Abstract：
Acute stress caused by short-term exposure to deleterious chemicals can induce the aggregation of RNA-binding proteins (RBPs) in the cytosol and the formation of stress granules (SGs). The cytoplasmic RBP, Ras GTPase-activating protein-binding protein 1 (G3BP1) is a critical organizer of SG, and its aggregation is considered a hallmark of cellular stress. However, assembly of SG is a highly dynamic process that involves RBPs; hence, existing methods based on fixation processes or overexpression of RBPs exhibit limited efficacy in detecting the assembly of SG under stress conditions. In this study, we established a G3BP1- Green fluorescent protein (GFP) reporter protein in a human neuroblastoma cell line to overcome these limitations. GFP was introduced into the G3BP1 genomic sequence via homologous recombination to generate a G3BP1-GFP fusion protein and further analyze the aggregation processes. We validated the assembly of SG under stress conditions using the G3BP1-GFP reporter system. Additionally, this system supported the evaluation of bisphenol A-induced SG response in the established human neuroblastoma cell line. In conclusion, the established G3BP1-GFP reporter system enables us to monitor the assembly of the SG complex in a human neuroblastoma cell line in real time and can serve as an efficient tool for assessing potential neurotoxicity associated with short-term exposure to chemicals.
摘要：
短期暴露于有害化学物质引起的急性应激可诱导RNA结合蛋白（RBP）在细胞质中的聚集和应激颗粒（SGs）的形成。细胞质RBP，RasGTP酶激活蛋白结合蛋白1（G3BP1）是SG的关键组织者，它的聚集被认为是细胞应激的标志。然而,SG的组装是一个高度动态的过程，涉及RBP；因此，基于固定过程或RBP过表达的现有方法在检测应激条件下SG的组装方面表现出有限的功效。在这项研究中,我们在人神经母细胞瘤细胞系中建立了G3BP1-绿色荧光蛋白（GFP）报告蛋白以克服这些限制。通过同源重组将GFP引入G3BP1基因组序列以产生G3BP1-GFP融合蛋白并进一步分析聚集过程。我们使用G3BP1-GFP报告系统在应激条件下验证了SG的组装。此外,该系统支持在已建立的人神经母细胞瘤细胞系中评估双酚A诱导的SG反应.总之,已建立的G3BP1-GFP报告系统使我们能够实时监测人类神经母细胞瘤细胞系中SG复合物的组装,并且可以作为评估与短期暴露于化学物质相关的潜在神经毒性的有效工具.
