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

作　　者：代薇露 胡欢 覃燕春 吴家红 商正玲 DAI Weilu;HU Huan;QIN Yanchun;WU Jiahong;SHANG Zhengling(Department of Immunology,School of Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China;Key Laboratory of the Biology and Characteristics of Modern Pathogens,School of Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China;Department of Parasitology,School of Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[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 [3]贵州医科大学基础医学院寄生虫教研室,贵州贵阳550025

出　　处：《贵州医科大学学报》2024年第9期1259-1268,共10页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81960295)。

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。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 pretreatment on virus susceptibility was analyzed.The relative mRNA expressions of intracellular interleukin-1β(IL-1β),IL-10,tumor necrosis factorβ(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 time

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 

分 类 号：R375.1[医药卫生—病原生物学]