Kim Doeun, Lee Min Seo, Sim Hyunchae, Lee Sangkyu, Lee Hye Suk
BK21 FOUR Community-Based Intelligent Novel Drug Discovery Education Unit, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, 41566 Republic of Korea.
BK21 Four-sponsored Advanced Program for SmartPharma Leaders, College of Pharmacy, The Catholic University of Korea, Bucheon, 14662 Republic of Korea.
Toxicol Res. 2022 Dec 22;39(2):251-262. doi: 10.1007/s43188-022-00163-z. eCollection 2023 Apr.
In the human body, proteins secreted into peripheral blood vessels are known as the secretome, and they represent the physiological or pathological status of cells. The unique response of cells to toxin exposure can be confirmed secretome analysis, which can be used to discover toxic mechanisms or exposure markers. Alpha-amanitin (α-AMA) is the most widely studied amatoxin and inhibits transcription and protein synthesis by directly interacting with RNA polymerase II. However, secretory proteins released during hepatic failure caused by α-AMA have not been fully characterized. In this study, we analyzed the secretome of α-AMA-treated Huh-7 cells and mice using a comparative proteomics technique. Overall, 1440 and 208 proteins were quantified in cell media and mouse serum, respectively. Based on the bioinformatics results for the commonly downregulated proteins in cell media and mouse serum, we identified complement component 3 (C3) as a marker for α-AMA-induced hepatotoxicity. Through western blot in cell secretome and C3 ELISA assays in mouse serum, we validated α-AMA-induced downregulation of C3. In conclusion, using comparative proteomics and molecular biology techniques, we found that α-AMA-induced hepatotoxicity reduced C3 levels in the secretome. We expect that this study will aid in identifying new toxic mechanisms, therapeutic targets, and exposure markers of α-AMA-induced hepatotoxicity.
The online version contains supplementary material available at 10.1007/s43188-022-00163-z.
在人体中，分泌到外周血管中的蛋白质被称为分泌组，它们代表了细胞的生理或病理状态。细胞对毒素暴露的独特反应可以通过分泌组分析来证实，该分析可用于发现毒性机制或暴露标志物。α-鹅膏毒肽（α-AMA）是研究最广泛的一种鹅膏毒素，它通过直接与RNA聚合酶II相互作用来抑制转录和蛋白质合成。然而，由α-AMA引起的肝衰竭期间释放的分泌蛋白尚未得到充分表征。在本研究中，我们使用比较蛋白质组学技术分析了α-AMA处理的Huh-7细胞和小鼠的分泌组。总体而言，在细胞培养基和小鼠血清中分别定量了1440种和208种蛋白质。基于细胞培养基和小鼠血清中共同下调蛋白质的生物信息学结果，我们确定补体成分3（C3）是α-AMA诱导肝毒性的标志物。通过细胞分泌组中的蛋白质印迹法和小鼠血清中的C3 ELISA测定，我们验证了α-AMA诱导的C3下调。总之，使用比较蛋白质组学和分子生物学技术，我们发现α-AMA诱导的肝毒性降低了分泌组中的C3水平。我们期望这项研究将有助于识别α-AMA诱导肝毒性的新毒性机制、治疗靶点和暴露标志物。
在线版本包含可在10.1007/s43188-022-00163-z获取的补充材料。
