Human papillomavirus type 16 (HPV16) belongs to viruses of the high-risk type and is associated by overexpression of E6 and E7 oncoproteins, which determine the oncogenic properties of the virus, such as immortalization and malignant transformation of proliferating epithelial cells. The biogenesis of redox-sensitive proteins E6 and E7 at the early stages of viral infection leads to blocking of the cell antioxidant defense system and ubiquintin-dependent degradation of p53 and Rb tumor suppressors. Maintaining high rates of tumor cell proliferation contributes to an increase in the level of reactive oxygen species (ROS) and a shift in the redox balance towards oxidative processes. Reduced glutathione (GSH) provides antioxidant protection to tumor cells through S-glutathionylation of thiol groups of redox-sensitive proteins, which leads to the appearance of multidrug-resistant forms of cancer. In this regard, drugs restoring redox balance and increasing susceptibility to antitumor therapy are of particular importance. We have established that, Bacillus pumilus RNase (binase) modulates the redox-dependent regulatory mechanisms that ensure tumor cell resistance to apoptosis in HPV-16-positive SiHa cells of cervical squamous cell carcinoma,. Binase in nontoxic concentrations initiates a number of pre-apoptogenic changes, i.e., decreases ROS and reduced glutathione (GSH) levels, suppresses the expression of the E6 oncoprotein, activates the expression of the p53 tumor suppressor, and reduces the mitochondrial potential of tumor cells. Binase-induced disruption of the integrity of the mitochondrial membrane is a signal for activation of the mitochondrial apoptosis pathway.

BACKGROUND: One of the main problems of modern oncotherapy is the lack of selectivity of the antitumor drugs, leading to their systemic toxicity on the body. Targeted expression of therapeutic genes represents a new approach in cancer gene therapy. One of the ways to achieve the selectivity of action of the therapeutic genes towards tumor cells is the use of promoters that are active only in tumor cells, but not in normal cells. AIM: To evaluate the functional activity and specificity of the promoter of the human secretory leukocyte protease inhibitor hSLPI. MATERIALS AND METHODS: The promoter of the hSLPI gene was cloned into the promotorless vector pTurboGFP-PRL. The functional activity and specificity of the promoter were assessed by the expression of the mRNA of the TurboGFP reporter gene and the intensity of its luminescence in A549, MCF-7, HeLa tumor cells and WI-38 normal cells using real-time polymerase chain reaction with reverse transcription and fluorescence analysis, respectively. RESULTS: Despite the literature data, the promoter of the hSLPIa gene demonstrated high transcriptional activity only against HeLa cervical cancer tumor cells. At the same time, in the cells of lung adenocarcinoma A549 and breast cancer MCF-7, as in normal WI-38 cells, a reduced level of promoter activity was observed. CONCLUSION: The data obtained confirm the need for a preliminary assessment of the functional activity of tumor-specific promoters in relation to tumor and normal cells.

Bacillus pumilus ribonuclease (binase) exhibits cytotoxic and oncolytic properties, while causing genotoxic effects at high concentrations. Mutants that have reduced catalytic activity and preserve the antitumor properties of the native enzyme could exert lower toxic side effects. Mutant binase forms with the Lys26Ala and His101Glu single substitutions were obtained by site-directed mutagenesis. A comparative analysis of Escherichia coli- and Bacillus subtilis -based expression systems demonstrated that the latter is better to use to produce the binase mutants. The binase mutants with reduced catalytic activity were isolated and purified to homogeneity by ion exchange chromatography; the maximum yield was 25 mg/L. Catalytic activities of the mutants toward natural RNA-substrates in comparison with those for native binase were estimated at 11% and 0.02%, respectively. Like native binase, the Lys26Ala mutant was found to be cytotoxic to the A549, BT-20, and HuTu 80 tumor cell lines, but did not substantially affect normal WI-38 cells. The His101Glu mutant did not show cytotoxicity.

Small cationic guanyl-preferring ribonucleases (RNases) produced by the Bacillus species share a similar protein tertiary structure with a high degree of amino acid sequence conservation. However, they form dimers that differ in conformation and stability. Here, we have addressed the issues (1) whether the homologous RNases also have distinctions in catalytic activity towards different RNA substrates and interactions with the inhibitor protein barstar, and (2) whether these differences correlate with structural features of the proteins. Circular dichroism and dynamic light scattering assays revealed distinctions in the structures of homologous RNases. The activity levels of the RNases towards natural RNA substrates, as measured spectrometrically by acid-soluble hydrolysis products, were similar and decreased in the row high-polymeric RNA >>> transport RNA > double-stranded RNA. However, stopped flow kinetic studies on model RNA substrates containing the guanosine residue in a hairpin stem or a loop showed that the cleavage rates of these enzymes were different. Moreover, homologous RNases were inhibited by the barstar with diverse efficiency. Therefore, minor changes in structure elements of homologous proteins have a potential to significantly effect molecule stability and functional activities, such as catalysis or ligand binding.

Bacterial ribonuclease binase exhibits a cytotoxic effect on tumor cells possessing certain oncogenes. The aim of this study was to identify the structural parts of the binase molecule that exert cytotoxicity. Out of five designed peptides, the peptides representing the binase regions 21–50 and 74–94 have the highest cytotoxic potential toward human cervical HeLa and breast BT-20 and MCF-7 cancer cells. The peptides B21–50 and B74–94 were not able to enter human lung adenocarcinoma A549 cells, unlike BT-20 cells, explaining their failure to inhibit A549 cell proliferation. The peptide B74–94 shares similarities with epidermal growth factor (EGF), suggesting the peptide’s specificity for EGF receptor overexpressed in BT-20 cells. Thus, the binase-derived peptides have the potential of being further developed as tumor-targeting peptides.

The important role of miRNA in cell proliferation and differentiation has raised interest in exogenous ribonucleases (RNases) as tools to control tumour-associated intracellular and extracellular miRNAs. In this work, we evaluated the effects of the RNase binase from Bacillus pumilus on small non-coding regulatory RNAs in the context of mouse RLS40 lymphosarcoma inhibition. In vitro binase exhibited cytotoxicity towards RLS40 cells via apoptosis induction through caspase-3/caspase-7 activation and decreased the levels of miR-21a, let-7g, miR-31 and miR-155. Intraperitoneal injections of binase in RLS40-bearing mice resulted in the retardation of primary tumour growth by up to 60% and inhibition of metastasis in the liver by up to 86%, with a decrease in reactive inflammatory infiltration and mitosis in tumour tissue. In the blood serum of binase-treated mice, decreases in the levels of most studied miRNAs were observed, excluding let-7g, while in tumour tissue, the levels of oncomirs miR-21, miR-10b, miR-31 and miR-155, and the oncosuppressor let-7g, were upregulated. Analysis of binase-susceptible miRNAs and their regulatory networks showed that the main modulated events were transcription and translation control, the cell cycle, cell proliferation, adhesion and invasion, apoptosis and autophagy, as well as some other tumour-related cascades, with an impact on the observed antitumour effects.

In this work, a comparative analysis of structural organization and biological activity of low-molecular weight ribonucleases (RNases) secreted by Bacillus altitudinis (balnase) and B. licheniformis (balifase) with Bacillus pumilus RNase binase was performed. All three RNases are close homologs; however, binase and balnase differ by only one amino acid residue: polar uncharged threonine at position 106 in the binase molecule is replaced by a non-polar hydrophobic alanine in balnase. RNases share similar physicochemical properties (MW, pI, aliphatic index) but differ in stability of three-dimensional structures that reflects in their ability to inhibit proliferation of cancer cells. Binase forms more stable dimers as compared with balnase and balifase due to the swapping interactions between its molecules that possibly leads to a prolonged antiproliferative effect towards human lung adenocarcinoma A549 cells. The impact of oligomerization in biological effects of RNases is discussed.

Migration of cancer cells from the primary tumor site to nearby tissues is the starting point of the metastatic process. The invasive properties of cells are especially important for carcinomas, since tumor cells need to overcome the basement membrane and go beyond its boundaries to the underlying tissues. Substances that reduce the invasive ability of malignant cells are promising as antimetastatic agents. In the present work, the possibility of inhibiting the ability of different cancer cell lines to migrate under the influence of the Bacillus pumilus ribonuclease (binase) was analyzed using the scratch-wound assay. It was established that binase at non-toxic concentrations (10 µg/mL) reliably suppressed the migratory ability of HuTu 80 human duodenum adenocarcinoma cells incubated with RNase for 48‒72 h. The antimetastatic potential of binase is confirmed by molecular modeling data demonstrating the ability of binase to inhibit cellular metalloproteinases that determine the migration of tumor cells.

Migration of cancer cells from the primary tumor site to nearby tissues is the starting point of the metastatic process. The invasive properties of cells are especially important for carcinomas, since tumor cells need to overcome the basement membrane and go beyond its boundaries to the underlying tissues. Substances that reduce the invasive ability of malignant cells are promising as antimetastatic agents. In the present work, the possibility of inhibiting the ability of different cancer cell lines to migrate under the influence of the Bacillus pumilus ribonuclease (binase) was analyzed using the scratch-wound assay. It was established that binase at non-toxic concentrations (10 μg/mL) reliably suppressed the migratory ability of HuTu 80 human duodenum adenocarcinoma cells incubated with RNase for 48-72 h. The antimetastatic potential of binase is confirmed by molecular modeling data demonstrating the ability of binase to inhibit cellular metalloproteinases that determine the migration of tumor cells.

Aim: Currently, in connection with the deterioration of the epizootic situation of rabies in the world, the question of improving the means of routine laboratory diagnostics of rabies remains highly controversial to obtain results of a high degree of reliability. Especially in demand diagnostic rapid test systems based on enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IFA) methods, the sensitivity and specificity of which depend on the degree of purification of antigenic components. Materials and Methods: In this article, a method is given for obtaining a rabies antigen and the results of an experimental study of the activity of its highly purified fractions were obtained by separation in a stepwise gradient of sucrose (20-60%) by ultracentrifugation at 30,000 g. Results: The study of electrophoretic profiles of antigenic fractions in 12.5% polyacrylamide gel showed that the major polypeptide fractions are located in the range of 44.6-70.0 kDa. According to the results of the tests in ELISA and IFA, Fraction 1 was the most active. We optimized the conditions for the formulation of the reactions, and the o-ptimal working concentrations were determined. Conclusion: The revealed immunochemical characteristics of this fraction will allow using it not only as an antigenic component for diagnostic test systems but also as an immunizing material for obtaining highly specific rabies immunoglobulins.

The N1/T1 RNase superfamily comprises enzymes with well-established antitumor effects, such as ribotoxins secreted by fungi, primarily by Aspergillus and Penicillium species, and bacterial RNase secreted by B. pumilus (binase) and B. amyloliquefaciens (barnase). RNase is regarded as an alternative to classical chemotherapeutic agents due to its selective cytotoxicity towards tumor cells. New RNase with a high degree of structural similarity with binase (73%) and barnase (74%) was isolated and purified from Bacillus licheniformis (balifase, calculated molecular weight 12421.9 Da, pI 8.91). The protein sample with enzymatic activity of 1.5 × 106 units/A280 was obtained. The physicochemical properties of balifase are similar to those of barnase. However, in terms of its gene organization and promoter activity, balifase is closer to binase. The unique feature of balifase gene organization consists in the fact that genes of RNase and its inhibitor are located in one operon. Similarly to biosynthesis of binase, balifase synthesis is induced under phosphate starvation; however, in contrast to binase, balifase does not form dimers under natural conditions. We propose that the highest stability of balifase among analyzed RNase types allows the protein to retain its structure without oligomerization.