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

Alexander Burov,Daria Spasskaya,Elena Shyrokova,Ekaterina Grigorieva,Timofey Lebedev,Alexander Rezvykh,Vladimir Popenko,Sergei Funikov,Pavel Spirin,Elmira Vagapova,Alexey Morozov,Vladimir Prassolov,Vadim Karpov,Olga Leonova,Alexandra Dalina

doi:10.3390/cells10113049

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.

Highlights

Most proteins in cells are degraded by the ubiquitin-proteasome system (UPS)
TZM-bl cells were derived from HeLa cells—one of the most frequently used cell lines
In these cells iP subunit expression is efficiently activated upon stimulation with cytokines

Summary

Introduction

Most proteins in cells are degraded by the ubiquitin-proteasome system (UPS). A core element of UPS is the 20S proteasome complex comprising four heptameric rings of either alpha or beta subunits arranged according to the αββα symmetry. Three of seven beta subunits (β1, β2, and β5) perform protein hydrolysis and exert caspase-like, trypsin-like, and chymotrypsin-like catalytic activities, respectively [1]. Constitutive catalytic subunits are replaced by the so-called immune catalytic subunits: β1i (encoded by PSMB9 gene), β2i (encoded by PSMB10), and β5i (encoded by PSMB8) [2]. Due to the structural peculiarities of the immune subunits, immunoproteasomes (iPs) demonstrate lower caspase-like activity, but higher chymotrypsin-like activity than the constitutive proteasomes, which is essential for the production of MHCI-compatible peptides [3]. IPs play an important role in inflammation and immune response [2,4]

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Discussion

Conclusion