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

ABSTRACT

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 ß1 (PSMB6), ß2 (PSMB7), and ß5 (PSMB5) subunits. Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, inflammation, and oxidative stress can induce the replacement of ß1, ß2, and ß5 with their respective immuno-subunits ß1i (PSMB9), ß2i (PSMB10), and ß5i (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.