上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2022 , Vol. 42 >Issue 8: 987 - 996

DOI: https://doi.org/10.3969/j.issn.1674-8115.2022.08.003

创新团队成果专栏

巨胞饮途径介导的新冠原始株病毒蛋白入胞的细胞水平初步研究

  • 江淦 ,
  • 杨于权 ,
  • 陈曜星 ,
  • 侯照远 ,
  • 高小玲 ,
  • 陈红专 ,
  • 贾浩
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  • 1.上海交通大学基础医学院药理学与化学生物学系，上海高校转化医学协同创新中心，上海 200025
    2.上海交通大学基础医学院生物化学与分子细胞生物学系，上海市肿瘤微环境与炎症重点实验室，上海 200025
    3.上海中医药大学交叉科学研究院，上海 201203
江淦（1987—），男，副教授，博士；电子信箱：scmcyidingkaixin@163.com。
陈红专，电子信箱：hongzhuan_chen@hotmail.com。
贾浩，电子信箱：fonney@sjtu.edu.cn

收稿日期: 2022-03-16

  录用日期: 2022-05-18

  网络出版日期: 2022-06-29

基金资助

国家自然科学基金(82171358);上海市青年科技启明星计划(19QA1405000);上海市高水平地方高校创新团队(SHSMU-ZDCX20211801)

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Preliminary study on the cellular level of SARS-CoV-2 proteins mediated by macropinocytosis pathway

  • Gan JIANG ,
  • Yuquan YANG ,
  • Yaoxing CHEN ,
  • Zhaoyuan HOU ,
  • Xiaoling GAO ,
  • Hongzhuan CHEN ,
  • Hao JIA
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  • 1.Shanghai Universities Collaborative Innovation Center for Translational Medicine, Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
    2.Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Department of Biochemistry & Molecular Cellular Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
    3.Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
CHEN Hongzhuan, E-mail: hongzhuan_chen@hotmail.com. #Corresponding authors.
JIA Hao, E-mail: fonney@sjtu.edu.cn

Received date: 2022-03-16

  Accepted date: 2022-05-18

  Online published: 2022-06-29

Supported by

National Natural Science Foundation of China(82171358);Shanghai Rising-Star Program(19QA1405000);Innovative Research Team of High-level Local Universities in Shanghai(SHSMU-ZDCX20211801)

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摘要

目的·探讨严重急性呼吸综合征冠状病毒2（severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）原始株的几种关键蛋白对多种细胞模型巨胞饮途径的影响。方法·①利用免疫共沉淀技术探索SARS-COV-2原始株的刺突蛋白受体结合域（spike protein receptor-binding domain，S-RBD）、核衣壳蛋白（nucleocapsid protein，N）和非结构蛋白7（non-structural protein-7，NSP7）等病毒蛋白与HEK-293T细胞内蛋白的相互作用。②于体外，将SARS-CoV-2原始株的S-RBD、N、NSP7病毒蛋白分别与HEK-293T/bEnd.3/Beas-2b细胞（正常细胞模型）共孵育，利用巨胞饮标志物——异硫氰酸荧光素（fluorescein isothiocyanate，FITC）标记的70 kDa葡聚糖（FITC-70 kDa-葡聚糖）水平的变化观察上述细胞巨胞饮水平的改变。③利用脂多糖（lipopolysaccharide，LPS）诱导的炎症细胞模型，分析SARS-CoV-2原始株的病毒蛋白对该炎症细胞的巨胞饮水平的改变。④在正常细胞模型和炎症细胞模型中，利用5-（N-乙基-N-异丙基）阿米洛利（EIPA）或载带Rab5小干扰RNA（small interfering RNA，siRNA）的脂蛋白纳米药物载体分别抑制由SARS-CoV-2病毒蛋白诱导的巨胞饮作用，进一步观察细胞对S-RBD、N、NSP7病毒蛋白的摄取情况。结果·① SARS-CoV-2原始株的3个病毒蛋白在被摄取入胞后，可与Rab蛋白家族发生结合。②研究发现，SARS-CoV-2原始株的S-RBD、N、NSP7病毒蛋白均可刺激HEK-293T/bEnd.3/Beas-2b细胞产生巨胞饮作用。③在炎症细胞模型中，3个病毒蛋白均可增强细胞的巨胞饮作用。④经EIPA（75 μmol/L）或载带Rab5 siRNA的脂蛋白纳米药物载体处理后，2类细胞对S-RBD、N、NSP7病毒蛋白的摄取均有减少。结论·SARS-CoV-2原始株的S-RBD、N、NSP7病毒蛋白可上调多种细胞模型的巨胞饮水平，尤其是在合并炎症感染的情况下；同时，巨胞饮抑制剂/脂蛋白纳米药物载体均可抑制由上述病毒蛋白上调的巨胞饮作用，继而减少病毒蛋白的入胞水平。

关键词： 巨胞饮; 严重急性呼吸综合征冠状病毒2; 5-（N-乙基-N-异丙基）阿米洛利; Rab5

本文引用格式

江淦 , 杨于权 , 陈曜星 , 侯照远 , 高小玲 , 陈红专 , 贾浩 . 巨胞饮途径介导的新冠原始株病毒蛋白入胞的细胞水平初步研究[J]. 上海交通大学学报（医学版）, 2022 , 42(8) : 987 -996 . DOI: 10.3969/j.issn.1674-8115.2022.08.003

Abstract

Objective ·To investigate the effects of several key proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on macropinocytosis in various cell models. Methods ·① The interactions between spike protein receptor-binding domain (S-RBD), nucleocapsid protein (N) and non-structural protein-7 (NSP7) of SARS-COV-2 and HEK-293T intracellular proteins were explored by co-immunoprecipitation assay. ② In vitro, S-RBD, N and NSP7 proteins of SARS-CoV-2 were incubated with HEK-293T/bEnd.3/Beas-2b cells (normal cell models), respectively, and the changes of macropinocytosis level of cells labeled with fluorescein isothiocyanate (FITC)-70 kDa-dextran were observed. ③ In vitro, S-RBD, N and NSP7 proteins of SARS-CoV-2 were incubated with inflammatory cells induced by lipopolysaccharide (LPS), respectively, and the changes of macropinocytosis level of inflammatory cells were analyzed. ④ In the normal cell models and inflammatory cell model, EIPA or lipoprotein nano-drug carriers loaded with Rab5 small interfering RNA (siRNA) were used to inhibit the macropinocytosis induced by SARS-CoV-2 proteins, respectively, and the uptake of S-RBD, N and NSP7 proteins by cells were further observed. Results ·① The three proteins of SARS-COV-2 could bind to Rab small GTPase proteins after being absorbed into cells. ② It was found that S-RBD, N and NSP7 proteins of SARS-COV-2 could induce the macropinocytosis after entering the HEK-293T/bEnd.3/Beas-2b cells. ③ Furthermore, the three proteins of SARS-COV-2 could enhance the megapinocytosis of the inflammatory cell. ④ After treatment with EIPA (75 μmol/L) or lipoprotein nano-drug carriers loaded with Rab5 siRNA, the uptake of S-RBD, N and NSP7 proteins were decreased in both types of cells. Conclusion ·S-RBD, N and NSP7 proteins of SARS-CoV-2 can up-regulate megapinocytosis levels in various cell models, especially in the case of combined inflammation infection. At the same time, macropinocytosis inhibitor / lipoprotein nano-drug carrier can inhibit the macropinocytosis up-regulated by the above proteins, and then reduce the entry levels of viral proteins.

Key words： macropinocytosis; severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); EIPA; Rab5

参考文献

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