Next-generation cell lines for profiling different proteasome forms and their implications in cancer

Alexander Burov¹Anastasia Yakovleva^2,3,4Georgy Linovsky^5,6Andrei Fedotov^5,6Vladimir Popenko⁷Olga Leonova⁷Aleksandr Lanin^5,6Marina Drutskaya^2,4,8Vadim Karpov¹Alexey V. Morozov^1*

• ¹Laboratory of Regulation of Intracellular Proteolysis, Engelhardt Institute of Molecular Biology (RAS), Moscow, Russia
• ²Laboratory of Molecular Mechanisms of Immunity, Engelhardt Institute of Molecular Biology (RAS), Moscow, Russia
• ³M.V. Lomonosov Moscow state university, Biology Department, Moscow, Russia
• ⁴Center for Precision Genetic Technologies for Medicine, Engelhardt Institute of Molecular Biology (RAS), Moscow, Russia
• ⁵M.V. Lomonosov Moscow State University, Physics Department, Moscow, Russia
• ⁶Life Improvement by Future Technologies (LIFT) Center, Moscow, Russia
• ⁷Department of Cancer Cell Biology, Engelhardt Institute of Molecular Biology (RAS), Moscow, Russia
• ⁸M.V. Lomonosov Moscow state university, Biology Department, Mosocow, Russia

Background. Most intracellular proteins undergo degradation by proteasomes, which exist in constitutive, immune, and intermediate forms. The diversity arises from the presence of different catalytic β subunits incorporated into the 20S core particle. Constitutive proteasomes contain β1, β2 and β5 subunits, whereas immunoproteasomes integrate β1i, β2i and β5i proteins. Intermediate proteasomes comprise combinations of constitutive and immune subunits, but typically lack the β2i subunit. Distinct proteasome configurations have widespread impact on cellular metabolism, gene expression, immune signaling, stress adaptation and tumorigenesis. However, the biological functions of proteasome subtypes remain incompletely defined, underscoring the need for refined experimental systems to uncover their specific activities. Methods. Previously, we generated cancer cell lines expressing mCherry-tagged β5i subunit under the control of endogenous regulatory elements. Using CRISPR/Cas9 nickase technology, we further modified the genomes of the colon adenocarcinoma SW620B8-mCherry and cervical adenocarcinoma TZM-blB8-mCherry cells. In SW620B8-mCherryB5-GFP cells, the eGFP gene was inserted at the 3’ end of the PSMB5 gene, which encodes the β5 subunit. Similarly, the β2i-encoding gene (PSMB10) was fused with the photo-switchable cyan fluorescent protein gene (PS-CFP2) in TZM-blB8-mCherryB10-CFP cells. Results. Efficient integration of fluorescent protein-encoding sequences into target genomes provided robust expression and integration of chimeric subunits into proteasomes. In TZM-blB8-mCherryB10-CFP cells, photo-conversion of PS-CFP2 allowed visualization of both intermediate and immunoproteasomes within the cellular nuclei following treatment with IFN-γ/TNF. In SW620B8-mCherryB5-GFP cells, analysis of intracellular proteasome localization revealed discrete regions enriched in β5i-containing proteasomes. Moreover, IFN-γ/TNF exposure induced a fluorescence shift from green to red, accompanied by redistribution of intracellular proteasomes in SW620B8-mCherryB5-GFP cells. Multiphoton microscopy of tumors grafted into immunocompromised mice showed the formation of β5i-containing, proteasome-enriched inclusions and suggested their potential release from cancer cells. Collectively, the TZM-blB8-mCherryB10-CFP cell line provides a tool to interrogate immunoproteasome functions independently of intermediate proteasomes, whereas SW620B8-mCherryB5-GFP cells enable visual discrimination between constitutive and β5i-containing proteasomes both in vitro and in vivo. Conclusions. Generated cell lines provide a novel platform for dissecting proteasome functions, addressing the distinct properties of individual proteasome forms, and assessing the impact of diverse compounds on the proteasome repertoire in cultured cells and in tumor xenografts.