A Cell-Based Platform for the Investigation of Immunoproteasome Subunit β5i Expression and Biology of β5i-Containing Proteasomes
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 attr...
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2021
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oai:doaj.org-article:19082ea56f0d4d82a9b1d7c06365760f2021-11-25T17:10:49ZA Cell-Based Platform for the Investigation of Immunoproteasome Subunit β5i Expression and Biology of β5i-Containing Proteasomes10.3390/cells101130492073-4409https://doaj.org/article/19082ea56f0d4d82a9b1d7c06365760f2021-11-01T00:00:00Zhttps://www.mdpi.com/2073-4409/10/11/3049https://doaj.org/toc/2073-4409The 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.Alexander BurovSergei FunikovElmira VagapovaAlexandra DalinaAlexander RezvykhElena ShyrokovaTimofey LebedevEkaterina GrigorievaVladimir PopenkoOlga LeonovaDaria SpasskayaPavel SpirinVladimir PrassolovVadim KarpovAlexey MorozovMDPI AGarticleproteasomeimmunoproteasomeintermediate proteasomenon-constitutive proteasomereporter cell lineBiology (General)QH301-705.5ENCells, Vol 10, Iss 3049, p 3049 (2021) |
| institution |
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| collection |
DOAJ |
| language |
EN |
| topic |
proteasome immunoproteasome intermediate proteasome non-constitutive proteasome reporter cell line Biology (General) QH301-705.5 |
| spellingShingle |
proteasome immunoproteasome intermediate proteasome non-constitutive proteasome reporter cell line Biology (General) QH301-705.5 Alexander Burov Sergei Funikov Elmira Vagapova Alexandra Dalina Alexander Rezvykh Elena Shyrokova Timofey Lebedev Ekaterina Grigorieva Vladimir Popenko Olga Leonova Daria Spasskaya Pavel Spirin Vladimir Prassolov Vadim Karpov Alexey Morozov A Cell-Based Platform for the Investigation of Immunoproteasome Subunit β5i Expression and Biology of β5i-Containing Proteasomes |
| description |
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. |
| format |
article |
| author |
Alexander Burov Sergei Funikov Elmira Vagapova Alexandra Dalina Alexander Rezvykh Elena Shyrokova Timofey Lebedev Ekaterina Grigorieva Vladimir Popenko Olga Leonova Daria Spasskaya Pavel Spirin Vladimir Prassolov Vadim Karpov Alexey Morozov |
| author_facet |
Alexander Burov Sergei Funikov Elmira Vagapova Alexandra Dalina Alexander Rezvykh Elena Shyrokova Timofey Lebedev Ekaterina Grigorieva Vladimir Popenko Olga Leonova Daria Spasskaya Pavel Spirin Vladimir Prassolov Vadim Karpov Alexey Morozov |
| author_sort |
Alexander Burov |
| title |
A Cell-Based Platform for the Investigation of Immunoproteasome Subunit β5i Expression and Biology of β5i-Containing Proteasomes |
| title_short |
A Cell-Based Platform for the Investigation of Immunoproteasome Subunit β5i Expression and Biology of β5i-Containing Proteasomes |
| title_full |
A Cell-Based Platform for the Investigation of Immunoproteasome Subunit β5i Expression and Biology of β5i-Containing Proteasomes |
| title_fullStr |
A Cell-Based Platform for the Investigation of Immunoproteasome Subunit β5i Expression and Biology of β5i-Containing Proteasomes |
| title_full_unstemmed |
A Cell-Based Platform for the Investigation of Immunoproteasome Subunit β5i Expression and Biology of β5i-Containing Proteasomes |
| title_sort |
cell-based platform for the investigation of immunoproteasome subunit β5i expression and biology of β5i-containing proteasomes |
| publisher |
MDPI AG |
| publishDate |
2021 |
| url |
https://doaj.org/article/19082ea56f0d4d82a9b1d7c06365760f |
| work_keys_str_mv |
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