Wang Jing, Zhang Xuemei, Li Zheng, Li Xiayu, Ma Jian, Shen Shourong
Department of Gastroenterology, Third Xiangya Hospital, Central South University, Changsha 410013; Hunan Key Laboratory of Non-resolving Infl ammation and Cancer, Third Xiangya Hospital, Central South University, Changsha 410013; Cancer Research Institute, Central South University, Changsha 410078, China.
Hunan Key Laboratory of Non-resolving Infl ammation and Cancer, Third Xiangya Hospital, Central South University, Changsha 410013; Cancer Research Institute, Central South University, Changsha 410078, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2017 Apr 28;42(4):365-373. doi: 10.11817/j.issn.1672-7347.2017.04.001.
To identify the interacting proteins with S100A8 or S100A9 in HEK293 cell line by flag-tag affinity purification and liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS). Methods: The p3×Flag-CMV-S100A8 and p3×Flag-CMV-S100A9 expression vectors were constructed by inserting S100A8 or S100A9 coding sequence. The recombinant plasmids were then transfected into HEK293 cells. Affinity purification and LC-MS/MS were applied to identify the proteins interacting with S100A8 or S100A9. Bioinformatics analysis was used to seek the gene ontology of the interacting proteins. Co-immunoprecipitation (Co-IP) was applied to confirm the proteins interacted with S100A8 or S100A9. Results: Fourteen proteins including pyruvate kinase, muscle (PKM), nucleophosmin (NPM1) and eukaryotic translation initiation factor 5A (EIF5A), which potentially interacted with S100A8, were successfully identified by Flag-tag affinity purification followed by LC-MS/MS analysis. Six proteins, such as tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (14-3-3ε) and PKM, which potentially interacted with S100A9, were successfully identified. Gene ontology analysis of the identified proteins suggested that proteins interacted with S100A8 or S100A9 were involved in several biological pathways, including canonical glycolysis, positive regulation of NF-κB transcription factor activity, negative regulation of apoptotic process, cell-cell adhesion, etc. Co-IP experiment confirmed that PKM2 can interact with both S100A8 and S100A9, and 14-3-3ε can interact with S100A8. Conclusion: PKM2 is identified to interact with both S100A8 and S100A9, while 14-3-3ε can interact with S100A9. These results may provide a new clue for the role of S100A8 or S100A9 in the progression of colitis-associated colorectal cancer.
通过Flag标签亲和纯化和液相色谱串联质谱法（LC-MS/MS）鉴定HEK293细胞系中与S100A8或S100A9相互作用的蛋白质。方法：通过插入S100A8或S100A9编码序列构建p3×Flag-CMV-S100A8和p3×Flag-CMV-S100A9表达载体。然后将重组质粒转染到HEK293细胞中。应用亲和纯化和LC-MS/MS鉴定与S100A8或S100A9相互作用的蛋白质。使用生物信息学分析来寻找相互作用蛋白质的基因本体。应用免疫共沉淀（Co-IP）来确认与S100A8或S100A9相互作用的蛋白质。结果：通过Flag标签亲和纯化后进行LC-MS/MS分析，成功鉴定出14种可能与S100A8相互作用的蛋白质，包括丙酮酸激酶、肌肉型（PKM）、核磷蛋白（NPM1）和真核翻译起始因子5A（EIF5A）。成功鉴定出6种可能与S100A9相互作用的蛋白质，如酪氨酸3-单加氧酶/色氨酸5-单加氧酶激活蛋白ε（14-3-3ε）和PKM。对鉴定出的蛋白质进行基因本体分析表明，与S100A8或S100A9相互作用的蛋白质参与了多种生物学途径，包括经典糖酵解、NF-κB转录因子活性的正调控、凋亡过程的负调控、细胞间粘附等。Co-IP实验证实PKM2可与S100A8和S100A9都相互作用，而14-3-3ε可与S100A8相互作用。结论：鉴定出PKM2与S100A8和S100A9都相互作用，而14-3-3ε可与S100A9相互作用。这些结果可能为S100A8或S100A9在结肠炎相关结直肠癌进展中的作用提供新线索。
