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Self-organized patterning and architecture construction studying is a priority goal for fundamental developmental and stem cell biology. To study the spatiotemporal patterning of pluripotent stem cells of different origins, we developed a three-dimensional embryoid body (EB) differentiation model quantifying volumetric parameters and investigated how the EB architecture formation, patterning, and scaling depend on the proliferation, cavitation, and differentiation dynamics, external environmental factors, and cell numbers. We identified three similar spatiotemporal patterns in the EB architectures, regardless of cell origin, which constitute the EB archetype and mimick the pre-gastrulation embryonic patterns. We found that the EB patterning depends strongly on cellular positional information, culture media factor/morphogen content, and free diffusion from the external environment and between EB cell layers. However, the EB archetype formation is independent of the EB size and initial cell numbers forming EBs; therefore, it is capable of scaling invariance and patterning regulation. Our findings indicate that the underlying principles of reaction-diffusion and positional information concepts can serve as the basis for EB architecture construction, patterning, and scaling. Thus, the 3D EB differentiation model represents a highly reproducible and reliable platform for experimental and theoretical research on developmental and stem cell biology issues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gordeeva, Gordeev and Erokhov.)

Proteasomes hydrolyze most cellular proteins. The standard reaction to determine proteasome activity in cellular lysate or elsewhere contains AMC-conjugated peptide substrate, ATP, Mg 2+ , and DTT. ATP and Mg 2+ 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 Mg 2+ 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 Mg 2+ . Moreover, the substrate degradation efficacy was proportional to the Mg 2+ /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 Mg 2+ on the hydrolysis of AMC-conjugated fluorogenic substrates by the 20S proteasomes should be considered.

Ferritins comprise a conservative family of proteins found in all species and play an essential role in resistance to redox stress, immune response, and cell differentiation. Sponges (Porifera) are the oldest Metazoa that show unique plasticity and regenerative potential. Here, we characterize the ferritins of two cold-water sponges using proteomics, spectral microscopy, and bioinformatic analysis. The recently duplicated conservative HdF1a/b and atypical HdF2 genes were found in the Halisarca dujardini genome. Multiple related transcripts of HpF1 were identified in the Halichondria panicea transcriptome. Expression of HdF1a/b was much higher than that of HdF2 in all annual seasons and regulated differently during the sponge dissociation/reaggregation. The presence of the MRE and HRE motifs in the HdF1 and HdF2 promotor regions and the IRE motif in mRNAs of HdF1 and HpF indicates that sponge ferritins expression depends on the cellular iron and oxygen levels. The gel electrophoresis combined with specific staining and mass spectrometry confirmed the presence of ferric ions and ferritins in multi-subunit complexes. The 3D modeling predicts the iron-binding capacity of HdF1 and HpF1 at the ferroxidase center and the absence of iron-binding in atypical HdF2. Interestingly, atypical ferritins lacking iron-binding capacity were found in genomes of many invertebrate species. Their function deserves further research.

A special problem in the surgery of rectal cancer is connected with a need for appropriate removal of intestine parts, along with the tumor, including the fragment close to the sphincter. To determine the length of fragments to remove, it is necessary to reveal areas without changes in molecule functioning, specific for tumor. The purpose of the present study was to investigate functioning the proteasomes, the main actors in protein hydrolysis, in patient rectal adenocarcinoma and different intestine locations. Chymotrypsin-like and caspase-like activities, open to complex influence of different factors, were analyzed in 43-54 samples by Suc-LLVY-AMC- and Z-LLE-AMC-hydrolysis correspondingly. Both activities may be arranged by the decrease in the location row: cancer→adjacent tissue→proximal (8-20 cm from tumor) and distal (2 and 4 cm from tumor) sides. These activities did not differ noticeably in proximal and distal locations. Similar patterns were detected for the activities and expression of immune subunits LMP2 and LMP7 and expression of 19S and PA28αβ activators. The largest changes in tumor were related to proteasome subtype containing LMP2 and PA28αβ that was demonstrated by native electrophoresis. Thus, the results indicate a significance of subtype LMP2-PA28αβ for tumor and absence of changes in proteasome pool in distal fragments of 2-4 cm from tumor.

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Tumor growth is associated with elevated proteasome expression and activity. This makes proteasomes a promising target for antitumor drugs. Current antitumor drugs such as bortezomib that inhibit proteasome activity have significant side effects. The purpose of the present study was to develop effective low-toxic antitumor compositions with combined effects on proteasomes. For compositions, we used bortezomib in amounts four and ten times lower than its clinical dose, and chose menadione sodium bisulfite (MSB) as the second component. MSB is known to promote oxidation of NADH, generate superoxide radicals, and as a result damage proteasome function in cells that ensure the relevance of MSB use for the composition development. The proteasome pool was investigated by the original native gel electrophoresis method, proteasome chymotrypsin-like activity-by Suc-LLVY-AMC-hydrolysis. For the compositions, we detected 10 and 20 μM MSB doses showing stronger proteasome-suppressing and cytotoxic in cellulo effects on malignant cells than on normal ones. MSB indirectly suppressed 26S-proteasome activity in cellulo, but not in vitro. At the same time, MSB together with bortezomib displayed synergetic action on the activity of all proteasome forms in vitro as well as synergetic antitumor effects in cellulo. These findings determine the properties of the developed compositions in vivo: antitumor efficiency, higher (against hepatocellular carcinoma and mammary adenocarcinoma) or comparable to bortezomib (against Lewis lung carcinoma), and drastically reduced toxicity (LD50) relative to bortezomib. Thus, the developed compositions represent a novel generation of bortezomib-based anticancer drugs combining high efficiency, low general toxicity, and a potentially expanded range of target tumors.

The protein VCP/p97 (also named CDC48 and TER94) belongs to a type II subfamily of the AAA+ATPases and controls cellular proteostasis by acting upstream of proteasomes in the ubiquitin-proteasome protein degradation pathway. The function of VCP/p97 in the baculovirus infection cycle in insect cells remains unknown. Here, we identified VCP/p97 in the fall armyworm Spodoptera frugiperda (Sf9) cells and analyzed the replication of the Autographa californica multiple nucleopolyhedrovirus, AcMNPV, in Sf9 cells in which the VCP/p97 function was inhibited. The specific allosteric inhibitor of the VCP/p97 ATPase activity, NMS-873, did not deplete VCP/p97 in infected cells but caused a dose-dependent inhibition of viral DNA synthesis and efficiently suppressed expression of viral proteins and production of budded virions. NMS-873 caused accumulation of ubiquitinated proteins in a manner similar to the inhibitor of proteasome activity, Bortezomib. This suggests the essential function of VCP/p97 in the baculovirus infection cycle might be associated, at least in part, with the ubiquitin-proteasome system., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., Competing Interests: CONFLICTS OF INTEREST There is no any conflicts of interest.

Accumulation of amyloid-β (Aβ) in neurons accompanies Alzheimer's disease progression. In the cytoplasm Aβ influences activity of proteasomes, the multisubunit protein complexes that hydrolyze the majority of intracellular proteins. However, the manner in which Aβ affects the proteolytic activity of proteasomes has not been established. In this study the effect of Aβ42 and Aβ42 with isomerized Asp7 on activity of different forms of proteasomes has been analyzed. It has been shown that Aβ peptides efficiently reduce activity of the 20S proteasomes, but increase activity of the 20S proteasomes capped with the 19S and/or 11S regulators. Modulation of proteasome activity is mainly determined by the C-terminal segment of Aβ (amino acids 17-42). This study demonstrated an important role of proteasome regulators in the interplay between Aβ and the proteasomes.

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.