Ribonucleases are considered as promising tools for anticancer treatment due to their selective cytotoxicity against tumor cells. We investigated a new RNase from B. altitudinis termed BALNASE (Bacillus altitudinis RNase). Balnase is a close homolog of the well-known cytotoxic binase, differing by only one amino acid residue: nonpolar hydrophobic alanine at position 106 in the balnase molecule is replaced by a polar uncharged threonine in binase. The most exciting question is how the physico-chemical properties and biological effects of RNase might be changed by A106T substitution. Here, we have developed a chromatography-based rapid and modern technique for the purification of this new RNase which allowed us to get a protein sample of high quality with specific activity of 1.2×106 units in preparative amounts, suitable for further investigation of its biological properties.

The biological effects of ribonucleases (RNases), such as the control of the blood vessels growth, the toxicity towards tumour cells and antiviral activity, require a detailed explanation. One of the most intriguing properties of RNases which can contribute to their biological effects is the ability to form dimers, which facilitates efficient RNA hydrolysis and the evasion of ribonuclease inhibitor. Dimeric forms of microbial RNase binase secreted by Bacillus pumilus (former B. intermedius) have only been found in crystals to date. Our study is the first report directly confirming the existence of binase dimers in solution and under natural conditions of enzyme biosynthesis and secretion by bacilli. Using different variants of gel electrophoresis, immunoblotting, size-exclusion chromatography and mass-spectrometry, we revealed that binase is a stable natural dimer with high catalytic activity.

The potential of microbial ribonucleases as promising antitumor and antiviral agents, determines today's directions of their study. One direction is connected with biodiversity of RNases. We have analyzed completed and drafted Bacillus genomes deposited in GenBank for the presence of coding regions similar to the gene of an extracellular guanyl-preferring RNase of Bacillus amyloliquefaciens (barnase). Orthologues of the barnase gene were detected in 9 species out of 83. All of these belong to "B. subtilis" group within the genus. B. subtilis itself, as well as some other species within this group, lack such types of RNases. RNases similar to barnase were also found in species of "B. cereus" group as a part of plasmid-encoded S-layer toxins. It was also found that taxonomic states of culture collection strains, which were initially described based on a limited set of phenotypic characteristics, can be misleading and need to be confirmed. Using several approaches such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), sequencing of genes for 16S ribosomal RNA and RNA polymerase subunit beta followed by reconstruction of phylogenetic trees, we have re-identified two RNase-secreting Bacillus strains: B. thuringiensis B-388 which should be assigned as B. altitudinis B388 and B. intermedius 7P which should be renamed as B. pumilus 7P. Therefore, small secreted guanyl-preferring RNases are the feature of "B. subtilis" group only, which is characterized by distinctive lifestyle and adaptation strategies to environment.

Unpredictable influenza pandemics, annual epidemics, and sporadic poultry-to-human avian influenza virus infections with high morbidity and mortality rates dictate a need to develop new antiviral approaches. Targeting cellular pathways and processes is a promising antiviral strategy shown to be effective regardless of viral subtypes or viral evolution of drug-resistant variants. Proteomics-based searches provide a tool to reveal the druggable stages of the virus life cycle and to understand the putative antiviral mode of action of the drug(s). Ribonucleases (RNases) of different origins not only demonstrate antiviral effects that are mediated by the direct RNase action on viral and cellular RNAs but can also exert their impact by signal transduction modulation. To our knowledge, studies of the RNase-affected cell proteome have not yet been performed. To reveal cellular targets and explain the mechanisms underlying the antiviral effect employed by the small extra-cellular ribonuclease of Bacillus pumilus (binase) both in vitro and in vivo, qualitative shotgun and quantitative targeted proteomic analyses of the influenza A virus (IAV) H1N1pdm09-infected A549 cells upon binase treatment were performed. We compared proteomes of mock-treated, binase-treated, virus-infected, and virus-infected binase-treated cells to determine the proteins affected by IAV and/or binase. In general, IAV demonstrated a downregulating strategy towards cellular proteins, while binase had an upregulating effect. With the help of bioinformatics approaches, coregulated cellular protein sets were defined and assigned to their biological function; a possible interconnection with the progression of viral infection was conferred. Most of the proteins downregulated by IAV (e.g., AKR1B1, AKR1C1, CCL5, PFN1, RAN, S100A4, etc.) belong to the processes of cellular metabolism, response to stimulus, biological regulation, and cellular localization. Upregulated proteins upon the binase treatment (e.g., AKR1B10, CAP1, HNRNPA2B1, PFN1, PPIA, YWHAB, etc.) are united by the processes of biological regulation, cellular localization, and immune and metabolic processes. The antiviral activity of binase against IAV was expressed by the inversion of virus-induced proteomic changes, resulting in the inhibition of virus-associated processes, including nuclear ribonucleoprotein export (NCL, NPM1, Nup205, and Bax proteins involved) and cytoskeleton remodeling (RDX, PFN1, and TUBB) induced by IAV at the middle stage of single-cycle infection in A549 cells. Modulation of the immune response could be involved as well. Overall, it seems possible that binase exerts its antiviral effects in multiple ways.

One of the approaches used to eliminate tumor cells is directed destruction/modification of their RNA molecules. In this regard, ribonucleases (RNases) possess a therapeutic potential that remains largely unexplored. It is believed that the biological effects of secreted RNases, namely their antitumor and antiviral properties, derive from their catalytic activity. However, a number of recent studies have challenged the notion that the activity of RNases in the manifestation of selective cytotoxicity towards cancer cells is exclusively an enzymatic one. In this review, we have analyzed available data on the cytotoxic effects of secreted RNases, which are not associated with their catalytic activity, and we have provided evidence that the most important factor in the selective apoptosis-inducing action of RNases is the structural organization of these enzymes, which determines how they interact with cell components. The new idea on the preponderant role of non-catalytic interactions between RNases and cancer cells in the manifestation of selective cytotoxicity will contribute to the development of antitumor RNase-based drugs.