GSE183592
A cell-based platform for the investigation of immunoproteasome subunit ß5i expression and biology of ß5i-containing proteasomes
The degradation of intracellular proteins is a dynamic and tightly regulated process. Most of such substrates is hydrolyzed by proteasomes. Different forms of proteasomes are known. The role of immunoproteasomes (iPs) and intermediate proteasomes (intPs) is emerging. Here, using a CRISPR-Cas9 nickase technology a panel of four cell lines was obtained. Cells of histiocytic lymphoma, colorectal adenocarcinoma, cervix adenocarcinoma and hepatocarcinoma origin were modified to express proteasomes with mCherry-tagged ß5i subunit, which is a catalytic subunit of iPs and intPs. Importantly, the chimeric gene expression in modified cells is under control of endogenous regulatory mechanisms and was increased following IFN-? and/or TNF-a stimulation. Fluorescent proteasomes are catalytically active and were distributed within the nucleus and cytoplasm of the modified cells. RNAseq revealed marginal differences in gene expression profile between modified and initial cell lines, predominate metabolic pathways and a pattern of expressed receptors were determined for each cell line. Using obtained cell-lines it was shown that anti-cancer drugs ruxolitinib, vincristine and gefetinib stimulate the expression of ß5i-containing proteasomes which might have an impact on the disease prognosis. Taken together, obtained cell lines represent convenient platform to study the immunoproteasome gene expression, localization of iPs and intPs, association of proteasomes with other proteins and many other aspects of proteasome biology under the influence of various drugs and conditions in real time and in living cells. Overall design: RNA-seq analysis of modified and control cell lines