A Cell-Based Platform for the Investigation of Immunoproteasome Subunit β5i Expression and Biology of β5i-Containing Proteasomes

Abstract

The degradation of most intracellular proteins is a dynamic and tightly regulated process performed by proteasomes. To date, different forms of proteasomes have been identified. Currently the role of non-constitutive proteasomes (immunoproteasomes (iPs) and intermediate proteasomes (intPs)) has attracted special attention. Here, using a CRISPR-Cas9 nickase technology, four cell lines: histiocytic lymphoma, colorectal adenocarcinoma, cervix adenocarcinoma, and hepatocarcinoma were modified to express proteasomes with mCherry-tagged β5i subunit, which is a catalytic subunit of iPs and intPs. Importantly, the expression of the chimeric gene in modified cells is under the control of endogenous regulatory mechanisms and is increased following IFN-γ and/or TNF-α stimulation. Fluorescent proteasomes retain catalytic activity and are distributed within the nucleus and cytoplasm. RNAseq reveals marginal differences in gene expression profiles between the modified and wild-type cell lines. Predominant metabolic pathways and patterns of expressed receptors were identified for each cell line. Using established cell lines, we demonstrated that anti-cancer drugs Ruxolitinib, Vincristine and Gefitinib stimulated the expression of β5i-containing proteasomes, which might affect disease prognosis. Taken together, obtained cell lines can be used as a platform for real-time studies of immunoproteasome gene expression, localization of iPs and intPs, interaction of non-constitutive proteasomes with other proteins, proteasome trafficking and many other aspects of proteasome biology in living cells. Moreover, the established platform might be especially useful for fast and large-scale experiments intended to evaluate the effects of different conditions including treatment with various drugs and compounds on the proteasome pool.

Multikinase inhibitors modulate non-constitutive proteasome expression in colorectal cancer cells

“…Human colorectal adenocarcinoma cell line SW480 and embryonic kidney HEK 293T cell line were kindly provided by Dr. Vladimir Prassolov. The SW620B8-mCherry cell line was obtained previously ( Burov et al, 2021 ). The SW480, SW480B8-mCherry and SW620B8-mCherry and HEK 293T cells were maintained in DMEM (Thermo Fisher Scientific, Paisley, Renfrewshire, Scotland, UK).…”

“…The plasmid encoding Cas9D10A, GFP and gRNAs, the donor plasmid used for recombination, as well as the protocol for validation of the obtained cell line were generated previously ( Burov et al, 2021 ). SW480 cells were co-transfected with the plasmids using Mirus TransIT-LT1 reagent (Mirus Bio LLC, Madison, WI, USA) according to manufacturer’s instructions.…”

“…To verify the presence of specific insert in the genome of SW480B8-mCherry cells we used two sets of primers that were obtained previously ( Burov et al, 2021 ) ( Supplementary Table S2 ). The fragments of genomic DNA were amplified with primer pairs A-B and C-D ( Burov et al, 2021 ) using Q5 High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA). The nucleotide sequence integrity was confirmed by bi-directional sequencing.…”

“…The cDNA was obtained from two microgram of total RNA using oligo(dT)20 primer and Magnus Reverse Transcriptase (Evrogen, Moscow, Russia). To estimate chimeric gene ( PSMB8-mCherry ) expression levels we used the G-H pair of primers ( Burov et al, 2021 ) ( Supplementary Table S2 ). For the amplification of the wild-type PSMB8 , PSMB5 , PSMB9 and PSMB10 genes fragments, primer pairs reported in ( Morozov et al, 2019 ) were utilized ( Supplementary Table S2 ).…”

“…Except use of pro-inflammatory cytokines with pleiotropic effects, currently, no specific drugs that facilitate immunoproteasome synthesis are known. At the same time, several reports indicate altered immunoproteasome subunit expression following treatment of cancer cells with other types of anti-cancer drugs - protein kinase inhibitors that are currently widely used in clinical practice ( Burov et al, 2021 ; Takahashi et al, 2021 ).…”