[The Effects of the Hydrogen Sulfide Donor GYY4137 on the Proteasome Pool of Colorectal Cancer 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 ß5 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.

[Structures of the Mouse Central Nervous System Contain Different Quantities of Proteasome Gene Transcripts].

Proteasomes are multisubunit complexes that degrade most intracellular proteins. Three of the 14 subunits of the 20S proteasome, specifically ß1, ß2, and ß5, demonstrate catalytic activity and hydrolyze peptide bonds after acidic, basic, and hydrophobic amino acids, respectively. Within proteasome, the constitutive catalytic subunits ß1, ß2, and ß5 can be substituted by the immune ßli, ß2i, and ß5i subunits, respectively. However, proteasomes do not always contain all the immune subunits at once; some proteasomes contain both immune and constitutive catalytic subunits simultaneously. Incorporation of immune subunits modifies the pattern of peptides produced by proteasomes. This is essential for antigen presentation and cellular response to stress as well as for a number of intracellular signaling pathways. We have developed a quantitative PCR-based system for the determination of the absolute levels of murine constitutive and immune proteasome subunits gene expression. Using the obtained system, we have estimated the expression levels of genes encoding proteasome subunits in the mouse central nervous system (CNS) tissues. We have shown that the quantity of transcripts of proteasome catalytic subunits in different CNS structures differed significantly. These data allow us to assume that the studied brain regions can be divided into two groups, with relatively "high" (cerebral cortex and spinal cord) and "low" (hippocampus and cerebellum) levels of proteasome subunit genes expression. Moreover, it was possible to distinguish structures with similar and significantly different gene expression profiles of proteasome catalytic subunits. Thus, the gene expression profiles in the cortex, spinal cord, and cerebellum were similar, but different from the expression profile in the hippocampus. Based on the obtained data, we suggest that there are differences in the proteasome pool, as well as in the functional load on the ubiquitin-proteasome system in different parts of the CNS.

[Dynamics of the Functional Activity and Expression of Proteasome Subunits during Cellular Adaptation to Heat Shock].

The ubiquitin-proteasome system (UPS) performs proteolysis of most intracellular proteins. The key components of the UPS are the proteasomes, multi-subunit protein complexes, playing an important role in cellular adaptation to various types of stress. We analyzed the dynamics of the proteasome activity, the content of proteasome subunits, and the expression levels of genes encoding catalytic subunits of proteasomes in the human histiocytic lymphoma U937 cell line immediately, 2, 4, 6, 9, 24, and 48 h after a heat shock (HS). The initial decrease (up to 62%) in the proteasome activity in cellular lysates was revealed, then 10 h after HS the activity began to recover. The amount of proteasomal α-subunits in the cells decreased 2 h after HS, and was restored to 24-48 h after HS. Fluctuations in the levels of mRNAs encoding proteasome catalytic subunits with the maximum expression 2 h after HS and a gradual decrease to 48 h after HS were observed. The average estimated number of mRNA copies per cell ranged from 10 for weakly to 150 for highly expressed proteasome genes. Thus, the recovery efficiency of UPS functionality after HS, which reflects the important role of proteasomes in maintaining cell homeostasis, was evaluated.

[C-terminal lysosome targeting domain of CD63 modifies cellular localization of rabies virus glycoprotein].

The glycoprotein of rabies virus is the central antigen elicited the immune response to infection; therefore, the majority of developing anti-rabies vaccines are based on this protein. In order to increase the efficacy of DNA immunogen encoding rabies virus glycoprotein, the construction of chimeric protein with the CD63 domain has been proposed. The CD63 is a transmembrane protein localized on the cell surface and in lysosomes. The lysosome targeting motif GYEVM is located at its C-terminus. We used the domain that bears this motif (c-CD63) to generate chimeric glycoprotein in order to relocalize it into lysosomes. Here, it was shown that, in cells transfected with plasmid that encodes glycoprotein with c-CD63 motif at the C-terminus, the chimeric protein was predominantly observed in lysosomes and at the cell membrane where the unmodified glycoprotein is localized in the endoplasmic reticulum and at the cell surface. We suppose that current modification of the glycoprotein may improve the immunogenicity of anti-rabies DNA vaccines due to more efficient antibody production.

[Heat-shock protein HSP70 decreases activity of proteasomes in human neuroblastoma cells treated by amyloid-beta 1-42 with isomerized Asp7].

Experimental evidences indicate that heat-shock protein 70 (HSP70) can serve as a prospective therapeutic agent to treat Alzheimer's disease (AD). It has demonstrated a neuroprotective effect in vivo on mice models of AD. Moreover, HSP70 decreases oxidative stress in neurons induced by amyloid-ß (Aß42) and its more toxic form with isomerized Asp7 (isoAß42). The dysfunction of Ubiquitin-proteasome system (UPS) is observed in AD. UPS is responsible for the degradation of the majority of cellular proteins and plays an important role in protecting cells from oxidative stress. Here, we have shown that the incubation of human neuroblastoma cells SK-N-SH with isoAß42 increases the activity of intracellular proteasomes, which are the principal elements of the UPS. On the contrary, the proteasomal activity was decreased in isoAß42-treated cells in the presence of exogenous HSP70. These results highlight the existence of an interplay between Aß peptides, proteasomes, and HSP70.

[A role of long noncoding RNAs in carcinogenesis].

The review describes the changes observed in long noncoding RNA (lncRNA) content and function at various stages of carcinogenesis, as well as the prospects of lncRNA application in cancer prognosis.

[A new immuno-PCR format for serological diagnosis of colon cancer].

Anew immuno-PCR format is described that is based on detection of membrane protein CDH17 in serum exosomes. Format application allows distinction between sera samples of healthy donors and colon cancer patients. Obtained results open a possibility of serological colon cancer diagnosis in high risk groups.

[RPN4 the yeast transcription factor promotes the complex defence against methyi, methanesulfonate].

Methyl methanesulfonate (MMS) is an alkylating agent commonly used in models of genotoxic stress. It methylates bases in DNA but also leads to oxidative stress. The transcription factor Rpn4 protects yeast cells from toxic effect of MMS. Although Rpn4 is a major regulator of ubiquitin-proteasome system (UPS), a number of data points to its participation in the stress response regardless of the UPS. We have demonstrated that under the methyl methanesulfonate stress Rpn4 promotes the regulation of several genes involved in DNA repair, antioxidant response and glucose metabolism. We suggest a mechanism of complex action of Rpn4 in the stress response.

[Activation of immunoproteasome subunit genes transcription in mice monocytes infected with different strains of mycobacteria].

Immunoproteasomal processing of mycobacterial antigens is necessary to control the infection and to protect the organism from development of active form of tuberculosis. Here we investigate the activation of immunoproteasome subunit genes transcription in peritoneal monocytes of C57Bl/6 mice infected with vaccine M. bovis BCG and virulent strain M. tuberculosis H37Rv. The level of transcription of LMP2, LMP7, MECL1 subunits didn't increase for one and two days after a single infection. Two rounds of infection with BCG strain M. bovis led to enhancement of the only LMP7 subunit gene transcription. However after subsequent infection of monocytes with vaccine followed by virulent strain infection the dramatic rise of all immunoproteasomal subunit genes transcription was observed. Activation of transcription of the gene coding the PA28alpha subunit of regulatory complex PA28 was observed only after a single infection of monocytes with strain M. bovis BCG. Thus, vaccination with strain M. bovis BCG promotes effective activation of immunoproteasomal genes in case of subsequent contact with virulent strain M. tuberculosis H37Rv.

[Serological diagnosis of tumors: methods of marker's search].

This review describes the most popular methods of search for serological markers of tumors that are used in clinical setting, provided with comparison of their efficiency.

[Enteric alpha defensin 5 is secreted into the blood stream by colon tumors].

It was demonstrated that enteric alpha-defensin 5 is undetectable in five blood serum samples of healthy donors, whereas its processed form is present in two out of five serum samples of colon cancer patients. Obtained results open a possibility of serological diagnosis of colon tumors in high risk cancer patients.

[The role of exosomes and microvesicles in carcinogenesis].

This review summarizes current knowledge on the role of tumor exosomes and microvesicles in progression, metastasis, and angiogenesis of tumors, as well as in suppression of adaptive and innate immunity.

[A new construct of dna reporter for immuno-PCR].

A new construct of DNA reporter has been designed for protein quantification by immuno-PCR. It has been shown that amplification efficiency of a reporter that contains a fragment of human adenovirus 2 flanked by homoprimer sequences is much higher vs. standard PCR format based on use of two different primers. Application of a new construct and its homoprimer-based detection opens a way to a significant increase in the immuno-PCR sensitivity and the efficiency of single molecule PCR.

[DNA vaccine encoding alpha-fetoprotein with fused ornithine decarboxylase degradation signal significantly suppresses hepatocellular carcinoma growth in mice].

Hepatocellular carcinoma (HCC) is among the most frequent malignancies in humans. HCC therapy is not efficient and the usual outcome is poor. In this regard, novel approaches to treat and prevent HCC are urgently needed. The Alpha-fetoprotein (AFP) is a serological marker of HCC. Recently it has been shown, that the DNA vaccines expressing AFP are capable in generating immune response against AFP. However, both, the immunization procedures and DNA vaccines used before were complex and not always very effective. We have shown that DNA vaccine encoding HIV-1 reverse transcriptase (RT) with fused ornithine decarboxylase (ODC) degradation signal induced a strong Th-1 immune response against RT in mice. Using this approach we designed a set of novel DNA vaccines bearing AFP and ODC degradation signal. Results obtained on transfected cells demonstrated efficient expression and fast proteasomal degradation of the recombinant AFP. The anti-tumor immune response stimulation was shown in immunized animals and most importantly a notable retardation of tumor growth was observed as a result of protective vaccination.

[Identification of proteins overexpressed in malignant gastric tumors: comparison of the results of 2-De and bioinformatics search].

Comparison of protein expression in intestinal and diffuse stomach tumors by 2D gel electrophoresis led to identification of three proteins (SOD2, S100A6, and TXN), which are overexpressed in tumors as compared to normal controls. It was shown, that overexpression of proteins SOD2 and TXN occurs much more frequently in diffuse tumors than in intestinal ones. A control panel of eleven proteins overexpressed in stomach tumors has been selected based on the data of comparative 2D analysis described in the literature. Bioinformatics search for mRNAs encoding proteins from the control panel in Oncomine database (which contains the results of determination of mRNA transcription level in tumor vs. normal samples) demonstrated the coincidence of proteomic and transcriptomic data for seven out of 11 proteins.

[Critical amino acids of ornitin decarboxylase degron: the presence and C-terminal arrangement is insufficient for alfa-fetoprotein degradation].

Mouse ornithine decarboxylase (ODC) degrades in proteasome in an ubiquitin-independent manner with an averagehalf-life of 2 h. The 37 amino acid long C-terminal fragment known as a degradation signal (degron) is responsible for the effective degradation of ODC. Recently, amino acids being critical for degradation in the ODC-degron have been mapped. Mutations of Cys441 and Ala442 led to protein stabilization, while a substitution of other amino acids composing ODC-degron had almost no effect on the protein turnover; whereas insertions or deletions in region between Ala442 and ODC C-terminus diminished greatly rate of protein degradation, e.g. positioning of the key amino acids from the C-terminus was shown to be crucial. Using these data we introduced both key amino acids into the alfa-fetoprotein with truncated exportation signal (deltaAFP), at the same distance from the C-terminus as they being in the ODC (deltaAFPCAG and deltaAFPLCAG). Removal of N-terminal exportation signal prevented secretion of modified proteins. Using in silico approach we demonstrated no significant difference in hydrophobicity or secondary structure between C-terminus of deltaAFP and mutated proteins. The degradation kinetics of deltaAFP, deltaAFPCAG, deltaAFPLCAG in cyloheximide-chase and proteasome inhibition assay (using MG132) was identical. Obtained results suggest that introduced substitutions are insufficient for effective recognition of mutated deltaAFP by26S proteasome. We assume thatadditional amino aci ds composing ODC-degron or their combine action could also affect degradation. Besides that, one cannot exclude that conformation of the mutated deltaAFP limits its C-terminus accessibility to proteasome.

[Identification of protein markers for serum diagnosis of cancer based on microRNA expression profiling].

A new algorithm has been developed for bioinformatics search of putative serum markers of cancer, which includes: 1) identification of microRNAs that are most often and most significantly overexpressed in tumors; 2) selection of mRNA targets regulated by microRNAs; 3) identification of mRNA targets encoding secreted proteins; 4) comparative analysis of mRNA transcription levels in normal and tumor tissues. Application of the algorithm led to discovery of seven putative serum markers of colon cancer: ADAMTS14, ANGPT2, CCL7, DEFA5, MMP11, MMP14, and PLAU. Experiments demonstrated that production of two out of seven proteins (MMP14 and DEFA5) is significantly increased in colon tumors vs. normal samples.

Identification of proteins overexpressed in papillary thyroid tumors.

A modified method of proteome comparative analysis based on preliminary removal of cell structural proteins by extraction using salt buffer and subsequent separation of extracts by two-dimensional gel electrophoresis was developed. Identification of differentially expressed proteins by mass spectrometry has revealed three proteins with noticeably increased level of synthesis in most samples of papillary thyroid tumors compared to normal tissues. An increase in ubiquitin content was found for the first time. Oncomarker search efficiencies by two-dimensional gel electrophoresis and bioinformatic search were compared.

[Comparison of 2D analysis and bioinformatics search efficiency for colon cancer marker identification].

Modification of 2D analysis protocol was developed, based on preliminary removal of major cellular proteins by extraction with buffer saline and elimination of high molecular weight proteins by gel filtration. This approach allowed identification of 12 proteins with increased expression levels in tumors versus normal tissues. Increase in expression levels of the eight proteins in colon tumors was discovered for the first time. We performed comparison of marker search efficiency by 2D analysis and SAGE in a control panel of 19 putative colon cancer markers, discovered by us previously and at the same time independently identified by other authors. Results of 2D analysis of control panel completely coincided with published data, as compared to search in SAGE database, which allowed identification of only one third of markers.

Regulation of Immunogen Processing: Signal Sequences and Their Application for the New Generation of DNA-Vaccines.

Immunization with naked genes (DNA-immunization) is a perspective modern approach to prophylactic as well as therapeutic vaccination against pathogens, as well as cancer and allergy. A panel of DNA immunogens has been developed, some are already in the clinical trials. However, the immunogenicity of DNA vaccines, specifically of those applied to humans, needs a considerable improvement. There are several approaches to increase DNA vaccine immunogenicity. One approach implies the modifications of the encoded immunogen that change its processing and presentation, and thus the overall pattern of anti-immunogen response. For this, eukaryotic expression vectors are constructed that encode the chimeric proteins composed of the immunogen and specialized targeting or signal sequences. The review describes a number of signals that if fused to immunogen, target it into the predefined subcellular compartments. The review gives examples of their application for DNA-immunization.