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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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Autores principales: Burov, Alexander, Funikov, Sergei, Vagapova, Elmira, Dalina, Alexandra, Rezvykh, Alexander, Shyrokova, Elena, Lebedev, Timofey, Grigorieva, Ekaterina, Popenko, Vladimir, Leonova, Olga, Spasskaya, Daria, Spirin, Pavel, Prassolov, Vladimir, Karpov, Vadim, Morozov, Alexey
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8616536/
https://www.ncbi.nlm.nih.gov/pubmed/34831272
http://dx.doi.org/10.3390/cells10113049
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author Burov, Alexander
Funikov, Sergei
Vagapova, Elmira
Dalina, Alexandra
Rezvykh, Alexander
Shyrokova, Elena
Lebedev, Timofey
Grigorieva, Ekaterina
Popenko, Vladimir
Leonova, Olga
Spasskaya, Daria
Spirin, Pavel
Prassolov, Vladimir
Karpov, Vadim
Morozov, Alexey
author_facet Burov, Alexander
Funikov, Sergei
Vagapova, Elmira
Dalina, Alexandra
Rezvykh, Alexander
Shyrokova, Elena
Lebedev, Timofey
Grigorieva, Ekaterina
Popenko, Vladimir
Leonova, Olga
Spasskaya, Daria
Spirin, Pavel
Prassolov, Vladimir
Karpov, Vadim
Morozov, Alexey
author_sort Burov, Alexander
collection PubMed
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.
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spelling pubmed-86165362021-11-26 A Cell-Based Platform for the Investigation of Immunoproteasome Subunit β5i Expression and Biology of β5i-Containing Proteasomes Burov, Alexander Funikov, Sergei Vagapova, Elmira Dalina, Alexandra Rezvykh, Alexander Shyrokova, Elena Lebedev, Timofey Grigorieva, Ekaterina Popenko, Vladimir Leonova, Olga Spasskaya, Daria Spirin, Pavel Prassolov, Vladimir Karpov, Vadim Morozov, Alexey Cells Article 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. MDPI 2021-11-05 /pmc/articles/PMC8616536/ /pubmed/34831272 http://dx.doi.org/10.3390/cells10113049 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Burov, Alexander
Funikov, Sergei
Vagapova, Elmira
Dalina, Alexandra
Rezvykh, Alexander
Shyrokova, Elena
Lebedev, Timofey
Grigorieva, Ekaterina
Popenko, Vladimir
Leonova, Olga
Spasskaya, Daria
Spirin, Pavel
Prassolov, Vladimir
Karpov, Vadim
Morozov, Alexey
A Cell-Based Platform for the Investigation of Immunoproteasome Subunit β5i Expression and Biology of β5i-Containing Proteasomes
title 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_short 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
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8616536/
https://www.ncbi.nlm.nih.gov/pubmed/34831272
http://dx.doi.org/10.3390/cells10113049
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