| 刘萧然,邢美宁,陈沛渝,孙 薇,应万涛.肝癌细胞中NME1功能研究及调控的pHis修饰底物发现[J].现代生物医学进展英文版,2024,(13):2401-2407. |
| 肝癌细胞中NME1功能研究及调控的pHis修饰底物发现 |
| Identification of pHis Modified Substrates Regulated by NME1 and its Function in Hepatocellular Carcinoma Cells |
| Received:February 18, 2024 Revised:March 15, 2024 |
| DOI：10.13241/j.cnki.pmb.2024.13.001 |
| 中文关键词: 肝细胞癌 NME1 组氨酸磷酸化 细胞表型 蛋白质组学 磷酸化蛋白质组学 |
| 英文关键词: Hepatocellular carcinoma NME1 Histidine phosphorylation Phenotype Proteomics Phosphoproteomics |
| 基金项目:国家自然科学基金面上项目（32271500）；国家重点研发计划项目（2020YFE0202200）；蛋白质组学国家重点实验室自主研究课题（SKLP-K202202） |
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| 中文摘要: |
| 摘要 目的：研究核苷二磷酸激酶1（Nucleoside diphosphate kinase A, NME1）在肝癌细胞HCCLM3中的功能作用及其调控的组氨酸磷酸化（Histidine phosphorylation, pHis）修饰底物。方法：采用划痕愈合实验和Transwell小室实验以及蛋白质组学和磷酸化蛋白质组学分析方法，对NME1敲除后细胞表型与分子水平变化进行分析。结果：在肝癌细胞中敲除组氨酸激酶NME1能降低细胞运动能力，全蛋白组差异分析发现NME1敲除后差异表达蛋白主要参与细胞底物黏附调控、细胞周期调节及蛋白激酶活性调控等生命活动过程。Western blot检测发现敲除NME1表达使细胞整体pHis修饰水平显著下调。随后使用整合pHis修饰位点鉴定策略，即通过双甲基标记、强阳离子交换色谱（Strong cation exchange, SCX）分离磷酸化修饰肽段与非修饰肽段、铜珠（Cu-iminodiacetic acid, Cu-IDA）富集组氨酸肽段以及质谱检测等过程，首次在肝癌细胞系中鉴定到242个pHis修饰位点及206个pHis修饰蛋白。大约25 %的pHis修饰蛋白为首次发现，其中NME1敲低组pHis修饰水平显著下调的蛋白有（Cell adhesion molecule 3, CADM3）、（Cytochrome C, CYCS）、（Glyceraldehyde-3-phosphate dehydrogenase, GAPDH）及（Phosphofructokinase, PFKM）等近30个，表明NME1可能通过这些蛋白及代谢酶的组氨酸磷酸化修饰水平的变化影响肿瘤细胞间的黏附及代谢能力。结论：肿瘤细胞中NME1表达水平的变化会影响肿瘤细胞的运动能力，细胞内pHis修饰水平的失调可能全面参与疾病进程。 |
| 英文摘要: |
| ABSTRACT Objective: Study the function of Nucleoside diphosphate kinase A (NME1) in hepatocellular carcinoma (HCC) cell line HCCLM3 and the regulated pHis substrates of NME1. Methods: The wound healing assay, Transwell chamber assay, and proteomics and phosphoproteomics analysis methods were used to analyze the phenotype and molecular changes of cells after NME1 knockout. Results: Herein, our results showed that histidine kinase NME1 depletion in HCC cell lines inhibited cell motility. Moreover, the proteome analysis found that the differentially expressed proteins after NME1 knockout were primarily involved in the regulation of cell adhesion, cell cycle, and regulation of protein kinase activity. Western blot analysis shows that knockout of NME1 decreases the overall level of pHis modification. Subsequently, using the strategy for unbiased histidine phosphoproteome profiling of dimethyl labeling, Strong cation exchange (SCX) separation of modified and non-modified naked peptides, Cu-iminodiacetic acid (Cu-IDA) enrichment, and mass spectrometry detection, we identified a total of 242 pHis-peptides, corresponding to 206 proteins in the HCC cell line. About 25 % of the pHis-modified proteins were reported for the first time, among which about 30 proteins with significantly down-regulated pHis modification levels in response to NME1 knockout include Cell adhesion molecule 3 (CADM3), Cytochrome C (CYCS), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Phosphofructokinase (PFKM), etc. Conclusion: These findings suggest that NME1 can influence the phenotype changes of tumor cells, such as cell motility, and the dysregulation of the intracellular pHis modification may be widely related to disease procession. |
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