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.

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.

Introduction: Proteasomes are multi-subunit protein complexes responsible for protein degradation in cells. Immunoproteasomes and intermediate proteasomes (together non-constitutive proteasomes) are specific forms of proteasomes frequently associated with immune response, antigen presentation, inflammation and stress. Expression of non-constitutive proteasome subunits has a prognostic value in several types of cancer. Thus, factors that modulate non-constitutive proteasome expression in tumors are of particular interest. Multikinase inhibitors (MKIs) demonstrate promising results in treatment of cancer. At the same time, their immunomodulatory properties and effects on non-constitutive proteasome expression in colorectal cancer cells are poorly investigated.Methods: Proteasome subunit expression in colorectal cancer was evaluated by bioinformatic analysis of available datasets. Two colorectal cancer cell lines, expressing fluorescent non-constitutive proteasomes were treated with multikinase inhibitors: regorafenib and sorafenib. The proteasome subunit expression was assessed by real-time PCR, Western blotting and flow cytometry. The proteasome activity was studied using proteasome activity-based probe and fluorescent substrates. Intracellular proteasome localization was revealed by confocal microscopy. Reactive oxygen species levels following treatment were determined in cells. Combined effect of proteasome inhibition and treatment with MKIs on viability of cells was estimated.Results: Expression of non-constitutive proteasomes is increased in BRAF-mutant colorectal tumors. Regorafenib and sorafenib stimulated the activity and synthesis of non-constitutive proteasomes in examined cell lines. MKIs induced oxidative stress and redistribution of proteasomes within cells. Sorafenib stimulated formation of cytoplasmic aggregates, containing proteolyticaly active non-constitutive proteasomes, while regorafenib had no such effect. MKIs caused no synergistic action when were combined with the proteasome inhibitor.Discussion: Obtained results indicate that MKIs might affect the crosstalk between cancer cells and immune cells via modulation of intracellular proteasome pool. Observed phenomenon should be considered when MKI-based therapy is applied.

Proteasomes are barrel-like cellular protein complexes responsible for the degradation of most intracellular proteins. Earlier, it has been shown that during assembly, hundreds of different cellular proteins are incorporated into retro-and herpes viruses. Among detected cellular proteins, there were different proteasome subunits (PS). Previous reports postulated the incorporation of 20S proteasome subunits and subunits of proteasome regulator complexes inside retroviruses. Here, we demonstrated the association of functional 20S proteasome with gammaretroviruses, betaretroviruses, and lentiviruses. Cleaved proteasome subunits β1, β2 and β5 were detected in tested viruses. Using fluorescent peptides and a cell-permeable proteasome activity probe, proteasome activity was detected in endogenous and exogenous retroviruses, including recombinant HIV-1. Taken together, our data favors the insertion of functional proteasomes into the retroviruses during assembly. The possible role of proteasomes in retroviruses is discussed.

Proteasome inhibitors are used in the therapy of several cancers, and clinical trials are underway for their use in the treatment of glioblastoma (GBM). However, GBM becomes resistant to chemotherapy relatively rapidly. Recently, the overexpression of ribonucleotide reductase (RNR) genes was found to mediate therapy resistance in GBM. The use of combinations of chemotherapeutic agents is considered a promising direction in cancer therapy. The present work aimed to evaluate the efficacy of the combination of proteasome and RNR inhibitors in yeast and GBM cell models. We have shown that impaired proteasome function results in increased levels of RNR subunits and increased enzyme activity in yeast. Co-administration of the proteasome inhibitor bortezomib and the RNR inhibitor hydroxyurea was found to significantly reduce the growth rate of S. cerevisiae yeast. Accordingly, the combination of bortezomib and another RNR inhibitor gemcitabine reduced the survival of DBTRG-05MG compared to the HEK293 cell line. Thus, yeast can be used as a simple model to evaluate the efficacy of combinations of proteasome and RNR inhibitors.

The members of the Flaviviridae family are becoming an emerging threat for public health, causing an increasing number of infections each year and requiring effective treatment. The consequences of these infections can be severe and include liver inflammation with subsequent carcinogenesis, endothelial damage with hemorrhage, neuroinflammation, and, in some cases, death. The mechanisms of Flaviviridae pathogenesis are being actively investigated, but there are still many gaps in their understanding. Extracellular vesicles may play important roles in these mechanisms, and, therefore, this topic deserves detailed research. Recent data have revealed the involvement of extracellular vesicles in steps of Flaviviridae pathogenesis such as transmission, immune evasion, and inflammation, which is critical for disease establishment. This review covers recent papers on the roles of extracellular vesicles in the pathogenesis of Flaviviridae and includes examples of clinical applications of the accumulated data.

Various external and internal factors damaging DNA constantly disrupt the stability of the genome. Cells use numerous dedicated DNA repair systems to detect damage and restore genomic integrity in a timely manner. Ribonucleotide reductase (RNR) is a key enzyme providing dNTPs for DNA repair. Molecular mechanisms of indirect regulation of yeast RNR activity are well understood, whereas little is known about its direct regulation. The study was aimed at elucidation of the proteasome-dependent mechanism of direct regulation of RNR subunits in Saccharomyces cerevisiae. Proteome analysis followed by Western blot, RT-PCR, and yeast plating analysis showed that upregulation of RNR by proteasome deregulation is associated with yeast hyper resistance to 4-nitroquinoline-1-oxide (4-NQO), a UV-mimetic DNA-damaging drug used in animal models to study oncogenesis. Inhibition of RNR or deletion of RNR regulatory proteins reverses the phenotype of yeast hyper resistance to 4-NQO. We have shown for the first time that the yeast Rnr1 subunit is a substrate of the proteasome, which suggests a common mechanism of RNR regulation in yeast and mammals.

—The ubiquitin-proteasome system (UPS) provides hydrolysis of most intracellular proteins in proteasomes. There are various forms of proteasomes that differ, among other things, in the set of proteolytic subunits and the presence of activators. Alzheimer’s disease (AD) is characterized by disturbances in the functional state of the UPS. At the same time, an increase in the expression of certain forms of proteasomes, in particular, proteasomes containing immune subunits (nonconstitutive proteasomes), has been shown. Here, we studied dynamic changes in the expression of catalytic proteasome subunit genes and corresponding proteins in the cerebral cortex of animals using a mouse model of AD (5xFAD transgenic mice). Increases by 4 and 6 folds in transcripts of the PSMB9 and PSMB8 genes encoding immune proteasome subunits were detected, as well as a significant increase in the content of immune β-subunits (by 2.8 folds, β1i; 2.2 folds, β2i) in samples from 5xFAD mice at the age of 380 days, compared with samples from mice at 60 days of age. Moreover, the activation of both 20S and 26S proteasomes containing immune subunits were revealed in samples from 380 days old 5xFAD mice by electrophoresis in native conditions. This indicates activated synthesis of the immune subunits and assembly of nonconstitutive proteasomes at the terminal stage of pathology development. The obtained data, in combination with the available literature, indicate that the activation of nonconstitutive proteasomes is a universal phenomenon characteristic of various animal models of AD, which may reflect both the development of neuroinflammation and adaptive processes in tissues induced by the accumulation of toxic protein aggegates.

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.

—The pathology of diseases arising from infections by viruses of Flaviviridae is largely determined by the development of systemic inflammation. The cytokines interleukin-1beta and interleukin-18 play a key role in triggering inflammation. Their secretion from cells, in its turn, is induced upon activation of inflammasomes. Activation of NLRP3 (NLR pyrin domain-containing family 3) inflammasomes was detected in cells infected with Flaviviridae. Some nonstructural proteins of these viruses have been shown to be able to activate or to inhibit the NLRP3 inflammasome, in particular, through interaction with its components. In this study, a functional NLRP3 inflammasome was reconstructed in human HEK293T cells and the effect of some nonstructural proteins of individual Flaviviridae viruses on it was studied. This model did not reveal any impact of nonstructural NS1 proteins of the West Nile virus, NS3 of hepatitis C virus, or NS5 of tick-borne encephalitis virus on the inflammasome components content. At the same time, in the presence of the NS1 of the West Nile virus and NS5 of the tick-borne encephalitis virus, the level of secretion of interleukin-1beta did not change, whereas in the presence of the NS3 protein of the hepatitis C virus, it increased by 1.5 times. Thus, NS3 can be considered as one of the factors of NLRP3 inflammasome activation and inflammatory pathogenesis in chronic hepatitis C virus infection.

Selective degradation of cellular proteins by the ubiquitin–proteasome system (UPS) is one of the key regulatory mechanisms in eukaryotic cells. A growing body of evidence indicates that UPS is involved in the regulation of fundamental processes in mammalian stem cells, including proliferation, differentiation, cell migration, aging, and programmed cell death, via proteolytic degradation of key transcription factors and cell signaling proteins and post-translational modification of target proteins with ubiquitin. Studying molecular mechanisms of proteostasis in stem cells is of great importance for the development of new therapeutic approaches aimed at the treatment of autoimmune and neurodegenerative diseases, cancer, and other socially significant pathologies. This review discusses current data on the UPS functions in stem cells.

Cancer cells are characterized by an increased level of metabolism and are highly dependent on the correct functioning of the processes that ensure homeostasis. Reactive sulfur species (RSS) are important molecular modulators of metabolic processes in both healthy and tumor cells. The effect of RSS and, in particular, H2S, on key cellular systems, including the ubiquitin-proteasome system (UPS), which provides the destruction of most intracellular proteins, has been shown. The main components of the UPS are proteasomes, multisubunit protein complexes, within which proteolysis occurs. At the same time, data on the effect of H2S directly on the pool of proteasomes in tumor cells are insufficient. Here, we studied the effect of incubation of SW620B8-mCherry colorectal adenocarcinoma cells expressing a fluorescently labeled proteasome subunit with 50, 100, and 200 μM of the hydrogen sulfide donor GYY4137. The effect of the substance on the proteasome pool was assessed 6, 24, 48, and 72 h after administration. It was shown that the chymotrypsin-like and caspase-like proteasome activity decreases in cells incubated with 200 μM of the GYY4137 for 24 h. This coincided with an increase in the expression of proteasome subunit genes. In lysates of cells incubated with 200 μM GYY4137 for 48 h an increase in the content of the constitutive β5 subunit was observed and the activity of proteasomes leveled off. Following prolonged incubation with GYY4137 (72h), an increase in the expression levels of some proteasome genes was also observed, although this did not have a significant effect on the activity and subunit composition of proteasomes. Thus, the obtained data indicate the modulation of proteasome activity by the hydrogen sulfide donor and the effect of GYY4137 on transcription and translation of proteasome genes.

Tick-borne encephalitis (TBE) is an emerging zoonosis that may cause long-term neurological sequelae or even death. Thus, there is a growing interest in understanding the factors of TBE pathogenesis. Viral genetic determinants may greatly affect the severity and consequences of TBE. In this study, nonstructural protein 1 (NS1) of the tick-borne encephalitis virus (TBEV) was tested as such a determinant. NS1s of three strains with similar neuroinvasiveness belonging to the European, Siberian and Far-Eastern subtypes of TBEV were studied. Transfection of mouse cells with plasmids encoding NS1 of the three TBEV subtypes led to different levels of NS1 protein accumulation in and secretion from the cells. NS1s of TBEV were able to trigger cytokine production either in isolated mouse splenocytes or in mice after delivery of NS1 encoding plasmids. The profile and dynamics of TNF-α, IL-6, IL-10 and IFN-γ differed between the strains. These results demonstrated the involvement of TBEV NS1 in triggering an immune response and indicated the diversity of NS1 as one of the genetic factors of TBEV pathogenicity.

Abstract—Proteasomes are key components of the ubiquitin–proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes have been revealed, as well as increased expression of their particular forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in cerebral cortex samples from C57BL/6 mice at the ages of 60, 190, 380, and 720 days. In addition, the contents of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the β5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the β5i subunit of different forms of proteasomes separated by electrophoresis in native gel were characterized. Compared with samples from young animals, in the cerebral cortex of animals at an age of 720 days the following changes in the expression patterns of proteasome genes were revealed: a decreased expression of the PSMB5 gene encoding constitutive proteasome subunit β5; increased expression of genes encoding immune proteasome subunits β5i and β1i. In tissue homogenates of aged mice, an increase in the content of immune subunits β1i and β2i was shown. In samples from old animals, chymotrypsin-like activity was decreased and a tendency to a decrease in caspase-like activity of proteasomes as well as the β5i subunit activity was revealed. Analysis of the activity of native complexes in tissues obtained from old animals revealed decreased chymotrypsin-like activity of 26S and 20S proteasomes containing the β5i subunit. Based on the obtained data, it can be assumed that changes in the pool of nonconstitutive proteasomes reflect aging-associated adaptive processes in the mouse brain.

Members of the Flaviviridae family are posing a significant threat to human health worldwide. Many flaviviruses are capable of inducing severe inflammation in humans. Flaviviridae nonstructural proteins, apart from their canonical roles in viral replication, have noncanonical functions strongly affecting antiviral innate immunity. Among these functions, antagonism of type I IFN is the most investigated; meanwhile, more data are accumulated on their role in the other pathways of innate response. This review systematizes the last known data on the role of Flaviviridae nonstructural proteins in molecular mechanisms of triggering inflammation, with an emphasis on their interactions with TLRs and RLRs, interference with NF-κB and cGAS-STING signaling, and activation of inflammasomes.

The ubiquitin-proteasome system is involved in the control of all essential molecular processes under normal conditions and the response of cells to stress. Rpn4p serves as a key transcriptional regulator of the proteasome in Saccharomycetes yeast and is also involved in the cellular response to various stresses. In addition to proteasomal genes, Rpn4 affects the expression of several hundred other genes, including genes involved in DNA repair and oxidative stress response. At the same time, the molecular mechanisms used by Rpn4 in controlling target genes and its functioning as a regulator of the cellular response to stress remain largely unclear. The aim of this work was to determine the Rpn4 domains required to ensure cell resistance to stress. It was shown that the N-terminal and central regions of the protein contain sites required for resistance to all types of stresses. The putative nuclear localization signal does not affect the functioning of Rpn4. Unexpectedly, a protein with the deletion of both zinc finger motifs that form the DNA-binding domain provides yeast resistance to oxidative stress and cycloheximide. Moreover, we showed that Rpn4 can be recruited to the promoter regions of the regulated genes even if they do not contain its binding sites. Based on these data, it can be assumed that Rpn4 is involved in gene regulation and the cellular response to stress due to protein-protein interactions.

Proteasomes hydrolyze most cellular proteins. The standard reaction to determine proteasome activity in cellular lysate or elsewhere contains AMC-conjugated peptide substrate, ATP, Mg2+, and DTT. ATP and Mg2+ are included to maintain 26S proteasome functionality. However, most cellular proteasomes are 20S proteasomes, and the effects of ATP on the turnover of fluorogenic substrates by 20S complexes are largely unknown. Here, we evaluated the effect of ATP alone or in combination with Mg2+ on the degradation of AMC-conjugated fluorogenic substrates by purified 20S proteasomes. Degradation of substrates used to determine chymotrypsin-, caspase- and trypsin-like proteasome activities was gradually decreased with the rise of ATP concentration from 0.25 to 10 mM. These effects were not associated with the blockage of the proteasome catalytic subunit active sites or unspecific alterations of AMC fluorescence by the ATP. However, ATP-induced peptide degradation slowdown was rescued by the addition of Mg2+. Moreover, the substrate degradation efficacy was proportional to the Mg2+/ATP ratio, being equal to control values when equimolar concentrations of the molecules were used. The obtained results indicate that when proteasome activity is assessed, the reciprocal effects of ATP and Mg2+ on the hydrolysis of AMC-conjugated fluorogenic substrates by the 20S proteasomes should be considered.

The ubiquitin-proteasome system is involved in the control of all essential molecular processes under normal conditions and the response of cells to stress. Rpn4p serves as a key transcriptional regulator of the proteasome in Saccharomycetes yeast and is also involved in the cellular response to various stresses. In addition to proteasomal genes, Rpn4 affects the expression of several hundred other genes, including genes involved in DNA repair and oxidative stress response. At the same time, the molecular mechanisms used by Rpn4 in controlling target genes and its functioning as a regulator of the cellular response to stress remain largely unclear. The aim of this work was to determine the Rpn4 domains required to ensure cell resistance to stress. It was shown that the N-terminal and central regions of the protein contain sites required for resistance to all types of stresses. The putative nuclear localization signal does not affect the functioning of Rpn4. Unexpectedly, a protein with the deletion of both zinc finger motifs that form the DNA-binding domain provides yeast resistance to oxidative stress and cycloheximide. Moreover, we showed that Rpn4 can be recruited to the promoter regions of the regulated genes even if they do not contain its binding sites. Based on these data, it can be assumed that Rpn4 is involved in gene regulation and the cellular response to stress due to protein-protein interactions.

Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.

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.