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.

Proteasomes are intracellular structures responsible for protein degradation. The 20S proteasome is a core catalytic element of the proteasome assembly. Variations of catalytic subunits generate different forms of 20S proteasomes including immunoproteasomes (iPs), which are present mostly in the immune cells. Certain cells of the immune system are primary targets of retroviruses. It has been shown that several viral proteins directly affect proteasome functionality, while inhibition of proteasome activity with broad specificity proteasome inhibitors stimulates viral transduction. Here we specifically addressed the role of the immunoproteasomes during early stages of viral transduction and investigated the effects of specific immunoproteasome inhibition and activation prior to infection using a panel of cell lines. Inhibition of iPs in hematopoietic cells with immunoproteasome-specific inhibitor ONX-0914 resulted in increased infection by VSV-G pseudotyped lentiviruses. Moreover, a tendency for increased infection of cloned cells with endogenously decreased proteasome activity was revealed. Conversely, activation of iPs by IFN-γ markedly reduced the viral infectivity, which was rescued upon simultaneous immunoproteasome inhibition. Our results indicate that immunoproteasome activity might be determinative for the cellular antiretroviral resistance at least for the cells with high iP content. Finally, therapeutic application of immunoproteasome inhibitors might promote retroviral infection of cells in vivo.

Background: Proteasomes degrade intracellular proteins. Different proteasome forms were identified. Proteasome inhibitors are used in cancer therapy, and novel drugs directed to specific proteasome forms are developed. Breast cancer (BC) therapy depends on the subtype of the tumor, determined by the expression level of Ki67, HER-2, estrogen and progesterone receptors. Relationships between the presence of specific proteasome forms and proteins that determine the BC subtype remain unclear. Here, using gene expression data in 19,145 tumor samples from 144 datasets and tissues from 159 patients with different subtypes of BC, we investigated the association between the activity and expression of proteasomes and levels of BC subtype markers. Methods: Bioinformatic analysis of proteasome subunit (PSMB1-10) gene expression in BC was performed. Proteasome heterogeneity in BC cell lines was investigated by qPCR. By Western blotting, proteasome composition was assessed in cells and patient tissue lysates. Proteasome activities were studied using fluorogenic substrates. BC molecular subtypes were determined by immunohistochemistry. Results: BC subtypes demonstrate differing proteasome subunit expression pattern and strong PSMB8-10 co-correlation in tumors. A significant increase in chymotrypsin- and caspase-like proteasome activities in BC compared to adjacent tissues was revealed. The subunit composition of proteasomes in tumor tissues of BC subtypes varied. Regression analysis demonstrated a positive correlation between proteasome activities and the expression of Ki67, estrogen receptors and progesterone receptors. Conclusion: BC subtypes demonstrate differences within the proteasome pool. Correlations between the proteasome activity, hormone receptors and Ki67 indicate possible mutual influence. Obtained results facilitate development of novel drug combinations for BC therapy.

Most intracellular proteins are degraded by the ubiquitin–proteasome system (UPS), with proteasomes directly hydrolyzing protein substrates. Specific forms of proteasomes (non-constitutive proteasomes), implicated in antigen presentation, cellular homeostasis maintenance and stress response have been described. However, proteasomes were also identified outside cells, where their function remains unclear. Proteasome secretion via extracellular vesicles (EVs) have been reported, though the direct transmission of non-constitutive proteasomes between cells has not been shown. Using genetically modified cells, including a human adenocarcinoma cell line SW620B8-mCherry expressing the β5i subunit of non-constitutive proteasomes fused to the mCherry protein, and a number of techniques, such as differential centrifugation, affinity isolation, unspecific precipitation, NTA and microscopy, EVs containing non-constitutive proteasomes were obtained and characterized. Different cell lines were shown to secrete varying amounts of vesicles containing non-constitutive proteasomes. The content of these proteasomes in EVs was increased after the stimulation of cells with IFN-γ. The interaction of vesicles secreted by SW620B8-mCherry cells with recipient cells was demonstrated. The β5i-mCherry chimera was detected in lysates of different recipient cells following incubation with EVs secreted from SW620B8-mCherry cells. The obtained results highlight the transfer of non-constitutive proteasomes from one cell to another via EVs.

Proteasomes degrade most intracellular proteins. Several different forms of proteasomes are known. Little is known about the role of specific proteasome forms in the central nervous system (CNS). Inhibitors targeting different proteasome forms are used in clinical practice and were shown to modulate long-term potentiation (LTP) in hippocampal slices of untreated animals. Here, to address the role of non-constitutive proteasomes in hippocampal synaptic plasticity and reveal the consequences of their continuous inhibition, we studied the effect of chronic administration of the non-constitutive proteasome inhibitor ONX-0914 on the LTP induced by two different protocols: tetanic stimulation and theta-burst stimulation (TBS). Both the tetanus- and TBS-evoked potentiation contribute to the different forms of hippocampal-dependent memory and learning. Field-excitatory postsynaptic potentials (fEPSPs) in hippocampal slices from control animals and animals treated with DMSO or ONX-0914 were compared. LTP induced by the TBS was not affected by ONX-0914 administration; however, chronic injections of ONX-0914 led to a decrease in fEPSP slopes after tetanic stimulation. The observed effects correlated with differential expression of genes involved in synaptic plasticity, glutaminergic synapse, and synaptic signaling. Obtained results indicate that non-constitutive proteasomes are likely involved in the tetanus-evoked LTP, but not the LTP occurring after TBS, supporting the relevance and complexity of the role of specific proteasomes in synaptic plasticity, memory, and learning.