泛素/蛋白酶体系统(UPS)在维持细胞蛋白质稳态中起着至关重要的作用。UPS中蛋白酶体的催化活性受β1(PSMB6)调控,β2(PSMB7),和β5(PSMB5)亚基。干扰素(IFN)-γ,肿瘤坏死因子(TNF)-α,炎症,和氧化应激可以诱导β1,β2和β5被各自的免疫亚基β1i(PSMB9)取代,β2i(PSMB10),和β5i(PSMB8),可以组装成免疫蛋白酶体。与标准蛋白酶体相比,免疫蛋白酶体对免疫反应发挥增强的调节作用,如加工和呈递MHCⅠ类抗原,促炎细胞因子的产生,和T细胞分化和增殖。免疫蛋白酶体的异常聚集可导致神经退行性疾病,如帕金森病,老年痴呆症,和肌萎缩侧索硬化症.探讨PSMB9在细菌感染后的作用,在本研究中,我们构建了过表达PSMB9-eGFP-His的慢病毒质粒,并使用三重质粒系统将质粒转染到HEK293T细胞中进行包装。用嘌呤霉素筛查后,我们获得了稳定表达PSMB9融合蛋白的人白血病单核细胞THP-1细胞系。Westernblotting(WB)和荧光显微镜验证了融合蛋白在稳定的THP-1细胞中的表达。采用定量PCR(qPCR)来测量THP-1细胞中PSMB9-eGFP的拷贝。免疫荧光结果发现eGFP-His不影响PSMB9的亚细胞定位。用镍亲和色谱纯化证实融合蛋白可以组装成20S免疫蛋白酶体并表现出对荧光肽底物的切割活性。这些结果表明PSMB9-eGFP融合基因已整合到染色体中,并能在构建的THP-1细胞系中稳定表达。该细胞系可用于亚细胞定位研究,动态表达式,和PSMB9在不同感染条件和疾病阶段的活细胞中的活性。它还为其他免疫蛋白酶体亚基PSMB8和PSMB10的稳定细胞系构建提供了模型。
The ubiquitin/proteasome system (UPS) plays a crucial role in maintaining cellular protein homeostasis. The catalytic activity of proteasome in the UPS is regulated by β1 (PSMB6), β2 (PSMB7), and β5 (PSMB5) subunits. Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, inflammation, and oxidative stress can induce the replacement of β1, β2, and β5 with their respective immuno-subunits β1i (PSMB9), β2i (PSMB10), and β5i (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.