ralb34k2-1刺激小鼠RAW264.7巨噬细胞对蛋白质PTM代谢物修饰及DENV-2易感性的影响
Effects of ralb34k2-1-stimulated mouse RAW264.7 macrophages on protein PTM and susceptibility to DENV-2
代薇露 1胡欢 1覃燕春 1吴家红 2商正玲1
作者信息
- 1. 贵州医科大学基础医学院免疫学教研室,贵州贵阳 550025
- 2. 贵州医科大学基础医学院现代病原生物学特色重点实验室,贵州贵阳 550025;贵州医科大学基础医学院寄生虫教研室,贵州贵阳 550025
- 折叠
摘要
目的 探讨白纹伊蚊唾液蛋白34k2-1(ralb34k2-1)刺激的小鼠单核巨噬细胞白血病细胞(RAW264.7细胞)胞内蛋白翻译后代谢修饰(PTMs)的变化及对2型登革病毒(DENV-2)易感性的影响.方法 利用抗乙酰化、乳酰化、琥珀酰化、巴豆酰化和丙二酰化的泛特异性抗体,通过免疫印迹法(Western blot)检测ralb34k2-1蛋白刺激后不同时相的RAW264.7细胞蛋白质翻译后代谢物修饰的情况,酶联免疫吸附双抗体夹心法(ELISA)检测细胞内外的乳酸含量;用2 mg/L ralb34k2-1预处理RAW264.7细胞24 h后、继续培养2 d、再用DENV-2感染细胞,通过实时荧光定量多聚核苷酸链式反应(RT-qPCR)检测感染18 h和36 h后的病毒核酸变化,分析ralb34k2-1预处理后对病毒易感性的影响;检测感染前后胞内白细胞介素1β-(IL-1β)、白细胞介素10-(IL-10)、肿瘤坏死因子β-(TGF-β)、诱导性一氧化氮合酶(iNOS)和精氨酸酶1(Arg-1)mRNA的相对表达情况,分析ralb34k2-1预处理对细胞免疫状态及细胞极化的影响.结果 胞内不同的代谢物对蛋白质的修饰程度主要表现为乳酰化修饰＞乙酰化修饰＞琥珀酰化修饰;在时相上,除琥珀酰修饰外,随时间的延长其余代谢物对蛋白质的修饰有增多的趋势,其中以6 h乳酰化修饰最多;ralb34k2-1刺激对细胞的乳酸产生无明显影响;经ralb34k2-1预处理后胞内DENV-E相对表达量较对照组高5倍以上,IL-10 mRNA的相对表达在感染前后均保持高水平(6～8倍);在感染前iNOS/Arg比值为3.46±1.59,但受ralb34k2蛋白预处理的细胞在感染后表现iNOS表达下调的趋势.结论 蚊唾液ralb34k2蛋白可能通过调控PTM代谢物修饰的方式,改变细胞的免疫状态从而增强登革病毒的易感性.
Abstract
Objective To investigate the changes in intracellular protein post-translational modifications(PTMs)stimulated by Aedes albopictus 34k2-1 salivary protein(ralb34k2-1)in mouse monocytic macrophage leukemia cell line(RAW264.7 cells)and susceptibility to Dengue virus type 2(DENV-2).Methods Protein post-translational modifications in RAW264.7 cells stimulated by ralb34k2-1 protein at different time points were detected by Western blot via using pan-specific antibodies against acetylation,lactylation,succinylation,crotonylation and malonylation,respectively.Enzyme-linked immunosorbent assay(ELISA)was used to detect the intracellular and extracellular lactate contents.After RAW264.7 cells were treated with 2 mg/L ralb34k2-1 for 24 hours,cultured for 2 days and then infected with DENV-2 viruses.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the changes in viral nucleic acids at 18 h and 24 h after infection.The effect of ralb34k2-1 pre treatment on virus susceptibility was analyzed.The relative mRNA expressions of intracellular interleukin-1 β(IL-1 β),IL-10,tumor necrosis factor β(TGF-β),inducible nitric oxide synthase(iNOS),and arginase 1(Arg-1)before and after infection was detected to analyze the effect of ralb34k2-1 pretreatment on cellular immune status and cell polarization.Results The degrees of protein modifications by different intracellular metabolites mediated by ralb34k2-1 protein were mainly manifested as lactylation modification＞acetylation modification＞succinylation modification.In terms of time phase,except for succinylation modification,there was an increasing trend of other metabolites-modified protein modifications over time,with acetylation modification being the most at 6 h.Ralb34k2-1 stimulation had no significant effect on cellular lactate production.After pretreatment with ralb34k2-1,the relative expression level of intracellular DENV-E was 5 times higher than that of control group.The relative expression of IL-10 mRNA remained high before and after infection(6-8 times).The iNOS/Arg ratio was 3.46±1.59 before infection.iNOS expression exhibited a downregulation trend in ralb34k2-pretreated cells after infection.Conclusion Mosquito salivary ralb34k2 protein may enhance the susceptibility to Dengue virus by regulating PTM metabolite modifications and changing the cell immune state.
关键词
白纹伊蚊/蚊唾液蛋白34k2-1/巨噬细胞/RAW264.7细胞/蛋白翻译后修饰/2型登革病毒/免疫调控Key words
Aedes albopictus/mosquito 34k2-1 salivary protein/macrophages/RAW264.7 cells/protein post-translational modification/DENV-2/immunoregulation引用本文复制引用
出版年
2024
NETL
NSTL
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