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

AbstractBaculovirus 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 (24–48hpi), 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

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

© The Author(s). 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 brainderived proteasomes from SJL mice with experimental autoimmune encephalomyelitis (EAE) in an ubiquitinindependent 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

ВОЗМОЖНАЯ РОЛЬ ПРОТЕАСОМ ПЕЧЕНИ В РЕАЛИЗАЦИИ МЕХАНИЗМОВ ТРАНСПЛАНТАЦИОННОЙ ТОЛЕРАНТНОСТИ

In contrast to the majority of organs in liver non-specific immunity predominates over adaptive one, and in response to the antigen presentation develops preferably not immune reaction but immunological tolerance. Tolerance is considered to provide some processes, such as apoptosis of reactive T-cells, immune deviation and active suppression of immune reactions. At the same time there are the grounds for believing that an important role in regulation of liver immune response is played by proteasomes, intracellular multiprotease protein complexes. This is confirmed by the fact of application of proteasome inhibitor bortezomib as immune suppressor in transplantology. Immune 26S- and 20S-proteoasomes participate in the formation of antigen oligopeptides and play a key role in T-cell immune response. It has been shown that the pool of proteasomes is subjected to significant changes during ontogenesis of immune competent organs. The changes in the pool of proteasosmes occur likely during the development of specific tolerance in transplantation too. The knowledge of the peculiarities of proteasome functioning and regularities of alterations of their shapes will enable the revealing of the mechanisms responsible for either graft rejection or acceptance. В отличие от большинства органов в печени неспецифический иммунитет преобладает над адаптивным, а в ответ на презентацию антигена предпочтительнее развивается не защитная иммунная реакция, а им- мунологическая толерантность. Считается, что толерантность обеспечивают ряд процессов, таких, как апоптоз реактивных Т-клеток, уклонение их от иммунного ответа и активная супрессия иммунных ре- акций. В то же время есть основания полагать, что важную роль в регуляции иммунного ответа печени играют протеасомы, внутриклеточные мультипротеазные белковые комплексы. Об этом свидетельствует факт применения протеасомного ингибитора бортезомиба в трансплантологии в качестве иммуносупрес- санта. Иммунные 26S- и 20S-протеасомы участвуют в образовании антигенных олигопептидов и выпол- няют ключевую роль в Т-клеточном иммунном ответе. Было показано, что пул протеасом подвергается существенным изменениям в процессе онтогенеза иммуно-компетентных органов. Знание особенностей функционирования протеасом и закономерностей изменения соотношения их форм позволит выявить механизмы, отвечающие за направленность вектора иммунного ответа на отторжение или принятие трансплантата.

THE PHARMACOLOGICAL VARIATION OF THE MORPHINE BY THE INHIBITORS OF THE CALCIUM-DEPENDENT ENDOPEPTIDES

The object of investigation: 670 he-rats of the Wistar line. The purpose of the work: searching for the ways of the directed correction of the morphine effect by acting the inhibitors of the calcium-dependent endopeptidases. The inhibitor Ca*992*99+-dependent endopeptidase - antipaine reduces the increment of the chromatinetranscription activity, to be induced by the morphine in neurons of the Central Nervous System (CNS) different structures. The density and sensitivity of delta -opiate receptors in the state of rejection of the morphine-dependent rats change; lupeptine prevents from the change of state of the delta -opiate structures. Antipaine and lupeptine intensify the analgetic properties of morphine with the combined introduction, reduce the expression of the morphine rejection syndrome of the morphine-dependent rats. The subject of introduction: the means for elimination of abstinent syndrome with the opia narcomanias, the preparation for analgesia on the base of morphine. The field of application: the narcology, the oncology, the military medicineAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

P13

The tumor microenvironment plays an important role in the progression of cancer and may be regulated by proteolysis, including the proteasomes. The proteasomes modify the biologically important molecules involved in the pathogenesis and progression of a variety of malignancies, including breast cancer (BC). The aim of this study was to investigate the features of the subunit composition of proteasomes in the tumor and its microenvironment of breast cancer. Material and Methods: The material for investigation was samples of tumor tissue of invasive ductal breast cancer. The study was conducted to estimate the distribution of the total pool of proteasome, proteasomes activator PA700, immune proteasome forms containing LMP7 and/or LMP2 subunit in tumor cells and stromal component using immunofluorescence. Furthermore, there was evaluated the distribution of the proteasome in the tumor. The expression of immune proteasomes and proteasomes activators in the cells was studied by immunofluorescence labeling of the cells by antibodies to immune proteasome subunits and cell markers. Fluorescence was analyzed by using a fluorescence microscope DM RXA2 (“Leica”, Germany) and confocal microscope TCS SP (“Leica”, Germany). The specificity of the primary antibodies was confirmed by check samples, at which the reaction was carried out only with the second antibody. No cross reaction between the first and second antibodies was tested by incubation of each primary antibodies with the opposing second antibodies. In addition, there was carried out labeling the cell nuclei by reagent Hoechst 33,342. Results: It was found that the tumor cells comprise immune proteasomes, also PA700 and PA28αb activators. Activator PA28αb and immune proteasomes are localized in the cytoplasm of tumor cells, whereas α1, 2, 3, 5, 6, 7 subunits and PA700 activator detected in the cytoplasm and in the nuclei of tumor cells. Availability of α1, 2, 3, 5, 6, 7 subunits in the nuclei of tumor cells shows the expression of constitutive proteasome subunits. Stromal cells are characterized by a high ratio of the α1, 2, 3, 5, 6, 7 subunits to the immune LMP2 subunit as compared to cells of invasive ductal carcinoma. This means that the pool of proteasome in tumor cells of invasive ductal cancer is enriched by the immune proteasomes as compared to stromal cells. Thus, samples of invasive ductal breast cancer contain predominantly tumor cells enriched by immune proteasomes, activators PA700 and PA28αb. The presence of proteasomes in stromal component indicates that the tumor microenvironment also has active processes of proteolysis, with involving proteasome system. Probably the processes occurring in the stromal component contribute to the output of the proteasome into the extracellular space, which is confirmed by other researchers about the existence of circulating proteasome pools and their further participation in dissemination of cancer. This work was supported by the Russian Foundation for Basic Research (Grant numbers 13-04-00169)

POSSIBLE ROLE OF LIVER PROTEASOMES IN THE REALIZATION OF MECHANISMS OF TRANSPLANTATION TOLERANCE

In contrast to the majority of organs in liver non-specific immunity predominates over adaptive one, and in response to the antigen presentation develops preferably not immune reaction but immunological tolerance. Tolerance is considered to provide some processes, such as apoptosis of reactive T-cells, immune deviation and active suppression of immune reactions. At the same time there are the grounds for believing that an important role in regulation of liver immune response is played by proteasomes, intracellular multiprotease protein complexes. This is confirmed by the fact of application of proteasome inhibitor bortezomib as immune suppressor in transplantology. Immune 26S- and 20S-proteoasomes participate in the formation of antigen oligopeptides and play a key role in T-cell immune response. It has been shown that the pool of proteasomes is subjected to significant changes during ontogenesis of immune competent organs. The changes in the pool of proteasosmes occur likely during the development of specific tolerance in transplantation too. The knowledge of the peculiarities of proteasome functioning and regularities of alterations of their shapes will enable the revealing of the mechanisms responsible for either graft rejection or acceptance

Iron metabolic pathways in the processes of sponge plasticity

© 2020 Finoshin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The ability to regulate oxygen consumption evolved in ancestral animals and is intrinsically linked to iron metabolism. The iron pathways have been intensively studied in mammals, whereas data on distant invertebrates are limited. Sea sponges represent the oldest animal phylum and have unique structural plasticity and capacity to reaggregate after complete dissociation. We studied iron metabolic factors and their expression during reaggregation in the White Sea cold-water sponges Halichondria panicea and Halisarca dujardini. De novo transcriptomes were assembled using RNA-Seq data, and evolutionary trends were analyzed with bioinformatic tools. Differential expression during reaggregation was studied for H. dujardini. Enzymes of the heme biosynthesis pathway and transport globins, neuroglobin (NGB) and androglobin (ADGB), were identified in sponges. The globins mutate at higher evolutionary rates than the heme synthesis enzymes. Highly conserved iron-regulatory protein 1 (IRP1) presumably interacts with the iron-responsive elements (IREs) found in mRNAs of ferritin (FTH1) and a putative transferrin receptor NAALAD2. The reaggregation process is accompanied by increased expression of IRP1, the antiapoptotic factor BCL2, the inflammation factor NFκB (p65), FTH1 and NGB, as well as by an increase in mitochondrial density. Our data indicate a complex mechanism of iron regulation in sponge structural plasticity and help to better understand general mechanisms of morphogenetic processes in multicellular species

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

AbstractEight 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

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