Zhu Chensi, Zhang Liangyan, Yu Wenjing, Tu Yeqing, Yang Xiaolan, Li Deyu, Wang Hui, Li Tao
State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China.
Int J Mol Sci. 2025 May 28;26(11):5168. doi: 10.3390/ijms26115168.
Botulinum neurotoxin type A (BoNT/A), among the most potent known toxins, is widely used in cosmetic medicine. However, its toxicity mechanisms remain poorly understood due to a lack of suitable models. Here, we generated a doxycycline (DOX)-inducible Neuro-2a cell line stably expressing the BoNT/A light chain (ALC). ALC expression was confirmed by GFP and FLAG tag antibodies, and its activity was validated through cleavage of the substrate SNAP-25. Using this model, combined with natural toxin infection of cells, phospho-antibody microarray analysis revealed significant alterations in host phosphorylation networks in both ALC-expressing and toxin-infected cells. Among the shared phosphorylation changes, 75 proteins showed upregulation, while 27 were downregulated. Upregulated phosphorylation events were enriched in pathways such as PI3K-AKT signaling, EGFR tyrosine kinase inhibitor resistance, and Ras signaling, whereas downregulated events were associated with the ERBB and thyroid hormone signaling pathways. Key alterations were observed in AKT signaling, with protein-protein interaction analysis identifying Hsp90ab1 and Map2k1 as central hub molecules for upregulated and downregulated proteins, respectively. This study establishes a robust Neuro-2a-based model system to study BoNT/A toxicity and provides insights into toxin-induced phosphorylation network changes, offering a valuable platform for therapeutic screening and mechanistic exploration.
A型肉毒杆菌神经毒素（BoNT/A）是已知毒性最强的毒素之一，在美容医学中广泛应用。然而，由于缺乏合适的模型，其毒性机制仍未得到充分了解。在此，我们构建了一种强力霉素（DOX）诱导的稳定表达BoNT/A轻链（ALC）的Neuro-2a细胞系。通过绿色荧光蛋白（GFP）和FLAG标签抗体证实了ALC的表达，并通过底物SNAP-25的裂解验证了其活性。利用该模型，结合细胞的天然毒素感染，磷酸化抗体微阵列分析揭示了在表达ALC的细胞和毒素感染的细胞中宿主磷酸化网络均有显著改变。在共同的磷酸化变化中，75种蛋白质上调，27种下调。上调的磷酸化事件富集于PI3K-AKT信号传导、表皮生长因子受体（EGFR）酪氨酸激酶抑制剂抗性和Ras信号传导等途径，而下调事件与ERBB和甲状腺激素信号传导途径相关。在AKT信号传导中观察到关键改变，蛋白质-蛋白质相互作用分析分别确定热休克蛋白90α（Hsp90ab1）和丝裂原活化蛋白激酶激酶1（Map2k1）为上调和下调蛋白质的中心枢纽分子。本研究建立了一个强大的基于Neuro-2a的模型系统来研究BoNT/A的毒性，并深入了解毒素诱导的磷酸化网络变化，为治疗筛选和机制探索提供了一个有价值的平台。
