Yulia V. Lyupina

New insights into the induction of the heat shock proteins in baculovirus infected insect cells.

Eight members of the HSP/HSC70 family were identified in Spodoptera frugiperda Sf9 cells infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by 2D electrophoresis followed by mass spectrometry (MALDI/TOF) and a Mascot search. The family includes five HSP70s induced by AcMNPV-infection and three constitutive cognate HSC70s that remained abundant in infected cells. Confocal microscopy revealed dynamic changes in subcellular localization of HSP/HSC70s in the course of infection. At the early stages (4 to 10 hpi), a fraction of HSPs is localized in distinct speckles in cytoplasm. The speckles contained ubiquitinylated proteins suggesting that they may be aggresomes where proteins targeted by ubiquitin are sequestered or processed for proteolysis. S. frugiperda HSP90 was identified in the 2D gels by Western blotting. Its amount was unchanged during infection. A selective inhibitor of HSP90, 17-AAG, decreased the rate of viral DNA synthesis in infected cells suggesting a supportive role of HSP90 in virus replication.

An important role of the heat shock response in infected cells for replication of baculoviruses.

Baculoviruses serve as a stress factor that can activate both death-inducing and cytoprotective pathways in infected cells. In this report, induction of heat shock proteins (HSPs) of the 70-kDa family (HSP/HSC70) in Sf-9 cells after infection with AcMNPV was monitored by Western blot analysis. Two-dimensional electrophoresis in polyacrylamide gel revealed changes in the cellular pattern of HSP/HSC70s and synthesis of a new member of the HSP/HSC70 family in the infected cells. Although infection with AcMNPV moderately increased the HSP/HSC70 content in cells under standard conditions, the infection potentiated the response to heat shock boosting the HSP/HSC70s content in infected cells several-fold in comparison with uninfected cells. Addition of KNK437, a known inhibitor of inducible HSPs, decreased the rate of viral DNA synthesis in infected cells more than one order of magnitude and markedly suppressed the release of budded viruses indicating the importance of the heat shock response for baculovirus replication.

Ubiquitin-independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity

Recent findings indicate that the ubiquitin‐proteasome system is involved in the pathogenesis of cancer as well as autoimmune and several neurodegenerative diseases, and is thus a target for novel therapeutics. One disease that is related to aberrant protein degradation is multiple sclerosis, an autoimmune disorder involving the processing and presentation of myelin autoantigens that leads to the destruction of axons. Here, we show that brain‐derived proteasomes from SJL mice with experimental autoimmune encephalomyelitis (EAE) in an ubiquitin‐independent manner generate significantly increased amounts of myelin basic protein peptides that induces cytotoxic lymphocytes to target mature oligodendrocytes ex vivo. Ten times enhanced release of immunogenic peptides by cerebral proteasomes from EAE‐SJL mice is caused by a dramatic shift in the balance between constitutive and β1ihigh immunoproteasomes in the CNS of SJL mice with EAE. We found that during EAE, β1i is increased in resident CNS cells, whereas β5i is imported by infiltrating lymphocytes through the blood‐brain barrier. Peptidyl epoxyketone specifically inhibits brain‐derived β1ihigh immunoproteasomes in vitro (kobs/[I] = 240 M‐1s‐1), and at a dose of 0.5 mg/kg, it ameliorates ongoing EAE in vivo. Therefore, our findings provide novel insights into myelin metabolism in pathophysiologic conditions and reveal that the β1i subunit of the immunoproteasome is a potential target to treat autoimmune neurologic diseases.—Belogurov Jr., A., Kuzina, E., Kudriaeva, A., Kononikhin, A., Kovalchuk, S., Surina, Y., Smirnov, I., Lomakin, Y., Bacheva, A., Stepanov, A., Karpova, Y., Lyupina, Y., Kharybin, O., Melamed, D., Ponomarenko, N., Sharova, N., Nikolaev, E., Gabibov, A. Ubiquitin‐independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity. FASEB J. 29, 1901‐1913 (2015). www.fasebj.org

Proteasome Functioning in Breast Cancer: Connection with Clinical-Pathological Factors

Breast cancer is one of four oncology diseases that are most widespread in the world. Moreover, breast cancer is one of leading causes of cancer-related deaths in female population within economically developed regions of the world. So far, detection of new mechanisms of breast cancer development is very important for discovery of novel areas in which therapy approaches may be elaborated. The objective of the present study is to investigate involvement of proteasomes, which cleave up to 90% of cellular proteins and regulate numerous cellular processes, in mechanisms of breast cancer development. Proteasome characteristics in 106 patient breast carcinomas and adjacent tissues, as well as relationships of detected proteasome parameters with clinical-pathological factors, were investigated. Proteasome chymotrypsin-like activity was evaluated by hydrolysis of fluorogenic peptide Suc-LLVY-AMC. The expression of proteasome subunits was studied by Western-blotting and immunohistochemistry. The wide range of chymotrypsin-like activity in tumors was detected. Activity in tumors was higher if compared to adjacent tissues in 76 from 106 patients. Multiple analysis of generalized linear models discovered that in estrogen α-receptor absence, tumor growth was connected with the enhanced expression of proteasome immune subunit LMP2 and proteasome activator PA700 in tumor (at 95% confidence interval). Besides, by this analysis we detected some phenomena in adjacent tissue, which are important for tumor growth and progression of lymph node metastasis in estrogen α-receptor absence. These phenomena are related to the enhanced expression of activator PA700 and immune subunit LMP7. Thus, breast cancer development is connected with functioning of immune proteasome forms and activator PA700 in patients without estrogen α-receptors in tumor cells. These results could indicate a field for search of new therapy approaches for this category of patients, which has the worst prognosis of health recovery.

Proteotoxic stress induced by Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda Sf9 cells

Baculovirus AcMNPV causes proteotoxicity in Sf9 cells as revealed by accumulation of ubiquitinated proteins and aggresomes in the course of infection. Inhibition of proteasomes by lactacystin increased markedly the stock of ubiquitinated proteins indicating a primary role of proteasomes in detoxication. The proteasomes were present in Sf9 cells as 26S and 20S complexes whose protease activity did not change during infection. Proteasome inhibition caused a delay in the initiation of viral DNA replication suggesting an important role of proteasomes at early stages in infection. However, lactacystin did not affect ongoing replication indicating that active proteasomes are not required for genome amplification. At late stages in infection (24-48 hpi), aggresomes containing the ubiquitinated proteins and HSP/HSC70s showed gradual fusion with the vacuole-like structures identified as lysosomes by antibody to cathepsin D. This result suggests that lysosomes may assist in protection against proteotoxicity caused by baculoviruses absorbing the ubiquitinated proteins.

New approach to study of T cellular immunity development: parallel investigation of lymphoid organ formation and changes in immune proteasome amount in rat early ontogenesis.

The expression pattern and distribution of proteasome immune subunits LMP7 and LMP2 in the developing rat spleen and liver as well as the periarterial lymphoid sheath formation were investigated. LMP7 and LMP2 were detected by immunoblotting in the spleen on the 21st embryonic day and during the first postnatal days in equal amounts. Their levels increased by the 8th and 18th postnatal days. Double immunofluorescent labeling the spleen cells revealed LMP7 and LMP2 in T and B lymphocytes localized in the red pulp in embryogenesis. Few T lymphocytes were discovered in periarterial zones on the 8th postnatal day. T lymphocytes filled these zones and formed lymphoid sheaths by the 18-19th day. In the liver, LMP7 and LMP2 were revealed by the 17-19th postnatal day. Immunofluorescent analysis showed their presence in hepatocytes at this period. The data suggest that T cell-mediated immune response in relation to hepatocytes is possible beginning from 18th to 19th postnatal day.

Changes in proteasome pool in human papillary thyroid carcinoma development

Searching the antitumor drug targets among proteasomes, “ubiquitous” enzyme systems, may provide a new impulse to the antitumor drug discovery. In this study, changes in the proteasome pool in the development of human papillary thyroid carcinoma were determined. Proteasome activities were evaluated by hydrolysis of commercial fluorogenic peptides. Changes in the expression of the total proteasome pool, proteasome 19S activator and proteolytic constitutive subunits X(β5), Y(β1) and immune subunits LMP7 (β5i) and LMP2 (β1i) were investigated by Western blotting. The distribution of the proteasome subunits in thyroid gland cells was detected by immunohistochemistry. It was shown that the chymotrypsin- and caspase-like activities as well as the expression of the total proteasome pool, proteasome 19S activator and immune subunits increased gradually in the tumors at the T2N0M0 and T3N0M0 stages in comparison with the control tissues. Among the structures studied, the expression of the 19S activator and immune proteasomes, which contain the LMP2 (β1i) subunit, was enhanced to the largest degree in tumor cells. The data obtained may be implicated in a new therapeutic strategy. Taking into consideration the antitumor function of the immune proteasomes, we advance the 19S activator as the target for the development of a novel antitumor therapy.

Proteomics of the 26S proteasome in Spodoptera frugiperda cells infected with the nucleopolyhedrovirus, AcMNPV.

Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 β subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection.

Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones.

The induction of heat shock proteins in baculovirus infected cells is well documented. However a role of these chaperones in infection cycle remains unknown. The observation that HSP70s are associated with virions of different baculoviruses reported by several researchers suggests that HSPs might be structural components of viruses or involved in virion assembly. These hypotheses were examined by using a novel inhibitor of the ATPase and chaperoning activity of HSP/HSC70s, VER-155008. When VER-155008 was added early in infection, the synthesis of viral proteins, genome replication and the production of budded virions (BV) were markedly inhibited indicating the dependence of virus reproduction on host chaperones. However, BV production was unaffected when VER-155008 was added in the mid-replication phase which is after accumulation of products required for completion of the viral DNA replication. These results suggest that the final stages in assembly of BV and their egress from cells do not depend on chaperone activity of host HSP/HSC70s.

Detection of active proteasome structures in brain extracts: proteasome features of August rat brain with violations in monoamine metabolism

The aim of this work was to detect changes in proteasome pools of brain parts of August rats with monoamine metabolism violations in comparison with that of control Wistar rats. To reveal active proteasome structures, a method of native electrophoresis for the analysis of crude tissue fractions was developed. By means of this method and following Western blotting, the most pronounced changes in reorganization of proteasome structures were detected in proteasome pool of the brain cortex of August rats. Main findings are the enhanced expression of immune proteasome subtypes containing proteolytic subunit LMP2 and activator PA28αβ as well as immune proteasome subtypes containing proteolytic subunit LMP7 and activator PA700 and simultaneously decreased expression of subtypes with subunit LMP2 and activator PA700 in the brain cortex of August rats compared to that of Wistar rats. These results were indirectly confirmed by SDS PAGE method followed by Western blotting, which showed the increased quantities of immune subunits and proteasome activators in the brain cortex of August rats compared to that of Wistar rats. Immune proteasomes were revealed by immunohistochemistry in neurons, but not in glial cells of August and Wistar rat cortex. The detected reorganization of proteasome pools is likely to be important for production of special peptides to provide the steady interaction between neurons and adaptation of central nervous system to conditions caused by monoamine metabolism deviations.