Structural and Functional Differences between Homologous Bacterial Ribonucleases.

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.

Anti-Influenza Activity of the Ribonuclease Binase: Cellular Targets Detected by Quantitative Proteomics.

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.

Direct inhibition of oncogenic KRAS by Bacillus pumilus ribonuclease (binase).

RAS proteins function as molecular switches that transmit signals from cell surface receptors into specific cellular responses via activation of defined signaling pathways (Fang, 2015). Aberrant constitutive RAS activation occurs with high incidence in different types of cancer (Bos, 1989). Thus, inhibition of RAS-mediated signaling is extremely important for therapeutic approaches against cancer. Here we showed that the ribonuclease (RNase) binase, directly interacts with endogenous KRAS. Further, molecular structure models suggested an inhibitory nature of binase-RAS interaction involving regions of RAS that are important for different aspects of its function. Consistent with these models, phosphorylation analysis of effectors of RAS-mediated signaling revealed that binase inhibits the MAPK/ERK signaling pathway. Interestingly, RAS activation assays using a non-hydrolysable GTP analog (GTPγS) demonstrated that binase interferes with the exchange of GDP by GTP. Furthermore, we showed that binase reduced the interaction of RAS with the guanine nucleotide exchange factor (GEF), SOS1. Our data support a model in which binase-KRAS interaction interferes with the function of GEFs and stabilizes the inactive GDP-bound conformation of RAS thereby inhibiting MAPK/ERK signaling. This model plausibly explains the previously reported, antitumor-effect of binase specific towards RAS-transformed cells and suggests the development of anticancer therapies based on this ribonuclease.

Two Main Cancer Biomarkers as Molecular Targets of Binase Antitumor Activity.

Cancer is frequently coupled with the disturbance of key signaling pathways. Aberrant activation of the mitogen-activated protein kinase (MAPK) signaling cascade, occurring in over 85% of cancers, is mainly caused by the genetic alterations of its main components-oncogenes EGFR and RAS, and plays a crucial role in cell fate. The importance of EGFR and RAS proteins in a variety of tumors suggests that they would be good therapeutic targets, but at present, no effective targeted therapy against these two oncogenes has been proven. Here, we show that ribonuclease from Bacillus pumilus (binase) inhibits MAPK signaling through direct interaction with EGFR and RAS proteins. This effect contributes to the antitumor potential of binase along with its enzymatic activity. Multitargeticity of binase prevents the development of drug resistance, which is considered a major obstacle to effective anticancer treatment.

The Cytotoxicity of RNase-Derived Peptides.

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 Native Monomer of Bacillus Pumilus Ribonuclease Does Not Exist Extracellularly.

Supported by crystallography studies, secreted ribonuclease of Bacillus pumilus (binase) has long been considered to be monomeric in form. Recent evidence obtained using native polyacrylamide gel electrophoresis and size-exclusion chromatography suggests that binase is in fact dimeric. To eliminate ambiguity and contradictions in the data we have measured conformational changes, hypochromic effect, and hydrodynamic radius of binase. The immutability of binase secondary structure upon transition from low to high protein concentration was registered, suggesting the binase dimerization immediately after translocation through the cell membrane and leading to detection of binase dimers only in the culture fluid regardless of ribonuclease concentration. Our results made it necessary to take a fresh look at the binase stability and cytotoxicity towards virus-infected or tumor cells.

Phylogenetic distribution of extracellular guanyl-preferring ribonucleases renews taxonomic status of two Bacillus strains.

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.

Three-step procedure for preparation of pure Bacillus altitudinis ribonuclease.

Ribonucleases are considered as promising tools for anticancer treatment due to their selective cytotoxicity against tumor cells. We investigated a new RNase from Bacillus altitudinis termed BALNASE (B. 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 × 10(6) units in preparative amounts, suitable for further investigation of its biological properties.

Draft Whole-Genome Sequence of Bacillus altitudinis Strain B-388, a Producer of Extracellular RNase.

Here, we present a draft genome sequence of Bacillus altitudinis strain B-388, including a putative plasmid. The strain was isolated from the intestine of Indian meal moth, a common pest of stored grains, and it is characterized by the production of extracellular RNase, similar to binase, which is of interest for comparative studies and biotechnology.

New insight into secreted ribonuclease structure: binase is a natural dimer.

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.

Draft Whole Genome Sequence of Bacillus pumilus Strain 3-19, a Chemical Mutant Overproducing Extracellular Ribonuclease.

Here, we present a draft genome sequence of Bacillus pumilus strain 3-19. It was derived from soil-isolated B. pumilus 7P using chemical mutagenesis and is characterized by elevated production of extracellular ribonuclease which is known to possess different biological activities with potential of applications in experimental research, medicine, and biotechnology.