PPARγ激活和抑制状态下调控IFNβ表达的互作蛋白鉴定与分析  

Identification and Analysis of Interacting Proteins Regulating IFNβExpression in PPARγActivated and Inhibited States

作　　者：马卓媛 杨玢 陈云龙 樊港 吴晓龙 王希音 华进联[1,2] 王妍 张永强[3] 张仕强[1,2] MA Zhuo-Yuan;YANG Bin;CHEN Yun-Long;FAN Gang;WU Xiao-Long;WANG Xi-Yin;HUA Jin-Lian;WANG Yan;ZHANG Yong-Qiang;ZHANG Shi-Qiang(College of Veterinary Medicine/Shaanxi Stem Cell Engineering Research Center,Northwest A&F University,Yangling 712100,China;Key Laboratory of Livestock Biology,Northwest A&F University,Yangling 712100,China;China Animal Health and Epidemiology Center,Qingdao 266032,China)

机构地区：[1]西北农林科技大学动物医学院/陕西省干细胞工程技术研究中心,杨凌712100 [2]西北农林科技大学家畜生物学重点实验室,杨凌712100 [3]中国动物卫生与流行病学中心,青岛266032

出　　处：《农业生物技术学报》2024年第5期1081-1092,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32072856);国家级大学生创新创业训练计划项目(202210712123)。

摘　　要：过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptorγ,PPARγ)在肺泡巨噬细胞中特异性高表达,具有代谢和免疫调控作用。在先天免疫诱导产生Ⅰ型干扰素过程中,PPARγ究竟与哪些互作蛋白共同参与该过程的调控目前尚不清楚。本研究利用慢病毒载体系统构建了稳定过表达PPARγ-3×Flag的野猪(Sus scrofa)肺细胞系(wild boar lung cell line,WSL)。以该细胞为模型,通过荧光定量PCR检测发现,添加PPARγ激动剂罗格列酮能显著降低双链DNA模拟物Poly(dA:dT)刺激诱发的干扰素β(interferonβ,IFNβ)表达水平;相反,添加PPARγ抑制剂T0070907能显著提高Poly(dA:dT)刺激引起的IFNβ表达水平。进一步利用抗Flag蛋白的免疫磁珠富集沉淀调控上述过程的PPARγ-3×Flag蛋白复合物,将细胞质蛋白和细胞核蛋白富集的PPARγ-3×Flag复合物分别消化成肽段,利用液相色谱-串联质谱联用(liquid chromatograph-tandem mass spectrometer,LC-MS/MS)进行检测分析。结果发现,在添加PPARγ激动剂的条件下,与PPARγ特异互作的胞质蛋白有101个,胞核蛋白有95个;在添加PPARγ抑制剂的条件下,与PPARγ特异互作的胞质蛋白有24个,胞核蛋白有14个。GO和KEGG分析表明,与PPARγ特异互作的胞质蛋白和核蛋白均与翻译调控相关。本研究为深入探究PPARγ调控IFNβ表达机制提供了新线索。Peroxisome proliferator activated receptorγ(PPARγ)is specifically overexpressed in alveolar macrophages and has metabolic and immunoregulatory roles.It is not clear which interacting proteins of PPARγare involved in the regulation of typeⅠinterferon production induced by innate immunity.In this study,wild boar(Sus scrofa)lung cell line(WSL)with stable overexpression of PPARγ-3×Flag was constructed using lentiviral vector system.Taking this cell strain as a model,fluorescence quantitative PCR assay revealed that the addition of the PPARγagonist rosiglitazone significantly reduced the level of interferonβ(IFNβ)expression induced by stimulating with the dsDNA mimic Poly(dA:dT);In contrast,addition of the PPARγinhibitor T0070907 significantly increased the level of IFNβexpression induced by Poly(dA:dT)stimulation.Further,the PPARγ-3×Flag protein complex regulating the above process was precipitated by immunomagnetic beads of anti-FLAG protein.The PPARγ-3×Flag protein complexes enriched in cytoplasmic proteins and nuclear proteins were digested into peptides and analyzed by liquid chromatography and tandem mass spectrometry(LC-MS/MS),respectively.The results showed that there were 101 cytoplasmic proteins and 95 nucleoproteins interacting specifically with PPARγwhen the PPARγagonist rosiglitazone was added.Under the condition of adding PPARγinhibitor T0070907,there were 24 cytoplasmic proteins and 14 nuclear proteins that specifically interacted with PPARγ.GO and KEGG analysis showed that specific PPARγinteractions of cytoplasmic proteins and nuclear proteins were related to translation regulation.This study provides a new clue to further study the mechanism of regulating of IFNβexpression by PPARγ.

关 键 词：过氧化物酶体增殖物激活受体γ(PPARγ) 干扰素β(IFNβ) 免疫沉淀-质谱联用 先天免疫 翻译调控 

分 类 号：S852.4[农业科学—基础兽医学] R392.1[农业科学—兽医学]