. Therefore, an alternative strategy for detection of interacting proteins is necessary. Here, we developed a novel system employing TurboID-fusion proteins within an integrative and inducible expression vector to investigate the interactome during stem cell differentiation. We validated our system by using U2AF2 and GFP as bait proteins, generated two distinct cell lines, and determining the minimum induction time required for optimal protein expression. Our results confirmed that the system did not alter the expected localization of U2AF2. Applying our system, we identified significant differences in the interactome of U2AF2 between the pluripotent and mesodermal differentiation stages, demonstrating that U2AF2 interacts with distinct protein sets following cell fate commitment. Our study successfully unveils a new tool for studying protein-protein interaction in human stem cells.
蛋白质-蛋白质相互作用的鉴定对于理解生物过程中的蛋白质功能至关重要。虽然免疫沉淀传统上用于分离蛋白质及其相互作用伙伴，但它在捕获瞬时相互作用方面存在局限性。邻近标记，特别是与生物素连接酶TurboID一起使用时，通过在体内快速有效地鉴定相互作用蛋白质来应对这一挑战。人类诱导多能干细胞是研究人类发育的宝贵模型，然而某些生物过程，如分化，可能难以分析，因为传统的转染方法具有挑战性。因此，需要一种检测相互作用蛋白质的替代策略。在这里，我们开发了一种新系统，在整合的可诱导表达载体中使用TurboID融合蛋白来研究干细胞分化过程中的相互作用组。我们通过使用U2AF2和GFP作为诱饵蛋白验证了我们的系统，生成了两种不同的细胞系，并确定了最佳蛋白质表达所需的最短诱导时间。我们的结果证实该系统没有改变U2AF2的预期定位。应用我们的系统，我们发现多能和中胚层分化阶段之间U2AF2的相互作用组存在显著差异，表明U2AF2在细胞命运决定后与不同的蛋白质组相互作用。我们的研究成功揭示了一种研究人类干细胞中蛋白质-蛋白质相互作用的新工具。
