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.

Binase Immobilized on Halloysite Nanotubes Exerts Enhanced Cytotoxicity toward Human Colon Adenocarcinoma Cells.

Many ribonucleases (RNases) are considered as promising tools for antitumor therapy because of their selective cytotoxicity toward cancer cells. Binase, the RNase from Bacillus pumilus, triggers apoptotic response in cancer cells expressing RAS oncogene which is mutated in a large percentage of prevalent and deadly malignancies including colorectal cancer. The specific antitumor effect of binase toward RAS-transformed cells is due to its direct binding of RAS protein and inhibition of downstream signaling. However, the delivery of proteins to the intestine is complicated by their degradation in the digestive tract and subsequent loss of therapeutic activity. Therefore, the search of new systems for effective delivery of therapeutic proteins is an actual task. This study is aimed to the investigation of antitumor effect of binase immobilized on natural halloysite nanotubes (HNTs). Here, we have developed the method of binase immobilization on HNTs and optimized the conditions for the enzyme loading and release (i); we have found the non-toxic concentration of pure HNTs which allows to distinguish HNTs- and binase-induced cytotoxic effects (ii); using dark-field and fluorescent microscopy we have proved the absorption of binase-loaded HNTs on the cell surface (iii) and demonstrated that binase-halloysite nanoformulations possessed twice enhanced cytotoxicity toward tumor colon cells as compared to the cytotoxicity of binase itself (iv). The enhanced antitumor activity of biocompatible binase-HNTs complex confirms the advisability of its future development for clinical practice.

Secretome of Intestinal Bacilli: A Natural Guard against Pathologies.

Current studies of human gut microbiome usually do not consider the special functional role of transient microbiota, although some of its members remain in the host for a long time and produce broad spectrum of biologically active substances. Getting into the gastrointestinal tract (GIT) with food, water and probiotic preparations, two representatives of Bacilli class, genera Bacillus and Lactobacillus, colonize epithelium blurring the boundaries between resident and transient microbiota. Despite their minor proportion in the microbiome composition, these bacteria can significantly affect both the intestinal microbiota and the entire body thanks to a wide range of secreted compounds. Recently, insufficiency and limitations of pure genome-based analysis of gut microbiota became known. Thus, the need for intense functional studies is evident. This review aims to characterize the Bacillus and Lactobacillus in GIT, as well as the functional roles of the components released by these members of microbial intestinal community. Complex of their secreted compounds is referred by us as the "bacillary secretome." The composition of the bacillary secretome, its biological effects in GIT and role in counteraction to infectious diseases and oncological pathologies in human organism is the subject of the review.

Extracellular Ribonuclease from Bacillus licheniformis (Balifase), a New Member of the N1/T1 RNase Superfamily.

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.

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.

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.

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.

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.

Barnase and binase: twins with distinct fates.

RNases are enzymes that cleave RNAs, resulting in remarkably diverse biological consequences. Many RNases are cytotoxic. In some cases, they attack selectively malignant cells triggering an apoptotic response. A number of eukaryotic and bacterial RNase-based strategies are being developed for use in anticancer and antiviral therapy. However, the physiological functions of these RNases are often poorly understood. This review focuses on the properties of the extracellular RNases from Bacillus amyloliquefaciens (barnase) and Bacillus intermedius (binase), the characteristics of their biosynthesis regulation and their physiological role, with an emphasis on the similarities and differences. Barnase and binase can be regarded as molecular twins according to their highly similar structure, physical-chemical and catalytic properties. Nevertheless, the 'life paths' of these enzymes are not the same, as their expression in bacteria is controlled by diverse signals. Binase is predominantly synthesized under phosphate starvation, whereas barnase production is strictly dependent on the multifunctional Spo0A regulator controlling sporulation, biofilm formation and cannibalism. Barnase and binase also have some distinctions in practical applications. Barnase was initially suggested to be useful in research and biotechnology as a tool for studying protein-protein interactions, for RNA elimination from biological samples, for affinity purification of RNase fusion proteins, for the development of cloning vectors and for sterility acquisition by transgenic plants. Binase, as later barnase, was tested for antiviral, antitumour and immunogenic effects. Both RNases have found their own niche in cancer research as a result of success in targeted delivery and selectivity towards tumour cells.