稳定表达PSMB9-eGFP-His蛋白的THP-1细胞系建立及免疫蛋白酶体活性检测

Establishment of a stable THP-1 cell line expressing PSMB9-eGFP-His protein and detection of immunoproteasome activity

泛素/蛋白酶体系统(ubiquitin/proteasome system,UPS)在调控细胞内蛋白质稳态过程中发挥重要功能,UPS中蛋白酶体的催化活性受β1(PSMB6)、β2(PSMB7)和β5(PSMB5)亚基调控,在干扰素-γ(interferon-γ,IFN-γ)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、炎症反应、氧化应激等条件诱导下,β1、β2和β5可被相应的β1i(PSMB9)、β2i(PSMB10)和β5i(PSMB8)替换,组装形成免疫蛋白酶体(immunoproteasome)。与标准蛋白酶体相比,免疫蛋白酶体具有更强的免疫调节作用,如加工递呈MHCⅠ抗原、调节促炎细胞因子的产生及T细胞分化、增殖等。免疫蛋白酶体的异常聚集会导致帕金森病、阿尔茨海默病、肌萎缩侧索硬化等神经退行性疾病的发生。为探究PSMB9在细菌感染后发挥的功能,本研究通过构建过表达PSMB9-eGFP-His的慢病毒质粒,利用三质粒系统转染HEK293T包装慢病毒,嘌呤霉素筛选,获得稳定表达PSMB9融合蛋白的人髓系白血病单核细胞THP-1细胞系,利用蛋白免疫印迹(Western blotting,WB)及荧光显微镜验证了PSMB9融合蛋白的表达,通过实时荧光定量PCR(quantitative PCR,q PCR)计算了稳转细胞系中PSMB9-eGFP基因的拷贝数。免疫荧光结果发现eGFP-His不会影响PSMB9的亚细胞定位,镍柱亲和纯化实验证实PSMB9融合蛋白可组装至20S免疫蛋白酶体中,且具有底物切割活性。上述结果证明PSMB9-eGFP基因成功整合入THP-1细胞基因组并稳定表达,可用于探究PSMB9亚基在活细胞模型中不同感染条件及疾病进程中的细胞定位、动态表达和活性的变化,并为其他2个免疫蛋白酶体亚基PSMB8和PSMB10的稳转细胞系构建提供了参考。

The ubiquitin/proteasome system(UPS)plays a crucial role in maintaining cellular protein homeostasis.The catalytic activity of proteasome in the UPS is regulated byβ1(PSMB6),β2(PSMB7),andβ5(PSMB5)subunits.Interferon(IFN)-γ,tumor necrosis factor(TNF)-α,inflammation,and oxidative stress can induce the replacement ofβ1,β2,andβ5 with their respective immuno-subunitsβ1i(PSMB9),β2i(PSMB10),andβ5i(PSMB8),which can be assembled into the immunoproteasome.Compared with the standard proteasome,the immunoproteasome exerts enhanced regulatory effects on immune responses,such as processing and presenting MHC class I antigens,production of pro-inflammatory cytokines,and T cell differentiation and proliferation.Abnormal aggregation of immunoproteasomes can cause neurodegenerative diseases like Parkinson’s disease,Alzheimer’s disease,and amyotrophic lateral sclerosis.To explore the function of PSMB9 after bacterial infection,we constructed a lentivirus plasmid overexpressing PSMB9-eGFP-His and transfected the plasmid into HEK293T cells for packaging by using a triple-plasmid system in this study.After screening with puromycin,we obtained a stable human leukemia monocytic THP-1 cell line expressing the fusion protein of PSMB9.Western blotting(WB)and fluorescence microscopy verified the expression of the fusion protein in the stable THP-1 cells.Quantitative PCR(qPCR)was employed to measure the copies of PSMB9-eGFP in THP-1 cells.Immunofluorescence results found that eGFP-His did not affect the subcellular localization of PSMB9.The purification with nickel affinity chromatography confirmed that the fusion protein could be assembled into the 20S immunoproteasome and exhibited cleaving activity for fluorescent peptide substrates.These results indicated that the PSMB9-eGFP fusion gene was integrated into the chromosome,and could be stably expressed in the constructed THP-1 cell line.This cell line can be utilized for the research on subcellular localization,dynamic expression,and activity of PSMB9 in live cells at different infection conditions and disease stages.It also provides a model for the stable cell lines construction of other immunoproteasome subunits PSMB8 and PSMB10.